Theworksho pwa sorganize db y the InternationalUnio no fForestr yResearc h Organizations and sponsoredb yth eNort hAtlanti c Treaty Organization,th e Heidemij (RoyalNetherland s LandDevelopmen t andReclamatio n Society)an d theMinistr y ofAgricultur e andFisherie s ofth eNetherlands . 'O Resistance to diseases and pests infores t trees

Proceedings of the Third International Workshop on the Genetics of Host-Parasite Interactions in Forestry, Wageningen,th e Netherlands, 14-21 September 1980

Editedb y H.M. Heybroek, B.R.Stepha n and K.vo nWeissenber g

X pudoc Y Centre for Agricultural Publishing and Documentation Wageningen - 1982

YS "v lb1-*. "2- 2-fc? - -£> ' PrecedingWorkshops :

Genetic improvement fordiseas e andinsec tresistanc e of foresttrees , 30Augus tt o 11Septembe r 1964;a NAT O andNS FAdvance d StudyInstitute . Pennsylvania StateUniversity ,Universit y Park,Pennsylvania ,USA . Proceedings:H.D .Gerhold ,E.J . Schreiner,R.E .McDermot t& J.A .Winiesk i (Eds):Breedin gPest-Resistan t Trees.Pergamo nPress ,Oxford . 1966.50 5p .

Basicbiolog y and international aspectso frus tresistanc e infores t trees, 17-24Augus t 1969;a NATO-IUFR OAdvance d Study Institute. University of Idaho,Moscow , Idaho,USA . Proceedings:R.T .Bingham , R.J.Hof f& G.I .McDonal d (Eds):Biolog y ofRus t Resistance inFores tTrees .USD A ForestService ,Miscellaneou s Publication No 1221.1972 .68 1p .

ISBN 9022 0 07944

© Centre forAgricultura l Publishing andDocumentatio n (Pudoc), Wageningen,1982 .

'Nopar to fthi spublicatio nma yb ereproduced , stored ina retrieva l sys­ tem,o rtransmitte d inan y formo rb y anymeans ,mechanical ,photocopying , recording,o rotherwise ,withou tth eprio rwritte npermissio n ofth e publisherPudoc ,P.O . Box4 ,670 0A AWageningen ,Netherlands .

Printed inth eNetherlands . Contents

Breeding for balance? An introduction ix

TOPIC 1: INTRODUCTORY

B.J. Zobel: Theworld' s need forpest-resistan t foresttree s 1 I.A.S. Gibson, J. Burleu <£ M. R. Speight: The adoptiono f agricultural practices forth e developmento fheritabl e ' resistance topest s andpathogen s in forestcrop s 9

TOPIC 2: HOST-PARASITE INTERACTION AND THE ENVIRONMENT

W.H. Parry: Therol eo fth eenvironmen t inhost-insec t interactions 22 W.H. Parry: Variability inconife rneedl e freezing asa resistance factort o aphids 32 P. Schutt: Trees,fungi ,an denvironmen t 39 M. Ridé: Environmental factors affectinghost-bacteri a inter­ actions inbacteria l diseases of foresttree s and shrubs 48 H.S. McNabb Jr., R.B. Hall &M.E. Ostry: Biological and physical modifications ofth eenvironmen t andth eresult ­ ing effectupo nth ehost-parasit e interactions inshort - rotation treecrop s 60

TOPIC 3: HOST-PARASITE INTERACTIONS AND GENETICS ON THE INDIVIDUAL PLANT LEVEL

M.K. Harris: Genes forresistanc e to insects inagricultur e witha discussio n ofhost-parasit e interactions in Carya 72 R.R. Nelson: Ongene s fordiseas e resistance inplant s 84 TOPIC 4: RESISTANCE MECHANISMS AND THEIR GENETIC CONTROL

G.S. Puritch <£G.D. Jensen: Non-specifichost-tre eprocesse s occurring inbar k inrespons et odamag e andthei rrol e indefens e 94 A.L. Shigo: Treesresistan tt osprea d ofdeca y associated withwound s 103 F.F. Jewell, D.C. Jewell & C.H. Walkinshaw: Histopathology ofanatomica lmechanism s forresistanc e to fusiform rusti nslas hpin e 110 B. B. Kinloch: Mechanisms and inheritance ofrus t resistance inconifer s 119 R. Siwecki, A. Werner, Z. Krzan & F. Miodzianowski: Resistance mechanisms in interactionsbetwee npoplar s andrus t 130 D.M. Elgersma: Resistance mechanisms ofelm st oDutc hel m disease 143 Y. Mamiya: Pinewil tan dpin ewoo dnematode : histopathological aspects ofdiseas e development 153

TOPIC 5: BIOCHEMICAL ASPECTS IN RESISTANCE BREEDING

M.R. Bridgen & J.W. Hanover: Indirect selection for pest resistanceusin gterpenoi d compounds 161 H.J. Schuck: Monoterpenes andresistance.o fconifer s to fungi 169 F. Scholz & B.R. Stephan: Buffering ofp H inplan torgan s and resistance against fungi 176 Z.C. Chen & P.D. Manion: Histoenzymologicalapproac hfo rde ­ tectinghost-parasit e interactions in foresttree s caused byheart-ro t diseases 187 G.I. Forrest: Preliminary work onth erelatio nbetwee nresis ­ tancet o Fomes annosus andth emonoterpen e composition ofSitk a spruce resin 194 E.I. Ladejtschikova & G.M. Pasternak: Biochemical aspects of the resistance of Pinus sylvestris to Fomes annosus 198 P.J. Charles, A. Delplanque, A. Marpeau, C. Bernard-Dagan & M. Arbez: Susceptibility ofEuropea nblac kpin e (Pinus nigra) toth eEuropea npin e shootmot h (Rhyacionia buoliana): variations of susceptibility atth eprovenanc e andindividua l levelo fth epin e andeffec to fterpen e composition 206

TOPIC 6: GENETIC VARIATION IN FUNGAL PATHOGENICITY

CE. Caten: Geneticbasi s forvariatio n in fungi 213 CM. Brasier: Genetics ofpathogenicit y in Ceratocystis ulmi andit ssignificanc efo rel mbreedin g 224 H. R. Powers, Jr. :Pathogeni cvariabilit ywithi nth egenu s Cronartium 236 G.A. Snow, W.L. Nance & E.B. Snyder: Relativevirulenc e of Cronartium quercuum f. sp. fusiforme on loblollypin e fromLivingsto nparis h 243 R.D. Raabe: Variationi npathogenicit yan dvirulenc ei n Fomes annosus and Armillaria mellea 251 L. Dimitri: Somehost/parasit e relationshipsbetwee nNorwa y spruce (Picea abies) and Fomes annosus 260 C. Delatour: Behaviouro f Fomes annosus inth este mo fNorwa y sprucean di nth elaborator y 268 C.H. Driver, D.R. Morse & R.L. Edmonds: Detectiono fdiseas e resistancei nDouglas-fi r seedlingsan dvariatio ni n pathogenicityi n Phellinus weirii bymonitorin gwate r stress after inoculation 275 L.D. Dwinell & J.B. Barrows-Broaddus: Variability ofhos tan d pathogeni nth epitc h canker complex 283

TOPIC 7: POPULATION GENETICS AND BREEDING FOR BALANCE

N. Lorimer: Pestoutbreak sa sa functio no fvariabilit yi n pestsan dplant s 287 W.J. Mattson, N. Lorimer &A.A . Leary: Roleo fplan tvariabil ­ ity (traitvecto r dynamicsan ddiversity )i nplant/herbi ­ vore interactions 295 E.G. Brittain: Stabilityi na multi-componen thost-pathoge n model 304 D. Krusche: Selectioni nhost-parasit e systems 312 C. Person, R. Fleming & L. Cargeeg: Non-specific interaction basedo npolygene s 318 H.M. Heybroek: Monocultureversu smixture :interaction s between susceptiblean dresistan ttree si na mixe d stand 326 W.J. Libby: Whati sa saf enumbe ro fclone spe rplantation ? 342 A. Segal, J. Manisterski, J. A. Browning, G. Fischbeck & I. Wahl: Balancei nindigenou splan tpopulation s 361 J. A. Browning, J. Manisterski, A. Segal, G. Fischbeck

K. Ohba: Breedingo fpine s forresistanc et oth ewoo dnematode , Bursaphelenchus lignicolus 387 G. Bazzigher: TheSwis s£ndothia-resistanc ebreedin gprogra m 396 G.I. McDonald & R.J. Hoff: Engineeringbliste rrust-resistan t westernwhit epin e 404 C. Delatour & Y. Birot: Theinternationa lIUFR Oexperimen to n resistanceo fwhit epine st obliste r rust (Cronartium ribicola). TheFrenc htria l 412 I. Blada: Relativebliste r rustresistanc eo fnativ eand * introduced whitepine si nRomani a 415 B. Zobel: Developing fusiform-resistant treesi nth esouth ­ easternUnite d States 417 H.R. Powers, Jr., S.D. Hubbard & R.L. Anderson: Testing for resistancet ofusifor m rusto fpin e 427 P. Raddi & A. Ragazzi: Italianstudie so nresistanc et opin e blisterrus t (Cronartium flaccidum) 435 R. Jalkanen: Lophodermella sulcigena inclone s andprogenie s ofScot spin ei nFinlan d 441 I. Baumanis, D. Pirags & Z. Spalvins: Resistance trials of Scotchpin eclone si nth eLatvia nSS R 448 0. Martinsson: Impacto fwester n gall rusti nnatura l stands oflodgepol epin e ' 450 R.J. Dinus: Integrating resistancebreedin g andtre eimprove ­ ment 452 G.I. McDonald: Progeny testingo fDouglas-fi r seedlingsfo r resistancet owester n sprucebudwor m 460 1. Blada: Testing larch clonesfo r Adelges laricis resistance . 466 W.H. Fogal, E.K. Morgenstern, P. Viidik & C.W. Yeatman: Varia­ tioni nsusceptibilit yo fnativ ean dintroduce d coniferous treest osom e insectso feaster nCanad a 472 M. Ridé : Internationalbacteria l cankertestin gprogramm eo n poplar 478

TOPIC 9: CONCLUDING REMARKS

H.D. Gerhold: Acoevolutionar y viewo fresistanc e breeding andresearc h 487 Recommendations 496

Index of hosts and parasites 498

Vlll Breeding for balance? An introduction

Breeding forresistanc e to aparasit e isbreedin g against aflexibl e adversary: onecanno ttak e itfo rgrante d thatresistance ,onc e obtained, will remain atth e same level.Th eparasit ema y comeu pwit hrar e orne w geneticvariant s thatcircumven tth eresistanc e andrende r thehos tsuscep ­ tible.Therefore ,breedin g forresistanc e toa parasit e is fundamentally different frombreedin g forresistanc e to frost,drought ,sal to rsom e otherenvironmenta l extreme. ' Foresttre ebreeding , at firstsight ,i sparticularl y vulnerablet o changes inth eparasite , asa quic kchang e fromon etre e species to another isdifficul t to achieve infores tmanagemen twithou tbi g losses.Thi si s causedb y the facttha t forestryha s alon gtim e scale compared toagricul ­ ture:th e agriculturalplan tbreede rma yconside r someresistanc e tob e 'stable'i fi tdoe sno terod ewithi ntwent y years,whic h ismor e thanth e normal lifetime ofa cultivar ; formos t foresttrees ,however ,thi s isonl y halfth e lifetime ofa singl ecrop . There isn ocaus e fordespair ,however , forther eclearl y is aceilin g toth epotentia l ofparasite s toadap tt o 'resistant'hosts .I tlook s asi f notal lresistanc e isovercom eb yvariatio no fth eparasite .Man y caseso f resistance inagricultura l cropshav eprove d tob e stableu p tonow .Natur e showstha tresistanc ema yhol d overmuc h longerperiods :popla r rustha s notye tadapte d tooak ,no teve nt o aspen.Fo rth ebreede r iti smos tim ­ portantt o find outi fther e isa patter nt othi sgeneti c adaptability: which changes arepossibl e forth eparasite ,whic h arenot ? Whenw ecompar e the three international gatherings concernedwit hfor ­ esttre ebreedin g forresistance , some differences stand out.A tth e first meeting inPennsylvani a in1964 ,whe nth e artwa s stillyoung ,th epossi ­ bility ofa break-dow n ofresistanc ewa sn omor e thana fa rech o from agriculturalplan tbreedin g experience.Technica lmatter s such as screening techniques hadmos tinterest .Th e secondmeetin gwa s held inth ede no fth e breeders forbliste r rustresistance , inMoscow , Idaho,i n1969 .Thi swa s oneyea r afterVanderplank' s treatise 'DiseaseResistanc e inPlants 'ap ­ peared. Onth ebasi s ofexperience s gained inagricultura l plantbreeding , thatboo kpredict s thatgros s adaptation ofth eparasit e canreadil yb e expected incase s of 'vertical'resistance ,bu ti shighl y unlikely incase s of 'horizontal'resistance .Consequently ,participant s hotly discussed the danger ofephemera l resistances andho wt oavoi d them. Firsthan dexperi ­ encewit hne w strains of forestparasite s was still lacking,however . Thisha s changed, alas. Inth eeleve nyear sbefor e thethir dmeetin g in1980 ,severa l changes inpathogenicit y of foresttre e fungihav ebee n noted: a resistance genei nsuga rpin ewa s overcomeb y ane wstrai ni n Cronartium ribicola; - new and specific strains ofth epopla r rust Melampsora larici-populina havebee n found inAustrali a andNe w Zealand, soon after the fungus appeared inthos ene w territories; infusifor m rust,a certai nincreas eo fpathogenicit ywa s observed onhos tpopulation s thatha dbee nselecte d forhighe r resistance toth e disease; in Ceratocystis ulmi, ahighl y aggressive strain (withtw ovariants ) was found spreading havoc among theelm s inEurop e and adjacentAsia ; a newaggressiv e straino f Gremmeniella abietina seemst opos e abi g threatt o severalpine s innortheaster nNort hAmerica . Itma yb e significanttha t atleas tth e lastthre e examples dono t fit thenea tpatter n ofagricultura l cropswit hthei r stable 'horizontal' and instable 'vertical'resistances .Perhap s insom ecase sw e arewit ­ nessing afine rgeneti c adaptationo fa colonizin gpioneer-parasit e to itsne whabita to na ne wcontinent . Where a finalvictor y overth eparasit eb y anabsolut e andpermanen t resistance doesno t seempossible ,som e sorto fbalanc ebetwee nhos tan d parasite evidently isth ebes ttha tca nb e attained: some situation in whichhos tan dparasit e are inequilibrium ,perhap sb yth epresenc e in thehos tplan to f acombinatio n ofresistanc e genes thatca nb e less• easily overcomeb y theparasite ,o rb yth epresenc e inth ehos tpopulatio n ofgeneti cvariabilit y thatma ydiminis h chances forparasit e change and specialization.Ecologica l factors can also influence sucha balance . Thethem eo fth e198 0Worksho p thereforewas : 'Breeding forbalance' . This turned outt ob e astimulatin g conceptwhic h intrigued andprovoke d theparticipants .Th emeetin gwa s structured sotha tth e earlier sessions laid abasi s forth eday-lon g sessionname d 'breeding forbalance' . Itwa s concluded that abalanc ebetwee nhos t andparasite ,a ta leve ltha ti s acceptable forth e forester,ma yb e avision ,a nanticipatio n and anideal , buttha t iti sa nelusiv e concepttoo ,no t sufficiently concrete to serve asa breedin g goal.A sProfesso rDr .J.C .Zadok s ofth eAgricultura l Uni­ versity inWageninge n askedu s inhi swelcom e address:Doe sbalanc e really exist? Ifso ,ca nyo umeasur e itan dexplai n itquantitatively ? Isi ta staticbalanc eo ra dynami c equilibrium? From the floor cameth equestion : balance forwhom ?Apparentl y severalbalance s orequilibri a arepossible , butno t all aredesirable . We stillhav emuc h to learnabou tth e interactionbetwee nhos tan d parasite, also ata populatio n level.I ti sdifficul tt oloo k intoth e future.Th e leading concepts oftomorro w mayb ebase d ondetail s thatar e nearly overlooked to-day orthe yma y revive discredited theories ofyester ­ day. Even ifw e avoid thewor d 'balance', somegenera l observations onth e currentthinkin g aboutresistanc ebreedin g in foresttree s canb egiven . The search for "theimmun egenotype "i sn o longerth e soleo r final goalo f resistancebreedin g -i fi teve rwas .I ti swidel y realized thateve na moderate degree ofresistanc e can suffice to save acro p andtha t itma yb e anessentia l prerequisite fordesignin g anintegrate d control system, in which adapted spacing,th e avoiding ofcertai n sites,specie smixture ,san ­ itation,chemica l controlmeasures ,thinnin g regime and other silvicultural treatments can allpla y apart . Iti swel l known thatsuc hsystem s tend to fail ifth ehos ti sto o susceptible.Further ,resistanc ebreedin g should be anintegra lpar ti nmos tgenera lbreedin gprograms ,becaus e attention should alwaysb e givent oth epossibilit y ofreducin g known susceptibili­ ties and avoidingne w ones. Ithappen s that innatura lpopulation s offor ­ esttre e species,resistanc e toa certai nparasit e isth erule ,whil econ ­ siderable susceptibility presents itself inselecte d orne w genotypesdur ­ inggeneti c improvemento fth e species. Insuc hcase s itseem squit epos ­ sible torestor e the original and stable resistance.Lastly , immunity or absolute resistance,wheneve r found, isa preciou spropert ywhic h shouldb e used anddeploye dwit h care andcaution . Iti snot ,a ssom ehav ethought , theconclusio n ofa resistanc ebreedin gprogram . TheWorksho p (72participant s from 21countries ,6 5papers )provide da massivebod y ofinformation , theory, and ideas.Thes e added toou runder ­ standingo fhost s andparasite s as abasi s for futurework ,bot h forbreed ­ ingan d for integrated controlo rpes tmanagement . Without liberal funds,i twoul d havebee nimpossibl e tobrin g sucha wide-ranging group ofdistinguishe d specialists and generalists together, sotha t anear-complet e state-of-the-art couldb e developed.W e arever y grateful toou r sponsors forthei r contribution, aswel l ast oou rhost s forthei rmassiv e support:th e 'Dorschkamp'Researc h Institute for Forestry andLandscap ePlanning , andth e International Agricultural Centre,bot h at Wageningen. The atmosphere of interestan dhapp y interactionwhic h reigned during theworksho p regrettably cannotb e transmitted inthi sbook .

TheOrganizin g Committee, HenryD . Gerhold Bent S0egaard HansM .Heybroe k Richard Stephan RobertF .Patto n Kimvo nWeissenber g The world's need for pest-resistant forest trees

BruceJ . Zobel

ProfessorEmeritu san dForestr yConsultant ,Schoo lo fFores tResources ,Nort hCarolin a StateUniversity ,Raleigh ,Nort hCarolina ,US A

ABSTRACT

Thepatter no fpes t attack andth egeneti c flexibility and stronggeneti c control ofresistanc e toman ypest s in forest trees, alongwit h thehig hcos t andeve nimpossibilit y of directchemica l controlsan dth euncertaint y ofbiologica l controls, all indicate thenecessit y fordeveloping'pes tre ­ sistant foresttrees . Thecommonl y cited danger frommonoculture s resulting from useo fgeneticall y improved treeswil lno tb evali d for agoo d foresttre e improvementprogra m inwhic h awid e adaptivebas e hasbee n amajo r objective ofbreeding .Geneti c controlo f economically importantcharacteristic s isgenerall y independent fromtha t foradaptabilit y orpes tresistance ;mos t important characteristics of foresttree s aremultigeni c and areinher ­ ited independently from another.Therefor e iti shighl yprac ­ tical tobree d fornarrowin g the geneticbas e for economic characteristics such astre e straightness orwoo d specific gravitywhil e atth esam etim ebroadenin g thebas e foradapt ­ ability todivers eenvironment s orresistanc e topests . Everybreedin gprogra m involves astruggl ebetwee n gainan d risk;thi s isespeciall y sowhe ndealin gwit hpes t control.W e have found thatther e isconsiderabl e genetic resistancet o mostpest s in foresttree s although sometype s of resistance aremuc heasie rt obree d fortha nothers .

INTRODUCTION

Pests arealway s aproble m inan y forestenterprise .A s forestmanage ­ mentbecome smor e intensive,pest s appeart obecom emor enumerous ;th esam e result seems tob e evidenta srotatio n ages aremad e shorter.Thi s apparent increase ispartiall y the resulto fclose r observation aswel l as agreate r concern aboutpest s and theireffec to n intensive forestmanagement ;unde r this situation,pes t attacks thatwer e formerly overlooked orwer e consid­ ered tob e amino r nuisance suddenlybecom e important.A good examplei s spidermite s on Pinus taeda h. This insectwa shardl ynotice d innatura l stands orplantation s but isconsidere d tob e aseriou spes to ngraft s in seed orchards and to someexten to nyoun gtree s inprogen y tests.Also ,th e greater apparentincidenc e ofpest s ispartiall y area l increase resulting frommor e intensivemanagement .A good example is Cronartium fusiforme Hedge.& Hun to n P. taeda; thisdiseas ebecome sprogressivel y more serious whenmanagemen tpractice s such assit epreparatio n are improved ornitroge n fertilizers areused . The damage andwound s that sometimes result from cultivation equipment andothe r activities related to intensive forest managementca nbecom e infectioncourt s forpes tattack . Another reason fora nincreas e inpest sunde rintensiv e forestmanage ­ ment, oftenoveremphasize d buto fimportanc eunde r some conditions, isth e establishment oftree swit h restricted genotypes over large contiguous acreages. Initself ,larg eplanting s ofa singl e species areno t serious unless thegenotype s used ares ounifor m that atru emonocultur eresults . There is aspecia l dangerwhe nvegetativ e propagules ofrestricte d genetic background areuse d foroperationa l planting;vegetativ epropagatio n in itself isno tba d ifa broa d enough geneticbas e isensure d throughus eo f several cloneswhic hwil l discourage wholesalepes tattacks . Thetru e situation related togeneti c diversity in foresttree s is generallymisunderstood . Trees arealmos t alwayshighl y heterozygousgen ­ etically, and recent studies have indicated that foresttree shav e the greatestgeneti cvariabilit y of anyorganisms .A largeadaptabilit y to adverse environments andpest swoul db eexpecte d since atre emus t survive manyyear s and reproduce over sometimes drastically changing conditions from dayt oda y and fromyea r toyear .Thus , though afores tma yb eplante d as amonocultur e with all individuals having similar genotypes,each,indi ­ vidual tree isneve rgeneticall y homozygous ina wa y similar to some agri­ cultural crops.Eac hrame to fa clon e ofa vegetativel y reproducedplan ­ tationwil lb e quiteheterozygou s for adaptability andpes tresistance .Th e danger frompest s occurswhe n allramet so fa clon e are susceptible toa givenpest . Iti softe ndifficul t to initiate forestplantatio nprograms ,especial ­ ly inarea swher e growing foresttree s operationally isno t acommo n and acceptedpractice . Iti sa rea l setback,therefore , if theplantation s are lostt opest s or adverse environments after aprogra m hasbee n initiated withmuc h effort,cos t and fanfare. Iworke d closelywit h sucha progra m in the three-county area inon e ofou r southern states.Larg e sumso fmone y andgrea teffort swer e expended onpropagand a to inform the layman andt o gethi m interested and excited aboutth eimportanc e ofplantin gpines . Everythingwa s favorable andplan swen twel l excepttha tth enurseryma ndi d notpa y attentiont oth eprovenanc e orgeographi c source differences within thepin e speciesh eproduce d inth enursery .Th e seedlings supplied for planting wereno twel l adapted toth ethree-count y area.A severe drought followedb y a Pales weevil attackwipe d outmos to fth e several hundred thousand acreso fplantations . (As Iremember , final survivalwa s less than 20%. )Thos e fewplanting s thatdi d survivewer e laterdamage db y anic e storm followedb ybar kbeetl e attacks.Althoug h this incidentoccurre d over 25year s ago,i tstil l isdifficul tt othi sda yt oge tpeopl et oplan t pines inth e area involved.The y cite theearlie r disaster as 'proof'tha t pineswil lno tgro wther e and almostcompletel y ignoreth ebiologica l explanation ofwron g seed source followed by destructionb ypests . Similar incidents arecontinuousl y occurringwit h exoticplantations , and the forestry future ofwhol e countries canb e injeopard yunles spest s canb e controlled.A prim e example occurred incentra l Brazil.Ver y large plantingswer emad eo f p. radiata D.Don ;thes ewer e laterdestroye db y Dothistroma, Only recently has itbee npossibl e to induce the localpeopl e to againplan tpine s in anare aideall y suited togro w P. caribaea Morelet and P. oocarpa Schiede. Inan y intensive forestryprogra m there is always atradeof fbetwee n gain achievable and risk encountered toobtai ngreate rgains .Thi sproble m isespeciall y evidentwhe npest s are involved.Decision smus tconstantl yb e maderelativ et oth eoptio no fobtainin g greater gain fromth eus eo f better trees alongwit hmor e intensivemanagemen t ofth e forests,an dth e possibly increased dangero fpes t attacks thatmigh tresul t fromdoin gso . Onlywhe npest-resistan t strains oftree s areuse d can forestry come close tooptima lproductio n ofdesire dproduct s andthu s its full contribution to Society.

PESTS AND EXOTICS

Some ofth emos tintensiv e andwidesprea d forestry operations nowbein g established depend uponplantation s ofexoti c species.Althoug h themajo r concern and emphasis related toexotic s ist oth etropica l and subtropical regions, there isals oconsiderabl e usageo fexotic s incoole rclimates ; examples are P. contorta Dougl. inSwede no r Pseudotsuga menziesii (Mirb.) Franco insevera lEuropea ncountries .N omatte rwher e exotic speciesar e used, amajo r consideration inthei r success relates tothei rsusceptibili ­ tyt o attack andthei rpotentia l ashost s forpests . Exotics aregenerall y planted inlarg eblock s ofsingl e specieswit h the seed forth eplanting s sometimesbein g from restricted sources orfro m smallnumber s ofparents .Fo r some Eucalyptus andpoplars ,vegetativ e propagules mayb euse d forregeneration , creating an ideal situation for attackb ypest s ifcar e isno ttake nt omaintai n asuitabl ybroa d genetic base. Inalmos t all instances the exotic isno tto owel l adjusted toit s new environment,wit hth e resulttha tth etree s inth e forest are growing under considerable stress.Exotic s aresometime suse db yperson swh o are uninformed ord ono tcare ,s otha ta larg e amounto f flagrant 'off-site' plantingha sbee ndon e and isstil lbein g done.Thi s isespeciall y true for some P. radiata, P. caribaea andcertai n Eucalyptus plantingswher e these outstanding foresttree shav ebee nplace d inquit eunsuitabl e environments because of their fineperformanc e elsewhereunde rprope rconditions . There areno tto oman y 'absolutes' inbiology ,bu ton etha tcome sth e closestt ocertaint y istha texoti cplantation s willb e attackedb ypest s ofon e kind or another.Th e situation isespeciall y insidious intha tofte n the exotic growspest-fre e init searl yyear s inth ene w environment,to o often leading toa euphori a and falseprojection s aboutit sproductio n potentials,base dupo nth epest-fre e growth.Bu t Ihav eneve r seeni t fail -i tma y take 2year s or itma y take 10- bu tpest s ofsom etype , often serious,wil lbecom e established inexoti cplantations . Ihav e recom­ mended toclient s intropica l areasusin g exotics thatthe y reduce their initial estimates ofproductiv e potential by about3 0% inanticipatio n of pest attacks asth eprogram sbecom e older.Man ytime spes tattac kwil l follow adverse environmental conditionswhic hhav eplace d thetree sunde r severe stress.Th epoo rphysiologica l conditiono fth eexoti ctree s that results enhances the spread anddamag eb ypest s thatma yhav e previously eitherbee nunknow no rwhic hwer e considered tob e ofonl ymino rimpor ­ tance. Iti ssa dan do fgrea tconcer nt om e forth e futureo fhealth y and profitable forests tovie wth emagnitud e and seriousness of losses from pestattack s onexoti c foresttre eplantings .Massiv eprogram shav e failed from suchpes t attacks;example s areth e large P. radiata plantations destroyedb y Dothistroma insevera l regionso fth eworld , includingBrazil , Rhodesia andEast-centra l Africa.Th e losso fconfidenc e in forestry that has occurred asa resul to fdestructio nb y thisdiseas ecoul dhave ,bee n prevented andmillion s ofdollar s couldhav ebee nsave d ifthos ewh oestab ­ lished theexoti c P. radiata hadheede dth e adviceo fa fe wpathologist s whowarne d that Dothistroma probablywoul dbecom e akille r inradiat apin e plantations whenplante d where there arewar m andmois tsummers .Eve nwhe n the adverse results ofdiseas e attacks resulting fromplantin g insuc h environments areno w soclearcut ,ther e still areinstance s ofplantin g P. radiata insuc hdisease-pron e climates.Man ypersons ,includin gmyself , havebee npuzzle d ast oho w Dothistroma, found on P. radiata in itsindige ­ nous range inCaliforni awher e iti sconsidere d tob e only anuisance ,ha s managed tobecom e established insuc hwidel y separated regions throughout theworld .Althoug hw ema yno tunderstand , the important facti stha ti t has spread to these areas andther e isn o reason tobeliev e itwil l not occur inothe r areaswit h environments suitable fordevelopmen t ofth e disease. Exotics are attackedb y 3genera l types ofpests : 1.Thos e indigenous toth e area inwhic h theexoti cplan t isplante d but neverbefor e known to attack theexotic .Suc hpest s often adjustt oth e exotic andca nbecom e amajo r destructive force.Thi s adjustment toth e exotic iso frathe r commonoccurrence ,especiall ywit h leaf-eating beetles andworms .Severa l species ofthes epest s havebecom emajo r defoliators of eucalypts inBrazi l andNe w Zealand ando f Cupressus lusitanica Mill,i n Colombia. 2.Pest s ofth e exotic inth e exotic'snativ e range follow itt oth ene w area.A prim e example ofthi s is Dothistroma on P. radiata. This disease appears tohav e followed P. radiata whenever itwa splante d ina nenvi ­ ronment suitable toth edevelopmen t of Dothistroma. 3.A pes tno tnativ e toth e areawher e theexoti c isplante d but alsono t indigenous toth ehom e environment ofth eexoti c candevelo p asa majo r pest inth e foresti nit sne wrange .A n insecto n P. radiata serves asa good example.Thi s insect, Sirex, seriously damagesplantations ,especiall y thesuppresse d andcodominan ttrees . Sometimes thepes t isharmful ,no tbecaus e itkill sth eexoti cbu tb y deforming its oi ti so fvalu e only for limited low-qualityproducts .A good example forthi s isth eso-calle d cypress stem canker on Cupressus planted inKeny a andothe r areas.Ste mdeformatio n is sosever e thatth e only suitable use for thetre e is for fiberproduct s and theycanno tpro ­ duceth egoo d quality solidwoo dproduct s forwhic h theplantation s were established.

PESTS AND INTENSIVE FOREST MANAGEMENT

As forestmanagemen tbecome smor e intensivew emus t learnt o adaptt o and reduceth e inevitable increased pressures frompes t attacks.Th ehig h costso f intensivemanagemen t and thegrea tpotentia lvalu e ofth eproduct s make itmandator y topreven tpest s from seriously reducing thevolum e and quality, and thus thevalue ,o fth e forestproduct s grown. Iti sno tpos ­ sible for anintensiv e forestmanagemen tprogra m tooperat e assom eun ­ thinking environmentalists have suggested, i.e., 'letth epest shav e their share'.Historically , growing foresttree sha syielde d alo wretur n onth e investment andpes t attacks caneasil y turnwha twoul db e aprofitabl e venture into alosin g enterprise.A sth e demand for forestproduct s becomes greater iti so fparamoun t importance forSociet y tomak eeac h forested acremor eproductive .Lan d onwhic h foresttree s canb e grown forwoo d products isbecomin g less available.Additionally , thebes t lands arebein g taken forus e inagricultura l production, forcing forestproductio nt o shifttowar d themor emargina l sites.Thi s intur nresult s inth etrees ' being grownunde r greater stress,an d thus theybecom emor e susceptible to pestattack . Certainpest s areincrease d by activities such as fertilization, site preparation orthinning .Fo r example, fusiform rust (Cronartium fusiforme) ismuc hwors e on fertilized and intensivelyprepare d sitestha n inplanta ­ tions that areno t intensivelymanaged .Thinnin g ofplantation s canresul t inmassiv e destructionb y Fomes annosus (Fr.)Cook ebecaus e the stumpso f the cuttree sbecom e aninfectio n court forth e disease.Cultivatio n of plantations cuts roots that arethe n sometimes attacked by insects and diseases. Ifoptima l gains through tree improvement aret ob eobtained , areduce d geneticbas e forth e economically important characteristics willresult . Onth e surface thiswoul d appear todevelo p inth e directiono f amonocul ­ ture thatwoul db e ideal forth e spread ofpests .Bu t greaterpes t attacks will notoccu r ifcar e istake n inth edevelopmen t ofth e tree improvement program. Iti smos t fortunate thati nfores ttree s almostal l economically important traits such astre e straightness orwoo d quality are genetically independent from characteristics ofpes tresistance .Wit hpracticall y no exceptions,thes e two sets ofcharacteristic s arecontrolle d bymultipl e genes and areinherite d independently andthu s areno t strongly correlated. Iti stherefor epossibl e tobree d forbette r economic characteristics by narrowingth egeneti cbas e forthes ewhil e at-th esam etim ebreedin g for greater adaptability andpes tresistanc eb ybroadenin g the geneticbase . Ourprogra m inth e southeasternUnite d Statesha sbee nusin g such adua l breeding system mosteffectivel y fornearl y thirtyyears .

WHY RESISTANT TREES ARE NEEDED AND WHEN SHOULD ONE BREED FOR RESISTANCE

Anybreedin gprogra m with foresttree s islong-ter m andexpensive .Why , therefore,doe son e spend alo to ftim e andmone y oncontrollin gpest s throughdevelopin g resistance rather thanusin g silvicultural, chemicalo r naturalpredato r controls?Th e fear isofte nexpresse d that 'pestswil l defeatus ' andtha tw ewoul db ebes tof ft od onothing . Such anegativ e attitude issilly ,becaus e any forest,manage d ornot ,wil lb e attackedb y pests.Whe n intensive forestry isapplied , 'new'pest s are found orsudden ­ lybecom e important,no treall ybecaus e they arene wbu tbecaus e theyha d notbee nclosel y observed previously.Althoug h intensive sitepreparation , cultivation, thinning and fertilization sometimes doresul t intree sbecom ­ ingmor epest-susceptible , sometimes theopposit e resultoccur swher e trees becomemor epest-tolerant .A good example ofthi s relates topin ebar k beetles: thehealthy ,well-tende d stands aremor e resistantt o insects than areth eoff-site ,th e over-age,th eover-dens eforests . Whetherbreedin g forpes tresistanc e shouldb e done depends uponth e availability and suitability ofothe rmethod s ofpes tcontrol .Fo rexample , onewoul d nottr yt odevelo p alobloll ypin e tob e resistant to fusiform rustonl y inth enurser ybed ; fungicide sprays are so simple and economical inth enurser y that thismetho d ofsprayin g isth epreferre d control.Bu t in forestplantation s the situation isreversed .Althoug h actions sucha s reducing the alternate hostca nb e applied andwil lhel p some,n o known practical and safemethod e is available forefficien t control of therus t inlarg e forestplantings . Iffusifor m rust ist ob econtrolle d successful­ lyi nlarg eplantations ,th eonl ypractica l way is tobree d forresistance . Thepin ehos t shows largevariabilit y insusceptibilit y toth e disease,an d inheritance ofresistanc e iso fa magnitud e such that iti srelativel y easy todevelo pusefu l resistant strains.Th epes t alsovarie s geneticallybut , although this complicates theproblem , itappear s tob epossibl e toover ­ come itb y resistancebreeding . Iti sunfortunat e thatth e improved silvi- culturalmethod s ofbette r sitepreparation , fertilization and sometimes cultivation currently usedt o improve tree growth andyiel d are allcon ­ ducive toincrease d rust infection.Thi s indicates thatth enee d for fusi­ form rustcontro l andthu sresistanc ebreedin g isconstantl y increasing as forestmanagemen t gets 'better'. Inth emor e 'hotspot 'rus t areas inth e southeasternUnite d States,man yo fu s feel thatwithou tus eo f resistant pines thegreate r growth resulting from sitepreparatio n and improved forestmanagemen t ismor e thanoffse tb y thegreate r losses from increased fusiformrust . Opposite to fusiform rust,i nm y opinion, Fomes annosus is an important pestagains twhic hbreedin g forresistanc ewil lprobabl y notb e ofprimar y value.Althoug h someprogres s hasbee nmad e inbreedin g forresistanc e to thispest ,th emajo r controlmechanism s shouldb e through silvicultural and speciesmanipulation . Inth e southernpines ,fo rexample , Fomes hasgener ­ allybee nkep tunde rreasonabl e controlb y good standmanagement . Although developing resistant strains isexpensiv e and takes special skills andmuc h time,i tha s theadvantag e that,onc e obtained, resistance isrelativel y permanent towhic h additional andbette r resistance canb e added.Th e long-term costo fcontro l isusuall y lesswit hbreedin g than with directcontrol sbecaus e costs ofth e latterusuall y reoccur frequently ora tleas tsevera l times throughout therotation .A good example is Dothistroma onradiat apine .Severa l studies have indicated thatther e isa reasonable resistance toth e disease.Despit e this, some organizations have chosent ocontro l Dothistroma bymakin g thenecessar y sprays duringth e mostsusceptibl eperio d of theplantation' s life rathertha ncarr y on resistancebreeding . Ifonl y onegeneratio n ofplanting s is considered, there isn o doubttha tresistanc ebreedin g wouldb e less efficient than sprays for Dothistroma control,bu t Igreatl y doubttha tthi swil l bes o for several generations.Additionally , anyus e ofchemical s inth e forest environment isno tdesirabl e and shouldb e avoidedwhe npossible .Th e advantage ofresistanc ebreedin g istha tn oundesirabl e substances are inserted into theenvironment .Biologica l control fills thisnee dwhe n it worksbut ,althoug hmuc h discussed andpromoted , effective biological control of forestpest s israr e and failures areth enorm .A good example isth e sprucebudwor m innortheaster n Canada.Muc h effortha sbee n extended forbiologica l control,wit hhardl y satisfactory results.Larg ean dcostl y spray control efforts havebee nundertake nwit h fairt ogoo d success,bu t the social and environmental objections are strong.Resistanc ebreedin g has beenessentiall y ignored but,althoug h Ia mobviousl ybiased , Ifee lresis ­ tancebreedin g for sprucebudwor m shouldb e given agoo d trial.Th epatter n of attack,th e occasional noninfected tree,th e ability toasses s attacko n young trees,al l indicate resistancebreedin gmigh tb e aworthwhil eap ­ proach. A common reasongive n forconcer n aboutresistanc ebreedin g in forest trees istha tne w andmor evirulen t strains ofdisease swil l developo r that insectswil lb e evolved thatca n attackth e resistant trees. (This same argumenthold s forcontro lb y sprays orbiologica l control.)Thi s potential, although real, hasbee nemphasize d outo fal lproportio nt o reality.Becaus e ofoutstandin g examples ofdevelope d tolerance, like flies toDD To r rusts onwheat ,th e layattitud e istha t overcoming of resistance byne w strainswil lb eregula r andnormal .Thi s isno t so ifprope rprecau ­ tions aretaken ,especiall y inlong-lived , heterozygous foresttrees .Th e concern toth e foresterwoul db emor e real if*hismanagemen tmethod s and treesplante d remained static over consecutive rotations,bein g the same for succeeding generations ofplante d trees.Bu t forestry isneve r static andw ehav e aspecia l advantage sotha t inou r longrotatio n ages, succeed­ ing cropswil lb e genetically different from thepreviou s cropplante d ona given site.Unles s the forestmanager san d geneticists are completely incompetent, thetree splante d ona sit ewhic hha dbee nplante d 30year s previously willb e genetically improved anddifferen t from the original planting.Durin g the30-yea rperio d required forth ecro p tomature ,ne w and improved strains oftree swil l havebee ndeveloped .Therefore ,th e fear of apes tevolvin g from the firstcro p to destroy the succeeding cropsma y betheoreticall y correctbu tdoe sno t fitwit h facts. Such an adaptation and evolving ofpest s certainly happens on annual cropsbu tshoul dno to n long-lived perennials ifa nactive ,ongoin gbreedin gprogra m isunderway , soth ene wplantin g stockwil l differ from theoriginal . Oftenth emagnitud e and complexity ofcontrol s required,whethe r they bechemica l orbiological ,mak e itimpossibl e toefficientl y use anything other than aresistanc eprogram .Althoug hbiologica l control currently is the 'darling'o fth e environmentalist, and somethingw ewoul d allb ede ­ lighted to seeperfected , iti sa so fno w generallymor e inth emind so f mentha n ametho d that isusefu l ona commercia l scale inmos t forest trees.Al l ofu swan tt obecom e as free aspossibl e from chemicals forth e control of insects anddiseases .Th e conclusionmus tbe :

Breeding for resistance to pests in forestry is absolutely mandatory. The adoption of agricultural practices for the development of heritable resistance to pests and pathogens in forest crops

I.A.S. Gibson, J. Burley & M.R. Speight

Department of Forestry, Oxford University, South Parks Road, Oxford, UK

ABSTRACT

Objectives, skills andmethod s of farmer and foresterhav e oftenconflicte d but annual cropbreedin g provides guidance for perennial crop improvementwhic h isparticularl y important for the rapidly increasing areas,product s andmanageria l diversity of foresttre eplantations .Th e dangers ofreduce d genetic variability that areinheren t inintensiv e selection programmes mayb e offsetb y theus e ofclona lmixture s andmultipl epopu ­ lationbreedin gbu tresistanc ebreedin gwil lpla y anincreas ­ ingly importantpar t inplantatio n development and itwil l call for inter-disciplinary co-operation. Development ofresistanc e intree sneed s long-termknowl ­ edgeo fth ebiolog y ofth epes t including itsvariatio n and taxonomy. Prediction and identification ofpes tproblem s are made difficultb y the lacko fpes t stability inspreadin g to new regions.Wherea s annual crops favour obligate,host-specif ­ icpathogens ,tree sprovid e substrate for saprophytic,unspe - cialized, facultativepathogen s and areofte n grown onmargina l sites thatreduc e theirhos tvigour . Whereas stability ofvertica l resistance intree sha sbee n detected, iti sboun d todeclin e andmixe d cropping should increase. Selection forhorizonta l resistance is likely tob e moreprofitabl e andma yb e extended togeneralize d fieldresis ­ tance sinceperennia l crops aresubjec tt oattac kb yman ypest s during acommercia l rotation.A tbot h levels,attentio nmus tb e paid toth eeffec to fth e environment onth eexpressio n ofre ­ sistance.Whil e seed orchard development accelerates theappli ­ cation ofvertica l resistance,vegetativ epropagatio n ismor e suitable forhorizonta l resistance.Technique s forearl ydetec ­ tiono fresistanc e arerequire d andma yb e adopted from agri­ culture. INTRODUCTION

Forestmanagement ,particularl y plantation development, arose largely inrespons e toth e erosion ofnatura l resourcesb y agriculture.Th eobjec ­ tives, skills andmethod s of farmer and foresterhav e often conflicted, mainlyfo rreason s entrenched inth e genesis ofthei r disciplines,ye tth e rapid rate of improvementpossibl ewit h annual cropsprovide s lessons and guidance forth edevelopmen t ofperennial ,an dparticularl y forest,crops . The farmer isconcerne d mainlywit h the intensivemanagemen t of single crop species ongoo d sites,yieldin g oneproduc t inwhic huniformit y isa t apremiu m and forwhic hvalu epe runi tyiel d ishigh . Incontrast ,th e forester commonly acts as anecologist ,maintainin g yields oflo wvalu e productsb y extensive management ofvariabl e crops onpoore r siteswit h severalobjective s includingprotectio n and amenity functions,a swel la s theproductio n of industrial rawmateria l which, initself ,cover s arang e ofpropertie s for anumbe r ofuses . The evolution,domesticatio n and some 50year s of intensivebreedin g of many annual cropshav e reduced their geneticbase , attaining aselectio n plateaubeyon d which further gaincall s fortfi eintroductio n ofgene s from wildpopulation s inthei rnatura l origin.Fores ttree s areonl y 2o r3 generations'remove d from thewil d type and,wit h theexceptio n ofsom e intra-specific,natura lpopulatio nvariant s and some restricted national breedingpopulations ,tre e specieshav econsiderabl e resources of genetic variation available.Nevertheless ,conservatio n ofgeneti c resources is essential here aswel l asi nagricultur e and, forman y species,i ti si n progress.

THE NEED FOR PLANTATIONS AND THE USE AND IMPROVEMENT OF EXOTIC SPECIES

Around the starto fth e20t hcentur y thedemand s ofrapidl y expanding and increasingly sophisticatedworl dpopulation s called for increases in forestproductio n thathav ebee nme t largelyb y the creation of even-aged plantations ofsingl e species,whic hhav e oftenbee nexotics . These species were selected fromnatura l standswit h environments similar tothos e ofth e areasunde r development and subsequent trials led toth eselectio no f2 o r3 provenance s orspecie s forestablishmen t ona large scale (seee.g . Burley &Wood , 1976). Once these specieswer e inuse , further selection and testingbrough tthe m towards anoptimu m forth e region,largel y through culling thewors t stock atth enurser ystage , selective thinning and seed collection from superiortrees . Thisha s retained aleve l ofgeneti cversatilit y inth e cropwhil e adapting itt oth e environment; itha sbee nsuccessfu l sotha ti ti sno w commonexperienc e to find that such crops are sowel l adjusted after 2o r3 generations thatthei rprogen y areusuall y superior to freshly introduced

10 r provenances.Durin g the last3 o r4 decade s thereha s alsobee n anincreas e inintra-specifi c improvement for forestplantatio n crops,simila rt otha t used forothe rperennia l crops,eithe rthroug h selection and vegetative propagation ofclona lmateria l ofoutstandin g merita si npoplars ,wher e this includespes tresistance ,o rNorwa y spruce (Picea abies (L.)Karst.) , orb y openo rcontrolle d pollination insee d orchardsbetwee nramet s of phenotypically superior trees.Th e latterha sha d considerable success for pines andeucalypt s and isno wbein g extended toothe rspecies . While these recent lines ofdevelopmen thav eprovide d immediatebene ­ fits, theymay , inth e longrun ,ris kth e sacrifice ofth ebenefit s ofth e field selection techniqueswhic h are associatedwit hplantatio nmanagement . The danger ofreducin g thegeneti cbas ema yb e offsetb y theus e ofclona l mixtures forvegetativel y propagated crops (Schreiner, 1939)an db ymul ­ tiplepopulatio nbreedin g (Namkoong et al., 1980).

JUSTIFICATION FOR BREEDING TREES WITH RESISTANCE

The development of pests in agriculture and forestry

The development ofeven-age d crops inagricultur e provided idealcon ­ ditions forman ypest s thathav e appeared, flourished anddecline d for reasons that,unti l recently,wer e littleunderstood . Now,however , there is anarra y ofexpedient s for their control,includin g cropvarietie s with high and stable levels of resistance to attackwhic h areno w important components ofintegrate d field controlprogrammes ,especiall y wherepesti ­ cideus e isno tacceptable . The intensivepractice s adopted by foresters during the last8 0year s havebee n accompanied by similar threats formpest s of allkinds .Befor e this,noxiou s insects andpathogen s occupied aplac e inth ebalance d dynam­ iccomple x of thenatura l forestwher e they seldom emerged asa threa tt o the survival ofthei rhos t species.Whil e these agenciesma yhav ecause d some serious economic losses,th ecos to f special controlmeasure s andth e practical problems thatthes e raisedmad e them generallyunacceptable . However, this situationha sno w changed radically and,wit hth ewidesprea d adoption of forestmonocultures ,idea l conditions havebee n created forth e establishment and spread ofpest s of allkinds .

Introduction of exotic pests

While these recent changes inth eemphasi s of forestpractic e took place, rapid advances inth evolum e and speed ofinternationa l transport greatly increased therisk so f introduction ofne wpest s into forests that hadpreviousl y beenprotecte d bynatura l barriers,threatenin g natural stands andplantation s alike,despit ephytosanitar y precautions.Th e reali-

11 tyo f thisthrea t isexemplifie d by the damage caused bywhit epin e blister rust (Cronartium ribicola J.C. Fischer), chestnutbligh t (Endothia parasi­ tica (Murrell)Anderso n& Anderson )amon gothe r introduced pathogens,an d thewinte rmot h (Operophtera brumata L.)o rth egyps ymot h (Lymantria dispar L.) inth einsects .Thi s threatcontinue s toexpan d asth e areao f new forestplantation s based onexotic sextends . Asw e shall seebelow ,thi s factor, inparticular ,no tonl y establishes ahig h level ofnee d for forestprotectio n againstpests ,i tals o creates animportan t areao fconstrain t onit s implementation.

The need for application of agricultural techniques in forestry and their limitation

Iti sbecaus e forestplantation s andnatura l standsno w representa majorpar to fth eresource s ofman y countries,a swel l asthei r increased vulnerability, thatther e isa nurgen tnee d tous eagricultura l technology toprotec t aswel l as improve them.However ,th edegre e towhic h thisi s possible depends on available funds andth enatur e ofth ecrop .Bot hthes e factors reduce the scope for supplementary protectionmeasure s inth e natural forestan dpos e smaller,bu t still significant limits onthei rus e inplantations .Th e accountingmethod s necessary forcrop swit h terminal yields andthe.difficultie spose db y the extento f forest areas,combine d with thesiz e and longevity oftrees ,reduc e theexten tt owhic hagricul ­ turalprotectio nmethod s canb e appliedunde r forestconditions . There are,therefore ,considerabl e attractions inth eus e ofheritabl e resistance toprotec t forests from damageb ybioti cagencies .

THE DEVELOPMENT OF HERITABLE RESISTANCE

The relation to resistance of other traits

Whileth ebreedin g ofpes tresistan t trees calls for ahig h levelo f interdisciplinary co-operation (Schreiner, 1966;Gerhold , 1970), theprac ­ tical difficulties inprovidin g abalance d combination ofpes t resistance andothe rdesirabl e featuresma yb e insuperable.Prioritie s are allocated toth eobjective s of acro p improvementprogramme ,base d on cost-benefit analysis and selection indexweighting ,wher eprotectio nma y rank low initially andwher egeneti cweight s areunknown . Further,pes tresistanc ema yno tb e correlated with other desirable crop features;o nth e simplest level,whil e hostvigou rma y increaseresis ­ tance to facultativepests ,thi sma yno t apply tomor e specialised organ­ isms.Pest s areno t astabl e feature of anycrop , asthei r impact depends onth e environment aswel l asth e geneticnatur e ofth ehost ;the yma y also take time toemerg e andofte n showa muc hhighe r genetic versatility than

12 theirhosts .Fo rthes e reasons,th e search forpes t resistance tendst o become secondary totha t forothe r aspects ofcro p improvement.

Constraints on developing resistance

Anydevelopmen t ofpes tresistanc e inplant s onmor e than anempirica l basis requires longter m knowledge ofth ebiolog y and life-cycle ofth e organisms concerned. While someprogres sha sbee nmad e inth eprognosi s of forestpest s for a fewinsects ,suc ha sth egyps ymoth , Lymantria dispar (Houston& Valen ­ tine, 1977)an dpin e loopermoth , Bupalus piniarius (L.)(Bevan , 1974), our information on forestpest s ingenera l isquit e inadequate to anticipate variations inthei r impact,onc ethe y areestablished , ort o supportthei r prognosis forne wregions . This islargel ybecaus e research into forestpest s hasbee nver y lim­ itedunti l recently sothat ,eve nnow ,ther e iseve ndoub to nth eprope r indentity of someo fou r commonest forestpathogens .Fo r example,Minte re t al. (1978)hav erecentl y showntha tman y ofth ecase s ofpin e needle dis­ easepreviousl y attributed to Lophodermium pinastri (Schrad. exHook. ) Chev. aredu et oa ne w species, L. seditiosum Minter, Staley &Millar ,an d thatothe r records refer to saprophytic species e.g. L. australe Dearness (seeals oMartinsson , 1979). There areman y other exampleswher e thetaxo ­ nomieposition , aswel l asth ebiology , of important forestpathogen s is stillpoorl y understood.Whil ether e isno tth e same lacko fbasi c infor­ mation on forestinsect s inth ewester nworld , this iscertainl y thecas e elsewhere,an dparticularl y inth ecas e of insectparasitoid s andth e habitat interactions ofspecialise d taxa.Ther e istherefor e considerable need toimprov e ourknowledg e of forest insects aswel l aspathogens .A useful review ofprogres s thatha sbee n achieved inthi s fieldha sbee n givenb yHanove r (1980). Our difficulties inth epredictio n and identification of future forest pestproblem s arecompounde d byth e facttha tthei r spread tone w regions is far from stabilised.Whil e this applies asmuc ht onatura l forests ast o plantations, species sucha spine s andeucalypts ,whic h areno wplante d in regions often far fromthei rnativ e areas,ma yb e expected tocontinu et o be atris k forman yyear s tocome . The spread ofdothistrom a blight (Scirrhia pini Funk& Parker ;imper ­ fectstate , Dothistroma septosporum (Doroguine)Morelet )fro m thenorther n hemisphere,wher e inman yplace s itwa s economically insignificant,t oth e south,wher e itha sprove d extremely damaging tocertai npin e species,i s anexampl e ofthi sthrea t (Gibson, 1972). Similarproblem s canb e found where forest insects havebee n introduced tone w areas and assumed quite differentbehaviou r patterns from those inthei rnativ e region.Bacteria , nematodes,viruses ,parasiti chighe rplant s andmite sma y allb eexpecte d

13 L topos eunpredictabl e threats to forestcrop s indu ecours e and inthes e casesw e shallprov e tob eeve nles swel lprepare d todea lwit hthem . Inadditio nt othes e limitations,th edifference s between thetype so f pathogens, and toa les s extent,insec tpests ,tha t arepredominantl y important to annual andperennia l crops respectively impose still further restrictions onth eus e of agricultural technology by the forester seeking toprotec thi s crops through heritableresistance . While iti sunrealisti c todra wto o sharp adistinctio nbetwee n obli­ gate and facultative parasitism, iti snevertheles s true thatannua l plants, largely composed of living tissues,includ e aver yhig hproportio n ofobligate ,host-specialise d pathogens inthei rpests .-Trees ,o n theothe r hand, areperennial s consisting of alarg e and increasing proportion of dead tissues (heartwood, dead roots,bark ,etc. )afte r they havepasse d the juvenile stages.Thi sprovide s asubstrat e forth e saprophyticbuild-u p of inoculum ofunspecialise d facultativepathogen s inclos eproximit y to living tissueswhic h canbecom e sufficient topermi tinfectio n inth e course oftim e ina wa y impossible inannuals . Inadditio n to this, forest crops areofte ngrow no nmargina l siteswhic hma y serve toreduc e their vigour and render them evenmor e susceptible to'attac kb y facultative organisms.Thus ,whil e treesma yb e subjectt o asimilar'rang eo fspecial ­ isedpathogen s asar e annuals,a muc hhighe rproportio n ofthei r diseases are likely tob e caused by less specialisedorganisms . The situation for insectpest s israthe rdifferent .Thes e show asimi ­ larrang eo f specialisation andhos tdependenc e buthav e little incommo n withth ephysiologicall y closerelationship s thatofte noccu rbetwee n fungi,bacteri a andviruse s (broadly speaking)an dthei rhosts .Neverthe ­ less, stress inth ehos tcause db y defoliation, droughto rothe renviron ­ mental factors canpredispos e toattac kb y such insects asbar k beetles (Scolytidae), longhornbeetle s (Cerambycidae), ambrosiabeetle s (Platy- podidae) andwoodwasp s (Siricidae), ata leve l thatha sn oparalle l in annual agriculturalcrops . Finally, there is alarg e.a neconomicall y important group of forest pestwhic hhav e little incommo nwit h agricultural problems andwher e heritable reactions ofhos ttissue s leading directly toresistanc e are particularly hard to find.Thi s includes the insects,deca y fungi andothe r organisms thatdestro y theheartwoo d oftrees ,ofte nwit h little external symptoms onth ehos t andwit h little selectionpressur e to reduce the potential ofth ehos tt o survive undernatura l conditions.A sw e shallsee , theprospec t of adapting agricultural technology toprovid e control through resistance tothi sclas s ofdiseas e is small indeed.

14 THE PLACE OF RESISTANCE BREEDING

Types of resistance

VERTICAL RESISTANCE The outstanding successes inagricultur e during thepresen t century thathav e gained remarkably high levels ofpes tresistanc e have largely involved annual species andhighl y specialised pathogens,wher e acombina ­ tiono f shortgeneratio ntim ei nth ehos tan d aphysiologicall y close host-parasite relationship (permitting directgeneti c control ofth e dis­ eased state)ver y largely contributed tothes eachievements . Thisha s generally involved atyp eo fresistanc eno w termed 'vertical' (Vanderplank, 1963), which isbase d onon e or afe wmajo r genes andi s distinguished fromhorizonta l resistance (whichw e shall discuss below) derived from the interactions ofa mino r genecomplex . Inth emajorit y ofcases ,vertica l resistance,whil eprovidin g ahig h level ofprotection , iseffectiv e only against somerace s ofth epest .I n the caseo f fungalpathogens ,i nparticular ,whic h often show ahig h level ofgeneti cversatilit y (Buxton, 1961), thisha s often ledt oth e 'break­ down' ofprotectio nwit h theemergenc e ofrace s ofth epes t ablet o attack thene w cropvariety .Thi sweaknes s isaggravate d furtherb y the relative simplicity ofth egenetic s ofth ehost-parasit e interactions where vertical resistance iseffective ,man yo fwhic h conform toth egene-for-gen e concept propounded by Flor (1953). Wherevertica l resistance hasbee n lost inimportan t annual crops the generation timeha s oftenbee nshor tenoug ht ocounterac t thisb y aflo wo f new resistantvarietie swhich ,whil e itha s led to acycli c situation,ha s oftenprove d acceptable. Inperennials ,howeve r this isno gpossible ,eve n where thegeneratio n timeca nb e reduced to aminimum . Instead, otherexpe ­ dients, such asth eus e ofpesticides ,hav eprove d more cost-effective. Examples canb e found inbliste rbligh to fte a (Exobasidium vexans Massée) andcoffe e rust (Hemileia vastatrix Berk.& Br.) . Although 'breakdown' isno t an inevitable resulto fth eus e ofvertica l resistance forcro pprotection , asRussel l (1978)ha spointe d out,th e dangers areenoug h to justify its avoidance for forestcrops ,particularl y asth eeconomi c hazard increaseswit h the intervalbetwee n the introduction ofth ene wvariet y and its eventual failurewit h the appearance of ane w raceo fpathogen .Th e extent anduniformit y ofindustria l forestcrops ,an d their lenghty rotations,mak ethe mparticularl y vulnerable tolat e failure ofvertica l resistance, although little evidence ofthi sha sbee n seeno na fieldscale . Fungal forestpathogen s asdivers e as Gremmeniella abietina (Lagerberg) Morelet, Ceratocystis ulmi (Buisman)Morea u and Melampsora larici-populina Kleb. (Gibbs,1978 ;Skilling , 1977;va nVloten , 1949)hav e all formed races

15 ofvaryin gpathogenity , sother e seems tob en o lack ofadaptabilit y in thisrespect . Iti spossible ,therefore ,tha tth e stability of resistance thatha sbee n found to forestpest sma yb e through the complexity ofth e host-parasite interaction.Thi sma yb e further stabilised by the relatively lowpopulation s ofexistin g tree crops,compare d to those ofcereal s and other annualswher e selectionpressure s forne wpathoge n strains isver y high andth eemergenc e ofne w races is almostcommonplace .Th ephysiologi ­ calcomplexit y ofresistanc e tocertai n rusts,suc h as Cronartium ribicola, may also contribute significantly toth e stability ofman y tree systemso f defence againsthighl y specialised pests (Bingham et al., 1971). Despite thepresen t situation,ther e isn o room forcomplacenc y and, with the increasing extento f forestmonocrops ,ther e isn o doubttha tth e stability ofvertica l resistance to forestpest swil l beboun d todecline . Safeguards againstthi sma yb e adopted through theus e ofmixture s of individuals with different inherentresistanc e factors,eithe r inseparat e plantations or,better , asmixture s insingl e stands.Mixe d cropping of thiskin dusuall y carrieswit h itsom e sacrifice ofcro pqualit ybu tthi s canno wb e avoided by theus e ofmultilin e cultivarswher e aunifor mgen ­ eticbas e iscombine d with arang e ofalternativ e resistance genes toa given disease.A tpresen t thisexpedien t isexpensiv ebu ti thas'potentia l value for forestprotectio n (Browning& Frey , 1969). Parallelswit h insectpest s arehar d to find althoughhighl yspecial ­ isedhos t relations andhig h levels ofresistanc e canb e found.Marke d variation occurs inth e colonisation of individual plants inuniform ;fie-ld s ofbrussel s sproutsb y Brevicoryne brassicae L.bu t iti sonl y effective in relation tocertai n aphid ecotypes (Dunn& Kempton , 1972)givin g ananalo ­ gous situationt omajo r gene resistance torusts .Simila r relationships are known infores t insectpest s (i.e. Cryptococcus fagi Baerensprung and Nuculaspis californica (Coleman)). While thesehav e the appearance ofver ­ tical resistance,thi sha s onlybee n shown ina fe wcases ,notabl y the Hessian fly (Mayetiola destructor (Say)) (Hatchett& Gallun , 1970).

HORIZONTAL RESISTANCE While thedistinctio n betweenhorizonta l andvertica l resistancewa sa t one timeregarde d asquit eclear-cut ,ther e isa growin gbod y of opinion now thatregard s the two asextreme s ofa grade d series ofgeneti c states ranging from simple relationships governedb y asingl emajo r gene tocom ­ plex states derived from anumbe r ofgenes ,man y ofwhic hma yb eminor . Indeed, somedoub tha sbee n casto nth evalidit y ofth edistinctio n between major andmino r genes inthi srespect . Whatever thecurren topinion ,th evie w thathorizonta l resistance arises from genetic complexes that formpar to fth egeneti cbas e ofth e host isstil l tenable (Vanderplank, 1963). Theprotectio n affordedb y horizontal resistance isneve r atth e samehig h level astha t from vertical

16 resistancebu ti tha sth eadvantag e ofstability . Ingenera l the protection thatthi s kind ofresistanc e offers covers awide r range ofpests ,includ ­ ingthos eweake r facultativeparasite s thathav e importance inforestry . The search forhorizonta l resistance isusuall y by selection ofindi ­ viduals for desirable characters from large fieldpopulations .Thes e are then screenedb yprogen y orclona l testst oensur e thatthei r special features are truly inherentbefor e fieldpropagation .Vegetativ epropa ­ gation isdesirabl e to avoid the risko fdispersa l ofdesirabl e combina­ tions ofgeneti c fasctor,whic hmigh toccu r through sexual reproduction. Field selection onthes e linesha sbee ndescribe d for resistance to He- terobasidion annosum (Fr.)Bref . (Driver& Ginns , 1966). All this is fully compatiblewit h forestplantatio npractic ewhic hi s often indirectly selective forincrease d crop quality andpes tresistance . Thebalanc e existingbetwee npest s andthei r hosts innatura l forestcommu ­ nities is largelybase d onhorizonta l resistance factors and itha sbee n advocated oftentha tlastin g solutions topes tproblem s ofplantation s may be found ina nunderstandin g ofth e ecological balance existing inth e communities whereplantatio n species arenativ e (Dinus, 1974). Possibly themos t important featureo fth eus e of field selection techniques istha tthe ypermi t selection towards thecontro l ofmor e than onepes t atth e same time.Th emajorit y ofcrop s area tris k from 3o r4 majorpest s inth ecours e ofth ecro pcycles 'an dperennials ,especiall y foresttrees ,ar eexpose d to aneve ngreate r number.Practica l cropim ­ provement should aim,therefore ,a tusefu lprotectio n levels againstal l themajo rpest s ina give ncro p situation and iti si nthi s respecttha t field selection towards horizontal resistance providesmuc hbette roppor ­ tunities than theus e ofvertica l resistance. Itmus tb e conceded,however , thatth ecombinatio n ofvertica l resistance factors toprovid e protection from anarra y ofpest s hasbee n successful ina nannua l crop such asric e (Khush, 1977)an dtha t someprogres s seems tohav e evenbee n achieved in this direction forpine s (seeGoddar d &Rockwood , 1980).

Tolerance

Hitherto, itha sbee n assumed thatther e isa napproximat e relation between symptom expression andlos s ofvalu e ofcro p frompes t attacks (Browning et al., 1977). This isno tnecessaril y soan d examples areknow n where symptom expressionma yb e severebu tpes timpac ti s relatively slight,notabl y incerea l rusts (Caldwell et al., 1958).

17 FUTURE NEEDS FOR RESISTANCE BREEDING IN FORESTRY

The role of the environment

While agricultural scienceha smuc ht ooffe r forth e improvemento f forestcrops ,muc h ofthi scall s fordevelopmen t within thecontex to fth e special circumstances of forestry. Forestplantation s arecharacteristicall y confined tomarkedl ysub - optimal sites andth e search forhorizonta l pestresistanc eb y fieldselec ­ tion automatically takes this into account.However ,vertica l resistance alsoneed s tob e sought attime s andher e allowancemus tb emad e forth e effects of adverse environmental influences inth e screening to select thesevarieties .Schoeneweis s (1975,1978 )ha spointe d outtha tt o conduct thescreenin g of forestvarietie s forpes tresistanc e under optimal growing conditions, andthu s follow some agricultural practices,coul d givedanger ­ ouslymisleadin g resultswher e thecro pma yultimatel yb e raised onsite s far from ideal. Itmus tb e accepted inprinciple ,therefore ,tha tdu e toth ecircum ­ stances of forestry,muc hmor e attentionneed st ob epai d toth einter ­ actionbetwee n environmental factors andhost-pes t reactionpattern s than isusua l inagriculture .Fiel d screening,particularl y forvertica lresis ­ tance, inspecies ,provenance s andintra-specifi c selections,shoul db e madeunde r the future growingcondition s forth ecrop .

Maintenance of genetic resources

Therelativel y recentdevelopmen to fmonocrop s and the later initiation of selectivebreedin g in forestry hasmean ttha tther ehav ebee nn omajo r declines atth egloba l level inth egeneti c resources ofth emos t important forest species.Nevertheless ,th ewarnin g onth edanger s ofgeneti c'over - refinement isclea r from agriculture and foresters arebeginnin g tohee d it. Excessive exploitation innatura l forests,particularl y intropica l rain forests,ha s threatened loss ofgeneti c resources and even losso f speciesbefor e theirutilit y hasbecom e fully known (Kemp,1978 ;Kem p & Burley, 1978;Burle y& Namkoong ,1980 )an d forsom ecommercia l plantation species importantnatura lpopulation s are atris k even ifth e species asa whole isnot . Forthi sreason , internationalprogramme s havebee nestablishe d by institutes indevelope d countries,ofte nwhit h support frommultilatera l andbilatera l agencies,t oexplore ,conserve ,evaluat e andus e tobes t advantage forestresource s ofal lkinds .However ,t odate ,i tha sno t provedpossibl e to initiate internationalbreedin gprogramme s forpine s (Kempe t al., 1972;FAO ,1979 )althoug h activities within IUFRO facilitate

18 exchange and testing ofnatura l and improvedpopulation s ina rang eo f species.

Accelerated breeding and resistance testing

There isoutstandin gnee d toreduc e thedisadvantage s posed by the size and longevity oftree s inal l aspects ofthei rgeneti c improvement,an d particularly inresistanc ebreeding . Inparticular ,technique s areneede d tospee d the testing ofselection s forcombination s ofdesirabl e qualities and,onc e found, forthei r rapid reproduction,withou t risk ofthei r dis­ persal. The development ofsee d orchards for seedproductio n (particularly if artificial pollination isused )ha s gone fart o accelerate seedproductio n withreliabl eheritabl e characters and isa suitabl emetho d forhandlin g vertical resistance factors.However ,vegetativ e propagation ismuc hmor e suitable forhorizonta l resistance andtechnique s for this,providin g quick utilisation ofdesirabl e geneticmaterial ,ar ealread ywel l established for many forestspecies . Much alsoneed s tob edon e toreduc e thedifficultie s posecKbyth e search forresistanc e in forestspecie s andth etestin g ofth eresults . Many ofthes edrawback s relate tochange s inhos treactio nwit h its agean d thenee d toachiev epresentatio n ofth epes tt oth ehos ti ntest swhic h correspond reasonablywit h field conditions.Som e advanceshav e already beenmad e to solvethes eproblems ,usin g techniques developed for agri­ cultural cropsusin geithe rexcise d tissues orindicator s (often empirical) based onth echemistr y ormorpholog y ofth ehost .Futur eproblem swil l call for special research tomee tth erequirement s of forestry. Forexample , diseasescause db ynematodes ,suc ha s Bursaphelenchus lignicolus Mamiya & Kiyohara (Mamiya, 1972), areonl yno wbein grecognise d outside the nursery anddamag e fromviruse s andvirus-lik e organisms to foresttree sha s scarcely beenrecognise d outsideEurop e andNort hAmerica . Indeed,no t only ascommercia l forestplantation s expend,bu t asthei r valuepe runi t area increases,s othe ywil lb e themor e closely scrutinised forpes t damage and awide rvariet y ofthes eproblem swil lb e revealed at aneconomi c levelmakin g themworth yo fcontrol .I nman ycase s themos tad ­ vantageous approach to thiswil lb e through theus eo finheren t resistance mechanisms.Technique s toexploi tthi sma y already existi n agricultural science insom e respects,bu t further research is always required to adapt these forus e in forestry.

REFERENCES

Bevan,D. ,1974 .Contro lo ffores tinsects :ther ei sa porpois eclos ebehin dus .In : D.Price-Jone s& K.E .Solomo n(Eds) :Biolog yi npes tan ddiseas econtrol .Proc .13t h Symp.Britis hEcol .Soc .p .302-312 .

19 Bingham,R.T. ,R.J .Hof f6 kG.I .McDonald ,1971 .Diseas eresistanc e infores ttrees .Ann . Rev.Phytopath .9 :433-452 . Browning,J.A .& K.J .Frey ,1969 .Multilin ecultivar sa sa mean so fdiseas econtrol . Ann.Rev .Phytopath .7 :355-382 . Browning,J.A. ,M.D .Simon s& E .Torres ,1977 .Managin ghos tgenes ;epidemiologi can d geneticconcepts .In :J.G .Horsfal l& E.B .Cowlin g (Eds):Plan tdisease .A nadvance d treatise.Vol .I .Ho wdiseas ei smanaged .Academi cPress ,London :p .191-212 . Burley,J .& G .Namkoong ,1980 .Conservatio no ffores tgeneti c resources.Invite dPape r 11thCommonwealt hForestr yConference ,Trinidad .CFI ,Oxford .2 5p . Burley,J .& P.J .Wood ,1976 .A manua lo nspecie san dprovenanc e researchwit hparticula r referencet oth etropics .Trop .For .Pap .No .10 ,CFI ,Oxford .22 6p . Buxton,E.W. ,1961 .Mechanism so fvariatio n inth epathogenicit yo fFusariu moxysporum . RecentAdvance si nBotan y1 ,Universit yo fToront oPress ,p .502-507 . Caldwell,P.S. ,J.F .Schäfer ,L.E .Compto n& F.L .Patterson ,1958 .Toleranc e tocerea l leafrusts .Scienc e128 :714-715 . Dinus,R.J. ,1974 .Knowledg eabou tecosystem sa sa guid et odiseas econtro li nmanage d forest.Proc .Amer .Phytopath .Soc .1 :189-190 . Driver,C.H .& J.H .Ginns ,1966 .A metho do fmas sscreenin go fsouther npine sfo rresis ­ tancet oa root-ro t inducedb yFome sannosu s (Fr.)Cooke .In :H.D .Gerhold , E.J.Schreiner ,R.E .McDermot t& J.A .Winiesk i (Eds):Breedin gpes tresistan ttrees . PergamonPress ,Oxford ,p .421-422 . Dunn,Z.A . &D.P.H .Kempton ,1972 .Resistanc e toattac kb yBrevicoryn ebrassica eamon g plantso fbrussel ssprouts .Ann .Appl .Biol .72 :1-11 . FA0,1979 .Geneti c improvemento ftropica llowlan dconifers .FO :MISC/79/15 ,Rome .8 6p . Flor,H.H. ,1953 .Epidemiolog y offla xrus ti nth eNort hCentra lStates .Phytopatholog y 43:624-628 . Gerhold,H.D. ,1970 .A decad eo fprogres si nbreedin gdisease-resistan t foresttrees . Unasylva 24(2-3):37-44 . Gibbs,J.N. , 1978.Intercontinenta lepidemiolog yo fDutc hel mdisea'se .Ann .Rev . Phytopath.16 :287-307 . Gibson,I.A.S. ,1972 .Dothistrom abligh to fPinu sradiata .Ann .Rev .Phytopath . 10:51-72 . Goddard,R.E .& D.L .Rockwood ,1980 .Co-operativ e forestgenetic sresearc hprogram . Twenty-secondProgres sReport .Univ .Fla .Schi .For .Res .Conservn. ,Res .Rept .No . 30.1 5p . Hanover,J.W. , 1980.Breedin gfores ttree sresistan tt oinsects .In :F.G .Maxwel l& P.R. Jennings (Eds):Breedin gplant sresistan t toinsects .Wiley ,Ne wYork .p .487-511 . Hatchett,J.H .& R.L .Gallun ,1970 .Genetic so fth eabilit yo fth eHessia nfl yMayetiol a destructor tosurviv eo nwheat shavin gdifferen tgene sfo rresistance .Ann .Entomol . Soc.Amer .63 :1406-1407 . Houston,D.R .& H.T .Valentine ,1977 .Comparin gan dpredictin gfores tstan dsuscepti ­ bilityt ogyps ymoth .Can .J .For .Res .7 :447-461 . Kemp,R.H. ,1978 .Exploration ,utilizatio nan dconservatio no ffores tgeneti cresources . Unasylva30 :10-16 . Kemp,R.H .& J .Burley ,1978 .Depletio nan dconservatio no ffores tgeneti cresources . In:J.C .Hawke s (Ed.):Conservatio nan dagriculture .Duckworth ,London ,p .171-185 . Kemp,R.H. ,J .Burley ,H .Keidin g& D.G .Nikles ,1972 .Internationa lco-operatio n inth e exploration,conservatio nan ddevelopmen to ftropica lan dsub-tropica lfores tgen e resources.Invite dpape r7t hWorl dFor .Congr .7 CFM/C:V/49 ,Bueno sAires ,Argentina . 16p . Khush,G.S. ,1977 .Diseas ean dinsec tresistanc ei nrice .Adv .Agron .29 :265-341 . Mamiya,Y. ,1972 .Pin ewoo dnematod eBursaphelenchu s lignicolusMamiy aan dKiyohara ,a sa causalagen to fpin ewiltin gdisease .Rev .Plan tProtect .Res .5 :46-50 . Martinsson,0. ,1979 .Testin gScot spin efo rresistanc e toLophodermiu mneedl ecast . Stud.For .Suec .Nr .150 .6 3p . Minter,D.W. ,J.M . Staley& CS . Millar,1978 .Fou rspecie so fLophodermiu m onPinu s sylvestris.Trans .Britis hMycol .Soc .71 :295-301 . Namkoong,G. ,R.D .Barne s& J .Burley ,1980 .A philosoph yo fbreedin gstrategy .Trop . For.Pape rNo .16 .Commonw .For .Inst. ,Oxford .6 7p . Rüssel,G.E. ,1978 .Plan tbreedin gfo rpes tan ddiseas eresistance .Butterworth ,London . 485p . Schoeneweiss,D.F. ,1975 .Predisposition ,stres san dplan tdisease .Ann .Rev .Phyto ­ pathology 13:193-211 . Schoeneweiss,D.F. ,1978 .Wate r stressa sa predisposin g factori nplan tdisease .In : T.T.Kozlowsk i (Ed.):Wate rdeficit san dplan tgrowth .Vol .5 .Academi cPress ,Lon ­ don,p .61-99 .

20 Schreiner,E. ,1939 .Th epossibilitie so fth eclon ei nforestry .J .For .37 :61-62 . Schreiner,E. ,1966 .Futur eneed sfo rmaximu mprogres si ngeneti c improvemento fdiseas e resistancei nfores ttrees .In :H.D .Gerhold ,E.J .Schreiner ,R.E .McDermot t& J.A.Winiesk i (Eds):Breedin gpest-resistan ttrees .Pergamo nPress ,Oxford ,p . 455-466. Skilling,D.D. , 1977.Th edevelopmen to fa mor evirulen t straino fScleroderri slager - bergiii nNe wYor kState .Europea nJ .For .Path .7 :297-302 . Vanderplank,J.E. ,1963 .Plan tdiseases ;epidemic san dthei rcontrol .Academi cPress , London.34 9p . Vloten,H .van ,1949 .Kruisingsproeve nme trasse nva nMelampsor alarici-populin aKlebahn . Tijdschr.PIZiekt .55 :196-209 .

21 The role of the environment in host-insect interactions

W.H. Parry

Departmento fForestry ,Universit yo fAberdeen ,St .Macha rDrive ,Ol dAberdeen , AB92UU , UK

ABSTRACT

Planthost/insec t interactions canb e extensively modified by changes inth eenvironment . Climate ison emajo r factorin ­ volved insuc hchanges ,temperatur e and rainfallbein g ofpar ­ ticularpotentia l importance inth ebreakdow n ofhos tresis ­ tancemechanisms .Parasite s andpredator s play an important role inmaintainin g lowpes tlevels .Th e survival ofpes t levels conducive to thecontinue d existence'o fthi s complexi s considered tob eo fprimar y importance during the adoptiono f resistance strategies.Hos tnutritiona l levels are important butthei r instability inrespons e toenvironmenta l changes minimises their effectiveness asresistanc e factors. Induced density-dependent resistance asdemonstrate d inbirc h inFin ­ landprovide s ausefu lmechanis m meriting further investigation inothe rtre e species.I ti sconclude d thatth e avoidance of host/insect synchronyb y the adoption ofa comple x ofresis ­ tancemechanism s aimed also atth emaintenanc e ofa non-econom ­ icpes t levelwoul d ensure long-term stability inmonocultura l forestry asi ti sno wpracticed .

INTRODUCTION

Planthost/insec t interactions havebee nreviewed.b y several authors in recentyears :e.g .McNeil l & Southwood (1978), Feeny (1976)an d Levin (1976). The role ofth eenvironmen t inth econtex to fresistanc e to forest insects hasbee nneglected , althoughbot hHanove r (1975)an d Stark (1965) havereviewe d thephysiolog y oftre eresistanc e toinsects . Theenvironmen t of anindividua l insectca nb e regarded asa combina ­ tion ofal l factorswhic h either directly orindirectl y influence thelif e oftha t individual.Miln e (1962)define s theeffectiv e environmenta s 'everythingi nth eUnivers ewhic hhelp s orhinder s fulfilmento fth e indi­ vidual'.Miln eplace d themai n factors acting on aninsec tpopulatio n into

22 Table 1. Classification ofth eenvironmen t according toMiln e (1962).

A.Density-independen t factors 1. Physical circumstances, i.e. weather 2. Indiscriminate actions ofothe r species B. Imperfectdensity-dependen t 1. Interspecific competition 2. Predators,parasites ,pathogen s C.Perfec tdensity-dependen t 1. Intraspecific competition

3 groupswhic h weredependen t onthei rrelationshi p todensit y (Table1) . Long-term co-evolutionary synchronization ofhos t and insect ideally leadst oth eoccurrenc e of anapparentl y stableplant/insec t relationship inwhic hneithe r ofth e two associates becomes dominant.Thi s stability couldb e destroyed by the appearance ofan yo fa variet y of disruptive factors.On e such factor lies inth e large-scale planting of artificial monocultures selected for factors other thanresistance . Theus e ofresistan t treesha s frequentlybee nsuggeste d asbein gth e ultimatemean s forpes tcontro l inbot h agriculture and forestry.Th eus e ofgeneticall y resistanthost s is frequently doomed to failure asgeneti c resistance will be expressed indifferen tway s under differing environmen­ tal conditions.Th e aimo fthi spresentatio n is toillustrat e thepossibl e effects ofenvironmenta l conditions onth eexpressio n ofplan t resistance suchtha tgeneti c resistance canb etailore d tomee tenvironmenta l condi­ tionsor ,i nspecifi c instances,th eenvironmen t couldb emodifie d tomee t therequirement s ofth eresistan thost .

CLIMATE

Elton (1958)wa s the firsto fman y observers toassociat e the absence ofpes toutbreak s intropica l forestswit hclimati c stability.Gra y (1972) concluded thatpes tproblem s intropica l situationswer etriggere d by cli­ matic instability.However , iti si nth eclimaticall y moreunstabl e temper­ ateregion s thatman y ofth epes tproblem s associated with afforestation areno w encountered. The 2mai nclimati c factors associated with insectoutbreak s arerain ­ fall andtemperature .Rainfal l affects theherbivorou s insect indirectly through itshos t andrainfal l conditions deviating strongly from normality will have thegreates teffec to nresistance .Suc h deviations areparticu ­ larly important intre e crops subjected torepeate d exposure tovariabl e seasonal and annualrainfall . White (1969)relate d 2outbreak s ofth epsylli d Cardiaspina densitexta

23 LÏ Taylor feedingo nth e leaves of Eucalyptus fasciculosa F.v.M.t o stress causedb y changes inth e rainfallpattern .Bot h outbreak periods werechar ­ acterizedb yunusuall ywe twinter s andunusuall y dry summers.Th e former caused the deathb y suffocation of feedingplan troots .Thi s reducedabili ­ tyt o take upwate rwa s exacerbated byth e lowavailabilit y ofwate r during the succeeding dry summers. Inorde rt oobtai n ameasur e ofth e stress imposed onth etree s under suchcondition s White calculated a 'stress index' (White, 1969). Inth e periods ofpsylli d outbreak ahighl ypositiv e serieso fstres s indexesas ­ sociatedwit h dry summers andwe twinter s were recorded. Outbreaks ofth e looper caterpillar, Selidosema suavis Butler feeding on shallow-rooted Pinus radiata D.Do n inNe w Zealand, Bupalus piniarius L. inth eNether ­ lands (Klomp, 1968)an d Choristoneura fumiferana (Clem.)population s in Canada (Morris,1963 )wer e alsocharacterize d bypositiv e stress indexes (White, 1974). White (1974)considere d that increases intota l and soluble nitrogen and amino acid levels inth e foliage oftree sunde r stresswa s the main factorchangin g suitability forherbivorou s insects. Ofparticula r interest inBritai n isth erecen toutbrea k ofth epin e beauty moth, Panolis flammea D. & S., onlodgepol e pine, Pinus contorta Dougl., invariou s areas of Scotland. InNave r forest,Sutherlan d the outbreak occurred in 1976 and 1977 (Stoakley, 1979). Calculation ofannua l stress indexes using rainfall data from thenearb yDavin a Lodgemeteorolog ­ ical station indicated thathighl ypositiv evalue s occurred inbot h 1975 and 1976 (Fig.1) .Bot h theseyear swer e characterised bywe twinters ,th e summero f 1976bein gparticularl y dry (Fig.1) .Stoakle y (1977)indicate d thatth eoutbrea kwa s associated withpin egrow no ndee punflushe d peat rather thano n freely drained ground. Substantial rootmortalit y bysuffo ­ cationwoul d have occurred onthes ewaterlogged , peaty sites during the winterprecedin g the outbreak. Theconife r oleoresin system isa poten tresistanc emechanis m against many insect species (Hanover,1975 ;Stark , 1965). Init shinderin g rolet o theentranc eo fvariou sbar kbeetle s the levelo fthi sresistanc e isclose ­ lyrelate d toth ewate r status ofth etrees .E.g. , theresistanc e of Pinus ponderosa Laws,t obar kbeetl e infestation is lower followingperiod s of deficient rainfall,injur y orothe r changes inth e environment (Vite, 1961). Water stress causes susceptibility by reducing the oleoresinexuda ­ tionpressur e (oep)whic hi sdependen to nth eturgidit y ofth e tree,speci ­ ficallyth eosmoti cpressur e ofth eepithelia l cells liningth eresi nduct s (Munch (1921)i nStark , 1965). Vite (1961)foun dtha toe p fluctuated diurnally andwa s affected during the summerb y temperature,ligh tintensit y andhumidity .However , seasonal fluctuationwa sprimaril y influenced by soilwate r anddecline d asth esea ­ sonprogressed . Thevariabilit y ofth epressur e and its dependence ona variety ofenvironmenta l factorsmake s itunlikel y thatoe p couldb euti -

24 T Standard normal deviates ® 2

®

Figure 1. Stress indices associatedwit h outbreaks of Panolis flammea in Sutherland,a :Standar d normal deviates,summe r (•) andwinte r (o)rain ­ fall,b :Stres s index (•).

used asa predictabl e genetic resistance mechanism. Both low andhig h temperatures can affectherbivorou s insectpopula ­ tionsb y killing insects,slowin g their rate ofmetabolism , modifying reproductive behavior or,changin g the susceptibility ofth ehos t toth e insectpest .Th e effecto flo wtemperatur e wasconsidere d by Parry (this book,pag e32) . Temperature has amarke d effecto nth eduratio n oflarva l stages.Wat t (1968)correlate d warm temperatures with outbreaks ofth egyps ymoth , Porthetria dispar inNe wYor k City,th egreates t increase innumber soccur ­ ringdurin g unusuallywar m summers.Unde r suchcondition s increased rates

25 L of fecundity and developmentminimise d the timeo fexposur e topredator s andparasites .Suc hconditions ,i ncombinatio nwit h thedrynes s usually associated withperiod s ofhig h temperature,woul dpu tth etree s under stress and increase theprobabilit y ofresistanc ebreakdown .

PARASITES AND PREDATORS

Iti sclea r thatth enumber s and oscillation range ofa ninsec t species ina give nhabita t isrelevan tt oth eregulator y ability of itsparasite s andpredators .Th eplantin g oftree swit h genotypes giving an increased resistance toa pes t insectwil l decrease theleve l oftha tpes t and,i n turn, affect itsparasite/predato r complex. Führer (1975,1978 )considere d thatth e survival of aspectru m of insectivorous insectswa s related to abasa l levelo fprey ,th e 'Eiserner Bestand', sufficiently high tomaintai n thecomple x withoutcausin gunac ­ ceptable damage.A reduction ofth ebasa l levelcoul d cause aloca lex ­ tinction ofthos e specialised predatory orparasiti c species operating at lowpre y density levels.A thighe rpre ydensit y levels,cause d eitherb y environmental or seasonal changes inth eresistanc e ofth ehos to rb y the absence ofth eregulator y effecto fth e lowdensit y complex,unspecialised , polyphagous insects are attracted.Th e sheernumbe r ofsuc h formsma y further reduce the level ofth elo wdensit y complex, ifi tstil lexists , through competition andthu scontribut e further to theeventua l breakdown ofresistance . Following resistant tree stand establishment aheavie r selectionpres ­ surewil lb e exerted onth epes tpopulation .W ehave ,o nth e onehand ,a n enormouslyplasti c insectgen epool , onth eother ,a stati c tree genepool . The eventual resulto fpes t selectionwil lb eth e emergence of aggressive races adapted toexis tunde r thene w conditions e.g. individual treedeme s ofth epine-lea f scale, Nuculaspis californica (Coleman)o nponderos apin e (Edmonds& Alstad , 1978). Under suchcondition s the existence ofspecial ­ ised lowdensit ypredator s andparasite sma yb ecrucial .

NUTRITION AS A RESISTANCE FACTOR

The amountan d availability of food is frequently themai n factor involved inintraspecifi c competition.Th eplan tca nrestric t food avail­ ability to insectherbivore s ina numbe r ofway s e.g. reducingprotei n digestibility with tannins.Competitio n for food isnormall y density-depen­ dentan d according toMiln e (1957)suc h arelationshi p isth eonl y truly density-dependent one.Despit e thehypothesi s ofSchwenk e (1962, 1963)tha t thenumbe r of leaf andneedl e eating insectswa s dependent onth e sugar contento fth e foliage iti sno w accepted thatdietar ynitrogen , inth e form ofa nadequat e andbalance d intakeo f amino acids andproteins ,i sa

26 critical factor indie t suitability forherbivorou s insects (McNeill & Southwood, 1978). Theus eo fresistan tplan t strains containing naturally lowo runbalance d proportions ofamin o acidswoul d appeart o represent an idealprotectiv e measure.Man y factors can contribute toth ebreakdow n of such aresistanc emechanis m e.g. climate, sitevariation , fertiliserappli ­ cationo r intraspecific and interspecific competition. Nitrogen isoccasionall y applied in forestmanagemen t e.g. application ofure a toboos tplan tgrowt hunde r certain conditions.Hube r &Watso n (1974)indicate d thatth e formo fnitroge n takenu pb yplant s influenced disease incidence and could alsob e criticalwit h regard to insectresis ­ tance.Th eeffec to fth e form ofN ondiseas e severity was dependent onth e specific host-parasite combination, rate and timing ofapplication , level ofresidua l soilN ,previou s cropping andth ephysica l environment.Th e level ofattac kb y the spruce aphid, Elatobium abietinum (Walker)i na young crop ofSitk a spruce (Picea sitchensis (Bong.)Carr. )emergin g from heather (Calluna vulgaris L.)chec kwa s considerablylowe r inchecke d plants than inothers .Spruc e aphidpopulation s aresensitiv e toplan t nitrogen levels (Parry, 1974,1976 ,1977 )an danalysi s ofthes eplant s indicated thatN levelswer emuc h higher inth e freegrowin gplant s (c. 1.2 % N ofdr yweight )i ncompariso nwit hplant s inchec k (c.0. 8 % N).Conse ­ quently,change s inN levels infores tcrop sma y resulti nincrease d tree susceptibility. Little isknow nconcernin g theeffect s ofinsec t feeding on thenutri ­ tional acceptability ofhos ttree s althoughbar kbeetle ,attac k is fre­ quently initiated bypionee r individuals which discriminate betweenresis ­ tantan dsusceptibl e trees.Thi s leads toth eeventua l colonization of suitable treesb y largenumber s ofbeetle s (Coulson, 1979). Themodifica ­ tiono f feeding sitenutritiona l quality isknow n tooccu r ine.g . Brevi- corgne brassicae h. where nutrient 'sinks' are formedb y alteration ofth e normal flowo fnutrient s inth ehos t (Way,1968 ;Wa y& Cammell , 1970). Uninfested rice-leafamin o acid levels averaged 13.4mg.g " while infested leaves averaged 79.3mg.g " (Cagampange t al., 1974). Similar reactionsma y occur intrees ,particularl y inrelatio n togal l formationwher enutrition ­ alcondition s arechange d by the feeding activities ofth e insect e.g. Adelgidae onconifers , Pemphigus spp.o npoplars . Theclassificatio n ofplant s into apparent andnon-apparen tb y Feeny (1975, 1976)marke d ausefu l step forward inth eexaminatio n ofth eco - evolution ofplant s and insects.Fores ttree s areclasse d as apparent plants i.e. they are long-lived and growni nassociatio nwit h largenumber s ofthei row nkind .Whil e conifers aregenerall y protected by resinsmos t deciduous treesutilis e high leaftanni n levels.Tannin s actb yprogres ­ sivelybindin gprotei n and lowering its availability to sap feeders and interferewit h thedigestiv eprocesse s ofchewin g insects (Feeny, 1969). Tannin levels inyoun g growing tissueswit hhig hnitroge n levels aregen -

27 erallymuc h inferior tothos e inmatur e tissueswit h lowernitroge nlevels . Consequently,man y insects feed andmatur e quickly onyoun g foliage.B y contrast,man y sawfly species feed onmatur e foliage inlat e summerwhe n the rateo f development isdecrease d inrespons e toth e lowereddigesti ­ bility ofth e food (Haukioja& Koponen , 1975;Haukioj a& Niemalä , 1974). The forestpractic e ofplantin g large areaswit h single clones oftree s allows aninsec tpes tt oquickl y synchronize itslif ecycl ewit hth ephe ­ nology ofth ehos twit h respectt operiod s ofmaximu m growthpotential . Where treephenolog yvarie s selection for synchronization canno toccu rt o such amarke d extent.Feen y (1975)indicate d that inmatur e stands ofoak , bud-burstma yrang e over 2weeks .Thi s limited the ability ofth eher ­ bivores to synchronize preciselywit h thetrees .Suc hsmal l differences in phenophase ofoa kstand sma yhav e largeeffect s onth epopulatio n levelso f wintermot h onoa k (Feeny,1975 ;Varle y et al., 1973). Themot hma yhav e respondedb y attempting tosynchroniz e with individual treesb y developing apterous femalesmor e inclined tore-colonis e trees onwhic h they feda s larvae. Iti stherefor e anessentia lpar to f any resistance control pro­ gramme topromot ephenologica l variability inth ehost .

INDUCED AND CONSTITUTIVE RESISTANCE

Induced resistance isth eresul to fa respons e ofth eplan tt oth e presence of afeedin g insectt oth edetrimen t oftha tinsect . Iti susuall y a density dependentphenomeno nb y contrastwit h constitutive resistance which isindependen t ofpes tlevels . Theproductio n of large quantities ofchemical s inorde r toreduc e the digestibility ofleave s imposes a 'strain'o nth ephysiologica l budgeto fa plant.Th eproportio n ofth e totalmetaboli c resources allocated to defence reflects the selectivepressure s imposed ontha tplan tspecie sb y insect pests during itsevolution .Heav y selectionwoul d result inhig hdefensiv e compound levels.Th emos t suitable sompromisewoul db e thatsituatio nwher e the chemicals areonl yproduce d inrespons e to attack. Such aninduce d response couldb e 'delayed' i.e. damageb y one insectgeneratio n gives plantresistanc e to subsequent generations; 'immediate'i.e . theplan t response decreases its susceptibility toth e insect inflicting thedamage , or 'indirect'wher e the damageb y one insect species increases treeresis ­ tance toothers . Induced resistance to Oporinia autumnata (Bkh.)ha sbee n demonstrated in Betula pubescens ssp. tortuosa byHaukioj a andhi sco-worker s inFinlan d where, inaddition , seasonal changes in leafquality , including decreasing water andnitroge n content and increasingphenoli c andtanni ncontent ,ar e associated with decreasing suitability anddigestibilit y ofbirc h leaves (Haukioja et al., 1978). O. autumnata responds tothes e constitutive changesb y feeding onyoun g leavesprio rt odevelopmen t of indigestibility

28 (Haukioja &Niemalä , 1974). Thebirc h response to leafdamag e retards the growth rate of 0. autum- nata larvae feedingo nyoun g foliagebot h inth e sameyea r and insubse ­ quentyears ,i.e . delayed andimmediat e induced resistance (Haukioja & Niemalä, 1977). Mechanical damage to leaves also resulted ina decrease d growth rate of larvae,feedin g onadjacen t leaves (Haukioja& Niemalä , 1979), a formo f indirect induced resistance.Althoug hbirche s areabl et o increase the amounto f leafphenolic s inrespons e tomechanica l damage,a processwhic h appears nott ooccu r inmatur e leaveswher e levels areal ­ ready high (Haukioja& Niemalä , 1979), iti s suggested thatothe rprocesse s may alsob e involved e.g., changes inproteinas e inhibitors (Haukioja & Niemalä, 1977). The levelo fconstitutiv e resistancema yb e connected toclimati c conditions e.g., inwar m summers the leavesmatur e earlier (Haukioja et al., 1978)an dcurtai l thegrowt hperio d of O. autvmnata by the early formation ofhig hphenoli c levels.Whethe r thiswa scritica lwit hregar d topopulatio n levels remainsunclear . The combination of induced and constitutive resistance mechanisms decreases theprobabilit y ofth eoccurrenc e of anoptimall y adaptedher ­ bivore.Th eus e ofbirc h cloneswit hhighl y developed density-dependent inductive responses offers animmediat e defencemechanis m responsive to increased pestnumber s causedb y environmental variation, such ashig h temperature.

CONCLUSION

The traditional concepto fth eutilisatio n ofplan tresistanc e asa simplemean s ofminimisin gpes tdamag epay s little attentiont oth e effects ofth eprolonge d selection imposed ona serie s of forest insectpes tgen ­ erations feeding onon ehos tgeneration . Selection leads to an inevitable diminution of theefficac y oftha tresistanc emechanism . Consequently, it issuggeste d thatresistanc emechanism s aiming atth e complete localelim ­ inationo f forest insectpes t species shouldb e rejected under forestplan ­ tationcondition s infavou r ofth e adoption ofa comple x ofweake rresis ­ tancemechanism s leading toth e formation ofa balance dplant-herbivor ere ­ lationship where neitherpartne rbecome s dominant. Inthi s contextMattso n etal.' s (thisbook ,pag e 000)concep to forganis m traitvector s could prove invaluable as itdescribe s how organisms interactwit h eachothe r in relation toth emanne rprescribe d by the seto ftrait s thateac hpossesses . Variation inthes e traitswoul d change theplant/insec t relationship ina predictablemanner . The development of suitable resistantvarietie s shouldb e coupledwit h environmental studies aimed atobtainin g information onth e effects of various factors onth eexpressio n of these genetic characteristics.Kalk -

29 stein (1976) has correlated Dendroctonus frontalis Zimmermann activity with various climatic variables in an attempt to develop.a predictive technique capable of evaluating future insect activity based upon past climatic con­ ditions. Similar climate-insect evaluations in conjunction with the use of resistant trees could provide practising foresters with the background in­ formation necessary to employ the most efficient silvicultural techniques in particular areas.

REFERENCES

Cagampang,G.G. ,M.D .Patha k& B.0 .Juliano ,1974 .Metaboli cchange si nth eric eplan t duringinfestatio nb yth ebrow nplant-hopper ,Nilaparvat a legensSti l(Hemiptera , Delphacidae).Applie dEntomolg yan dZoolog y9 :174-184 . Coulson,R.N. ,1979 .Populatio ndynamic so fbar kbeetles .Annua lRevie wo fEntomolog y 24:417-447 . Edmonds,G.F .& D.N .Alstad ,1978 .Co-evolutio ni ninsec therbivore san dconifers . Science199 :941-945 . Elton,C.S. ,1958 .Th eecolog yo finvasio nb yanimal san dplants .Methuen ,London .18 1p . Feeny,P.P. ,1969 .Inhibitor yeffec to foa klea ftannin so nth ehydrolysi so fprotein sb y trypsin.Phytochemistr y 8:2119-2126 . Feeny,P.P. ,1975 .Biochemica lco-evolutio nbetwee nplant san dthei rinsec therbivores . In: L.E.Gilber t& P.H .Rave n (Eds): Co-evolutiono fanimal san dplants .Universit y ofTexas ,Austin/London ,p .3-19 . Feeny,P.P. ,1976 .Plan tapparenc yan dchemica ldefence .In :J.W .Wallac e& R.L .Mansel l (Eds):Biochemica linteractio nbetwee nplant san dinsects .Recen tAdvance s inPhyto ­ chemistry10 .Plenum ,Ne wYork/London ,p .1-40 . Führer,E. ,1975 .Überlegunge nzu rWirkun gresistenzsteigernde rMassnahme n imWal dau f denMassenwechse lforstliche rSchadinsekten .Forstarchi v46 :228-233 . Führer,E. ,1978 .Forstzoologi ezwische nTheori eun dPraxis .II .Abschie dvo nvertraute n Vorstellungen.Forstarchi v49 :66-69 . Gray,B. ,1972 .Economi c tropicalfores tentomology .Annua lRevie wo fEntomolog y17 : 315-334. Hanover,J.W. , 1975.Physiolog y oftre eresistanc et oinsects .Annua lRevie wo fEnto ­ mology20 :75-95 . Haukioja,E .& S .Koponen ,1975 .Birc hherbivore san dherbivor ya tKevo' .In : F.E.Wielgolask i (Ed.):Fennoscandia ntundr aecosystem sPar t2 .Springer ,Berlin / Heidelberg/NewYork .Ecologica lStudie s17 :181-188 . Haukioja,E .& P .Niemalä ,1974 .Growt han denerg yrequirement so fth elarva eo fDineur a virididorsata (Retz.)(Hym .Tenthredinidae )an dOporini aautumnat a (Bkh.)(Lep. , Geometridae)feedin go nbirch .Annale sZoologic iFennic i11 :207-211 . Haukioja,E .& P .Niemalä ,1977 .Retarde dgrowt ho fa geometri dlarv aafte rmechanica l damaget oleave so fit shos ttree .Annale sZoologic iFennic i14 :48-52 . Haukioja,E .& P .Niemalä ,1979 .Birc hleave sa sa resourc efo rherbivores :seasona l occurrenceo fincrease dresistanc e infoliag eafte rmechanica ldamag eo fadjacen t leaves.Oecologi a39 :151-159 . Haukioja,E. ,P .Niemalä ,L .Iso-Iivari ,H .Ojal a& E.M .Aro ,1978 .Birc hleave sa sa resourcefo rherbivores .Repor to fth eKev osubarcti cResearc hStatio n14 :5-12 . Huber,D.M .& R.D .Watson ,1974 .Nitroge nfor man dplan tdisease .Annua lRevie wo fPhyto ­ pathology 12:139-165 . Kalkstein,L.S. ,1976 .Effect so fclimati c stressupo noutbreak so fth esouther npin e beetle.Environmenta lEntomolog y 5:653-658 . Klomp,H. ,1968 .A seventee nyea rstud yo fth eabundanc eo fth ePin eLooper ,Bupalu s piniariusL . (Lepidoptera:Geometridae) .In :T.R.E .Southwoo d (Ed.):Insec tabun ­ dance.Roya lEntomologica lSociet yo fLondo nSymposiu m4 :98-108 . Levin,D.A. , 1976.Th echemica ldefense so fplant st opathogen san dherbivores .Annua l Reviewo fEcolog yan dSystematic s7 :121-159 . McNeill,S .& T.R.E .Southwood ,1978 .Th erol eo fnitroge ni nth edevelopmen to finsect / plantrelationships .In :J.B .Harbourn e (Ed.):Biochemica laspect so fplan tan d animalcoevolution .Academi cPress ,Ne wYork/London ,p .77-98 . Milne,A. ,1957 .Th enatura lcontro lo finsec tpopulations .Canadia nEntomologis t89 : 193-213.

30 Milne,A. ,1962 .O na theor yo fnatura lcontro lo finsec tpopulations .Journa lo fTheo ­ reticalBiolog y3 :19-50 . Morris,R.F. ,1963 .Th edynamic so fepidemi c sprucebudwor mpopulations .Memoir so fth e EntomologicalSociet yo fCanad a31 :1-322 . Parry,W.H. ,1974 .Th eeffect so fnitroge nlevel si nSitk aspruc eneedle so nElatobiu m abietinum (Walker)population s inNorth-easter nScotland .Oecologi a 15:305-320 . Parry,W.H. ,1976 .Th eeffec to fneedl eag eo nth eacceptabilit yo fSitk aspruc eneedle s toth eaphid ,Elatobiu mabietinu m (Walker).Oecologi a23 :297-313 . Parry,W.H. ,1977 .Th eeffect so fnutritio nan ddensit yo nth eproductio no falat e Elatobium abietinumo nSitk aspruce .Oecologi a30 :367-375 . Schwenke,W. ,1962 .Neu eErkenntniss eübe rEntstehun gun dBegegnun gvo nMassenvermehrunge n anKiefer nun dFichtennadel nfressende rSchadinsekten .Zeitschrif tfü rangewandt e Entomologie50 :137-142 . Schwenke,W. ,1963 .Übe rdi eBeziehun gzwische nde mWasserhaushal tvo nBäume nun dde r Vermehrungblattfressende r Insekten.Zeitschrif tfü rangewandt eEntomologi e51 : 371-376. Stark,R.W. ,1965 .Recen ttrend si nfores tentomology .Annua lRevie wo fEntomolog y10 : 303-324. Stoakley,J.T. ,1977 .A sever eoutbrea ko fth epin ebeaut ymot ho nlodgepol epin ei n Sutherland.Scottis hForestr y31 :113-125 . Stoakley,J.T. ,1979 .Pin ebeaut ymoth .Forestr yCommission ,London ,Fores tRecor d120 . 11p . Varley,G.C. ,G.R .Gradwel l& M.P .Hassell ,1973 .Inspec tpopulatio necology, -a nana ­ lyticalapproach .Blackwel lScientific ,Oxford .21 2p . Vite,J.P. ,1961 .Th einfluenc eo fwate rsuppl yo noleoresi nexudatio npressur e andre ­ sistancet obar kbeetl eattac ki nPinu sponderosa .Contribution s fromBoyc eThompso n Institute21 :37-66 . / Watt,K.E.F. ,1968 .Ecolog yan dresourc emanagement .McGra wHill ,Ne wYork .p .62-65 . Way,M.J. ,1968 .Intraspecifi cmechanism swit hspecia lreferenc et oaphi dpopulations . In: T.R.E.Southwoo d (Ed.): Insectabundance .Roya lEntomologica lSociet yo fLondo n Symposium4 :18-36 . Way,M.J .& M.E . Cammell,1970 .Aggregatio nbehaviou ri nrelatio nt ofoo dutilizatio nb y aphids.In :A .Watso n (Ed.):Anima lpopulation si nrelatio nt othei rfoo dresources . Symposium ofth eBritis hEcologica lSociet y10 :229-247 . White,T.C.R. ,1969 .A ninde xt omeasur eweather-induce dstres so ftree sassociate dwit h outbreakso fpsyllid si nAustralia .Ecolog y50 :905-909 . White,T.C.R. ,1974 .A hypothesist oexplai noutbreak so floope rcaterpillars ,wit h specialreferenc et opopulation so fSelidosem asuavi si na plantatio no fPinu sradi - atai nNe wZealand .Oecologi a16 :279-301 .

31 Variability in conifer needle freezing as a resistance factor to aphids

W.H.Parr y

Departmento fForestry ,Universit yo fAberdeen ,St .Macha rDrive ,Ol dAberdeen , AB92UU , UK

ABSTRACT

A review ofth e literature onoverwinterin g mortality of the green spruce aphid indicated thata clos e relationship existed between spruceneedl e freezing temperatures and aphid freezing temperatures causingmortality . Resistant treeswer e found tob e thosewher eneedle s froze athighe r temperatures thanth eremainder , suchtree s exhibiting higher aphidmortal ­ ity inman ywinter s inN.E .Scotland .A s defoliation and aphid population levels arecorrelate d with overwintering aphidsur ­ vival thismechanis m isver y important inpreventin g spruce aphid outbreaks in allexcep tmil dwinters .Th e possibilities of incorporating thismechanis m into the geneticpoo l of forest trees isdiscusse d aswel l asit spossibl e existence inth ere ­ lationship between otherplant-suckin g insects andthei rhost .

INTRODUCTION

The existence ofSitk a spruce. Picea sitchensis (Bong.)Carr . resistant toth e green spruce aphid, Elatobium abietinum (Walker),ha sbee n observed by several authors includingMatthew s (1950), Behrndt (1961)an dParr y & Powell (1977).Thes e trees exhibited theclassica l resistancecharacteris ­ tic of foliage retention inperiod swhe n adjacenttrees ,frequentl ywit h intermingling foliage,exhibite d aheav y defoliation. Suchtree shav ebee n encountered frequently inN.E .Scotland . Long-term observations showed that this apparentresistanc ewa s occasionally lost in years followingmil d winters only toreappea r insubsequen t years. Studies onthi sphenomeno n indicated thatgrowt h rates of aphidpopula ­ tions insprin gwer e similar onal ltree s andn odifference s inth e rates ofeithe r symptom development ordefoliatio nwer e evident (Parry& Powell , 1977). Themai n difference between trees retaining needles (=resistant ) andtree s showingregula r defoliation (=susceptible )la y inth ehighe r number of aphids onth e latter inearl y spring, thisbein g particularly

32 marked on firstyea rneedle s exposed togreate r temperature extremes.Th e number ofaphid spresen t atpea kpopulatio n inlat e June/early July,an d consequently the amounto fdefoliation ,wa s correlated with thenumbe r of aphids survivingwinte r (Powell& Parry , 1976). Therefore, overwintering survivalwa s the critical factor inth edamagin gpotentia l ofth e aphid.

OVERWINTERING SURVIVAL OF E. ABIETINUM

Inoceani c regions such asBritai n the aphidprimaril y overwinters as apterous,viviparous ,parthenogeneti c females.I nth econtinenta l climate ofCentra l Europe overwintering eggspredominat e (Scheller, 1963;Klof te t al., 1961). Freezingmortalit y of active,overwinterin g E. abietinum hasbee n fre­ quently observed (Bevan, 1966 andFig .1) .E.g. , inN.E .Scotlan d atemper ­ ature of -5 °Cwa s recorded onth enigh to fMarc h 12/13, 1978whic h caused 63% mortalit y ofadult s and8 3% mortalit y of immature stages inon e Sitka spruce stand.Th e distribution ofth e live anddea d aphids indicated that complete aphidmortalit y occurred onsom e individual needles,complet esur ­ vival onother s (Fig.1) .Suc h anon-rando m distribution associates indi­ vidual aphidmortalit y with thedeat ho fneighbourin g aphids onth e same needle.

Figure 1. Non-random mortality of aphids onSitk a spruceneedle s following exposure to -5 °C (a= needle swit h live aphids,b =needle swit h dead aphids).

33 INITIATION OF FREEZING IN E. ABIETINUM

As freezingmortalit y of E. abietinum isdependen tno t only onth e temperature ofth e surrounding airbu t alsoo ntha t inth eneedl e feeding site,the nvariatio n inth elatte rcoul d resulti ndifferentia l freezing mortality. Thisprovide s abasi s fora resistanc emechanism . Inisolatio n from its sprucehos tth e ability of E. abietinum towith ­ stand lowtemperature s without freezingvarie s inrelatio nt o its feeding stage (Table 1).I nth e fieldheav y aphidmortalit yoccur s attemperature s well abovethos erequire d tokil l E. abietinum inisolatio n fromth enee ­ dles.E.g. , Carter (1972)observe d aheav y aphidmortalit y at- 8 °Cwhil e Powell& Parr y (1976)foun d that- 7 °Cgreatl y reduced overwinteringpopu ­ lations.Thi s fieldmortalit y atrelativel y hightemperature s mustb ea corollary of attachment toth eneedles .A comparison over4 hour s ofth e timerelate dmortalit y at-1 2 °Co f'tw ogroup s ofaphids ,on eremainin g undisturbed onth eneedles ,th e otherdetached , showed thatdetache d aphids survivedwhil e attached aphids exhibited a9 0% mortalit y (Powell, 1974). Carter (1972)indicate d thatsurfac ewate r hadn o effecto nth emortal ­ ityo f E. abietinum anddirec t inoculation from external icethroug hth e aphid cuticularpore s (Salt, 1963), can,therefore ,b eeliminated . Powell (1974)considere d aphid freezingt ob e theresul to fdirec t inoculationo f the aphid from the frozentissue s of Sitka sprucevi a the sap inth e stylet food canal. In Myzus persicae (Sulzer),whic h isroughl y comparable insiz e to E. abietinum, the diameter ofth e stylet food canal isapproximatel y 0.5 |jm(Hoof , 1958). Thiswoul dhav ea minima l effecto nth e freezingpoin t depression of liquidswithi n thecanal ,an dwoul d notrestric t ice forma-

Table 1. Theeffec to fgu tconten to n theresistanc e to freezing of (a) E. abietinum and(b ) Myzus persicae.

Aphid instar Gut , Mean supercooling (°C) Author

(a)Insta r I Empty -22.70± 1.11 Parry (1979) Instar I Empty -26.97± 1.02 Parry (1979) Instar I Empty -24.7 Powell (1974) Instar I Empty <-19 Carter (1972) Instar IIt o adult Full -15.3 ± 0.32 Powell (1974) Adults Full -11.0± 1.0 Carter (1972) Adults Full c. -9t o-1 0 Parry (1979)

(b)Insta r I Empty -22.67± 1.00 Parry (1978) Adults Full -12.00 ±0.87 Parry (1978)

34 tion. Salt& Kak u (1967)showe d thatth e initialnucleatio no fneedle s of Coloradoblu e spruce, Picea pungens Engelmann,occurre d inth estele , probably inth exylem ,th e endodermisactin ga sa barrie r toimped e ice propagation inth emesophyll . Observations of Sitka spruceneedle s during cooling indicated that freezing initially occurred within thevascula rbundl e andwa s visually observable as asudde n lightening ofth ebundl e colour following thefor ­ mation of ice.Th emesophyl l remained dark greendurin g subsequentcooling , probably due to the impedance toic epropagatio nb y theendodermis ,unti l similar colour changes occurred inthi stissu e atlowe rtemperatures . Visual observations of aphids cooledwhil e remaining undisturbed onth e needles indicated that freezingoccurre d atth e same time asic ebecam e visually apparenti nth eneedl e stele.Disturbe d aphids cooled on similar needles remained unfrozen onth eneedl e surface forsom etim e following stele, and frequentlymesophyll ,ic eformation . The supercoolingpoint s ofspruc eneedle s (Table 2)ar e considerably highertha nthos erecorde d for E. abietinum inisolatio n from itshos t (Table 1), and arecomparabl e toth e -7 °Ct o- 8 °Crequire d tocaus eheav y aphid fieldmortality .

FREEZING AS A RESISTANCE FACTOR

Inorde r totes t thehypothesi s thatvariatio n inth e ability ofnee ­ dles tosupercoo l couldb eutilize d asa resistanc e factortw ogroup s of treeswer e selected, one group showing high aphid overwintering survival, the second alo wwinte r survival ineac ho f3 successiv ewinters . Inorde r tominimis e environmental variation treepair swer e situated asphysicall y close together aspossibl e and frequentlypossesse d intermingling branches androo t systems.Th e temperature atwhic h ice formedwa s significantly higher inthos e trees showing low aphidoverwinterin g survival thani n

Table 2. Thetemperature s atwhic h ice formation commences inth eneedle s ofvariou s Picea species.

Species of Picea Temperature ofic e formation Author commencement (°C)

P. abies -4t o- 8 Pisek (1960) P. sitchensis -9t o-1 2 Powell (1974) P. sitchensis -5t o- 9 Parry& Powel l (1977) P. sitchensis -4t o- 6 Parry (1979) P. pungens -7t o-1 3 Salt& Kak u (1967)

35 those trees showing high aphid overwintering survival (Parry& Powell , 1977). Sitevariatio n inth e supercooling ability ofSitk a spruceneedle s can occur (Table 2). Thehig h supercooling ability ofsuc hneedle s onon e site (Powell, 1974)contrast swit h the lower level atth e second site (Parry & Powell, 1977), outbreaksbein gmor e severe andmor e frequent atth efirst .

EFFECTS ON A STAND OF SITKA SPRUCE

High levels of spruce aphid cancaus e amarke d fall inhe tleade r growth of Sitka spruce,particularl y young spruce during andimmediatel y following the establishmentperio d (Carter, 1977;G . de'Britt ,pers . comm.). Under thewinte r temperature regimes common inScotlan d (Parry & Powell, 1977)susceptibl e Sitka sprucewoul db e subjected to repeated spring attackscausin g asubstantia l depression inth eheigh t incremento f these trees.Therefore , asth eplantatio nmature s its susceptibility to attack decreases asthes e susceptible spruce are suppressed andeithe r die or arephysicall y removed. InBritai n the traditional practice of selective thinning resulted inth e removal ofa hig hproportio n ofsuc htrees . Inth e modernpractic e of line thinning the susceptible trees remain and thestan d retains its original characteristics. There is some evidence ofa nincrease d resistance to E. abietinum in maturing Sitka spruce crops.Beva n (1966)indicate d thatyoun g cropso f Sitkaspruc ewer epron e toattac kb y E. 'abietinum up toth e thicketstage . Powell (pers.comm. )surveye d Sitka spruce stands inScotlan d and concluded thatpol e stage treeswer e less susceptible thanyounge r crops.Schelle r (1963)indicate d thattree s oflowe r growth incurred thehighe r population density, althoughher e theeffect s ofshelte rwer e operative.B y contrast Ohnesorge (1959)indicate d that>2 0yea r old sprucewa smor e susceptible than <10yea r old spruce.Mor ewor k isneede d onth eeffect s of tree ageo n susceptibility inrelatio n tothinnin g technique,sit e characteristics and genetic variability.

SELECTION OF RESISTANT SITKA SPRUCE

Inth e selection ofresistan t trees thecritica lparamete r isth e inability of Sitka spruceneedle s to supercoolmuc hbelo w the depressed freezingpoin to f theextracellula r water,thi sbein g determined by anum ­ ber ofenvironmenta l and othervariables .Th emechanis m ofplan ttissu e freezingha sbee nreviewe d indetai lb yMazu r (1969),Alde n& Herman n (1971)an dBurk e et al. (1976). By selecting Sitka spruce trees inwhic h extra-cellular water freezes atrelativel y high temperaturesplantatio n susceptibility to E, abietinum

36 couldb e significantly decreased,particularl y duringmil d winters whenat ­ tacksnormall y develop. Suchtree swoul d be found inselectivel y thinned, mature Britishplantation s whichhav ewithstoo d several aphid attacks.Th e use oftree sgrow n from imported seed or seed orchards containing treesse ­ lected forothe rcharacteristic swoul db eo flittl evalu e inthi scontext . The selection of Sitka sprucewit hhig h temperature extra-cellular water freezingpropertie s offers aresistanc emechanis m which isunlikel y tob ebroke nb y genetic selection ofresistan t strains of aphids.However , selection could occur in favour ofholocycli c races of E. abietinum over­ wintering inth eeg g stage.Thi s lattermechanis m removes thepositiv e effects ofmil dwinter s onoverwinterin g aphidnumber s and considerably decreases thepossibilit y ofsprin goutbreaks .

OVERWINTERING MORTALITY IN OTHER SPECIES

Thevulnerabilit y ofth e active,feedin g aphid tolo w temperature mortality isillustrate d by the frequency withwhic h theeg g serves asth e overwintering stage, inth eAdelgida e overwintering normally occurs inth e form ofdiapausin g firsto r later instar sistentes capable of surviving temperatures downbelo w -30 °C (Parry, 1980). Whensa pimbibitio n commences these sistentesbecom e susceptible totemperature s ashig h as -5 °C.Ther e isn orelationsho p betweenmortalit y and the freezingcharacteristic s of thehos twhil e theAdelgida e remaini ndiapaus e (Parry, 1980). As theter ­ mination ofdiapaus e and consequent commencement ofsa puptak evarie swith ­ inth eAdelgida e those species emerging from diapause and commencing feed­ ing ata nearlie r time shouldb e lessresistan t to lowtemperature . Adelges prelli (Grosmann),whic h isconsidere d tob eparticularl y sensitive tolo w temperature, commences sapuptak e inmid-Januar y (Merker et al., 1957). By comparison A. coo leyi, inwhic h sapuptak ebegin s inmid-February , isex ­ posed to lowtemperature s for amuc h shorter time (Parry, 1980).Conse ­ quently there isa greate rpossibilit y ofmodifyin gAdelge smortalit yb y selection ofconifer s inwhic h the sap imbibedb y the feeding Adelgidae freezes athighe r temperatures thannorma l inthos e specieswhic hcommenc e feeding ata nearlie rtime . Inorde r to avoid icenucleatio nwithi n thegu tcontent smos tinsec t species overwinter invariou snon-feedin g states,e.g. , eggs,pupae ,o r adultswit h empty guts.Ver y little canb e done tomodif y the lowtempera ­ ture susceptibility ofsuc h forms.Therefore ,th emanipulatio n ofoverwin ­ teringmortalit yb y selectionwithi n thehos ttree s isrestricte d toa narrow spectrum of sap-sucking conifer aphids overwintering asactive , feeding forms.

37 REFERENCES

Alden,J .& R.K .Hermann ,1971 .Aspect so fth ecold-hardines smechanis m inplants . BotanicalRevie w37 :37-142 . Behrndt,V.G. ,1961 .Auftrete nun dBekämpfun gde rSitkalau si mFrühjah r196 7i mPrivat ­ waldde sWeser-Ems-Raumes .Allgemein eForstzeitschrif t16 :659-661 . Bevan,D. ,1966 .Th egree nspruc eaphi sElatobiu m (Neomyzaphis)abietinu mWalker .Scot ­ tishForestr y20 :193-201 . Burke,M.J. ,L.V .Gusta ,H.A .Quamme ,C.J .Weise r& P.H .Li ,1976 .Freezin gan dinjur yi n plants.Annua lRevie wo fPlan tPhysiolog y27 :507-28 . Carter,C.I. ,1972 .Winte r temperaturesan dsurviva lo fth egree nspruc eaphid ,Elatobiu m abietinum (Walker).Fores tRecor dNo .84 ,Forestr yCommission ,London .1 0p . Carter,C.I. ,1977 .Impac to fgree nspruc eaphi do ngrowth .Researc han dDevelopmen t Paper116 ,Forestr yCommission ,London .8 p . Hoof,H.A .van ,1958 .Onderzoekinge nove rd ebiologisch eoverdrach tva nee nnon ­ persistentvirus .Mededelin gNo .161 ,Instituu tPlanteziektenkundi gOnderzoek ,Wage ­ ningen,th eNetherlands .9 6p . Kloft,W. ,P .Erhard t& H .Kunkel ,1961 .Di eFichtenröhrenlaus ,Elatobiu mabietinu m (Walk.1849) ,ei nendemische sInsek tde rRotficht ePice aexcels ai nEuropa .Wald ­ hygiene4 :121-125 . Matthews,J.D. , 1950.Genetics .Repor to nFores tResearc hfo rth eyea rendin gMarc h1949 . ForestryCommission ,London ,p .26-29 . Mazur,P. ,1969 .Freezin ginjur yi nplants .Annua lRevie wo fPlan tPhysiolog y20 : 419-448. Merker,E. ,0 .Eichhor n& I .vo nKleist ,1957 .Di evernichtend eWirkun gvo nklimatische n Kälteeinbrüchenau fTannenläus ede rGattun gDreyfusia .Zeitschrif tfü rangewandt e Entomologie41 :333-352 . Ohnesorge,B. ,1959 .Di eMassenvermehrun gde rSitkalau si nNordwestdeutschland .Forst ­ archiv30 :73-78 . Parry,W.H. ,1978 .Supercoolin go fMyzu spersica e inrelatio nt ogu tcontent .Annal so f appliedBiolog y90 :27-34 . Parry,W.H. ,1979 .Acclimatisatio n inth egree nspruc eaphid ,Elatobiu mabietinum .Annal s ofapplie dBiolog y92 :299-306 . Parry,W.H. ,1980 .Studie so nth efactor saffectin gth epopulatio nlevel so fAdelge s cooleyi (Gillette)o nDougla sfir .3 .Lo wtemperatur emortality .Zeitschrif tfü r angewandteEntomologi e90 :133-141 . Parry,W.H .& W .Powell ,1977 .A compariso no fElatobiu mabietinu mpopulation so nSitk a sprucetree sdifferin gi nneedl eretentio ndurin gaphi doutbreaks .Oecologi a27 : 239-252. Pisek,A. ,1960 .Di ePhotosynthetische nLeistunge nde rimmergrüne nPflanze n(einschl . Koniferen).In :W .Ruhlan d (Ed.):Handbuc hde rPflanzenphysiologi eBd .V .Tei l2 . 439p . Powell,W. ,1974 .Supercoolin gan dth elo wtemperatur e survivalo fth egree nspruc e aphid,Elatobiu m abietinum.Annal so fapplie dBiolog y78 :27-37 . Powell,W.^ &W.H .Parry ,1976 .Effect so ftemperatur eo noverwinterin gpopulation so fth e greenspruc eaphid ,Elatobiu mabietinum .Annal so fapplie dBiolog y82 :209-219 . Salt,R.W. ,1963 .Delaye dinoculativ e freezingo finsects .Canadia nEntomologis t95 : 1190-1202. Salt,R.W .& S .Kaku ,1967 .Ic enucleatio nan dpropagatio ni nspruc eneedles .Canadia n Journalo fBotan y45 :1335-1346 . Scheller,H.D .von ,1963 .Zu rBiologi eun dSchadwirkun gde rNadelholzspinnmilbe ,Oli - gonychusunungui s (Jacobi),un dde rSitkafichtenlaus ,Liosomaphi sabietin aWalke r (Hom.Aphid.) .Zeitschrif tfü rangewandt eEntomologi e51 :258-284 .

38 Trees, fungi,an d environment

P. Schutt

Lehrstuhl für Forstbotanik, Universität München, Amalienstrasse 52, D-8000 München 40, BRD

ABSTRACT

Usingpathologica l events inth erhizospher e and inth e phyllosphere asexamples ,i ti spointe d outtha ta give npatho ­ logical situation often cannotb eexplaine db ymean s ofth ees ­ tablishedmode lwhic hsay stha tgeneticall y controlled defense traits aremodifie dwithi n certain limitsb y environmental in­ fluences.Ther e arenumerou s additional effects likecompeti ­ tion, allelopathy, antagonisms,synergism s and secondaryplan t substances,whic h arethemselve s influenced by the environment andwhic hobscur e the interrelationships between genetic and environmental factors.

INTRODUCTION

Inintroductor y paperspresente d atscientifi cmeeting s iti scustomar y andusefu l toprovid e afairl ybroa dbackgroun d dealingwit hne w results in related fields,i na n attemptt ocompar e and linkthe mwit h current aspects ofresearc h ofmor e directrelevanc e toth emai ntopics . The title ofthi spaper , 'Trees, fungi,an d environment', issuffi ­ cientlybroa d topermi t such anapproach .A t firstsight ,however ,thi s simultaneously createsbot h anattractiv e and aconfusin g feeling.Pathol ­ ogists and geneticistsma ywel l feel thati ti s farto o general atitl e since itembrace s analmos t limitlessnumbe r ofdee p and far-reaching problems, eacho fwhic h interact and so lead to anea rhopeles s situation fromwhic h toestablis h linkshavin g generalvalidity . Central Europeanpathologist s may recall thewell-know n relationship betweenclimat e and diseaseboldl y expressedby :

good wine years - good years for insects bad wine years - good years for fungi

Ingeneralitie s likethese ,environmen t isgive nequa lweigh tt oclimate ,

39 anover-simplificatio n which isuntrue . Ifw e define theter m 'environmen­ tal influences'a s 'parameterswhic h acto n treesexternally 'the nw ehav e to include inadditio n toclimat e theman ybiogeni c andnon-biogeni c fac­ tors.Thi s leadsu s into fieldswhic h areaffecte d byproblem so fcompeti ­ tion,o fallelopathy , and ofsymbiosis ,activitie s which areofte n regarded asperiphera l disciplines ofpathology .Observation s andresearc h inthes e fields,however ,ofte ncontribut e towards abette r understanding ofth e processes ofdiseas e outbreak anddiseas eprogression . Before Itr yt odemonstrat e thecomplexitie s ofestimatin g theinflu ­ ence ofsingl e environmental factors onhost ,parasite ,an d diseasepro ­ gression,perhap s Ishoul d remindyo u ofsevera l factswhic hma yb e trivial butnone-the-les s importantbiologica l keywords forth e followingobserva ­ tions: First, iti sth epredispositio n fora trait ,no tth etrai titself , which isunde r genetic control.The-expressio no f atrait ,however ,i s oftengreatl y influenced by theenvironment .Becaus e the same environmental factor can,i nprinciple , affectdifferen t components ofa necosyste m by different degrees andways , theinfluence s ofth eenvironmen t are difficult toanalys e and areeve nmor e difficult topredict . Second, theter m 'resistance'i sunderstoo d asth egeneticall ycon ­ trolled aspect ofstability .Dispositio n orpredisposition ,however ,ar e regarded as alterations of stability, duet oenvironmenta l factors. Third, trees areexpose d tovaryin g environmental factorsdurin gthei r long lifean d are,therefore ,differen t frommos t agricultural andhorti ­ cultural crops.Consequentl y the samehos tca nb e affected indifferen t yearsb y different environmental influences.Moreover , long-lived plants have developed anadditiona lmetho d ofsurvival ,namely , anabilit yt o recover. Perhapsyo uwoul d keepthes e3 rule s and definitions inmin d when trying tounderstan d the interactions between environmental and genetic factors during thedevelopmen t ofcertai n treediseases .

EXAMPLE 1: ROOT-ROT CAUSED BY FOMES OR ARMILLARIA

Thosewh ohav eha d long experience inresearc hwit h Fomes or Armillaria willprobabl y agreewhe n Isa ytha tthes e fungi are still fullo fmyster y forpathologist s with regard to:certai npart s of theinfectio nbiology ; themechanis m ofpredispositio n ofth ehost ;and , lastbu tno t least,t o the details andspecializatio n ofpathoge n reactions againstth e diverse biotic andnon-bioti c factors inth esubstrate . Our factual knowledge aboutenvironmenta l dependencies ofroo trot si s scarce and islittl emor e thanth eessenc e oflon gpractica lexperience . Thismeans ,fo r Fomes, higher infectionrate sunde rneutra l or alkaline soil conditions;an d for Armillaria, increased attack afterpronounce d

40 changes of soilmoisture . Beyond this, canw e really draw amor eprecis epictur e of environmental influences on fungal aggressiveness inspit eo fgreate r researchefforts , bettermethod s and improved facilities,an do fa nincreasin g demand for controlmethods ?Unfortunatel y we can't,an d iti sm y feeling thatth e reasons forthes e failures areno tto odifficul t todetermine . InNorwa y spruce, F. annosus (Fr.)Cook e is,i neconomi c terms,th e most important tree disease ofCentra l Europe -th e 'Fichten-Rotfäule'. Differing fromRishbeth' s findings inScot spine ,th e fungususuall yin ­ vades through theroo t andno tth e stump surfaces (Dimitri,1969 ;Käärik , 1975). Itis ,however ,ver y difficult to artificially copy the infection processes, todevelo pmethod s of artificial inoculation and toge t areal ­ istic impression ofth enatura l defencemechanism s ofth ehost-tree . Onepossibl e reason forthes edifficultie s appears tob e that Fomes of­ fersver y little competition againstothe rmember s ofth e soilmicroflora , sotha tit smyceliu m hasver y littlechanc et o survive.Thi s seemst ob e muchmor e thecas e inaci d rather thanneutra l or alkaline substrates.Th e fungus itself is largelypH-toleran t (Courtois, 1972). Itsmos t important antagonists,however ,usuall yprefe r alowe r soilpH , and soth ehydro - genionconcentratio n inth e soil isa n important environmental parameter. However,p H acts notdirectly ,bu t ina secondar y wayb y influencing the competitionwit h other soil-borne fungi. Thevitalit y andvirulenc e ofa soil-born e fungus such as Fomes isin ­ fluencedby , amongothe r factors,th e amount andth e quality ofnutrient s available inth e soil, especially inth e rootregion . Intur nth e avail­ ability ofthes enutrient s ist oa certai n degree dependent uponth equan ­ tity,th eminera l composition, andth e state ofdecompositio n ofth e lit­ ter.Thu s theenvironmen t directly affects thesituatio nbecaus e theminer ­ alnutrien t supplyo fth e soil, aswel l asclimati c factors controlling photosynthesis andmetabolism , interact and affectbot h the amountan d quality ofth elitter . Schuck& Lechle r (1980)fo rth e firsttim eprove d that aqueous litter- extracts canhav e arathe rdramati c effecto nth evegetativ e developmento f F. annosus. Depending on severalvariables ,eithe r astimulatio n ora n inhibition of fungus development occurred. The followingparameter sha d relatively pronounced effects: - species litter: ingenera l coniferspromote ,deciduou s treesinhibit , - different stands ofth e same species:sit eeffect , - duration of extraction andconcentratio n ofextract :amoun to fchemica l components dissolved, - timeo fextraction : autumn less thanspring . Surely,thes e results cannotonl yb e explained by differences inth e amounto fnutrient s available toth e fungus.Allelopathi c effects areals o involved since conifers oftenproduc e acontinuou s andpermanen t litter

41 layer theeffect s ofwhic h seem tob e ofconsiderabl e importance toth e soilmicroflora .Moreove r theleachin g ofchemica l compoundsha sbee n described forsevera lplan tcommunitie s including foresttrees ;i ti sknow n tob ehighl y important from anecologica l point ofview . To reiterate:i nou r case study of Fomes, important environmental factors affectth e developmento fmetaboli c compoundswhic h are liberated duringth emicrobiologica l decomposition ofth e litter;thes e compounds act ina nallelopathi c way.Naturall y theprocesse s areaffecte db y temperature andmoisture .Bu t forth e firsttim ew ehav et ocommen tals o thatgeneti c influences areembodie d inthi s interaction ofnumerou smodifyin g factors. Inessenc e iti sth e ability ofa tre eto : - absorb aspecia l spectrum ofminera lnutrients ; -produce ,distribute , and store aspecia l spectrum of secondary plant substances. There isn o doubttha tbot h these abilities are affectedb y the genetic variability ofeac htree .I fthi sassumptio n iscorrect ,the nth eallelo ­ pathic andnutritiona l components affecting theviabilit y andvitalit y of the fungus inth e soil are,t oa certai n degree,bot h qualitatively and quantitatively predetermined by genetic differences inthei r laterhos t plants. Allelopathy may alsopla y adirec trol e during the courseo f infection by Fomes and Armillaria. Thishappen s inth erhizosphere , aregio n ofhig h microbiological activity, causedb y theexudatio n of fluid andvolatil e substances through root surfaces.Amino-;acids ,sugars ,terpenes ,phenolics , andgrowt h substances mayb e liberated ina species-specifi c composition. Theywil l obviously affectth eenvironmen t close toth eroo tsurfac e and therefore affect directly thephytopathologica l activities of soil-borne fungi. We know thatrhizomorp h formationo f Armillaria isstimulate d byglu ­ cose inth epresenc e of IAA (Garraway, 1975)an d alsob y 1- asparagi n (Rykowski, 1976). The same fungus exhibitspronounce dmorphological , physiological, andpathologica l variations depending onth ebiochemica l complexwhic h ispresented . Moreover,ther e isconsiderabl e variationbe ­ tweendifferen t isolateswhe nkep tunde r the same environmentalconditions . Evidently Armillaria differs from other forestpathogen s and ismanifeste d inman ybiotype s eachwit h adifferen t ecological demand.Finally , every single isolate isabl e toreac tt ochange s inth e environment ina ver y plasticmanner . Thusw ehav et orecor d that Armillaria hasth epuzzlin g abilityt o reactt oenvironmenta l factors ina ver y complicated way. Furthermore,th e fungusha s additional and ratherunusua l featureswhic h cannotb e ignored. Forman yyear sw ehav e recognised the ability of Armillaria to live asa saprophyte aswel l as aparasite ,an doccasionall y eventh epossibilit y of a symbiotic existence asa mycorrhiza lpartne rha sbee n discussed.

42 From results of some limitedpilo tstudie s inth e laboratory, Armillaria hasbee nobserve d tohav e thecapabilit y ofreactin g allelopa- thically ontre e seedlings and,vic eversa ,ther e aregrowt h differences ofth e funguswit hrespec tt oroo texudate s ofvariou s coniferseedlings : 15m lbeaker s containing a3 c m longpiec e ofrhizomorp h atth ebotto m and coveredwit h quartz sandwer eplante dwit h 10-day-old seedlings ofdiffer ­ entconife r species.Afte r 6weeks ,th e following features were observed: -th epresenc e ofrhizomorph spromote d the formationo fsecondar y roots in Picea abies (L.)Karst , and Pseudotsuga menziesii (Mirb.)Franco ,bu tno t in Pinus sylvestris (L.)an d Larix decidua Mill.; -roo t length remained unaffected; -rhizomorp hyiel dvarie dwit hth econife r speciesplante d inclos eproxim ­ ity (pine> douglas-fi r> spruce). Although thesepreliminar y trials haveno t alwaysproduce d reproducible resultswit h different sources and ages ofrhizomorphs ,w e dare to express somepreliminar y suppositions: Obviously therewer emutual ,species-specifi c effectsbetwee n fungus and seedling eventhoug h directcontac tbetwee n roots andrhizomorph s did notoccur . Soquestion s arise ast owhethe r orno tthes e relationshipsar e therul e and ifthe y are the firststag eo f apathologica l event. Inan y case, thiskin d ofexperienc ewiden s anddeepen s ourpictur e ofth ebiolog ­ icalrole so f Armillaria. Parasite -saprophyt e -competito r -symbiont : allthes e functionsma yb e fulfilledb y Armillaria. What,however , areth e conditionswhic h influence achang e ofrole ? Returning to Fomes, which ismorphologicall y far lessvariabl e than Armillaria andmuc hmor e sensitive to antagonists,w ehav e some evidence thatthi s fungus too could acta sa mycorrhiza lpartne r oftre eroot s -a t leasttemporaril y andunde r favourable conditions.Kowalsk i (1970)dis ­ covered suchrelationship s inScot spin e roots and other exampleshav ebee n recorded. Especially in Picea abies, theroot s ofwhic h areregularl yas ­ sociated withmycorrhiza l fungi, Fomes doesno tsee mt opla y arol e asa symbiont (Bucking, 1979), thusdifferin g from Armillaria whichpossibl y starts itspathologica l activities after asymbioti cstage . Inspit eo fthis ,mycorrhiza e also seem tohav e some role inth ein ­ fectionbiolog y of Fomes. Butthi s isi n amor epassiv ewa yb y affecting thepre-infectiona l stageo f 'invading resistance'calle d axenie byGäu - mann,Krup a& Nylun d (1972).Als oKrup a et al. (1973)pointe d outtha t short-roots (roots associated withmycorrhiza l fungi)o f Pinus sylvestris and P. echinata Mill, liberated increased amounts ofvolatil emono -an d sesqui-terpenes whencompare d withroot swithou tmycorrhizae .Apparentl y theproductio n ofth eparticula r group ofcompound swa smarkedl y promoted, several ofwhic h arequit e toxic to Fomes and some other rootpathogens . This isconfirme db yman y invitr o investigations (Hintikka, 1970;Schuck , 1977). Thus itseem s that agoo dmantl e ofmycorrhiza e around aroo tsyste m

43 L canreduc e Fomes infectionb y fungicidal chemicalexudates . Thus,volatil e terpenes inth erhizospher e arever y relevantenviron ­ mental factors affecting F. annosus. Incontrast ,th e samecomponents , looked atfro m thehos tpoin to fview , aremetaboli c compounds formingpar t ofth edefenc e system. Again, the formationo fterpene s and their composition depend onenvi ­ ronmental conditions.A sw ehav e seenbefore ,the y are amixtur e of second­ aryplan t substances,th ebiogenesi s ofwhic h isonl y guaranteed underhig h photosynthetic activity.Als o therat e of liberation ofchemica l compounds having differingboilin gpoint s isa functiono ftemperature . Volatile terpenes arecomponent s ofconife r resin.The y areno tonl y liberated intoth e environment through root,stem , and leaf surfaces,bu t they also fill the space of intercellular systems inman y tissues.Thi si s thereaso nwh ythe y arepar to fth ehost-specifi c environment forever y invadingpathogen . Inth ecas e of 'Fichten-Rotfäule', themonoterpene sar e said tohav eresistance-formin g abilities,a tleas t inth e sense of 'spreading-resistance' afterGäumann . The composition ofth emonoterpen e fraction is,t osom e extent,indi ­ vidually fixed;certainl y iti sspecies-determined . Individualpattern s of monoterpene-compositionare ,however ,modifie d bynon-rando m differences withinth e stemwhic h aredifficul t toexplai n (Schuck& Schutt, 1975). Consequently for invadingpathogens ,w ehav e tocontend , apriori ,wit hth e existence ofregion s ofvaryin g toxicity.Additionall y there arealter ­ ations toth e individual monoterpene compositionwhic hma yb e inducedb y theparasit e itself,o rwhic h aret ob e explained by atraumati c reaction aftermechanica l wounding.Madziara-Borusiewic z & Strzelecka (1977)foun da marked decrease ofth e limonene andborny l acetateportio n inth emono ­ terpene-fractio no f Picea abies following infectionb y Armillaria. And Schuck (unpublished)reporte d thatth emonoterpene s originating fromtrau ­ maticresi nduct s followingwoundin ghav e adifferen tcompositio n incom ­ parisonwit h resincollecte d fromundamage d parts ofth e sametree . This indicates thatth emonoterpen e fraction,whic h affects thedevel ­ opmento froo tan dwoo dpathogeni c fungi,i sno tabsolutel y stablewithi n the samehost . Itvarie s for internal reasonsbetwee n differentpart so f the same stem, itvarie s after the invasion ofth epathogen , and iti s modified aftertraumati c reactions.Thu s againw e observe soman y interac­ tionsbetwee n external and internal influences that iti sdifficul t tous e the single term 'environmental factor' init sorigina l sense.Also ,a sa consequence ofth e interaction of factors,w esens esom eerosio no fth e boundarybetwee n disposition andresistance-cause d differences to disease attack.

44 EXAMPLE 2: FOLIAGE PATHOGENS

Itma yb eusefu l todiscus s furtherwhethe r this erodedmeanin g of proved terms is specific topathologica l events inth erhizosphere ,o r whether itca nb e discovered elsewhere,fo r instance inth ephyllosphere . Like therhizosphere ,th ephyllospher e represents aborderlin e between theplan t andth e environment. Iti scharacterize d byhig hbiologica l activity (Schutt, 1974). Also typical isth e rather complicated chemistry ofth eplant' s surface layer ofcuticula rwaxes .The y arebuil tu po f esters ofunsaturate d fatty acidswit hhighe r aliphatic alcohols,consist ­ ingo fparaffins ,alcohols ,esters ,ketones ,aldehydes ,an d fattyacids , eacho f the fractionsbein g represented by 10-15 single components.Th ene t result isa nincalculabl e number ofcombinations . Cuticular waxes arechemicall y andphysicall y heterogenous.The y crys­ tallize asplates ,rods , grains,an d intermediate formswhic hprovid e an uneven,microclimaticall y variable leaf surface.Th e chemical composition ofth ewaxes ,togethe rwit hthei r differences instructure , seem tob e species-determined andbot h aregeneticall y controlled. Becausew ehav eevidenc e that foliarpathogen s reactparti /negativel y andpartl ypositivel y to agive nchemica l composition of foliarwaxes , there isreaso nt obeliev e thatth e chemistry ofcuticula r waxes eventually has anaxeni c effecto n leaf-inhabiting fungi (Schutt, 1971). Inprincipl e this association isno t affectedb y thosevolatil e terpeneswhic h fillth e intercellular spaceswithi n conifer needlesbu twhic h are soluble inwa x andtherefor ema yb e incorporated astrue ,species-specifi c components in the chemistry ofth ecuticula rwaxes . Iti sprobabl e thatthi spredetermine d endogenous situation canb e modified by environmental factors such as light intensity,whic h canlea d toconsiderabl e changes inth ewa x structure.Th epathologica l situation, however, isextremel y influenced byproblem s arising from the activities of micro-organisms ofth ephyllosphere .The ymak e ithighl y complex andvery , very difficult toanalyze .Micro-organisms ,mainl yyeast s andbacteria ,oc ­ cur inimmens e numbers and inman y forms;fo rexample ,1 3millio nbacteri a canb e foundpe r cm2 of leafsurface . Wekno w thecompositio n ofth e flora ofth ephyllospher e isb yn omean s constant,an d successions inth e foliar colonization canb e observed.Thu s the spectrum of speciesvarie swit h leafage ,whic h isparallele d by an alteration inth echemica l composition of itswaxes .Antagonisti c effects areals o involved inthes eprocesses .Som eyeast s areabl e todecompos e the leafwaxes ,thu sconsiderabl y changing the chemical situation.Othe r spe­ ciesproduc e growth-substances, secrete amino-acids orcarbohydrates ,an d change theecologica l condition onth e leaf surfaces and inevitably alter their suitability forth eestablishmen t ofpathogens .This ,however ,lead s to analmos tradica l change inou r thinking.A t firsti tseem st oaccurate -

45 ly follow our established model, according to which genetically controlled defence traits (resistance) are modified within certain limits by environ­ mental influences. But as we learned, in practice, this model is not valid under all conditions. Both in the phyllosphere and rhizosphere there are borderline interactive systems affected by high microbiological activities where the inter-relationships between genetic and environmental factors are strati­ fied and obscure. For a proper estimation of a given pathological situa­ tion, much more is necessary than the knowledge of simple cause-and-effect relationships between environment and host or pathogen. Thus it is neces­ sary to have a deeper understanding of the complex ecological factors and their interactions, an aim which can only be met through familiarity with recent studies and findings in related disciplines. Pathology can no longer be regarded in isolation but rather as an integral part of a complex eco­ system in which countless abiotic and biotic factors are interwoven one with another: a single division into genetic and environmental components is no longer a wholly acceptable concept.

REFERENCES

Bucking, E., 1979. Fichten-Mykorrhizen auf Standorten der Schwäbischen Alb und ihre Beziehung zum Befall durch Fomes annosus. European Journal of Forest Pathology 9: 19-35. Courtois, H., 1972. Das Keimverhalten der Konidiosporen von Fomes annosus (Fr.) Cke. bei Einwirkung verschiedener Standortfaktoren. European Journal of Forest Pathology 2: 152-171. Dimitri, L., 1969. Untersuchungen über die unterirdischen Eintrittspforten der wichtig­ sten Rotfäuleerreger bei der Fichte (Picea abies Karst.). Forstwissenschaftliches Centralblatt 88: 281-308. Garraway, M.O., 1975. Stimulation of Armillaria mellea growth by plant hormones in relation to the concentration and type of carbohydrate. European Journal of Forest Pathology 5: 35-43. Hintikka, V., 1970. Selective effect of terpenes on wood-decomposing Hymenomycetes. Karstenia 11: 28-32. Käärik, A., 1975. Succession of microorganisms during wood decay. In: W. Liese (Ed.): Biological transformation of wood by microorganisms. Springer-Verlag, Berlin- Heidelberg-New York. p. 39-51. Kowalski, S., 1970. Mycorrhizal synthesis of Pinus sylvestris L. and Fomes annosus. Bulletin de l'Académie Polonaise des Sciences V 18: 405-408. Krupa, S. &J.E . Nylund, 1972. Studies on ectomycorrhizae of pine. III. Growth inhibition of two root pathogenic fungi by volatile organic constituents of ectomycorrhizal root systems of Pinus sylvestris L. European Journal of Forest Pathology 2: 88-94. Krupa, S., J. Andersson &D.H . Marx, 1973. Studies on ectomycorrhizae of pine IV. Volatile organic compounds in mycorrhizal and non-mycorrhizal root systems of Pinus echinata Mill. European Journal of Forest Pathology 3: 194-200. Madziara-Borusiewicz, K. &H . Strzelecka, 1977. Conditions of spruce (Picea excelsa LK.) infestation by the engraver beetle (Ips typographus L.) in mountains of Poland. I. Chemical composition of volatile oils from healthy trees and those infested with the honey fungus (Armillaria mellea [Vahl.] Québ.). Zeitschrift für Angewandte Entomologie 83: 409-415. Rykowski, K., 1976. Recherche sur la nutrition azotée de plusieurs souches de l'Armillaria mellea. I. L'utilization de diverses sources d'azote. European Journal of Forest Pathology 6: 211-221. Schuck, H.J., 1977. Die Wirkung von Monoterpenen auf das Mycelwachstum von Fomes annosus (Fr.) Cooke. European Journal of Forest Pathology 7: 374-384.

46 Schuck,H.J .& H.H .Lechler ,1980 .Di eWirkun gwäßriche rStreuextrakt everschiedene r Waldbäumeau fMycelwachstu mun dKonidiosporenkeimun gvo nFome sannosu s (Fr.)Cke . EuropeanJourna lo fFores tPatholog y 10:36-47 . Schuck,H.J .& P .Schutt ,1975 .Di eVerteilun gflüchtige rTerpen ei mFichtenstam mun d ihreBedeutun gfü rdi eAusbreitungsresisten z gegenüberFome sannosus .Biochemi eun d Physiologiede rPflanze n167 :65-77 . Schutt,P. ,1971 .Untersuchunge nübe rde nEinflu ßvo nCuticularwachse nau fdi eInfek ­ tionsfähigkeitpathogene rPilze .I .Lophodermiu mpinastr iun dBotryti scinerea . EuropeanJourna lo fFores tPatholog y 1:32-50 . Schutt,P. ,1974 .Di ePhyllosphäre ,ein eZon ehohe rbiologische rAktivität .Forstwissen ­ schaftlichesCentralblat t93 :1-10 .

47 Environmental factors affecting host-bacteria interactions in bacterial diseases of forest trees and shrubs

M. Ridé

Stationd ePathologi eVégétal ee tPhytobactériologi e INRA,Rout ed eSaint-Clément , Beaucouze,4 900 0Angers ,Franc e

ABSTRACT

Research achievements arereviewe d ofth e lastdecennia l period aboutenvironmenta l andepidemiologica l factorsaffect ­ inghost-phytopathogeni c bacteria interactions,particularl y bacterial tree diseases causedb y Erwinia, Pseudomonas, Kanthomonas, and Agrobacterium species.Variou s aspects are illustrated: influence of inoculumsources ,temperature ,an d rains on forecasting thepotentia l activity qffir eblight ; Pseudomonas ice-nucleationactivity ; effecto fedaphi cproper ­ tiesupo nth e development offores tbacteria l diseases;an d effects ofepiphyti c microflora and leachates of aerialstruc ­ tures on X. populi.

INTRODUCTION

Damages causedb yphytopathogeni c bacteria to foresttree s and shrubs areno ts o frequenta sthos einduce db y fungi.Nevertheless ,bacteria l diseases increasingly constitute apotentia l riskwhic hmus tb etake nin ­ toaccoun t in forestplantin gchoice s andbreedin gprogramme s forresis ­ tance.Th e evolution of forestry inth erecen tpas t isth ereaso n forthi s increased risk,e.g . use ofmonoclona l and same ageplanting s (Populus, Salix), the active interesto fsylviculturist s insom enobl e trees (cherry, walnut, ash)an dthei rvegetativ emicropropagation , andth emor e andmor e important intervention of foresters inchoosin g certain trees and shrubs tointegrat e into landscapeplannin g andwin dprotectio n (Crataegus, Cotoneaster, Sorbus). Thebacteri a damaging foresttree s areexposed , as areal lpathogeni c microorganisms,t ovariou s interactionsbetwee n climatic and edaphicfac ­ tors, genetic hostconstitutio n and itsphysiologica l development, and evolution ofth estructur e ofpathogeni c populations inside thecomple x microflora of aerial structures oftree s and rhizosphere. Iti sth epurpos e ofthi srevie wt osynthesiz e ourmai n findingso fth e lastdecennia lperio d

48 about some factors affectinghost-phytopathogeni cbacteri a interactions, limited toth eenvironmenta l andepidemiologica l aspects,an d excluding cellular andmolecula r host-pathogenrelationships .

PRINCIPAL BACTERIAL DISEASES OF FOREST TREES AND SHRUBS

Environmental host-bacteria interactions willb e illustrated pertaining toth ebacteria l diseasesmentione d inTabl e 1,whic h areo f interest for treebreeder s and forthei reconomi c impacts.Nevertheless ,le ti tb e remembered that somepathogeni c bacteria localized atth emomen t ina ver y restricted area,suc ha s Erwinia nigrifluens Wilson, Starr& Berge ro n walnut inUS A (Wilsone t al., 1957)o r Xanthomonas populi subsp. Salicis on Salix dasyclada Wimm. (DeKam , 1978), constitutepotentia l risks forth e future.Bacteri a which arepresen t incomple x infection chains or associated with insects (Ridé& Prunier , 1963;Danilewic z &Tomaczewski , 1970)o rwit h nematodes, such astoxinogeni c bacteria associated withth epinewoo dnema ­ tode (Bursaphelenchus lignicolus Mamiya& Kiyohara )o n Pinus densiflora S. & Z. and P. thunbergii Parlât (Okue t al., 1980), will notb etake nint o

Table 1. Principalbacteria l diseases of foresttree s andshrubs .

Causal agent Disease Hosts Agrobacterium radio- Crowngal l Various deciduous trees bacter var. turnefa- and conifers ciens Corynebacterium hale- Tumors Pinus halepensis pensoïdes Erwinia amylovora Firebligh t Pyrus, Malus, Crataegus, Cotoneaster, Pyracantha, Sorbus, andothe r Pomoideae Erwinia Salicis Watermark disease Salix Pseudomonas Twigblight , Cerasus, Citrus, Populus, syringae blossomblight , Pyrus, Prunus, Salix, shootwilt ,canke r Syringa, etc. Pseudomonas savas- European ashcanke r Fraxinus tanoï f.sp . fraxini

Pseudomonas mors- Bacterial canker, Pirunus avium prunorum dieback,shoo thol e Xanthomonas corylina Filbertbligh t Corylus Xanthomonas juglandis Walnutbligh t Juglans Xanthomonas populi Bacterial canker Populus

49 account inthi sreview , inspit eo fthei reviden tecologica l interest.Als o diseases inducedb ymycoplasmalik ebodie s andothe r formso fMollicute s will notb e evokedhere .

INOCULUM SOURCES, DYNAMICS OF PATHOGEN POPULATIONS AND CLIMATIC CONDITIONS

Fire blight Theetiolog yo fPomoidea e firebligh twa sdemonstrate d forth e first time 100year s ago (Burrill, 1882). Firebligh tcause db y Erwinia amylovora (Burrill)Winslo w et al. isno wpresen t inal lth e apple andpea r growing regions ofNort hAmerica . Itwa s introduced innorthwester n Europe probably about 1955,an ddurin g thepas t2 0year s itha sbecom e themos t serious bacterial disease inBelgium ,Denmark , England, France,Wes tGermany , and EastGermany . Itssprea d seems stopped inPoland . Though considered tob e themos t important disease oforchards ,fir ebligh t isals o ofinteres tfo r the forester, insofar ash e isconcerne d with landscape appointments and protectionmainl y with Crataegus spp., Pyracantha spp., Cotoneaster spp., Sorbus spp.,etc. ;thes e trees and shrubs are frequently dangerous inoculum sources. E. amylovora isprobabl y thephytopathogeni c bacterium onwhic hth e influence ofenvironmenta l conditions hasbee nexplore d most. Itoverwinter s inbar ktissue s alongth e edgeso fcanker s initiatedb y infections ofth e previous year. Insprin g thebacteri amultipl y atth ecanke rmargin s pro­ ducing theprimar y inoculum (oozeo r slime)disseminate d by rain,wind ,an d flying insects.Th e firstinfectio n takesplac e onblossom s orvegetativ e shoottips . Asmentione d byAldwinckl e &Bee r (1978), blossom infection occurs firsti nmos tregions , 'buti na coole r area such assouther nOntari o in Canada,blossom s oftenescap e infection andvegetativ e shoottip sbecom e infectedwel l afterbloo m (Dueck& Quamme , 1973).Disseminatio n ofth e bacteria ina warm , generally dry area such asCaliforni a ismainl yb y insects;bu t inmor ehumi d areas,disseminatio nb y rainca nb emor eimpor ­ tant' . After E. amylovora penetrates throughwound s ornatura l openings such asnectaries ,hydathodes ,o r lenticels,th e infected tissues frequently exudedroplet so fooz econsistin go fa matri x ofpolysaccharides .Exudat e production isofte nconsidere d tob e related tohig htemperatur e andrela ­ tivehumidity .Nevertheless ,i nclimati c chambers exudation takesplac e within awid erang eo ftemperatur e andRH ,betwee n 15 °Can d2 8 °Can d between 60an d 90% RH ,respectivel y (Paulin& Lachaud , 1978). Apparently, other factors such asplan tnutritio n andwate r supply could alsob eimpor ­ tant forexudat eproduction .Bacteri a contained inooz e function assecond ­ ary inoculum sources forlat eprimar y and secondaryblossoms ,vegetativ e shoots, andfruit .

50 The relationship between inoculum dose and infection developmentha s been studied onlyrecently .Th erat eo fsympto m development and thepropor ­ tiono f inoculated blossoms seemdirectl yproportiona l toth enumbe ro f E. amylovora cellswhic h areintroduce d intoth e floral cups (Beer& Norelli, 1975). Californianworker s tried tomonito r thepresenc e of E. amylovora onpea r and appleblossom s ina nattemp tt ocorrelat e bacteria populations withweathe r conditions (Thomsone t al., 1975). Thepresenc e of thepathoge nwa s demonstrated inblossom s ando nth e surface ofcanker s priort odevelopmen t of symptoms of flower infection.Moreove r itwa s found that E. amylovora multiplied inhealth y flowers andwa s detected 14day s before symptoms appeared. Based on 7year s ofmonitoring , aformul aha sbee npropose d topredic t when E. amylovora ismos t likely tob e detected inCaliforni aconditions , only after dailymea n temperature exceeded alin edraw n from 16.7 °Co n March 1t o 14.4 °Ci nMay .Goo d control ofth e disease isgenerall yob ­ tained after spraying streptomycin onlywhe nth emea ntemperatur e ofth e prediction line isexceeded . Incontras twit h observations inCalifornia , largeepiphyti c populations haveno tbee ndetecte d inpom e fruitblossom s ineaster nUSA .Th e same situation seems toexis tno w inEurope ,particularl y inth eNetherland s (Miller& va nDiepern , 1978)bu tperhap s also insouther nFrance ,wher e the varietal structure ofth eorchard s andtre e training techniques arever y different from California.Result s recently obtainedwit hver y late second­ aryblossom s onPass e Crassanevariet ywoul d emphasize thepossibl e role of very late and latent infections forth enex t spring. Soblosso m monitoring techniques used inCaliforni a seem difficult totransfe r toEuropea nclimat ­ icconditions . Forthi s reasonBillin g (1974, 1980)trie d torelat e the growth rateo f E. amylovora andth e rain requirement indevelopin g ametho d for assessing potential firebligh tactivit yi nth efiel d in south-easternEngland . Iti s interesting tonot etha tthi smetho d takes into accountth epotentia l doubling ofth ebacteri a related to temperature.Thi smetho d isno wbein g tested indifferen t countries inEurope ,base d onth e following system. Whenth e rain istake nint o accountaccordin g toth e followingscore : R = 0,n orain ;R = 0.5 , rain^ 2. 5 mm;R =1 ,rai n> 2. 5 mm; the incubation period (I)i nday si sende dwhen :

P.D. Ê3 6I -6 , whenP.D . =potentia l doublingpe r daydefine db y dailymaximu m andminimu m temperature.Thi s system makes itpossibl eto : - forecastth epotentia l activity of firebligh t ina give n areausin g simple and availabledata , - knowth emos tprobabl e appearance ofsymptoms ,b y drawing the graphs

51 daily,an d - assess the risks following arai n or anabnormall y high temperature duringth epresenc e ofprimar y orsecondar yblossoms . Buti twil lb enecessar y toadap tBilling' s system to areaswher e fire blighti sparticularl y dangerous,suc h assouth-wester n France. Itwil lb e especially important totak e into account other factorswhic h couldinflu ­ ence initiation to infection, i.e. heavy dewso rmists ,activit y andden ­ sity of flying insects,frequenc y of late secondary blossoms,etc .

Pathogenic pseudomonads Incontras tt o E. amylovora which seemst ohav e temporary epiphytic stages, Pseudomonas syringae vanHal l andothe rpathogeni c pseudomonads of fruitan d foresttree s arewidel y distributed asepiphyti c bacteria.Bu ti t isnecessar y todistinguis h between P. syringae, acollectiv e specieswhic h probably includesvariou s ecotypes adapted todifferen t genera sucha s Prunus, Pyrus, Juglans, Populus andvariou sherbaceou s species,livin gan d surviving uponnutrient swhic h leach from aerialpart s ofplants ; and other pseudomonads such as P. mors-prunorum Wormald oncherr y or P. mors-prunorum f.sp . persicae Prunier,Luisett i &Garda n (= P. persicae) onpeach ,whic h seemmor e specific and detectable only indisease dorchards . Bydifferen tmethod s (specificmedia ,antibioti c resistantstrains , immunofluorescence techniques)i ti spossibl e nowt o followth e dynamics and structure of Pseudomonas populations on leaf,petiole ,an dtwi gsur ­ faces, and insidehost-tissues .Th e evolution of P. syringae populations depends ontre evarietie s or species,an dorgans ,a son eca nnotic e on graphs drawn according toLuisett i &Pauli n (1972). P. syringae is always present onth e leaf surfacewithou t symptoms during thevegetativ e period. Itdisappear s onlywhe nhig h temperatures occur.Th e same situationexist s ondifferen t species of Populus such as P. nigra, P. maximowiczii, and varioushybrids .Th e influence ofhig h temperature anddrynes s onth e contraction of P. syringae populations ismor econtraste d anddrasti c on Citrus leaf surfaces inGreec e asmentione d byPanagopoulo s & Psallidas (1973). Inth e case of P. mors-prunorum onsweet-cherrie s the levelo f populations ismaintaine d by leaf spotinfection s during the season.Never ­ theless,Freigou n (1973)foun d apositiv e relationship betweenbacteria l counts and leafwetnes s and anegativ e relationship with sunshine duration. The same scheme isrelate d to P. persicae, the causal agento fpeac hbac ­ terialbligh t (Gardane t al., 1972); thisbacteriu m maintained ahig h inoculum level onth e leaf surface eveni nautumn , aperio dwithou t any 4 5 symptomsmacroscopicall yperceptibl e on leaves.Thi s level (10-1 0 bac­ teriape r leaf)i shig h enough toinfec t fresh leaf scars during the fall. The influence of lowtemperature s andchillin g isparticularl y remark­ ableo nth e development ofpeac hbacteria l blight.Frequentl y insouth ­ easternFrance ,negativ e temperatures interfere with thedormanc yperio d of

52 peach trees andon eca nobserv e the firstsymptom s after leaf fallinfec ­ tions during the first fortnighto fDecember .Th e influence ofchillin g is also importantwhe n cambial activity ofth e trees starts again (Vigouroux, 1974). Icenucleatio n activity inducedb y epiphytic populations of P. sy­ ringae, abacteriu m isolated from awid evariet y ofplan t species,offer s another curious and interesting aspecto flo wtemperature-pathogeni cbac ­ teria-host tissueinteractions . Fifteenyear s agoPanagopoulo s &Cross e (1964)i nEngland , andDuran d etal . (1967)i nFrance ,demonstrate d that P. syringae could developa rapid infection after the appearance ofhypodermi c cellmicrolesion sin ­ ducedb y light frostdamag e on flowers,twigs ,leaves ,an d fruitletso f pear.Mor erecentl yArn y et al. (1976)showe d onmaiz e that P. syringae cellsha d awel l defined rolei nth einductio n ofic enucleatio n inhos t tissues.Th e sameresult swer eobtaine db yPauli n& Luisett i (1978)o n tobacco ando npea r twigs. Iti spossibl e toobserv e the samephenomen a on poplar shoots.Amon gphytopathogeni c and saprophyticbacteri a livingo n leafsurfaces ,onl y P. syringae and sometimes some Erwinia herbicola strains seem able toinduc e icenucleatio n activity (Lindow et al., 1978). Thispropert y isdependen t onbacteria l strains,inoculu m dose,tem ­ perature, andnatur e ofcultur emediu m onwhic hbacteri a aremaintaine d (Paulin& Luisetti ,1978 ;Hiran oe t al., 1978). Thisne w aspecto fth e temperature-bacteria-host-interactions couldmodif y the control ofsom e bacterial diseases such asblosso m blighto fpea r inth efuture .

Agrobacterium spp. and soil Unlikephytopathogeni c bacteriawhos e ecology isrelate d to aerial plantstructures , Agrobacterium species aregenerall y associatedwit hroo t collars androo t systems ofconifer s anddeciduou s trees:proximat e envi­ ronments ofroot s (rhizoplan andrhizosphere )an d galls inducedb y virulent strains.However , some Agrobacterium strains areabl e togiv e riset ogall s ontrunk s orbranches , for instance on Populus grandidentata inUSA , or P. euramericana cv.Robust a or P. alba inFrance ,ye tthi s situation israr e (Dochinger, 1969;Rid é& Lopez, 1976). Tumorswhic h aredisaggregatin g insoi l constitute excellent sources of pathogenic Agrobacterium tumefaciens (Smith& Townsend )Conn ,bu tals o saprophytic A. radiobacter. Thesebacteri ahav eecologica l nichescompa ­ rablewit h those of Rhizobium species,whic h one can sometimes isolate at the same time as A. tumefaciens (Gaur& Sen, 1976). Agrobacterium species areremarkabl y concentrated inth erhizosphere , and iti sno trar e to find therepopulatio n levels 10 000time shighe r than inth esoil . Schrothe t al. (1971)hav e showntha t Agrobacterium hasa positive chemiotachism foractivel y growingroots . Thesebacteri a overwinter inth esoi l andtemperature shighe r than

53 34 °C ar e inhibiting forthem ;tha tcoul d explain the low frequency of crown-gall intropica l zones.Furthermor e the relativeproportion s of pathogenic to saprophytic Agrobacteria arevariabl e according toth eplan t root system and soilconditions :thu s on2 8 soil samples-inCalifornia , Schrothe t al. (1971)foun d 17o fthe mwit h anappreciabl e proportiono f pathogenic strainsvaryin g from 1/13 to1/500 .Thi sbalanc ebetwee npatho ­ genic and saprophyticAgrobacteri a probably shouldb e taken into accounti n thebiologica l control ofcrown-gal l inth e fieldb y using the antagonistic bacteriocinogen A. ra.dioba.cter K8 4strai npropose db yKer r (1972)i n Australia.

SOIL, NUTRITION, AND PHYTOPATHOGENIC BACTERIA

Information aboutth e effecto fedaphi c properties uponth e development of forestbacteria l diseases andth e conservation ofphytopathogeni cbacte ­ riai nsoi l isver ymeager ,still ,relationship sbetwee nwate r supply, nutrient level,succulenc e ofhos ttissues ,tre evigour , and susceptibility tobacteria l diseases havebee nknow nthroug h empirical observations fora longtime . Forexampl e inth eTherai nValle y ofnorther nFrance ,numerou spopla r plantings aregenerall y distributed on2 type s ofsoil .On eo fthe m consists of athi n layer of arable soil ona mixtur e ofcla y and turf,wit hbrow n turfdeepe r and incompletely decayed organicmatte r -plan t associations Cirsium, Eupatorium, Juncus, and Molinia -p H 6.Th eothe ri sa deepe r arable soil (60cm )o na blac k turf and-pHvaryin gbetwee n 6.5 and 7- plant association Phragmites, Epilobium, Consolida, and Spiraea (Ridé& Viart, 1966). Poplar growth isver ypoo ro nth e firstsoi lwhic h isassoci ­ atedwit h low activity of X. populi. Incontrast ,canke r expression isex ­ cellento nth e second typeo fsoil . Concerning Pseudomonas diseases of fruitan d foresttree s and fre­ quently also fireblight ,heavy ,poorl y drained, orhighl y acidic soils sustainmor e extensive and severedamage .Relationship s between treenu ­ trition and shootsucculenc eparticularl ywit h respectt o firebligh t susceptibility wereperceive db yParke r et al. (1961).Extr a applications ofnitroge n induce anincrease d susceptibility to fireblight ,ofte nexacer ­ batedb y excesses ofpotassiu m orphosphorus .Th ebes ttheoretica l workwa s probably doneb yLewi s &Kenworth y (1962):variou s combinations ofnutrient s were supplied topotte d trees growing inquart z sand ina greenhous e and artificially inoculated with E. amylovora. Just asParke r et al. (1961) observed, these authors noted thattree s suppliedwit h anexces s ofnitroge n weremor e susceptible thancontro l treeswhic h receivedbalance dnutrients . Butnitroge n deficiency also increased susceptibility to firebligh tresul ­ ting from deficiencies ofvariou s cations:calcium , copper,iron ,magnesium , manganese,molybdenum , orzinc .Whe na five-fol d excess ofcalciu m was

54 supplied, tree shoots couldno tb e infected.Unfortunately , nutrientinter ­ actions innatura l soilswoul d limitth etransfe r ofresult s obtained in sandy soil toth eorchard . Similar resultswer e obtained morerecentl yb yVigourou x &Hugue t (1977)o nth ebacteria l blighto fpeac h causedb y Pseudomonas persicae. Thisbacteria l disease ismainl y distributed onmor eo r less acidic soils andgraniti c arena ordiluviu m sand,generall ypoo r inorgani cmatter . Trials onvariou s sites andpotte dplant s demonstrated thebeneficia l effecto fcalciu m onth edecreas e of susceptibility tobacteria lblight . Ca effects onmembranes ,enzymes ,cel lwalls ,an dCa-phytochrom e interac­ tions areno w considered important, andthe yprobabl y interferewit hth e host-bacteria relationship.Nevertheless ,th e influence ofnutritio n andC a balancewit h othernutrient s remain always difficultt o interpret innature .

INTERACTIONS OF HOST, EPIPHYTIC MICROFLORA, LEACHED SUBSTANCES, ENVIRONMENT AND PARASITE

During spring,bacteria l poplar canker causedb y Xanthomonas populi Ridéi scharacterize d byproductio n ofbacteria l slimewhic h ismainl y dispersedb y rain,wind , and insects and constitutes aninfectio n source on thene w shoots.Infectio nsucces s depends onth e concordance between tissue receptivity, entryway s opent o X. populi, andquantit y ofinoculu mavail ­ ableo n aerial structures ofpopla r (Ridée t al., 1978). Many factorsma y contribute toeliminat e X. populi during itstemporar y epiphytic stage,an d thesehav e implications inth eepidemiolog y ofth edisease . Iti simportant , forexample ,t okno w ifth ebacteriu m ispresen t for alon gtim e and ata population levelhig h enough to instigate aneffectiv e infectionwhe nhos t tissuesbecom ephysiologicall y receptive. Investigations wereundertake n onth eevolutio n of an artificially introducedpopulatio n of X. populi on asusceptibl e clone (S.6-2 ) ando na resistanton e (P. nigra cv. Italica)durin g 2years ,197 5 and 1976,th e latteryea rbein g characterized by drought andver yhig h temperatures during spring and summer.Group s of saprophytic and epiphyticbacteria , yeasts, andyeast-lik e organismswer e evaluated periodically andthei rrol e considered in limiting theevolutio no f X. populi (Table2) . In1975 , X. populi rapidly decreased on P. nigra after3 0day s (itdis ­ appeared completely after 60 days); itremaine d approximately at1 0 per 4 leafo nth e susceptible clone S.6- 2 (itspopulatio nwa s still high, 10, atth een do f September).Durin g thesam eperiod ,population s offluores ­ centpseudomonad s (mainly P. suringae) were alwaysver y lowo n S.6-2 ,an d noteve ndetectabl e atth een do fJune . Incontrast ,the yvarie d from 10 inJun et o 10 inJul y onth eresistan t clone (invitr o this Pseudomonas exerted aclea r activity against X. populi). In1976 ,i nth e drasticcondi ­ tionsmentioned , X. populi decreased asslowl y onth eresistan t clone aso n

55 Table 2. Evolutiono fth enumber s (xiooo)o fepiphyti c bacteria (resp.yeas t cells)pe r leafo n asusceptibl e and aresistan t cloneo fpopla r after sprayingwit h X. populi on28.5.197 5 (upper table)-an d24.5.197 6 (lower table). Fluo.pseudo .= fluorescen tpseudomonad s including Ps. syringae, Ps. viridi- flave and Ps. fluoresceins types.Y . like =yeast-lik emicroorganism s (main­ lyincludin g Aureobasidium pullulans). S =susceptibl e clone (S.6-2) . R =resistan tclon e (cv. Italica).

Date of X. populi Fluo.pseudo . Yellowbact . Yeasts +Y . like sampling R

1975

28-5* 3.3 110 0 5.5 24 56 29-5 4700 2300 2.1 830 0 130 33 200 30-5 12000 5900 22 250 9.4 11 55 150 2-6 5700 1700 0 55 0 48 30 100 5-6 2200 600 6.6 150 0 • 130 15 72 12-6 860 320 3.3 490 25 60 • 150 180 18-6 1100 300 0 880 0 110 150 330 25-6 1000 350 0.55 840 0.55 <0.1 93 310 9-7 1300 5.6 0 1300 0 150 140 260 24-7 1200 1.0 0 2300 11 260 1100 490

1976

24-5* 0 0 0 0.34 3.8 25 25-5 35000 22000 0 3.0 0 0.7 4.4 5.9 26-5 25000 12000 0 0.34 0.50 0.34 5.0 8.3 28-5 46000 21000 0 0 <0.1 0.26 7.0 9.6 1-6 24000 10000 0 3.6 0.33 0.52 3.7 17 8-6 40000 15000 0 2 0.40 0.83 9.0 22 21-6 14000 3000 0 2.7 1.9 0.34 24 86 8-7 8000 5300 0 0.26 1.6 2.6 130 480 27-7 1400 1400 0 2.6 0 34 130 340

Countedbefor e spraying.

56 the susceptible one.Durin g thesam eperiod ,pseudomonad s wereno tdetect ­ ableo n S. 6-2 andthei rpopulation s werever ypoo ro n P. nigra andno t highenoug h toexer t anantagonisti c effect against X. populi; yellow bacteriawer e also affectedb y drought andtemperatur e onbot h clones,an d yeasts andyeast-lik e organisms to alesse r degree.Th e lack ofrain s during the lastexperimenta l period couldpartiall y explainwh y X. populi wasno teliminate d from thelea fsurfaces .Moreover , iti spossibl e that themucoi d polysaccharide of X. populi exerts aprotectiv e effect against droughtconditions ,simila r toothe r xanthomonads.Althoug h therol eo f someothe r components of leafmicroflor a isprobabl y notnegligibl e inth e regulation of X. populi, in 1976pseudomona dpopulation swer e nothig h enough toexer t anantagonisti c effect against X. populi. The qualitative evolution ofmicroflor a also depends onothe r factors whichinfluenc e quality and quantity ofsubstance s leached from foliage; genetic constitution, climatic conditions,bu tals ophysiologica l ageo f the leaves,soil , and fertilizersma y influence leafsurfac enutrient s and canopymicroclimate , and therebyma y alterphylloplan emicroflora . Initial investigations undertaken onbalsam sproduce db yepidermi c cells of stipules and leafexudate s during the firststag e ofth ene w shoots show/thes esub ­ stances contain flavonoid aglycones andterpenoid s and are likely to inhibit X. populi on P. nigra. Inadditio nt o inhibiting compounds localized onth e young shoots during the fastgrowin gperiod , somenutrient s areexcrete d at the sametim ewhic h are favourable tovariou s components ofmicroflora : carbohydrates, growth substances, and aromatic acids. Pseudomonas spp.ar e ablet ous e some aromatic compounds ascarbo n sources indegradatio n ina mixture ofphenoli c substances.Accordingl y thesebacteri a could havea complex role inth e regulation ofepiphyti c microflora and couldb eo f decisive importance toth enatura l control ofpathogeni cpopulations .

CONCLUSION

Ecological andepidemiologica l data concerningphytopathogeni c bacteria generally require alo to feffor tt oobtain .On e ofth emai n difficulties ofa bacteriologist' s work isprobabl y the absence of amacroscopicall yde ­ tectable reference stageo fth ebacteria l pathogens,unlik e fungiposses ­ sing someperfectl y defined stage and form of fructification andripening : ascospores, conidial forms,uredospores ,aeciospores ,etc . Twenty years ago the evolution ofbacteriologica l techniques using replication onspecifi c culturemedi a andmor e sophisticated methods,suc h asimmunofluorescenc e orimmunoenzymology ,mad e itpossibl e todetec t pathogenic bacteria invariou s complexmicrobia l ecosystems:evolvin g lesions,microflor a of aerial structures oftrees ,an dth erhizosphere . After Crosse (1963),man y researchworker shav ebee n interested in phytopathogenic bacteriapopulatio n dynamics.Impact s ofclimati c and

57 edaphic factors on host-bacteria relationships are just beginning to be understood. Genetic background of the host and components of phenotypic expression of the resistance to bacterial diseases of forest trees will be explored more and more in the near future, requiring good cooperation be­ tween phytopathologists, physiologists, climatologists, and geneticists.

REFERENCES

Aldwinckle,H.S .& S.V .Beer ,1978 .Fir ebligh tan dit scontrol .Hort-Review s1 :423-474 . Arny,D.E. ,Lindow ,S.E .& CD . Upper,1976 .Fros tsensitivit y ofZe amay sincrease db y applicationo fPseudomona ssyringae .Natur e262 :282-284 . Beer,S.V .& J.C .Norelli ,1975 .Factor saffectin gfir ebligh tinfection san dErwini a amylovorapopulation si npom efrui tblossoms .(Abstract. )Amer .Phytopath .Soc .Proc . 2:95 . Beer,S.V .& J.C .Norelli ,1977 .Fir ebligh tepidemiology :factor saffectin greleas eo f Erwiniaamylovor ab ycankers .Phytopatholog y 67:1119-1125 . Billing,E. ,1974 .Th eeffec to ftemperatur eo nth egrowt ho fth efir ebligh tpathogen , Erwiniaamylovora .J .Appl .Bact .37 :643-648 . Billing,E. ,1980 .Fir ebligh ti nKent ,Englan di nrelatio nt oweathe r (1955-1976).Ann . Appl.Biol .95 :341-364 .[And ]Fir ebligh t (Erwiniaamylovora )an dweather :a compari ­ sono fwarnin gsystems .Ann .Appl .Biol .95 :365-377 . Burrill,T.J. , 1882.Th ebacteria :a naccoun to fthei rnatur ean deffects ,togethe rwit h asystemati cdescriptio no fth especies .Illinoi sInd .Univ .Ann .Rpt .11 :93-157 . Crosse,J.E. ,1963 .Bacteria lcanke ro fston efruits .V. .A compariso no flea fsurfac e populationso fPseudomona smors-prunoru mi nautum no ntw ocherr yvarieties .Ann . Appl.Biol .52 :97-104 . Danilewicz,K .& M .Tomaszewski ,1970 .Degradatio no flignin ,lignol ean dphenoli cacid s inpoplar swoo dwit hPseudomonas ,a nintestinal ebacteri afro mth elarva eo fParan - threnetabaniformi sRott .Act aMicrobiologic aPolonica ,Ser .B ,2 :4-19 . Dueck,J .& H.A .Quamme ,1973 .Fir ebligh ti nsouther nOntari oi n1972 .Canad .Plan tDis . Survey53 :101-104 . DeKam ,M. ,1978 .Xanthomona spopul isubsp .Salicis ,caus eo fbacteria lcanke ri nSali x dasyclada.Eur .J .o fFores tPatholog y8 :334-337 . Dochinger,L.S. ,1969 .Agrobacteriu mgal lo fhybri dpopla r treesi nIowa .Phytopatholog y 59:1024 . Durand,R. ,J .Luisett i& M .Ridé ,1967 .Di eBeziehunge nzwische nde nFrühjahrsfröste n undde rdurc hPseudomona ssyringa eau fBirnbäume nverursachte nBakteriose .X .Agrar - meteorolog.Dreiländertreffe n (Abstract). Freigoun,S.O. , 1973.Relationshi pbetwee nenvironmenta lfactor san dinoculu mpotentia l ontw ocherr yvarieties .Proc .o fWorkin gpart yo n"Pseudomona ssyringa egroup" . Angers,p .79-90 . Gardan,L. ,J .Luisett i& J.P .Prunier ,1972 .Etude ssu rle sbactériose sde sarbre s fruitiers.IV .Recherch ee tétud ede svariation sd ePseudomona smors-prunoru m f.sp . persicaeà l asurfac ede sfeuille sd ePêcher .Ann .Phytopathol .4 :229-244 . Gaur,Y.D .& A.N .Sen ,1976 .Th eutilit yo fmanganes e inlactos emediu m todifferentiat e rhizobiafro magrobacteria .Curren tScienc e45 :865 . Hirano,S.S. ,E.A .Maher ,A .Kelma n& CD . Upper,1978 .Ic enucleatio nactivit yof - fluorescentplan tpathogeni cbacteria .Proc .4t hInt .Conf .Plan tPath .Bact .Anger s 2:717-724 . Kerr,A. ,1972 .Biologica lcontro lo fcrown-gall :see dinoculation .J .Appl .Bact . 35:493-497 . Lewis,L.N .& A.L .Kenworthy ,1962 .Nutritiona lbalanc ea srelate dt olea fcompositio n andfir ebligh tsusceptibilit y inth eBartlet tpear .Proc .Amer .Hort .Sei .81 : 108-115. Lindow,S.E. ,D.C .Arn y &CD . Upper,1978 .Erwini aherbicol aa bacteria lic enucleu s activei nincreasin gfros tinjur yt ocorn .Phytopatholog y68 :523-527 . Luisetti,J .& J.P .Paulin ,1972 .Etude ssu rle sbactériose sde sarbre sfruitiers .III . Recherchessu rPseudomona ssyringa e (VanHall )à l asurfac ede sorgane saérien sd u Poiriere tétud ed ese svariation squantitatives .Ann .Phytopath .4 :215-227 . Miller,H.S .& H .va nDiepen ,1978 .Monitorin go fepiphyti cpopulation so fErwini a amylovorai nth eNetherlands .Act aHorticultura e86 :57-63 .

58 Oku,H. ,T .Shiraishi ,S .Ouchi ,S .Kurozum i& H .Ohta ,1980 .Pin ewil ttoxin ,th e metaboliteo fa bacteriu m associatedwit ha nematode .Naturwissenschafte n 67:198-199 . Panagopoulos,CG . &J.E .Crosse ,1964 .Fros tinjur ya sa predisposin g factori nblosso m blighto fpea rcause db yPseudomona ssyringae .Natur e202 :1352 . Panagopoulos,CG . &P.G .Psallidas ,1973 .Studie so nth eepiphyllou spseudomonad san d otherbacteri a isolatedfro mCitrus .Proc .Workin gpart yo n"Pseudomona ssyringa e group".Angers ,p .67-78 . Parker,K.G. ,N.S .Luepsche n& E.G .Fischer ,1961 .Tre enutrition san dfir ebligh t development.Phytopatholog y 51:557-560 . Paulin,J.P .& J .Luisetti ,1978 .Ic enucleatio nactivit yamon gphytopathogeni cbacteria . Proc.4t hInt .Conf .Plan tPath .Bact .Angers ,p .725-731 . Paulin,J.P .& G .Lachaud ,1978 .Inoculation swit hErwini aamylovor aunde rcontrolle d environmentalconditions .Act aHorticultura e 86:31-38 . Ridé,M .& M .Lopez ,1976 .Crow ngal le tpeuplier sd'ornement .Group ed etravai lde s Maladiesd uPeuplier .FA OBordeaux-Angers .5 p . Ridé,M .& J.P .Prunier ,1963 .Manifestation spathologique sconsécutive sau xattaque sd e Dendromyzasp .su rPeuplier .CR . Congrèsd el'Associatio n Françaisepou r l'Avancement desSciences ,p .156-158 . Ridé,M .& M .Viart ,1966 .Etud ed el acontaminatio nd'un epeuplerai epa rl echancr e bactérien.Bull.Servic ecuit .Peuplie rSaul eSEIT A (1-2):43-61 . Ridé,M. ,S .Rid é& C .Poutier ,1978 .Factor saffectin gth esurviva lo fXanthomona s populio naeria lstructure so fpoplar .Proc .4t hInt .Conf .Plan tPath .Bact .Angers . p.803-814 . Schroth,M.N. ,A.R .Weinhold ,A.H .M cCai n& D. C Hildebrand,1971 .Biolog yan dcontro l ofAgrobacteriu mtumefaciens .Hilgardi a40 :537-552 . Thomson,S.V. ,M.N .Schroth ,W.J .Molle r& W.O .Reil ,1975 .Occurrenc e offir ebligh to f pearsi nrelatio nt oweathe ran depiphyti cpopulation so fErwini aamylovora .Phyto ­ pathology65 :353-358 . ' Vigouroux,A. ,1974 .Obtention sd esymptôme sd ebactérios ed uPêche r (Pseudomonasmors - prunorum f.sp .persicae )su rrameau xd ePêche rdétaché se tconservé se nsurvie : Effetd ufroid .Ann .Phytopathol .6 :95-98 . Vigouroux,A .& C .Huguet ,1977 .Influenc ed usubstra td ecultur esu rl asensibilit éd u Pêchera udépérissmen tbactérien .CR . Acad.Agric .Fr .63 :1095-1103 . Wilson,E.E. ,M.P .Star r& J.A .Berger ,1957 .Bar kcanker ,a bacteria ldiseas eo fth e Persianwalnu t tree.Phytopatholog y47 :669-673 .

59 Biological and physical modifications of the environment and the resulting effect upon the host-parasite interactions in short-rotation tree crops1

Harold S.McNab b Jr.,Richar d B.Hal l &Michae lE .Ostry 2

ABSTRACT

Environmental factors of the atmosphere and soil are pre­ sented in relation to their effects upon incidence of disease in short-rotation, intensive plantation culture of Populus. Environmental modifications inherent for this cultural system and added for pest management schemes are discussed. Tree density, a measure of spatial relationships within stands, is a recurring stand characteristic in the discussion. The term, spatial resistance, is proposed for the summation of the com­ plicated interactions of the many environmental factors asso­ ciated with stand density that affect disease expression toward decreased disease incidence.

INTRODUCTION

The continued increasing demand for wood fiber in North America has caused silviculturists to study the management of short-rotation intensive forest systems (Larson & Gordon, 1969) that approach the intensity of present-day agricultural systems. The pronounced reduction in rotation age with an accompanying increase in annual wood fiber yield per land unit allows increased management inputs that can make the short-rotation, in­ tensively managed forest ecosystems profitable for industries utilizing wood fiber (Isebrands et al., 1979); i.e., paper and fiberboard, or for energy production.

1. Journal Paper No. J-10120 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011 USA. Projects No. 2210, 2294, 2295. Also contribution from the North Central Forest Experiment Station, USDA Forest Service, 1992 Folwell Ave., St. Paul, MN 55108, USA. 2. Professor of Forest Pathology, Associate Professor of Forest Genetics, Iowa State University, Ames, IA 50011, and Associate Plant Pathologist, USDA-Forest Service, North Central Forest Experiment Station, St. Paul, MN 55108, respectively.

60 Selections andhybrid s ofth e genus Populus havebee nconsidere d ideal for short-rotation, intensivelymanage d systemsbecaus e ofthei rrapi d growth,eas e ofestablishmen t through stem orroo tcuttings ,apparen teas e ofcoppic e regeneration, andwid egeneti c diversitywithi n thegenus . Populus spp.ar econsidere d pioneer species inindigenou s forestecosys ­ tems, developing dense,even-aged ,pur e species stands duringth e early years ofth e stand.Durin g the firstdecad e ofnatura l standdevelopment , atleas t2 overlappin g periods ofnatura l thinning occur inwhic hpathogen s and insects are involved (Graham et al., 1963). Ourpape r considers the relationship ofenvironmen t tothes ehost-parasit e interactions asthi s environment ismodifie d inth e short-rotation, intensive culture of Populus spp. andho w this environment canb e furthermodifie d inth e development of integrated pestmanagemen t systems. Wewil l divide the environment into twobroa d categories, atmospheric and soil environments, fully realizing that these twoenvironment s interact closely,bu tthei r separate categorizationwil l allowmor e criticaldiscus ­ siono fth e individual factors involved.Th ediscussio nwil l includebot h biological andphysica l modifications ofth eenvironment .

THE ATMOSPHERIC ENVIRONMENT

Natural reproduction of anaspe n (Populus tremuloides Michx.)stan d after clear-cutting or fire isb y rootsprouts ,producin g an initially dense stand ofrelativel y fewgenotypes .Seedlin g reproduction of P. deltoïdes Bartr.i nopen ,moist ,bottomland s also isdens e initially. During the2 decade s afterreproduction ,natura l thinning drastically reduces stand density. Short-rotation, intensively managed systems of Populus selections andhybrid s arebase d uponth epremis e ofho wefficient , interm s oftim e andeconomi cbenefits ,th e silviculturist canmanag eth e photosynthesizing areao ftree st oproduc emaximu m quantity ofwoo d fiber peruni t area of land.Thu s density ofsuc hintensivel y cultured stands can approach initial densities ofnaturall y reproduced aspen and cottonwood stands.Littl e research directly relating stand density ofpoplar s to disease incidence isavailable ,bu tman y studies havebee nconducte dre ­ lating environmental conditions toproductio n and germination ofpropagule s ofpopla r leaf and stempathogens .Thes e latter studies canb euse d in development ofmodel s relating stand density topathoge n incidence and disease development. InSchipper 1s (1976a)stud ywit h4 clone s of P. x euramericana Guinier considered tob emoderatel y tohighl y resistant to leafrus t (Melampsora medusae Thum.), 3tre e densitieswer eused : 500 0 (1.4-m spacing), 1000 0 (1.0-m spacing), and 15 000 (0.8-m spacing)trees/h a at2 geographical locations.Rating s ofrus t severity andresultin g recommendation forclona l usewer ebase d onSchreiner' s (1959)work .A tRhinelander ,Wisconsin ,wher e

61 the alternate host, Larix laricina (DuRoi )K .Koch ,i spresent ,rus t ratings for 2clone swer ehig henoug h atal l densities toexclud e them from futureplanting s attha tlocation .Th e other 2clone swoul d havebee n excluded from futureplanting s only onthei r rustrating s atth e 1500 0 trees/ha density, trees at1 000 0an d 5000/h adensitie s giving ratings below the arbitrary limit setb y Schreiner.Result s atth eAmes , Iowa, location,wher e Larix spp.ar escarce ,wer e similarbu t less severe for rustdisease . Inaddition ,observation s onincidenc e of Marssonina brunnea (Ell.& Ev. )P .Magn .parallele d rustobservations .Altere d disease sever­ ity atdifferen tplantin g densitieswa s related to leaves remainingwe tfo r longerperiod s after rain,dew ,o r irrigation.Thi s longerperio d oflea f wetness resulted from reduction ofai rmovemen t indense r standswit h high canopy density.Thus , although actual inherenttre e resistancema y notb e altered, leaves ontree sgrow n atnarro w spacing areexpose d to longer periods ofidea l infection conditions than aretree s grown atwide rspa ­ cing.Further ,suc hinteraction sma yb e different fordifferen tclones . These observations weremad ebefor e firstcoppic e cuti nthes epopla r stands.Ou r observations on sproutgrowt h after firstcoppic e cut indicate thatth e firstcoppic e generation iseve nmor e dense thanth e original planted densitybecaus e ofmultipl e stem sprouting. This example illustrates therelationshi p ofcanop y and stand densities with airmovemen twithi n the stand andresultin g disease incidence.I n attempting tous eresult s ofothe rresearc h thatconside r individual envi­ ronmental factors andthei reffect supon-pathoge ngrowt h andreproduction , these individual factorswithi npopla r standsmus tb e separated and related tocanop y and stand density.Moistur e and temperature areth e 2factor s most involvedwit hproductio n andgerminatio n ofpropagule s ofpathogen s and,thereby , intensity ofdisease s instand s (Anselmi& Cellerino ,1980 ; Spiers, 1978;Heathe r & Sharma,1977 ;Pino n& Poissonnier ,1975 ; Castellani, 1966). Moisture isi n2 forms,fre ewate r onlea fan d stem surfaces andrela ­ tivehumidit y ofth e surrounding atmosphere.Rainfall ,dew ,guttation , and irrigation allcontribut e to leafwetness .Duratio n ofthi s freewate r dependsupo n aserie s ofcomplicate d interactions among thevapo r pressures withinplan ttissue ,th eboundar y layer,an d the surrounding air. Ingener ­ alterms ,ai rmovement ,temperature , andrelativ e humidity directly affect the durationo flea fwetness .Fo rmos tpathogens , freewate r is considered necessary forgerminatio n ofspore s andhos tpenetratio nb y theger mtube . Forcertai npathogens ,relativ ehumidit yvalue s above8 0% wil l allow spore productionwhil e relative humidities of 98% orgreate r areneede d for spore germination. Temperature directly affects themetabolis m ofth epathogen ,thereb y allowingu s todetermin e lower andhighe r limits forspor eproductio n and germination. Inaddition , for Marssonina brunnea, Castellani (1966)ha s

62 showna ninteractio nbetwee n temperature and rainfall.A thighe rtempera ­ tures,mor e rainfall isnecessar y forseriou s disease incidence.W ebeliev e this interaction tob edirectl y related toduratio n oflea fwetness .Bot h lightan d airmovemen t directly affecttemperature ,bu tunde r standcon ­ ditions, tend tocounterac t eachother .Dense r stands allow lesslight , thereby lessradian tenerg ymakin g forcoole r conditions.Dens estands , however,hav e less airmovemen t andmoistur e evaporation and, thus, reduced heat loss toth e atmosphere.Unde rusua l growing conditions inthes edens e Populus stands,moistur e relationships thereby affectth e host-parasite interactions toa greate r degreetha ntemperature . Whendiscussin g thecharacteristic s ofstan d density,mentio n shouldb e made ofclona l and speciesmixtures .A nexampl e of aspecie smixtur ewil l be discussed laterunde r soilenvironment . Clonalmixture s couldmodif y the atmospheric environmentb yusin gmixture s representing different crown architectures.Th e intensive culture system using Populus couldviolat e one ofth e important forestpes tmanagemen tprinciples :maintenanc e ofgeneti c diversitywithi n stands.O nth e otherhand ,b y judicial choice ofclone so r selections,w eca n incorporate awide r rangeo fgenotype s thanwoul db e presenti nnatura l stands thataris eb y rootsproutin g orsee ddissemi ­ nation from arestricte d localpopulation . Althoughmuc h researchha sbee ndon eo nmeasurin g atmospheric envi­ ronmental factors incro p fields and relatingthe m todiseas eincidence , littleha sbee ndon e inintensiv e forest culture forth e determination of relationships amongenvironmenta l effects and disease intensity.Ne win ­ strumentation isno w available thatrecord s leafwetness ,ai r temperature andmovement , andrelativ e humidity thatwil l greatly aid suchresearch .W e arebeginnin g studies of theserelationship s inou rshort-rotation ,inten ­ sivelymanage d Populus stands (Younge t al., 1980)s otha tw ewil lb e able tobette runderstan d the effects ofstan d density asi ti smanipulate d by tree spacing,clona l crown architecture andpes tresistance ,prunin g (nat­ ural and artificial), coppice treatments, species and clonalmixture , rotation age,an dth e artificial addition ofphysical ,chemical ,an dbio ­ logical factors (fertilization, irrigation,pesticides ,an dbiologica l control agents)o ndiseas e incidence. Before leavingth ephysica l atmospheric environment, furthermentio n should bemad e ofmoisture , airmovement , and light.Eithe r overhead or ground irrigationma yb euse dt o supplementnatura lrainfal l inthes e intensive-culture systems,especiall y ifpulpin g effluent isavailable . Overhead irrigationwil lmaintai n longerperiod s oflea fwetness ;however , itma yhav e onebenefi t ina pes trelationship .Th eCottonwoo d leafbeetl e (Chrysomela scripta F.), aseriou spes ti nplantatio n establishment and clonalnurseries ,i seffectivel ymanage db yperiodi c rains thatphysicall y dislodge larvae and adults from leaves (Caldbeck etal. , 1978). Overhead irrigationha s the same effect.An y irrigationpractic ewil l need tob e

63 integrated intopes tmanagemen t systems thatconside r allpests . Airmovemen t also is involvedwit hmovemen t ofpropagule s ofpathogens . Both sporemovemen t fromwithi n and fromwithou tth e stand areo fconcern . Primary inoculumma yb e initiatedwithi n the stand from overwintering pathogen ondea d leaves (usually sexual reproduction)o r from cankerso r lesions onstem s (usually asexual reproduction)an d from outside thestan d fromnatura l stands inth e locality or from stands some distance away (Kam, 1973, 1975;Kobayash i &Chiba ,1962 ;Chib a& Zinno, 1960;Waterman , 1954). Convection aircurrent swithi n stands contribute greatly tomovemen to f spores, especially inth eelevatio n ofspore s into thehorizonta l air stream.Ai rmovemen t isespeciall y important inpopla r culturebecaus e of the possibility ofgeographica l isolation ofspecifi cpests .W e find that Melampsora medusae, inarea swher e the alternatehosts , Larix spp.,ar e scarce,ma yno tbecom e seriousuntil,lat eseason .Thus , for Iowa,wher e Larix isno tnative ,thi s disease isno t significant,bu t fornorther n Wisconsin,growt h lossma y reach2 0% (Widin& Schipper, 1976). Windmove ­ mento furediospore s northward from southern cottonwood areas and southward brings theprimar y inoculum into Iowa. Thirteenpopla r planting sitescontainin g 33clone s replicated three times ateac h sitewer eplante d throughout thenorth-centra l United States ina nattemp tt odetermin e thegeographica l distribution ofpopla rpatho ­ gens and insects (Ostry, 1979). Initial disease readings indicated specific areadistributio n ofcertai n leafdisease s (Schipper, 1976b). Iowa plantings weremos t seriously diseased with Septoria musiva Peck and Marssonina brunnea. Septoria musiva was found forth e firsttim e inRhine - lander,Wisconsin ,planting s in198 0eve nthoug h thispathoge n iscommo no n leaves of Populus balsamifera L.i nnatura l stands innorther n areas of

Wisconsin andMichigan . Marssonina brunnea wasno tcommo n inth e St.Pau lF Minnesota,planting s from 1975throug h 1978,bu ti n1980 ,thi s leaf.diseas e hasbecom e serious atthi s location.Thi s situation istypica l ofman yo f the diseases thatw e found initially only ata fe wlocations .A spoplar s aregrow ni na nare a over time1,th emor e serious thepopla r leafan d stem diseasesbecome .Som eo fthi s spread intone w areasprobabl y is associated with distribution of diseased poplar cuttings,bu tw ebeliev e thatth e spread also isb y aerialmovemen t ofspore s from native and locally grown Populus spp.i nornamenta lplantings .W e shouldmention ,though ,tha t2 leafpathogen s haveremaine d somewhat localized, Phullosticta sp.i nth e southern sites and Septotinia populiperda Waterman &Cas h inth enorther n sites (Ostry, 1979). Webeliev e thatlea frus t isth eonl y serious disease forwhic h geographical distribution ofth epathoge n canb euse d as afacto r indevelopmen t ofpes tmanagemen t systems.Fo r example,moderatel y suscep­ tible clones canb e safelyplante d inIowa . Light,beside s interactingwit htemperature , alsoha sdirec t and indi­ recteffect supo n disease resistance.Hig h lightintensities ,suc h asthos e

64 found athig h elevations,cause d abreakdow n ofresistanc e inoat st o Puccinia graminis avenae (Browning et al., 1964). Higher incidence ofth e serious canker of Populus tremuloides causedb y Hypoxylon mammatum (Wahl.) Millerha sbee n associatedwit hstan d openings andedge s (Anderson & Anderson, 1968)an dunprune d trees inthes e locations (Ostry& Anderson , 1979). Aspen is shade intolerant and, indens e stands,self-prunes ,re ­ ducing thenumbe r of lowerbranche s thatotherwis emigh t serve as infection sites forth epathogen .Recently , 2insect s thatdesir ehig h lightcondi ­ tions forovipositio n sites ontree s (Saperda inornata Say and Magicicada septendecim L.)wer e foundassociate d with the incidence of Hypoxylon canker (Anderson et al., 1979). Suchovipositio nwound s andpossibl ebir d feeding atthes e oviposition sites seem tob eentranc epoint s forth e pathogen. Thus, light and,therefore , anope nstan dma ycaus etree s ofth e stand tob emor e susceptible tospecifi c diseases associated withlight - requiringinsects . Lightinteractin gwit htemperatur e alsoma ypredispos e trees toste m canker and decaypathogens .W eobserve d forth e firsttim e in1980 ,i n exposed situationswithi npopla r stands,seriou s sunscald wounds oncertai n poplar clones.Thi s sunscald damagewa s colonizedb y canker fungi (i.e., Cytospora chrysosperma Pers.)an ddeca y fungi (i.e., Armillaria mellea (Vahl.)Quel.) . Thus,whe nmakin gpes tmanagemen t decisions aswel la s silvicultural andeconomi c recommendations for ashort-rotation ,inten ­ sivelymanage d system, limitations onbot h ends ofth e stand-density spectrum arepresent . Wehav ediscusse dphysica l effects ofth e atmosphere onpathoge nactiv ­ ity.Ther e arechemica l andbiologica l factors thatshoul db e included. Among thechemica l factors arepesticides .Unde r field andnurser ycondi ­ tions, leafdisease s havebee nmanage dwit h fungicides (Sheridan et al., 1975; Carlson,1974 ;Cellerin o &Freccero , 1970). Preliminary results indicate thatth ecanke r disease causedb y Septoria musiva ina nexperi ­ mentalpopla rplantatio n incentra l Iowaplante d to adensit y of1 000 0 trees/hawa smanage dwit h the fungicide captifol (McNabb et al., 1980). A resistantpopla r clonewa s includedwit h3 moderatel y tohighl y susceptible clones.Captifo l reduced the incidence ofcanke r inthes e susceptible clones toth e level ofth eresistan tclone . Inaddition ,whe n a surfactant was addedt o another fungicide,a sligh tindicatio n of increased suscepti­ bilitywa snote d foral lclones ,includin g theresistan t one.Th eus eo f fungicides inmodifyin g thechemica l atmospheric environment isbein g investigated further forthes e Populus culturesystems . Relatively little isknow n aboutth e above-ground biological environ­ ment.Bot h insects and small animals areknow nt opredispos e treest o disease.Mic e and rabbit feedingwound s areentranc epoint s forcanke ran d decay fungi.I naddition ,anima l feeding can causetre e stress that,i n turn, increases the incidence ofstress-relate d cankers causedb y Cytospora

65 chrysosperma,Dothichiz a populea Sacc.& Br. ,an d Phomopsis macrospora Kobayashi & Chiba.On e insect-breeding predisposition, that for Hypoxylon canker,wa sdiscusse d earlier.Othe rtype s of insectpredispositio n include feedingo n leaves and stems thatrang e frompunctur ewound s totissu e loss (Palmberg, 1977), includingbranc h and stembreakage .Woun d pathogens regularly colonize such feeding andbreedin g sitesbu thav eno tbee nimpor ­ tantexcep t for Hypoxylon mammatum. Other fungi andbacteri a also arepar to fthi sbiologica l environment. Anumbe r ofworker s have suggestedusin g leaf fungitha tparasitiz e poplar leaf-rusturediospore s asbiologica l control agents (Omar& Heather , 1979). A currentstud y inPolan d associates specific leafbacteri a withrust - resistantpopla r clones (Danilewicz, 1980). This approach inmodifyin g the biological atmospheric environment shouldb epursued .

THE SOIL ENVIRONMENT

Aswit h atmospheric environment, stand density has animportan tinflu ­ ence onth eeffect s that soilenvironmen tha so ndiseas e incidence.Durin g a droughtperio d from1975-7 7 incentra l Iowa,-stress-relate dcanke rfungi , Dothichiza populea, Phomopsis macrospora, and Cytospora chrysosperma, caused serious disease inintensiv e culturepopla rplanting s (Schippere t al., 1977). Observations made in197 6 inpopla r stands containing 4 P. x euramericana clonesplante d at3 differen t initial densities (5000 , 10000 ,an d 1500 0trees/ha )indicate d greater stem death from girdling cankers for2 o fth e clones asstan d density increased.Th eplanting swer e of2 types ,pur e clonalblock s randomly distributed within each density plot, and alternating clonal rows (only2 o fth e4 clone s used)withi n each densityplot .Th e samepatter no fclona l susceptibility asinfluence db y spacingwa s observed inbot h cases.Therefore ,w e concluded that2 clone s had inherentresistanc e to stress-related cankers. Previous studies with these stress-related cankershav e indicateda close relationship betweenbar kmoistur e levels and resistance to these fungi (Butin, 1956). Barkmoistur ewa s related towoun dperider m develop­ ment and,therefore ,t ohos tabilit y to isolatepathogen s andt o callus injuredtissue . Further observations onth e alternate-row plantations during thenex t 2 years (1977an d 1978)differe d fromth e 1976data .Th e clonal differences of 1976disappeare d (Haywood &McNabb , 1979). Greenhouse and growth-chamber studieswit hthes e2 clone s indicated no differences inbar kmoistur e relationships.Th eonl ypossibl e knowndifference sbetwee n these 2clone s thatcoul d account forou r observations areroo tgeometr y and amounto f rootgrowth . Simply stated, the seeminglymor eresistan t clone in197 6ha d deeper roots.A sdrough tcondition s persisted duringth e secondyear , 1976-77, soilmoistur e continued tob edeplete d to alowe r depth.There -

66 fore,whe n allroot s ofth e seemingly resistant treesbecam e associated withmoisture-deficien t soils,stress-relate d pathogenswer e ablet ocaus e disease. Although differences amongdensitie s continued during 1977 and1978 , suchdifference s became less significant overthi speriod . Prolonged droughtconditions ,especiall y thosetha tcaus econtinue d fall andearl y winter soil-moisture deficiency, seemed to eventh edensit y and clonal effectswit hou r P. x euramericana clones,wit h the resultbein g serious stem kill ofal l clonesb y canker fungi. Asdiscusse d earlier, irrigationma ybecom e analternativ e inthes e intensive-culture systems.Unde r irrigation, soil-moisture deficiencies are eliminated duringperiod s ofdrough t ando ndrie rplantin g sites.Again , caution isneede d sotha t adverse effects ofhighe rmoistur e on leafan d stem diseases areminimized . With thehig hdensitie s andcoppic e rotations thatar e characteristic of intensive culture,th epotentia l fortree-to-tre e rootgraftin g is substantial.Thi sma yhav e asignifican t role indiseas e transmissioni n such stands.Alternatively , rootgraft smigh tb ebeneficia l inthi styp eo f forestry ifthe yprotec t the original spacing andcompositio n ofth e stand by allowingcross-feedin g between root systems.Ou r investigations have demonstrated thepossibilit y ofsuc hcross-feedin g buthav ebee ninconclu ­ sive onth epossibilit y ofwetwoo dbacteri a transmission (Paarmann, 1980), andhav e demonstrated geneticbarrier s toroo tgraf t formation in Populus hybrids. Aswoul d beexpected , stand density interactswit h other soil factors involved inpredispositio n oftree s topests .Obviou s factors areth eman y soilnutrient s absorbedb yplan troots .Drai n onsuc hnutrient sbecome s greater asstan d density increases.Althoug h literature isavailabl e on nutrientrequirement s forgrowt h ofpoplar s (Burg& Schoenfeld,1978 ; Zoltân, 1978), there are fewconfirme d examples ofdiseas emodifyin gef ­ fectso f specificnutrient swit h Populus (Brück& Manion , 1980;Garbay e & Pinon, 1973;Suzuki ,1973 ;Gremmen , 1964). Reports concerning theeffect s ofnitroge n onanothe r foresttre e suggestpossibl emodifyin g effects thatnutrient sma yhav e inhost-parasit e interactions in Populus plantations.Hig hnitrat e levelswer e associated with adecreas e of Poria weirii rootro ti nDouglas-fi r inOrego n (Shea, 1970). Whenre d alder, anitrogen-fixin g (actinorhizal)species ,occurre d withinDouglas-fi r stands,thi sroo tro twa s relatively scarce (Trappe, 1971). Thus,beside s applications ofnitroge n fertilizer, another optioni s indicated;biologica l modification ofth e soil chemical environmentwit h nitrogen-fixingorganisms . Manipulation ofsoi lmicroorganisms ,especiall y those associated with treeroots ,actinorhiza l bacteria,an dmycorrhiza l fungi,i s anapproac h thatw ebeliev e tob epromisin g formodifyin g nutrientrelationship s in

67 intensive,short-rotatio n systems (Halle t al., 1979). Inaddition , theus e ofactinorhiza l species inmixe dplanting sma yhav ebeneficia l modifying effects on spatial interactions within the stand atmosphere.Test shav e begunusin gEuropea nblac k alder (Alnus glutinosa (L.)Gaertn. )i nmixture s with Populus (Borders,1980) . Carefulmonitorin g ofbot h soilan datmo ­ spherewil lb e essential forcritica l assessment ofth e effects ofsuc h standmixtures . During thepas tdecade ,knowledg e ofmycorrhiza l fungi and therela ­ tionships thatthe y formwit htree sha s increased to aleve l thatallow s possiblemanipulatio n ofthes eorganism s undernurser y and standcondi ­ tions.Poplar s developbot hectomycorrhiza ean dendomycorrhiza e (Walker, 1979). Ectomycorrhizae maypreven t infection ofroot sb y certain root-rot organisms.Th eendomycorrhiza l relationships also seem tomodif y disease interactions. Interpretationo fstudie swit hendomycorrhiza e are difficult because these structures increase the efficiency ofphosphoru s uptakeb y trees. Interactions among soil organisms indicateothe rway s thatthes e root pathogens canb emanaged . Insoil sbelo wp H 6i nEas tAnglia ,England , Fomes annosus (Fr.)Cke .i sno t able tocompet e successfully with other soil fungi,thu scausin gonl ymino r root-rotincidenc e-i nScot spin eplan ­ tations on suchsite s (Rishbeth, 1950). Once anunderstandin g of suchsoi l interactions is available,specifi c soil treatmentsma yb epossibl e forth e manipulation ofsoi lmicrobiologica l populations. Indeed,w ema yb edoin g suchno wwithou t realizing it.Althoug h directherbicida l injury topoplar s hasbee nnote d (Anselmi, 1978), effecto fherbicide s onsoi lorganism s in poplar-plantation sitepreparatio n hasno tbee n examined thoroughly (Walker, 1979).

CONCLUSION

Ourdiscussio n ofth e atmospheric and soilenvironment s withinth e intensive short-rotation culture systems of Populus has indicated anumbe r ofenvironmenta l modifications resulting fromth e system itselfan dother s suggested forstan dmanagement .On e stand characteristic has recurred in our discussion,tre e density.Thi scharacteristi c isa measur e ofth e spatialrelationship swithi nth e stand.Practica lmodificatio n ofstan d environment isdirectl y or indirectly associated with these spatialrela ­ tionships.Th e facttha ttre edensit y hasbee n associated with reported changes inhost-parasit e interactions in Populus stands indicatesth e importance ofdensit ymodification s inth edevelopmen t ofpes tmanagemen t schemes. Thecomplicate d interactions amongth eman yenvironmenta l factors associated withth e spatial relationships ofstan d density present achal ­ lenge forpresen t and futureresearc hefforts . Ifth ebasi c factors are

68 chosen properly, the summation of these interactions affect disease expres­ sion producing a type of stand resistance. We propose the term, spatial resistance, for this stand effect. Spatial resistance should not be over­ looked when evaluating genetically controlled host-parasite interactions. In addition, the concept of spatial resistance will help to organize obser­ vations on stand-host-parasite relationships found in other forest stands. Such a systems approach will promote a better understanding of one of the many complex phenomena associated with forest and plantation ecosystems.

REFERENCES3

Anderson,G.W .& R.L .Anderson ,1968 .Relationshi pbetwee ndensit yo fquakin gaspe nan d incidenceo fHypoxylo ncanker .Fores tScienc e14 :107-112 . Anderson,N.A. ,M.E .Ostr y& G.W .Anderson ,1979 .Insec twound sa sinfectio nsite sfo r Hypoxylonmammatur no ntremblin gaspen .Phytopatholog y69 :476-479 . Anselmi,N. ,1978 .Fitotossit avers ol esalicace ed iprodott iormonic iutilizzat ine l diserbode lriso .Cellulos ae Cart a29 :17-36 . Anselmi,N .& G.P .Cellerino ,1980 .Relationshi pbetwee nth eresistanc eo fpopla rt o Marssoninabrunne aan dth eperiod so fincubatio nan do fpresporificatio no fth e parasite.In :Proc .F.A.0 .Internationa lPopla rCommissio nWorkin gPart yo nDisease s -I.U.F.R.0 .Workin gPart yS2.05.0 3meeting ,Kórnik ,Poland ,1- 5 September.1 0p . Borders,B.E. ,1980 .Firs tyea rgrowt ho ftw oPopulu sclone sgrow ni nmixture /withAlnu s glutinosa.Unpublishe dM.S .Thesis ,Iow aStat eUniversity ,Ames .4 4p . Browning,J.A. ,E .Bustamante ,J .Orjuel a& H.D .Thurston ,1964 .Th eeffec to fligh t intensityo nth ereaction so foa tseedling st oPuccini agramini savenae .(Abstract) . In:Agronom yabstracts ,America nSociet yAgronom ymeeting ,Kansa sCity ,Missouri , 15-19November ,p .62 . Brück,R.I .& P.D .Manion ,1980 .Interactin genvironmenta l factorsassociate dwit hth e incidenceo fHypoxylo ncanke ro ntremblin gaspen .Canadia nJourna lFores tResearc h 10:17-24 . Burg,J .va nde n& P.H .Schoenfeld ,1978 .Populierenteelt ,bodemvruchtbaarhei d engrond ­ onderzoek.Populie r15 :75-80 . Butin,H. ,1956 .Untersuchunge nlibe rResisten zun dKrankheitsanfälligkei t derPappe l genenüberDothichiz apopule aSacc .e tBr .Phytopathologisch eZeitschrif t28 :353-374 . Caldbeck,E.S. ,H.S .McNabb ,Jr .& E.R .Hart ,1978 .Popla rclona lpreference so fth e Cottonwood leafbeetle .Journa lEconomi cEntomolog y71 :518-520 . Carlson,L.W. ,1974 .Fungicida lcontro lo fpopla rlea fspots .Canadia nPlan tDiseas e Survey 54:81-85 . Castellani,E. ,1966 .Influenc ede sfacteur sclimatique ssu rle sinfection sde s Peuplierseuramericain spa rMarssonin abrunne a (Eli.e tEv. )P .Magn .Phytopathologi a Mediterranea 5:41-52 . Cellerino,G.P .& V .Freccero ,1970 .Contro lo fMarssonin abrunne aschedule daccordin g toth eflushin go fpoplars .In :Proc .F.A.0 .Internationa lPopla rCommissio nWorkin g Groupo nDisease smeeting ,Farnham ,U.K. ,2 7September- 3October .1 1p . Chiba,0 .& Y .Zinno ,1960 .Uredospore so fth epopla rlea frust ,Melampsor alarici - populinaKleb. ,a sa sourc eo fprimar yinfection .Journa lJapanes eForestr ySociet y 42:406-409 . Danilewicz,K. ,1980 .Erwini aherbicol aan dPseudomona sfluorescen sa sindicator sfo r examinationo fth eresistanc eo fpopla rclone st oMelampsor a larici-populinaKleb . In:Proc .F.A.0 .Internationa lPopla rCommissio nWorkin gPart yo nDiseases-I.U.F.R.0 . WorkingPart yS2.05.0 3meeting ,Kórnik ,Poland ,1- 5 September.2 p . Garbaye,J .& J .Pinon ,1973 .Nutritio nmineral ee tsensibilit éà Marssonin abrunne a (Eli.e tEv. )P .Magn .d ePopulu sX euramerican a (Dode)Guinier ,cv . '1-214':étud e préliminaire surjeune splant se nmilie ucontrôlé .Annale sde sScience sForestière s 30:423-431 .

3. Although11 7reference swer euse di npreparin gthi sreview ,spac elimitation s prevented theirinclusio ni nthi ssection .A complet elis ti savailabl efro mth e seniorauthor .

69 Graham,S.A. ,R.P .Harrison ,Jr .& CE . Westell,Jr. ,1963 .Aspen s- Phoeni xtree so f theGrea tLake sRegion .Th eUniversit yo fMichiga nPress ,An nArbor .27 2p . Gremmen,J. , 1964.Th eMarssonin adiseas eo fpoplar ,2 .Inoculatio nexperiment so nlea f discswit hascospore san dconidia .Nederland sBoschbouwtijdschrif t 36:149-157 . Hall,R.B. ,H.S .McNabb ,Jr. ,C.A .Maynar d& T.L .Green ,1979 .Towar ddevelopmen to f optimalAlnu sglutinos asymbioses .Botanica lGazett e14 0 (Suppl.):S120-126 . Haywood,W.F .& H.S .McNabb ,Jr. ,1979 .Moistur estres san dincidenc eo fste mcanker s inintensivel ygrow npoplars .In :Proc .Firs tNort hCentra lTre eImprovemen t Conference,Madison ,Wisconsin ,21-2 3August ,p .123-132 . Heather,W.A .& J.K .Sharma ,1977 .Som easpect so fpopla rrus tresearc hi nAustralia . AustralianForestr y40 :28-43 . Isebrands,J.G. ,J.A .Sturo s& J.B .Crist ,1979 .Integrate dutilizatio no fbiomass . TAPPI62 :67-70 . Kam,M .de ,1973 .Lif ehistory ,hos trang ean ddistributio no fSeptotini apodophyllina . EuropeanJourna lo fFores tPatholog y3 :1-6 . Kam,M .de ,1975 .Ascospor edischarg ei nDrepanopeziz apunctiformi si nrelatio nt oinfec ­ tiono fsom epopla rclones .Europea nJourna lo fFores tPatholog y5 :304-309 . Kobayashi,T .& 0 .Chiba ,1962 .Overwinterin go fsom eleaf-attackin gfung io fpoplars . JournalJapanes eForestr ySociet y44 :19-24 . Larson,P.R .& J. C Gordon,1969 .Photosynthesi san dwoo dyield .Agricultura lScienc e Review7 :7-14 . McNabb,H.S. ,Jr. ,M.E .Ostry ,P.A .Tipton ,R.S .Sonnelitter ,J.M .Zit o&E.S . Caldbeck, 1980.Fungicida lmanagemen to fSeptori acanke ro fPopulu sgrow nunde rshort-rotatio n intensiveculture .(Abstrac t 534).In :Progra man dabstrac tbooklet ,America nPhyto - pathologicalSociety-Canadia nPhytopathologica lSociet ymeeting ,Minneapolis , Minnesota,24-2 8August ,p .243 . Omar,M .& W.A .Heather ,1979 .Effec to fsaprophyti cphylloplan e fungio ngerminatio n anddevelopmen to fMelampsor alarici-populina .Transaction sBritis hMycologica l Society 72:225-231 . Ostry,M.E. ,1979 .Diseas eresearc ho fpoplar sgrow nunde r intensivecultur ei nth e northcentra lregio no fth eUnite dStates .In :Proc .Nort hAmerica nPopla rCounci l 16thmeeting ,Thompsonville ,Michigan ,14-1 6August ,p .83-91 . Ostry,M.E .& G.W .Anderson ,1979 .Hypoxylo ncanke r incidenceo nprune dan dunprune d aspen.Canadia nJourna lFores tResearc h9 :290-291 . Paarmann,S.C , 1980.Roo tgraft si nPopulus :Growt hrelation san dwetwoo ddiseas e transmissionbetwee ntrees .Unpublishe dM.S .Thesis ,Iow aStat eUniversity ,Ames . 42p . Palmberg,C. ,1977 .Introductio nan dimprovemen to fpoplar si nAustralia .Australia n Forestry40 :20-27 . Pinon,J .& M .Poissonnier ,1975 .Etud eépidemiologiqu e duMarssonin abrunne a (Ell.e t Ev.)P .Magn .Europea nJourna lo fFores tPatholog y5 :97-111 . Rishbeth,J. , 1950.Observation so nth ebiolog yo fFome sannosus ,wit hparticula r referencet oEas tAnglia npin eplantations .I .Th eoutbreak so fdiseas ean decolog ­ icalstatu so fth efungus .Annal so fBotan y14 :365-383 . Schipper,A.L. ,Jr. ,1976a .Popla rplantatio ndensit yinfluence sfoliag edisease .In : Intensiveplantatio ncultur e- fiv eyear sresearch .USD AFores tService ,Genera l TechnicalRepor tNC-21 ,Nort hCentra lFores tExperimen tStation ,St .Paul ,Minnesota . p.81-84 . Schipper,A.L. ,Jr. ,1976b .Hybri dpopla rdisease san ddiseas eresistance .In :Intensiv e plantationcultur e- fiv eyear sresearch .USD AFores tService ,Genera lTechnica l ReportNC-21 ,Nort hCentra lFores tExperimen tStation ,St .Paul ,Minnesota ,p .75-80 . Schipper,A.L. ,H.S .McNabb ,Jr .& W.F .Haywood ,1977 .Dothichiz aan dPhomopsi scanker s ofhybri dpopla r inIowa .(Abstrac t 128).In :Proc .America nPhytopathologica l Society4 :109-110 . Schreiner,E.J. ,1959 .Ratin gpoplar sfo rMelampsor a leafrus tinfection .USD AFores t Service,North-easter nFores tExperimen tStatio nResearc hNot eNE-90 .3 p . Shea,K.R. , 1970.Pori a rootrot :Problem san dprogres si nth ePacifi cNorthwest . In:T.A .Toussoun ,R.V .Beg a& P.E .Nelso n (Eds):Roo tdisease san dsoil-born epatho ­ gens,Universit yo fCaliforni aPress ,Berkeley ,p .164-166 . Sheridan,J.E. ,J.E .Harpe r& G .Stevenson ,1975 .Not eo nepidemiolog yan dcontro lo f poplar leafrust .Ne wZealan dJourna lScienc e18 :211-216 . Spiers,A.G. ,1978 .Effect so flight ,temperature ,an drelativ ehumidit yo ngerminatio n ofurediniospore sof ,an dinfectio no fpoplar sby ,Melampsor a larici-populinaan dM . medusae.Ne wZealan dJourna lScienc e21 :393-400 . Suzuki,K. ,1973 .Studie so nth esusceptibilit y topopla rlea frus tinfluence db y

70 differentnutrien tconditions .(I )Change so fsusceptibilit y inducedb ynutrien t deficiency.Journa lJapanes eForestr ySociet y55 :29-34 . Trappe,J.M. ,1971 .Biologica lcontro l- fores tdiseases .In :Proc .Wester nFores tPes t Committee,Wester nForestr yan dConservatio nAssociatio nmeeting ,Spokane , Washington,3 0November .2 p . Walker,C , 1979.Hybri dpopla rmycorrhiza ean dendogonaceou s sporesi nIowa .Unpublishe d Ph.D .Dissertation ,Iow aStat eUniversity ,Ames .25 8p . Waterman,A.M. ,1954 .Septori acanke ro fpoplar si nth eUnite dStates .USD ACircula r No.947 .2 4p . Widin,K.D .& A.L .Schipper ,Jr. ,1976 .Epidemiolog y andimpac to fMelampsor amedusa e leafrus to nhybri dpoplars .In :Intensiv eplantatio ncultur e- fiv eyear sresearch . USDAFores tService ,Genera lTechnica lRepor tNC-21 ,Nort hCentra lFores tExperimen t Station,St .Paul ,Minnesota ,p .63-74 . Young,K.R. ,H.S .McNabb ,Jr .& M.E .Ostry ,1980 .Aeria lpathogen si na centra lIow a poplarplantation . (AbstractG-14) . IowaAcadem yo fScienc eProceeding s87 :center ­ fold6 . Zoltân,T. ,1978 .Anyagforgalm ivizsgâlato knyârfâva lvégzet t szennyvizöntözéses tenyészedény kisérletekben.Agrokémi aé sTalajta n27 :399-415 .

71

t L Genes for resistance to insects in agriculture with a discussion of host-parasite interactions in Carya

Marvin K. Harris

Department of Entomology, Texas ASM University, College Station, Texas 77843, USA

ABSTRACT

Informationconcernin g thegenetic s ofresistanc e inin ­ sect/plant interactions inagricultur e isavailabl e foronl y someplant s and aminuscul enumbe r of insects.Th e scanty data presently available allowsnumerou s interpretations, oneo f which istha t agriculturally useful resistance toinsect si s usually anartifac to fplan tdomesticatio n rather than acapi ­ talization on acoevolve dproduc to f insect'an dplant .Close r studyo fnatura l defensemechanism s inindigenou ssystems , particularly systemsundergoin g domestication, canprovid ene w evidence todevelo p aholisti c theory ofhos tplan t resistance toinsects .Greate r emphasis oninsect/plan t genetics and insect/plantbiogeograph y wouldhel pprovid e thenecessar y framework for intra-a swel l as interdisciplinary communication of ideas concerning hostplan tresistance ;and , assisti n designing strategies forusin gthi stoo l inpes tmanagement .

ORIGIN AND PERMANENCE OF RESISTANCES

Aknowledg e ofth egenetic s ofhost-plan t resistance to arthropodsan d the reciprocal (virulence of arthropods toplants )i simportan tbecaus e of therol e itca npla y indiscovering , incorporating, deploying andmain ­ taining resistantplant s forus e inagriculture .Numerou s recentpublica ­ tionsprovid e significant spacet othi ssubjec t (Day,1977 ;Harris ,1980a ; Maxwell &Jennings ,1980 ;Russell , 1978). A close inspection ofTabl e 1an dth eplant/insec t interactions sug­ gests some generalities (alsonote db yRussell ,1978 ;Gallu n& Khush , 1980). First,quantitativel y inherited resistance isstable .Second ,bio - type development, ifi toccurs ,wil lb eprimaril y against antibiosismech ­ anismswhe narthropod s areexpose d tomonogeni c resistance.Third ,mono ­ genic resistance hasbee nquit e stable insom eplant s against someinsects . Fourth, agriculturally useful resistancema y ingenera lb e found inan yo f

72 Table 1. Inheritance,number s ofgene s involved inresistance , andprio r relationship ofresistance ,o fcrop st o insects (compiled from reviewsb y Carpenter et al. (1979), Gallun& Khus h (1980),Harri s (1975), andRussel l (1978)).

Crop Insect Resistance ,numbe r ofgene s Biotypes Prior known relation- domi- re- quanti- shiu- 2p nant cessive tative

Rice brownplanthoppe r 2 + s? green leafhopper 4 A white-backed planthopper 1 U gallmidg e X X? + U Sorghum corn leaf aphid X + U greenbug 1* + A shootfl y U chinchbu g X A / Wheat hessian fly 7 + A greenbug 1 S? cereal leaf beetle 7 S? stem sawfly 1* A Barley hessian fly 1 s? cereal leaf beetle s? greenbug 2 s? Corn european corn borer X A cornearwor m X U fallarmywor m X S? Cotton bollweevi l 3 U thrips 3* u tobaccobudwor m 2 u Empoasca spp. 1* u Apple Dys aphis devecta 1 A rosy apple aphid 1 A woody apple aphid1 A Raspberry Amphoraphora rubi X A Amphoraphora agathonica X A

73 Table 1. Continued Muskmelon melon aphid 1 U red pumpkin beetle 1 S? Squash striped cucumber beetle X S? squashbu g X S? Sweetclove r sweetclover aphid 1 U Alfalfa pea aphid 1 + U spotted alfalfa aphid X? X? + U Pear pearpsyll a X A Pecan pecantwi g phylloxera X? X? +? S 1. X =numbe r ofgene sunclear ; *= incomplete dominance;? = statu s questionable. 2. S= sympatric;A = allopatric ;U =unknown . the categories ofinheritance . Fifth,gene s for resistancema yb e obtained both fromplan tger mplas m whichha s coevolvedwit hth e arthropod aswel l as fromtha twhic hha sno tcoevolve dwit h the arthropod. The stability ofquantitativel y inherited resistanceha sbee n anindi ­ cation to some,mos tnotabl y Robinson (1980), thatresistanc e breeding efforts shouldb e concentratedprimaril y inthi sdirectio ndu et oth e instability,wit h occasionally catastrophic consequences,o fsom emonogeni c resistance (exhibitedprimaril y indisease s and ininsect swit hpartheno - genetic reproduction). Russell (1978)suggest s thatpes tresistanc ebase d onmonogenicall y inheritedmorphologica l characters such aspubescenc e orsoli d stemscon ­ fersdurability , citingwhea tste m sawfly,cerea l leafbeetl e and Empoasca sp. asexamples ;an d also arguestha tothe r types ofmonogeni c resistance mayhav ebee n insufficiently exposed topes tattac kt o allowbiotyp eex ­ pression tooccur ,o reve n ifbiotype s have occurred, their abilityt o disseminate mayb e low and thusthe yhav e so farescape d discovery.Agree ­ mentwit hRussel l (1978)woul d allow includingmorphologicall y basedresis ­ tantmonogene s toth epropose dplan tarchetype .Eve nthi s seemingly straightforward generality requires some qualification intha tmany ,per ­ hapsmost ,morphologica l characters arepolygenicall y controlled inth e plant. Data inTabl e 1summariz emos to fwha tha sbee nreporte d regarding

74 genetics ofresistanc e toarthropod s inagriculture .Ther e are3 majo r problemswit h interpretation ofthes edata . 1.Quantity :Th egeneti cbase s formos t agricultural resistances to arthropods haveye tt ob e described andsubsequen t studiesma y change present trends.Ther e are3 3 insects attacking 14crop s included inTabl e 1.Th etota l numbero f insect species known isabou t3/ 4 milliono fwhic h only a fewthousan d or so areconsidere d tob e croppest s (Metcalfe t al., 1962). Totalplan tspecie s knownnumbe r afe whundre d thousand ofwhic h only afe wthousan d areconsidere d tob e economically important(i.e . Hedrick, 1919). Thus,presen tknowledg e regarding genetics ofresistanc e to insects inagricultura l plants hasbee nobtaine d from lesstha n1 % ofth e species ineac hcategory . 2.Quality : Furthermore,n ocomplet e characterization ofth egenetic s ofresistanc e hasbee nconducte d foreve n 1insec t orplan t sotha teve n thebes t studies of insects onth emos twel l investigated crops are asye t incomplete.Also ,resistanc e geneswhic h areknow nhav e oftenno tbee n characterized ast o their origin (allopatric, sympatric-coevolved and sympatric-unselected,woul db e onesuc hsyste m ofclassificatio n (Harris, 1980a)). This isals o true forth emajorit y ofresistance s ingenera l (Leppik, 1970;Harlan , 1977;Harris ,1980a )s otha teve nwhe nthei r genetics aredescribed , this deficiency is likely toremain . 3.Perspective : Informationpresente d inTabl e 1wa sprimaril y obtained by agriculturists whosemajo r goalwa s obtainingplant swhic hwoul dproduc e highyield s ofgoo d quality ofa desire d resource ona regula rbasi s ata n acceptable cost inth epresenc e ofth e insect.Brownin g (1974)addresse sa similarproble m ofperspectiv e inpathology .Painte r (1968)observe s in regard topas t successes inbreedin g forHessia n flyresistanc e that 'these goalshav ebee nreache dwithou texac tknowledg e ofth emechanis m ofresis ­ tance and, inth eearl y stages,withou texac tinformatio n aboutth egeneti c factors involved'. Increased attentiont oth egenetic s of resistance has beenmad edurin gth epas tdecad eo r soo ncrop s likewheat ,raspberries , rice, alfalfa,sorghum , etc.,an dhopefull y abette r overall understanding willb eobtaine d inth e future.Th e inclusion ofbot hplan t and arthropod population geneticists inteam sbreedin g forresistanc ewoul d improve the perspective fromwhic h datahav ebee ngathere d inth epas t (seeGallu n & Khush (1980)fo rmethodology) . Data inTabl e 1represen t aver y small sample size from asye tincom ­ plete studies carried outwit hth eprimar y intento fproducin g better agricultural plants rathertha nunderstandin g thegenetic s ofresistance . Thus,whateve r interpretations aremad emus tb e ofa speculativ enatur e and tentativependin g further studies. Harlan& Starks (1980)observ e that 'Biological theorywoul d suggest thatth emos t likely region inwhic h to find resistance toa give n insect wouldb ewher e the insect isendemic.. . Inactua lpractice ,ther e mayo r

75 i mayno tb e acorrelation' .Presumably , thebiologica l theory referredt o implies that sufficientnatura l selectionpressur ewa s exerted onth eplan t populationb y the arthropodpopulatio n throughth ecours e ofcoevolutio ns o thatth epresen t situationha s resulted inplan t individuals andpopula ­ tionswhic h arebot hbiologicall y and agriculturally fit.Biologicall y fit? Yes. This is self-evidentbecaus e ofth econtinue d presence ofbot h species inth esam ehabitat .Agriculturall y fit?N o ormaybe .Agricultura l valuei s dependentupo nplant s orplan tpart s relating tohuma nresource swhic hnee d notb erelate d toplan tcharacter s important for survival atall . Infact , numerous defensemechanism s inplant s involvingescap e inspac e andtim e andbiologica l associationsma yb e extremely important inth ewil d forth e production of fertile offspring, andcompletel y unacceptable in agriculture (Harris, 1980b). Ofth e 69resistance s (Table 1), 27rel yprimaril y on allopatric resistance, 1o nsympatri c resistance, 16o npossibl y sympatric resistance and sources of2 5ar eunknown .Allopatri c resistanceha sbee n heavily relied upon inagricultur e forth e construction ofgeneti c defenses againstarthropod s andthi s showstha tpractic eha sno tbee n consistent withth e 'biological theory' addressed above. There are additional factorswhic h should-b econsidere d ifon ewer et o investigate biological foundations ofresistanc e inagriculture .Fo rexam ­ ple, resistance shouldb emos t abundant forthos e arthropodswhic hhav e exerted the greatestnatura l selectionpressure s onth eplan t throughtime . Genes forresistanc e shouldb edominant ,linke d andconsis to fmultipl e allelest o facilitate and stabilize inheritance.Wher emultipl e selection pressures areconsistentl y experienced by theplan tthroug h time,a s from anassociate d arthropod complex, again linkage amonggene s andmultialleli c inheritance would appear tob eexpecte d asth eprogen y ofgeneratio n after generation ofth eplan t are selectively attacked and their abilityt o produce fertile offspring is affectedb y these selectionpressures .Fur ­ thermore, arthropodswhic h areexpose d tothes edefense s should reflect similar geneticmakeup s asthei r own abilities toproduc e fertile progeny are affected (seePerson , 1959,1966 ;Perso n& Mayo , 1974;Grot h& Person , 1977, fordiscussion s inplant/pathoge n systems,an dLewontin , 1974). Agriculturally useful resistance hasno tbee n associated witharthro ­ pods which areo rhav ebee n observed toexer tth egreates t selectionpres ­ sure onth eplan tpopulation .Gene s forresistanc e to agive nspecie s are seldom linked andrarel y ifeve r isresistanc e tomor e thanon e species obtained from asingl e gene.Multipl e alleles for agriculturally useful resistance to arthropods areunknown .Furthermore , arthropod biotypes inheritthei rvirulenc e recessively. Only inth ematte r ofdominanc e of plant inheritance ofresistanc e doesbiologica l theory agreewit hagricul ­ tural experience.Thi s can alsob eexplaine d through traditional plant breedingpractice s andhos tplan tresistanc e investigations preferentially identifying dominantplan tgene srathe r thanrecessiv e ones, therebybi -

76 asingthes eresults . Ifon epostulate s thatresistanc e canb e anartifac to f agriculture and represents ane w genotype towhic h the arthropodha sno tpreviousl y been exposed, thenvirulenc e geneswil l endow individual arthropods possessing themwit h suddenly increased fitness inthi sne w circumstance.B y arapi d presentation ofresistanc e genes,correspondin g virulence genes (ifthe y exist at all), could increase infrequenc y inth e arthropodpopulatio nwit h almostequa l rapidity andresul ti nhomozygou s recessivelyvirulen tbio - typeswhic h stillposses smodifier s evolved forth edominan tbu tno w aviru- lentallele s atthes e loci.Further , ifbot h the 'avirulent'populatio n and the 'virulent'populatio n aremixe d andexpose d to ahos ttha ti ssuscepti ­ ble toboth ,th e 'avirulent'populatio n should exhibitgreate r fitness and predominate (seeMacKenzie , 1980,fo r further discussion). Reduced fitness ofbacteria l 'biotypes'resistan tt o antibiotics (i.e.McVeig h& Hobdy , 1952)an d arthropods resistantt opesticide s ascompare dt opopulation s in antibiotic andpesticide-fre e environments,respectively , indicate thisi s agenera lphenomeno n associated withman-manipulate d selection forces. MacKenzie (1980)observe s that 'moreresearc h emphasis on differential survival ...i sneeded' . ' Thisbecome sparticularl y importantwhe non e considers taking anindig ­ enousplan t and selecting anarchetyp e tob eproduce d ineven-age dmonocul ­ tures inth epresenc e ofit s coevolvedbiot a for anextende dperio d of time.Henr y Ford's2.5-million-acr erubbe rplantatio n failure inits ' nativeBrazi l inth e 1930'sprovide s abitte rexampl e ofth eproble m and mayb econtraste d toth emor e recentmultimillio nacr e endeavor ofDanie l Ludwig atJari ,Brazil ,wher enon-indigenou s Gmelina arborea VanHie s and Pinus caribaea Morelet treeplantation s arebein g grown successfully for the timebein g on anequall y vast scale.Th e latter effort ismor e typical oftraditiona l agricultural approaches intha tcenter s ofproductio n are usually removed fromplan tcenter s oforigin .Thi s istru e forothe rtre e crops aswel l sucha scoffee ,cacao ,apple ,banana ,coconut ,almond , and rubber.Onl y afe w important tree food cropshav eno t followed thispatter n and,o fcourse ,tre e fiber-crops areonl y recentlymovin g toward exotic plantations. Considering themongre l origins ofmos to fou r agriculturalplant s and theofte nunclea r relationships theseplant shav eha dwit h their attendant biota (Harris,1980b) , abette r understanding ofwha tdoe shappe n inplan t domestication toinfluenc e susceptibility to arthropodsma yb e obtained from aclose r inspection of aplan t ascultivatio n ofi tproceed swithi n its originalhabitat .

77 RESISTANCES IN THE PECAN: A PLANT UNDERGOING DOMESTICATION

Thepecan , Carga illinoensis (Wang.)K .Koc h (Juglandaceae), isindige ­ noust oth e southwesternU.S . (Little, 1971), asar eth earthropod swhic h commonly attack it (Harris,1980b , 1980c). Thepeca n isals oth emos t importanthorticultura l cropnativ e toth eU.S . (Brison, 1974), andi s presently undergoing domestication sotha t experimental situations exist varying fromwil decosystem s tomonoculture s of singlecultivar s grownove r hundreds ofhectares .Nut sproduce d fromwil d trees arehighl y palatable andpresen testimate s indicate thatca .8 5% ofth ehectarag e inTexa s (ca. 300 000)consist s oftree snatur eplante d and ca.7 5% ofth enut s sold in anaverag e year areproduce db y suchtrees .Thus , inthi s spectrum ofwil d ecosystem tomonoculture ,th e 'managednative ' (wildpeca ntree swhic hhav e beencleare d ofcompetin g trees andbrush ,thinned ,pruned , fertilized and sprayedwit hpesticide s invaryin g degrees)presentl y predominates.Consid ­ ering thatscarcel y acentur y ago,wil d trees inth enatura l ecosystem exclusively reigned, thepresen t status ofth epeca n appears torepresen ta dramatic change;however ,th enex t fewdecade s are likely towitnes s an evenmor e rapid transition toward themanage d dultivari nmonocultur e as 'managed natives'ar ereplace db y deliberately plantedmonoculture s of selectedpeca n cultivars andothe r crops such assoybeans .

The walnut caterpillar Three insectswhic h attackpeca nprovid e some insight intopeca nde ­ fensemechanisms .Th ewalnu tcaterpilla r Datana integerrima G. &R . (Lepidoptera:Notodontidae )i s alea f feeder ofplant s inth eJuglandaceae . Iti sdistribute d easto fth eRock yMountain s from Canada toMexic o andi s univoltine into the latitude of Illinois,bivoltin e toNorther nTexa san d trivoltine farther south.Massiv ewidesprea d defoliations havebee n reported caused bywalnu tcaterpilla r since'theearl y 1900's (Cutler,1976 ; Haseman, 1940;Hixson , 1941). One such infestation inTexa s was observed in 1973 anddocumentatio n ofpar to fth e affected areawa smad e using aerial infraredphotograph y (incooperatio nwit hBil lHart ,USDA-SEA , Citrus Insects Laboratory,Weslaco ,TX) .Studie s atth etim ethroughou t theare a and follow-up studiesusin gth e film indicatedn otree s (unless sprayed withpesticide )i nth e Juglandaceae inth e areawer e ablet owithstan d attack andcomplet edefoliatio n resulted (Harris, 1980c). Thiswa s asever e disappointment toth e investigators whoha dhope d to identify sourceso f walnutcaterpilla r resistantpeca nger mplas m fromth eaffecte d area.Al l indications fromthi s study and follow-up investigations indicate thatn o significantbiologica l differences occur amongth ethousand s ofpecan s examinedwhic h couldb euse d asa sourc eo fresistance .W e concluded that pecans are,fo ral lpractica lpurposes ,universall y susceptible towalnu t caterpillar and,conversely ,walnu tcaterpilla r areuniversall y virulento n

78 pecan.Harri s (1980c)ha s speculated thatthi s situationma y havebee nth e resulto fpreviou s selection; and, ifthi s istrue ,th epeca n genepoo l would notcontai ngene s forresistanc e (agriculturally speaking). Infact , individuals containing 'resistance'gene swoul db e less reproductively fit thanth epreponderantl y susceptible individuals and thecullin g ofresis ­ tancewoul d be acontinua l andongoin gproces s eventoday .

The pecan weevil A second insectwhic h attacks thepeca n isth epeca nweevil , Curculio caryae (Horn) (Coleoptera:Curculionidae) .Thi spredominantl y biennial obligatory nut feeder infestspeca n andhickor y inth eplan tgenu s Carya (Gibson, 1968). Pecannut s inth ewate r stageo fdevelopmen t are destroyed by adultfeeding , and subsequent oviposition inmor ematur e nuts results in nut destructionb y the larvae.Estimate s ofdamag e capacity indicate that onepeca nweevi l candestro y about6 nut sb y feeding,wit h the female destroying anadditiona l 22o r sothroug h oviposition andprogen ydevelop ­ mentdurin g one generation (2-3yea r life cycle). This attack occurs about 120day s after floweringwhe nth enu tha s reached full size andbegu nt o fillwit h energetically expensive oils, fats andproteins .N orfiacro- structure ofth epeca ntre e contains such alarg e amounto fenerg y ins o small apackag eno r represents amor e importantelemen ti nth eeffor tt o continue the species. Itappear s self-evident,give nthi s expenditure of energy and thebiologica l importance ofth enu tt oth epecan ,tha tth e pecanweevi l should represent oneo fth emos t importantbioti c selection pressures onth epecan .Consequently , this interactionha sbee n examined in order to identify thenatur e andexten to fpeca n defensemechanism s with regard topeca nweevil . Defensemechanism s arepresen t and appear toconsis to fescap e intim e and space exemplified atth enu tcluster ,th e individual plant andth e population level.Peca nnut s aresusceptibl e topeca nweevi l oviposition based onthei rphenology .Th eperio d fromge l staget o shuck split isth e window intim ewhe n successful oviposition ispossibl e (Harris& Ring , 1979). Nuts attacked duringth ewate r stage,jus tprio r toth eonse to fth e gel stage,ar eshe db y thetre e and iti squestionabl e ifovipositio n is ever seriously attempted insuc hnut s although adult feeding and subsequent nut shedding certainly occurs.Al lpeca nnut s studied havebee n found tob e acceptable forovipositio nbetwee n gel stage and shuck split andt o sustain larval developmentonc e ovipositionha soccurred . Confrontive defense mechanisms topreven tpeca nweevi l ovipositionbetwee nge l stage andshuc k splithav eye tt ob e found and Ibeliev e they dono toccu r inan ybiologi ­ callymeaningfu l capacity, ifa tall .Give n stable supplies ofnut s each year, thepeca nweevi l appears capable of infestingmos t ifno t allo fthe m sotha t few ifan ynut s escape (seeHarri s &Ring ,1980 ;Harri s et al., 1981, formor e discussion).

79 However,nu tsupplie s areno t stable fromyea r toyear . Infact ,nu t numbersvar ywidel y through time inwil d andnativ e-population so ftree s fromn omeasurabl e production insom eyear s toproductio ni n excesso f 100 000nuts/hectar e inothers .Furthermore ,nu tproductio n andbarrennes s appear tob e relatively synchronized from treet otre e overwid e areas (100km 2 ormore )s otha twhol e areasten d toproduc e nuts ina give nyea r followedb y 1o rmor ebarre nyears ,etc . Themagnitud e ofthi s fluctuation and itseffect s onth epeca nweevi l arereadil y apparentwhe nwil d trees areexamine d forpeca nweevil .Typi ­ cally, suchtree shav e few ifan ypecan s andthu sweevi lpopulation s are quite low.Th e fewpeca nweevil spresen t inth e areawhe n amas tyea r occurs arequit e successful inincreasin g theirpopulatio n sinceoviposi - tion sites aretemporaril y non-limiting,bu tth e synchronized lownu t productionwhic h follows starves thepopulatio nbac k to low levels.Effect s onth epeca n aretha tproductio n is sufficienti nmas tyear s tosatiat e the nutpredato rwit h only asmal lportio n ofth enut sproduced ,whil e theres t escape.N odoub tthi s defensemechanis m also reduces selectionpressure s fromothe rnu t feeders.

The pecan phylloxera A third insect,th epeca nphylloxer a Phylloxera devastatrix (Perg.) (Homoptera:Phylloxeridae )produce s conspicuous galls onpeca npetioles , leafletmidrib s andoccasionall y nutlets.Thes e galls are formedb y nymphs hatching from overwintering eggs shortly afterbudbrea kwhe nthe y settle and feedo nth eyoun grapidl y growing tissuewhic h isthe ninduce d to form a gall around them.Gall s splitope n7 5day so r so afterbudbrea k and affected leavesbegi nt odehisc e fromth etre e leavingonl y afe wgalle d leaves until leafdro p inth e fall.Heavil y galled treeshav e less leaf surface andwher e flowerso r flowering shoots aregalled , nutproductio ni s reduced oreliminated , buthig hnu t reduction duet o infestation only occurs inth e severestoutbreaks .Wil dtree s rarely canb e foundt ob e infestedwit h P. devastatrix although close inspectionwil l sometimes reveal agal lno w and again.Onl ytwic ei nth epas t8 year so fobservin g several thousands of genetically differentpecan shav e Ifoun d awil d tree tob eheavil y infested and thenth e infestationwa s limited to1 tree . Vegetatively reproduced cultivars,however , are anothermatter .Th e culti- vars 'Stuart', 'Schley' and 'Success' arereporte d susceptible by some authors and resistantb yother s (Table 2). J.W.Stewar t (TAEXAre aEntomol ­ ogist,Uvalde ,TX )ha s observed atre egrafte d to 'TexasProlific ' and 'Success' inwhic h the 'TexasProlific 'ha sbee nheavil y galled year after year,ye tth e 'Success'i svirtuall y gall-free.Thes e kinds of findings suggest adifferentiatio n ofth epeca nb yth ephylloxer a and adifferentia ­ tiono fth ephylloxer ab y thepeca n asha sbee nreporte d innumerou s plant/pathogen interactions, (Wood& Graniti ,1976 ;Day , 1977)a swel l as

80 Table 2. Responses ofvariou speca n cultivars to Phylloxera devastatrix fromvariou s locations atvariou stimes .

Location Cultivar Resistant Susceptible Reference

Georgia Stuart X Moznette et al. Schley X (1940) Success X Louisiana Stuart X Baker (1935) Schley X Louisiana Caspiana X Calcote & Schley X Bagent (1974) Success X Stuart X Louisiana Caspiana X Boethel,in : Stuart X Carpenter et al. (1979) Texas Texas Prolific X Stewart(pers . Success X comm.) Texas SanSab a Improved X Calcote (pers. Western Schley X comm.)an dper ­ Eastern Schley X sonalinvesti ­ Caddo X gations atBi g Cheyenne X Valley,T X Mahan X (seetext ) Sioux X Ideal X Burkett X

wheat/Hessian fly, Rubus/raspberry aphids,alfalfa/alfalf a aphids (Maxwell & Jennings, 1980); andconifer/scal e (Edmunds& Alstad , 1978)interactions .

DISCUSSION

Summarizing resistance towalnu tcaterpillar ,peca nweevi l and P. devastatrix inpecan :walnu tcaterpilla r susceptibility appears tooccu r among alltree s atsimila r levels sotha twhe n anoutbrea k occurs,defo ­ liation isevenl y spread acrossth e trees;peca nweevi l susceptibility appears tob emitigate dprimaril yb y the capacity ofindividua l treest o synchronize extendedbarre nperiod swit hyear s ofhig hnu tproductio n acrossth etre epopulatio n sotha tentir e areas areproducin gnut s orar e relativelybarre n atth e same time.Additionall y somewithi n tree and between treeescap e ispossibl e inbearin gyear s duet oth enumbe r and manner inwhic h clusters areproduce d andth edistributio n ofonse to fge l stages from tree totree ,respectively ,bu tthi s ismino r compared toth e contributionmastin gmake s toescap e frompeca nweevi l attack; P. devas­ tatrix resistance appears tob eunde rth e control ofmajo r genes andsub ­ jectt obiotyp e development inth einsect .

81 The pecan defense mechanism to each insect discussed appears to be phenotypically and, presumably, genotypically different and only resistance to P. devastatrix would fall within the realm of traditional host plant resistance to insects commonly selected for use in agriculture. Further­ more, this classic kind of resistance has already been shown to be vulner­ able to what appears to be biotype development, and the likelihood of developing a single cultivar which would remain resistant for 100 years or so seems extremely optimistic. In a recent review of host plant resistance of pecan to insects and mites, Carpenter et al. (1979) summarize reports from 1917-1979 on 22 in­ sects and note: 'Partial resistance to the pecan weevil ... was demon­ strated to result from host evasion. No basis for resistance to other pests had been confirmed'. This indigenous plant population has yet to provide us with agriculturally useful resistance to most endemic arthropods associated with it. However, major production of this commodity within its native range still relies primarily on managed natives and as this 'indige- nousness' (Browning, 1974) is lost, greater problems like P. devastatrix can be expected to emerge and better understanding of mechanisms of pecan/ arthropod interaction will, of necessity, result. Close attention to these harbingers of potential devastation may allow better anticipation of future problems and will certainly contribute to a better understanding of plant/insect/agriculture interaction in general.

ACKNOWLEDGEMENTS

Partial assistance for this research was supported by the Science and Education Administration of the U.S. Dept. of Agric. under Grant No. 7800156 from the Competitive Research Grants Office. Support from the sponsors of this workshop is appreciated. Thanks also to Dr. R.L. Gallun and Dr. Clayton Person for discussions on this manuscript. The author assumes responsibility for views taken.

REFERENCES

Baker, H., 1935. Phylloxera devastatrix Pergande on pecans. J. Econ. Entomol. 28: 681-5. Brison, F.R., 1974. Pecan culture. Capital printing. Austin, Tex. 294 p. Browning, J.A., 1974. Relevance of knowledge about natural ecosystems to development of pest management programs for agro-ecosystems. Proc. Am. Phytopath. Soc. 1: 191-9. Calcote, V.R. &. J.L. Bagent, 1974. Phylloxera control sought. Pecan South. 1: 8-9. Carpenter, T.L., W.W. Neel & P.A. Hedin, 1979. A review of host plant resistance of pecan, Carya illinoensis, to insecta and acarina. Bull. Entomol. Soc. Amer. 25: 251-7. Cutler, B.L., 1976. Instar determination and foliage consumption of the walnut cater­ pillar on pecan in Texas. M.S. Thesis. Texas A & MUniv . College Station. Day, P.R. (Ed.), 1977. The genetic basis of epidemics in agriculture. Ann. N.Y. Acad. Sei. Vol. 287. 400 p. Edmunds, G.F., Jr. & D.N. Alstad, 1978. Coevolution in insect herbivores and conifers. Science 199: 941-5.

82 Gallun,R.L .& G.S .Khush ,1980 .Geneti cfactor saffectin gexpressio nan dstabilit yo f resistance.In :Breedin gplant sresistan tt oinsects .Joh nWile yan dSons ,Inc . p.63-85 . Gibson,L.P. ,1968 .Th egenu sCurculi oi nth eUnite dStates .Entomol .Soc .Amer .Misc . Pub. 6:239-85 . Groth,J.V .& CO . Person,1977 .Geneti c interdependence ofhos tan dparasit ei n epidemics.In :P.R .Da y (Ed.):Th egeneti cbasi so fepidemic si nagriculture .NYA S 287:97-106 . Harlan,J.R. , 1977.Source so fgeneti cdefense .In. -P.R .Da y (Ed.):Th egeneti cbasi s ofepidemic si nagriculture .NYA S287 :345-56 . Harlan,J.R . &K.J .Starks ,1980 .Germplas mresource san dneeds .In :F.G .Maxwel l& P.R.Jenning s (Eds): Breedingplant sresistan t toinsects .Joh nWile yan dSons ,Inc . p.253-276 . Harris,M.K. ,1975 .Allopatri cresistance :searchin gfo rsource so finsec tresistanc efo r usei nagriculture .Environ .Entomol .4 :73-85 . Harris,M.K . (Ed.),1980a .Biolog yan dbreedin gfo rresistanc et oarthropod san d pathogenso fcultivate dplants .TAES ,M P1451 .60 5p . Harris,M.K. ,1980b .Arthropod-plan t interactionsrelate dt oagriculture ,emphasizin ghos t plantresistance . In:M.K .Harri s (Ed.):Biolog yan dbreedin gfo rresistanc et o arthropodsan dpathogen so fcultivate dplants .TAES ,M P1451 .60 5p . Harris,M.K. ,1980c .Searchin gfo rhost-plan tresistanc e toarthropod si nagriculture . EPPOBull .10 :349-355 . Harris,M.K .& D.R .Ring ,1979 .Biolog yo fpeca nweevi lfro movipositio n tolarva l emergence.Southwester nEntomol .4 :73-85 . Harris,M.K .& D.R .Ring ,1980 .Adul tpeca nweevi lemergenc e relatedt osoi lmoisture . J.Econ .Entomol .73 :339-343 . Harris,M.K. ,D.R .Ring ,L .Aguirr e& J.A .Jackman ,1981 .Longevit yo fpost-emergen t pecanweevi li nth elaborator yan dfield .Environ .Entomol .10 :201-205 ^ Haseman,L. ,1940 .Th ewalnu tcaterpillar .MO .Agri .Exp .Stn .Bull .418 :1-14 . Hedrick,V.P. ,1919 .Sturtevant' snote so nedibl eplants .N.Y .Agr .Exp .Stn .Stat eo f N.Y. Dept.o fAgr .27t hAnnua lRept .Geneva ,N.Y .2 .68 6p . Hixson,E. ,1941 .Th ewalnu tdatana .Okla .Agri .Exp .Stn .Bull .246 :1-30 . Johnson,C , 1976.Introductio nt onatura lselection .Univ .Par kPress ,Baltimore , MD. 213p . Leppik,E.E. ,1970 .Gen ecenter so fplant sa ssource so fdiseas eresistance .Annu .Rev . Phytopath.8 :324-344 . Lewontin,R.C. ,1974 .Th egeneti cbasi so fevolutionar ychange .Columbi aUniv .Press . 346p . Little,E.L. ,1971 .Atla so fUnite dState strees .Vol .1 .Conifer san dimportan t hardwoods.USD AFor .Serv .Misc .Publ .1146 . MacKenzie,D.R. , 1980.Th eproble mo fvariabl epests .In :F.G .Maxwel l& P.R . Jennings (Eds): Breedingplant sresistan tt oinsects .Joh nWile yan dSon sInc .p .183-213 . McVeigh,I .& C.J .Hobdy ,1952 .Developmen to fresistanc eb yMicrococcu spyogene svar . aureust oantibiotics :morphologica lan dphysiologica lchanges .Amer .J .Bot . 39:352-359 . Maxwell,F.G .& P.R . Jennings (Eds), 1980.Breedin gplant sresistan tt oinsects . JohnWile yan dSons ,Inc .68 3p . Metcalf,CL. ,W.P .Flin t& R.L .Metcalf ,1962 .Destructiv e andusefu linsects . McGraw-HillBoo kCo. ,Ne wYork .108 7p . Moznette,G.F. ,C.B .Nickels ,W.C .Pierce ,T.L .Bissell ,J.B .Demaree ,J.R .Cole , H.E. Parson& J.R .Large ,1940 .Insect san ddisease so fth epeca nan dthei rcontrol . USDAFarmer sBull .1829 .7 0p . Painter,R.H. ,1968 .Insec tresistanc e incro pplants .Univ .o fKansa sPress ,Lawrence . 520p . Person,C , 1959.Gene-for-gen e relationshipsi nhost :parasit e systems.Can .J .Botan y 37:1101-1130 . Person,C. ,1966 .Geneti cpolymorphis m inparasiti c systems.Natur e212 :266-267 . Person,CO . &G.M.E .Mayo ,1974 .Geneti c limitationso nmodel so fspecifi c interactions betweena hos tan dit sparasite .Can .J .Bot .52 :1339-1347 . Robinson,R.A. ,1980 .Th epathosyste mconcept .In :F.G .Maxwel l& P.R . Jennings (Eds): Breedingplant sresistan tt oinsects .Joh nWile yan dSons ,Inc . Russell,G.E. ,1978 .Plan tbreedin gfo rpes tan ddiseas eresistance .Butterwort hInc . Boston,Mass .48 5p . Wood,R.K.S .& A .Granit i (Eds), 1976.Specificit y inplan tdiseases .Plenu mPress , N.Y. 354p .

83 Ongene s for disease resistance in plants

R.R. Nelson

EvanPug hProfessor ,Departmen to fPlan tPathology ,Th ePennsylvani aStat eUniversity , UniversityPark ,Pennsylvani a16802 ,US A

ABSTRACT

Balancebetwee nhos t andparasit ema y have come abouti na process ofco-evolution , inwhic hman y genes forresistanc e and virulence respectively accumulated. Thedistinctio nbetwee n major andmino r genes isartificial :whethe r agen e acts 'major'o r 'minor'depend s onth e geneticbackground . Iti s showntha tresistanc e genes thathav ebee novercom eb y the pathogen canstil lb eusefu l forth eplan t and itsbreeder .

INTRODUCTION

TheOrganizin g Committee forthi sWorksho pha s askedm et oprovid ea summarypresentatio n aboutth ecurren tthinkin g inth e areao fhost - parasite interactions andgenetic so nth e individualplan tlevel .Th e Committeeviewe dm ypresentatio n ason etha twoul dhel p tose tth e stage foryou r laterdiscussion s onbreedin g forbalanc e tocertai npest s of foresttrees .Althoug h Ia m indeedhonore d topla y anactiv erol e inthi s Workshop, Imus tconfes s that Iapproac hm y taskwit hconsiderabl e'trepida ­ tion.A s amatte r of fact,i tha sbecom e extremely difficult inrecen t years to speako rwrit e about;gene s fordiseas e resistance inplants .Ther e isa nenormou s amounto fconfusio n anddisagreemen t aboutterminolog y and concepts.Ther e isa stron gundercurren to fdogm agenerate db ymany ,in ­ cludingmyself .W e relyo ndogm abecaus e thedogmati c approach requires few, ifany , facts;an dw ehav egenerate d remarkably few facts.W ehea rs o much aboutwea k genes and strong genes,majo r genes andmino r genes,dura ­ ble genes and defunctgenes ,rac e specific genes andrac e non-specific genes,vertica l genes andhorizonta l genes,an d soon .Fo r example,mor e than2 0term shav ebee n or arebein guse d tocharacteriz e a rate-reducing formo fdiseas e resistance e.g.horizontal ,partial ,durable ,non-specific , generalized,unifor m tomentio nbu t afew . Itseem st om etha tal lo fth e terms areessentially , ifno tabsolutely , synonymous.T omak ematter s worse, the listseem s togro wrathe rtha ndiminish .W egenerat e theory

84 after theory,bu trarel y conduct appropriate or adequate research totes t theirvalidity .Th eultimat ehighligh t ofth epresen t state ofconfusion , ifno tconflict , isth e saddening facttha tw e can'teve n agreet o dis­ agree.Thi s rather sobering state of affairwil l notdete rm e fromat ­ tempting tosummariz e thecurren tthinkin g ongene s fordiseas e resistance inplants . Iappreciat e thatsom eo fm y remarksma yhav en odirec trele ­ vancet othos eo fyo uwh o serve askeeper s ofth etrees .I fm y chapter whets even asingl e scientific appetite,m y effortswil l havebee nworth ­ while. Plant scientistshav e found itconvenien t toarbitraril y characterize disease resistance genes into 2majo r categories eventhoug hw e acknowledge thatresistanc e and susceptibility aremerel y extremes of abroa d continuum ofhost-parasit e interactions.Th edifferenc ebetwee n ahypersensitiv e reaction and asmal lnecroti c lesion isa relativ e indication ofhos t response toa parasite .Th e differencebetwee n asmal lnecroti c lesionan d a somewhat larger one is justa srelative .Differen t scientists classify the samehos trespons e asbein g aresistan t or asusceptibl e reaction dependingupo nthei rpersonal ,i fno tarbitrary ,opinio no fwha ti sresis ­ tance andwha t is susceptibility. Atan yrate ,on ecategor y ofresistanc e genesembrace s those genes that are said toenabl e thehos t torestric tth e successful establishment of infection sites and, thus, inhibitth e infectionprocess .Fro m the stand­ pointo fpopulatio n dynamics,thes e genes aresai d tob e extremelyeffec ­ tive againstcertai n races,bu ttotall y ineffective againstothe rraces . Placed ina nepidemiologica l framework, theserace-specifi c genes aresai d toaffec tth eexten to fdiseas e onsetb y restricting the amounto feffec ­ tive initial inoculum.Race s lackingvirulenc e genest oovercom erace - specific resistance genes aredisqualifie d from epidemic encounters.Race - specific genes areconsidere d tob e ofn ovalu ewhe nconfronte d bypathoge n populations withmatchin gvirulenc e genes.Fro m ageneti c standpoint, race-specific resistance isusually ,bu tno tnecessarily , conditioned bya single gene and amajo r gene attha t according tocurren tbelief .Playin g thewor dgame ,resistanc e conferredb ythi scategor y ofgene sha sbee n termedvariabl y ashypersensitivity , race-specific,non-uniform , major gene,vertical , and soon . The other category ofresistanc e genes is said toenabl e thehos tt o resist andretar d the colonization and reproduction ofth eparasit e follow­ ing successful infection.Fro m the standpoint ofpopulatio n dynamics,th e genes are said toretai n some degreeo feffectivenes s regardless ofracia l exposure. Itwoul db enaive ,however ,t o assume orexpec ttha t non-specific resistancewoul db euniforml y effective against all races orsub-popula ­ tionso frace s inligh to fth eknow n ability ofracia lpopulation s to differ invirulenc e andparasiti c fitness. Epidemiologically,non-specifi c resistance israte-reducin g innature ;

85 itaffect s the rate ofdiseas e increase (measured inon ewa yb y theappar ­ entinfectio n rate)and , thus, thetermina l amounto fdisease .Reductio ni n the amount and rate ofdiseas e increase isaccomplishe d byhos tmechanism s thatrestric tth enumbe r of successful infections,exten d the latentperio d (thetim e required from infectiont oth e subsequentproductio n ofinocu ­ lum), restrictth e amounto ftissu etha ti scolonize d from asingl einfec ­ tionsite ,an dreduc eth e amountan dduratio no finoculu mproductio no r sporulation. From ageneti c standpoint,non-specifi c resistance isusually ,bu tno t necessarily, conditioned by several genes andmino r genes attha t according tocurren tacceptance . My case forth e 2kind s ofresistanc eha sbee nmad e ina simplisti c fashionb y design.Naturally , all formso fresistanc ewhic h allow some levelo f sporulation areno trace-non-specific .Race-specifi c resistance doesoccu r inwhic h sporulation isno tinhibite d entirely. Ina simila r vein,hypersensitiv e infection sites areknow nt o increase insiz e and sporulate onmor ematur eplant swhe nth ehost' s resistancemechanis m ap­ pears toerode .I ti s further evidenttha tpre-infectio n factorspla ya role insom e forms ofrate-reducin g resistance.Thus , rate-reducingresis ­ tance isno t justa resistanc e togrowt h and sporulation;ther e areman y examples inwhic hnon-specifi c resistance canb e characterizedpartiall yb y thepresenc e of fewerlesions .

STABILIZING SELECTION: The saga of weak.and strong genes

Somepopulation s ofplan tparasiti c fungi aremor e fittha nothe r populations. Sucha biologica lprincipl e standswithou t challenge.Wh y some populations are less fittha nother smerit s some discussion. Iti stemptin g tode-fus e thetopi cb y simply statingtha tpopulation s are less fitbe ­ causethe y are less fit;tha tthe y areles s fitbecaus ethe y lackcertai n vitalgene s for fitness.Som e developments inrecen tyear shav eplace d certain complications inoptin g forsuc ha convenien t dismissal ofth e subject. There is aschoo l ofthough ttha tbelieve s thatnatur e tends toplac e someconstraint s onparasiti c populationsmunchin g happily towardsun ­ bridledvirulence .Natur e favorsth emor e rationalpopulation swh o conserve theirenergie s forbette r endeavors;o rs oth eargumen tgoes . The same school ofthough tconcede stha tnatur eha sn ocontrol ,befor eth efact , overpopulation s accumulatingmor evirulenc e genes than arenecessar y for survival and reproductive success.Wha tnatur e does, the schoolcontends , isse et o ittha tthe y areles s fitt o succeed thanthei r lessprecociou s relatives.Thus ,th ederivatio n ofth eVanderplankia naxio m thatpopula ­ tionswit hunnecessar y genes forvirulenc e are less fitt osurvive . There are atleas t2 theorie s regarding theultimat e fateo funnecess -

86 ary geneso f anykind .On e theorypresume s thatgene s that areno t called upont operfor m ina vita l functionwil l dropou to fth epopulatio n or remain atbes t inver y low frequencies.Th e other theory,whic h Ihav ecom e tosupport ,contend s that the issue isno twhethe r agen e isunnecessary , butrathe rwhethe r itspresenc e ina genom e is adetrimen t toth epopula ­ tioncarryin git . Thematte r ofwhethe r orno tpopulation s carryingunnecessar y genes for virulence are less fitt o succeed hasbecom e somethingmor e thana mer e exercise inintellectua l jousting. Itha sbecom e acontroversia l issue in plantpathology ,particularl y among thosewh o areconcerne dwit hplan t disease controlb ymean s ofdiseas e resistance.Th e ideatha tpopulation s with afe w (ifany )virulenc e genes aremor e fitt o succeed onhost swit h few (ifany )resistanc e genes than arepopulation swit hunnecessar yviru ­ lencegene s (incontex twit h thesam ehosts )ha spenetrate d ourthinkin g on thepotentia l value ofgen edeploymen t ando nth egeneti c composition of multilines.Muc hha sbee n said andwritten ,pr o andcon ,abou tth ehypoth ­ esis ofstabilizin g selection. Some,includin gmyself ,hav e givenu p all hopeo ftestin g thehypothesis ,sinc e thebuilt-i n escapehatche spreclud e anyvali d test.Nonetheless ,th e ideao fstabilizin g selection isuse db y somewh oreflec to npopulatio n dynamics andparasiti c fitness. Itha sbee n theorizedtha twithi n allparasiti c species there arepopulation s ofsupe r races thatcarr yvirtuall y everyvirulenc e gene the speciesha s evermanu ­ factured. Itlie s there innatur e asa slumberin g giant inminut e frequency because its gamuto fvirulenc e genesha srendere d itles s fitt o succeedo n hostpopulation s that aren omatc h fori to n agene-for-gen ebasis .Ther e arethos ewh obeliev e thata substantia l portion of amultilin emus tcon ­ sisto fknow n susceptible tissue sotha t stabilizing selection canprotec t the resto fth emultilin e fromth eravage s of acomple x race.Th epro ­ ponents ofdeployin g different resistance genes indifferen tbu t contiguous regionsbeliev e thatcomple x raceswil lb e stabilized againstan dwoul db e unable tocontinu e their travels to thenex tregion . Letu s suppose, forth emoment ,tha tther e aregene s that functiont o induce disease andtha tther e areothe rgene s thatconditio n overall fit­ ness. Iti sreall y not anunreasonabl e tenet;i n factther e is some evi­ dence for it.Le tu s further suppose that atleas t some ofthes e different kinds ofgene s are inherited independently ofon e another.Again ,no ta n unreasonable tenet;i n factther e issom e evidence for it.I fthes esuppo ­ sitions aretrue ,a s Ibeliev e them tobe ,the n onemigh texpec tt o find populations with fewgene s forvirulenc e thatar e fitan dothe r suchpopu ­ lations that areunfi tt o succeed. Onemigh tals o expect to findpopula ­ tionswit hman yvirulenc e genes,som eo rman y ofwhic hma yb e unnecessary byou r criteria,tha t are fitan dothe r suchpopulation s that areunfi tt o succeed. There isn oreason ,t om ywa y ofthinking ,wh y allo fthes ecombi ­ nations ofvirulenc e and fitnessgene s should notexis t innatura lpopula -

87 tions atsom epoin ti ntime .I fthi s istrue ,a s Ibeliev e itis ,the n Ia m forcedt orejec tth eequatio ntha tsimplicit y equals fitness andretur nt o thetene ttha t fitpopulation s are fitbecaus e they arefit ,pathogeni c simplicity aside.

NON-SPECIFIC RESISTANCE AND GENETIC EQUILIBRIUM: The natural way of life

Race-nonspecific resistance,whe nviewe d asa rate-reducin gresistance , appears tob e conditioned by several toman ygene s inmos tinstances . Intensityusuall y isa quantitiv e trait.A quantitive traiti sconditione d byman ygene sbecaus e 1gen ecanno taccomplis h thetask .Eac ho fth egene s conditioning aquantitativ e traitcontribute s something toa collectiv e venture thatnon e can accomplish alone.Removin g asingl egen e froma many-gene system should notdrasticall y alter theexpressio no fth etrait . Towha tdegre e thetrai tma yb e alteredwoul d depend onth e relativeim ­ portance ofth egen e inth e collectiveventur e and thetota lnumbe r of genes controlling thetrait .Greate r fluctuations inth e reactiono f ahos t todifferen t races should occur asmor e andmor e genes areremoved .A s all but1 gen e areremoved , thehos tprobabl ywoul d reactquit e differently thani tha dbefor et oth esam eraces . Itwoul d react so'differently , in fact, ast operhap s simulate arace-specifi c reactiont oth eraces ,i.e. , resistant to some and susceptible toothers . A reverse ofth e sequence ofevent s justdescribe dwould , as Ise eit , represent the evolution ofgene s fordiseas e resistance inplants .Co - epicenters, geographic areas inwhic hbot hhos t andparasit ehav eco - evolved,mos t accurately depictth e story ofth e evolution ofgene s for virulence and resistance.Th e story is anincredibl eDarwinia nclassic . Solanum demissum Lindl. andth e latebligh t fungus inMexico , Tripsacum and teosinte andth enorther n leafbligh tfungu s inMexico ,an dth eol dlan d raceso fric ean dth eric eblas tfungu svividl y depictth eclassi c sagao f theco-evolutio n ofhos tan dparasite .Th erelationshi p todaybetwee neac h ofthes ehost-parasit e combinations isno t sharply antagonistic.Eac ho f thehost s sustainsmodes tlevel s ofdisease ,bu tdiseas e iso fn omajo r concernt oth ehos tinsofa r as itsultimat e reproductive success andper ­ sistent survival areconcerned .Bot hhos tan dparasit e had learned thatth e price ofco-existenc ewa spreferabl e toth e alternating thrill ofvictor y andth e agonies ofdefeat .A relaxation ofselectio npressur e forbot h specieshighlighte d theirne w relationship.Neithe rwa s or istoda y in serious jeopardy ofextinction .The yha dbecome , ina sense ,th e 'odd couple'o fth ebiologica lworld . Philosophical orteleologica l asthes e speculations may seem,the y would seemt orepresen t the scientific explanation embodied inth eco - evolution andco-existenc e ofhos t andparasit e innatura l ecological niches.W e canretur nt oth ebeginnin g ofthei runiqu e affairt o learnho w

88 they arrived atthei r currentstatu s ofequilibrat e co-existence.Th e initial resistance of ahos tt o a 'new'parasit eprobabl ywa s accomplished by ageneti c change ata singl e locus.Plant swit h thatresistanc e gene probably reacted toth eparasit e ina hypersensitiv e manner;probabl y like amodern-da y cultivar whose resistance isconditione d by asingl e gene.I t isunconceivabl e tome.tha tthe y reached equilibrium atth ever y beginning ofthei rco-evolution . Subsequently, newpopulation s ofth eparasit e evolved and strainspathogeni c orvirulen tt oth ethe n resistanthos twer e selected.Th ehost ,i nturn ,generate d ane w source ofeffectiv eresis ­ tance; and soon .Suc hmus thav ebee nth ecase ,sinc ebot hhos t andpara ­ site stillexist . Theproces s ofco-evolutio nproceeded , perhaps stepwise ina gene-for - genemanne r orperhap s inanothe rway ;i treall y doesn'tmatter .Hos t genotypeswit h fewer genes forresistanc e andpathoge n genotypeswit h fewer genes forvirulenc eprobabl ywer e selected against andeithe r dropped out ofthei r respective populations orremaine d inlo w frequency.Ther e canb e little doubttha tth emos t fitt opersis twoul d dominatebot hpopulations . At any rate,i ti sdifficul t form e toimagin e that allgenotype s that existedthroughou t theproces s ofco-evolutio n remain apar to fth ecurren t populations ofhos t andparasite . How longth eproces s ofco-evolutio n has occurred andwha t stageo f co-evolution currently exists areno t atal l important.B y thetim eth e host andparasit e had reached astag e ofacceptabl e co-existence,eac hha d accumulated asubstantia l number ofresistanc e genes orvirulenc egenes . The facttha tthe yno w exist inrelativ e harmony indicates thatth e last resistance gene incorporated into thehos tgenom e didno tconfe r amassiv e orhypersensitiv e response againstth epathogen .No r did the lastvirulenc e genepos e aseriou s threatt oth ehost .Ther ewa s and isn o immediate selectionpressur e oneithe r adversary.Eac hha d found amutua l safety in numbers ofgene s forresistanc e orvirulence .Gene s thatonc e functioned separatelywit h only temporary success inth eearlie r stages ofco - evolution now function collectively with amor epermanen tsuccess . Ifth e assumption iscorrec ttha tth e longproces s of co-evolution resulted inth eultimat e accumulation ofman y resistance andvirulenc e genes inth ehos t andparasite ,geneti cprobabilitie s suggesttha tsubse ­ quentchange s inth e future course ofthei r co-evolutionwil lb e largely subtle ones.A t leasti tseem sunlikel y thateithe r opponentwil l enjoyth e massive superiority exhibited inth e earlier stages ofthei rco-evolution .

SOME THOUGHTS ON GENES FOR DISEASE RESISTANCE

Manha sno tonl y found itconvenien t tocharacteriz e disease resistance into2 majo r types,bu th eha s also found itconvenien t toconclud e that the2 type s ofdiseas e resistance areconditione d by different kinds of

89 L genes.Th e implications, forexample ,i nth eterm s 'majorgenes ' and 'minor genes' arereasonabl y obvious. Ifmajo r gene resistance isth e samea s race-specific resistance andmino r gene resistance isthe -sam e asnon ­ specific resistance,th e2 differen tkind s ofresistanc emus tb econdi ­ tionedb y differentgenes ;o rs oth e argument goes. Several years ago, Ipropose d theconcep ttha tgene s for race-specific andnon-specifi c resistance areth e same genes.Th e conceptsinc eha sbee n developed ingreate r detail.Th e2 majo r kindso fdiseas e resistance,m y argumentcontended , areno tindication s ofth e actiono fdifferen tgenes , butrathe r areexpression s ofdifferen t actions ofth e samegene s indif ­ ferentgeneti cbackground s orwhe n facedwit h differentgenotype s ofth e parasite.Ther e are,i n fact,n omajo r genes andmino r genes.Ther e are only genes fordiseas e resistance.Certainly , different genesma y confer varying levels ofexpressio no reffectivenes s and theexpressio no fresis ­ tance genesma yb e influenced ormodifie db yothe r resistancegene si n different geneticbackgrounds ;backgroun d isth eke yt o geneaction . The implication ofth e concept istha tw e recognize and characterize genes asbein g 'major'whe nthe y are separate andrace-specifi c andcharac ­ terize the same genes as 'minor'whe nth ehos trespons e suggests anon ­ specific orrate-reducin g typeo fresistance . I againsubmi ttha tgene s functionon ewa ywhe nthe y areseparat e and anotherwa ywhe nthe y aretogether .Again ,th e Solanum demissum—potato latebligh t storyha s arelevance .Niederhause r stated, 'InMexic ow ewer e forcedt oconcentrat e onth emultigeni c fieldresistanc ewhe n itwa s found thatn otuber-bearin g Solanum specieswa s immune orhypersensitiv e toth e pathogenwhe nexpose d inth e field'.H e also stated, 'Field resistance is characterized by slow-spreading lesions inwhic h sporulation issparse .Th e lesionspe rplan t are fewer,an dten d tob e onolder ,lowe rleaves' . Niederhauser apparentlywa s discussing aresistanc e identical towha ti s now considered tob e arate-reducin g type ofresistance . Man attempted tomak eus eo fth e impressive resistance demonstrated by S. demissum to amultitud e ofrace s of Phutophthora infestans (Montagne)d e Bary inth eToluc aValle y ofMexico .Althoug h S. demissum displayed a rate-reducing resistance,gene swer e extracted singly from the speciesan d a race-specific orhypersensitiv e resistancewa s theresult .Thus , the birth ofth eR-gene s whichwer e considered major genesbecaus ema nha sbee n scientifically andphilosophicall y schooled toreac twit hmajo r favort o genes eliciting sucha dramati chos tresponse . Iti sdifficul t form et o comprehend howma n canextrac t amajo r gene from ahos tgenotyp e expressing minor generesistance . The firstR-gene ,R- ., soo n fellpre yt o arac eo f P. infestans andma n wentbac kt o S. demissum andextracte d anotherR-gene ,R 2.Tha tgen ebrok e down andma nwen tbac k foranother .An d soon .On e gene couldno t contain thehighl yvariabl e latebligh t fungus.I fma nha dwante dhi s cultivated

90 potato, S. tuberosum L., tobehav e like S. demissum against P. infestans, he should have taken allo fth egene stha tprotecte d S. demissum andpu t thembac k together again.Majo r geneshav ebee nobtaine d fromwil d land varieties ofric etha texhibi tgeneralize d or slow-blastingtraits . One final comment. Ifa race-specifi c gene isineffectiv e againsta race, it isassume d nott o function atal l inresistin g thatrace .Simi ­ larly, if5 race-specifi c genes areal l ineffective against arace ,the y are allpresume d tob e functionless againstth erace . Itseem smor ereason ­ ablet om etha t5 singularl y ineffective genesca nb e collectively func­ tional and effective. Ifeac hcontribute s something inth ewa y ofresistin g theparasit e atsom epoin t after infection,th enet ,collectiv e resultlog ­ ically shouldb e acollectiv e resistance togrowt h and/orreproduction .

SOME THOUGHTS ON 'DEFEATED' RESISTANCE GENES

Theperiodi c arrival ofpathoge npopulation s with avirulenc e genet o overcome single,race-specifi c resistance genesha s sentman y suchgene st o thebiologica l cemetery.The y restther ewit h fullhonor shavin g donethei r task gallantly ifno t indefinitely. Itseem s implicittha tth etendenc y to bury defeated resistance genes isdon ewit hth epresumptio n thatthe yn o longer areo f anyvalu e atall ;wh y elsewoul d theyb eburied . My colleagues and Ihav ebee nworkin gwit h thewinte rwhea t -powder y mildew system toevaluat e themerit s anddemerit s ofcertai ngeneti c strat­ egies tomanag eplan tdisease s atsom e acceptable level.Havin g access to thenear-isogeni c lines developedb yBriggl e ina Chancello r background, eachcontainin g asingle ,differen tpowder y mildew (Pm)resistanc e gene,w e havebre d 2 four-genepyramid s totes tth etheor y thatth e additive effect ofsevera l defeated resistance geneswoul d create anequilibriu m witha pathogen genotypepossessin g allth evirulenc e genesneede d tomatc h allo f the resistance genes inth epyramid .Th eunbounded , ifno tbiased , faithi n thevalidit y ofm ypyrami d theoryprompte d ust oassum etha tth epyramide d resistance geneswoul d create anequilibrate , slow-mildewing relationship withth epathogen . Ifsuc hwer e thecase ,i twoul db enecessar y to evaluate eachresistanc e gene and allpossibl e two-gene combinations to ascertain whateac h genema yb e contributing, ifanything , toth e successful collec­ tiveventure .O nth e surface,i tma y seemnaiv et oevaluat e single,de ­ feated genes since iti s assumed thatthe y areo fn ovalu ewhe n oncede ­ feated.Nonetheless , 5near-isogeni c lineswit h differentP m geneswer e compared toth e susceptible recurrentparent ,Chancellor , forthei rreac ­ tiont o apathoge n isolatepossessin g all ofth evirulenc e genesneede dt o overcome the 5P m resistance genes.Thre e ofth e 5isogeni c lines,contain ­ inggene sPm3c ,Pm4 ,o r agen e identified asMichiga nAmber ,exhibite d a dramatic residual effectexpresse d asreduce d numbero f lesions andre ­ duced sporulationwhe ncompare d toChancellor .Thes edefeate d geneswer e

91 L nottotall y overcome ason emigh thav e assumed. Why did such adiscover y remainundetecte d fors o long?Defeate d genes arediagnose d asbein g defeated based on aqualitativ e reaction. Ifa susceptible typereactio n isobserve d ona genotype ,i ti s assumed thata genotype is susceptible.Th epresume d allo rnothin g syndrome associated withrace-specifi c genes dictates thatsuc hgene scanno thav e aninter ­ mediate reactiono rsom edegre e ofresidua leffectiveness . Theresidua l effectso fdefeate d resistancegene sprovid e abeautifu l and solidexplanatio n to accountfo rth e facttha tdefeate d geneswer e retained inwil dpopulations ,eve nafte rthei rdefea ta tsom eearl ypoin t inth eco-evolutio n ofhos tan dparasite .The ywer eretaine dbecaus e they contribute something ofvalue .

THE EFFECTS OF GENE DOSAGE ON THE RESIDUAL EFFECTS OF DEFEATED RESISTANCE GENES

TheF- .o fth e crossesbetwee n eacho fth eP m isolines andth e recurrent parentChancello rwer eheterozygou s foreac ho fth eP m resistancegenes . The availability oftha tF , seedpermitte d ust o assess theeffec to fgen e dosage onth eresidua l effects ofcertai n defeatedpowder ymilde wresis ­ tance genes.Three-wa y comparisons amongChancellor ,th ehomozygou s isoline andth eheterozygou s F..line sreveale d thatth e lines heterozygous forP m genes 3c,4 ,o rMichiga nAmbe r displayed approximately half ofth e residuality demonstratedb yth e isolineshomozygou s forthos egenes . Vanderplank states thatrace-specific ,vertica l resistance genes functionagains tepidemi c developmento fplan tdisease sb y reducingth e amounto feffectiv e initial inoculum (X )availabl e fordiseas eonset . Vanderplank further statestha tvertica l resistance geneshav en o influence ondiseas e increaseb y raceswit hvirulenc e genest omatc hthem .Mathe ­ matical analysis andmodelin go fplan tdiseas e epidemics areconducte d withthos e assumptions ofth erol eo fvertica l resistance genes.Th e dramatic effecto ndiseas eefficienc y andsporulatio nb y certainvertica l Pmgene s againstrace swit hvirulenc egene s toovercom e them clearly demonstrates thatrace-specifi c resistance alsoma yhav e aneffec to n disease increase and canindee d influence the apparent infection rate(r) , a traitheretofor e attributed solely togene s forhorizonta l resistance. Any researcherworkin gwit h thegeneti c lines ofwinte rwhea tuse d in this study,bu tno tknowin gtha tth e lineswer e isogenic for so-called 'major'P m genes,woul d haveconclude d quite logically thatth e lines carrying genesPm3c ,Pm4 ,o rM Apossesse d some levelo frate-reducin g or rate-limiting resistance.An dyet ,rate-reducin g resistance,wit h rare exception, isconsidere d tob eunde r thecontro l ofso-calle d 'minor' genes orpolygenes .Th epresen tresearc hdemonstrate s thata gen ema y perform ina qualitativ e or aquantitativ e manner depending uponth e

92 genotypeo fth epathoge ntha tconfront sit .

CONCLUDING REMARKS

As keepers ofth etrees ,yo uhav epondere d thequestio n ofbreedin g for balance.Ther e isn o simple,universa l answer toth e question.Breedin g for balance orequilibriu m isessentiall y acompromis ebetwee n feastan d fam­ ine.Ther e isn onee dt ocompromisin g tothos eparasite swhic hca nb esuc - cesfully containedb y imbalance.N o one,fo rexample ,woul d suggesttha t aworksho pb e convened todiscus sbreedin g forbalanc ewit h aparasit e for which asingl eresistanc e geneha s successfully controlled itou to fba ­ lance for6 5years .M ybes t advice onbreedin g forbalanc ewoul db e tod o so ifbreedin g for imbalance doesn'twork .M y treatise ends ontha tpro ­ foundthought .

93 Non-specific host-tree processes occurring in bark in response to damage and their role in defense

George S. Puritch & Garry D. Jensen

Environment Canada Pacific Forest Research Centre, 506 W. Burnside Rd., Victoria, B.C. Canada

ABSTRACT

Investigations ofhost-pathoge n interactions andwoun d healing inconife rbar kb y the lateDr .D.B . Mullick led toth e concepto f2 distinc tcategorie s ofperiderm . Periderms inth e exophylactic category function asth enorma l outer covering of stems andbranches ,protectin g living tissues fromth e external environment.Thos e inth enecrophylacti c category servea s barriersbetwee nnecroti c and living tissues'.Th euniqu easso ­ ciation ofnecrophylacti c periderm with successful healing followingbar knecrosis ,regardles s ofth enatur e ofth eexter ­ nalcasua l agent,indicate s that acommo nhos tproces s occurs inrespons et o diverse stimuli.Th e sameproces smus t also occur endogenously atabscissio n sites and inrhytidom eforma ­ tion.Thi s common,endogenou sproces s calledphelloge nrestora ­ tion, istriggere db y the loss ofa functiona l phellogen,an d isviewe d as amajo r non-specific componento fhos tdefense . Thenon-specifi c healingproces s can serve as abasi s for unravelling the complexities ofhos tpathoge ninteractions .

Oneo fth emajo r obstacles togeneti c selection oftree s resistanto r tolerantt opathogen s isth emeage rknowledg e ofth ehos trespons et o pathogen attack. Invasion ofa pathoge n throughbar knormall y triggersa sequenceo fcomple xphysiological ,chemica l and anatomical eventswithi n the living cells ofth e tree.Thes eevent svar y inbot htim e and spacean d areinfluence d by the specificpathoge n andhost ,a swel l asb y numerous other factors includingmetaboli c condition ofbot h organisms,environmen ­ talcondition s atth etim eo f attack,tim e ofyea r andnutrition . Identifi­ cationo fthi smyria d ofevent s and clarification ofwhic hevent s areth e cause oreffec to fresistanc e toth epathoge n isa tas ktha t ismonumenta l

1.Pathoge ni sdefine da san yagen tmicrobe ,insect ,parasiti cplan twhic hcause schroni c physiologicaldisorder s(pathogenesis )i nth ehos t(Treshow ,1970) .

94 butessentia l forth eunambiguou s selection ofgeneticall y resistant stock. During the course ofresearc h atthi s laboratory, investigationsb y the lateDr .D.B . Mullickprovide dne w insights into thenon-specifi c healing reactions oftree s andprovide d avaluabl e foundation for interpreting host-pathogen interactions.Thi spape rpresent s anovervie w ofMullick 1s work and itscontributio n toth e study ofhost-pathoge nrelationships . Mullick's investigations beganwit h anattemp tt ochemicall y categorize Abies spp.susceptibl e andresistan tt o attackb y Adelges piceae (Ratz.), thebalsa mwooll y aphid (bwa). This 'aphid' isa tiny ,parthenogeni c insect that feedsb y inserting its stylet into livingbar k cells and extracting their contents (Balch, 1952). During thecours e of its feeding, theaphi d secretes salivawhic hcontain svariou scompound s including an auxin-like component (Puritch,unpublishe d data). As achemica lmarke r the reddish purplepigment s thatoccurre d inth ephelle m ofperider m around healed bwa feeding siteswer e selected (Fig.1) .Utilizin g newchromatographi c tech­ niques (Mullick,1969a ;1969b )i twa s discovered thatth epigment swer ea new classo fnon-anthocyani c compounds thatoccurre d asmixture s of several distinctpigment s (Mullick, 1969c). Further investigation revealed that similarpigment s occurred ata variet y ofothe r sites inth ebcir ksuc ha s deadresi nblisters ,lea fabscissio nzones ,heale d injuries and areaso f oldpathoge n attack (Mullick& Jensen , 1973b). Inthes e sites,pigment s always occurred inth ephelle m ofth e sequentperiderm . Thus, although the pigmentswer eno tuniqu e tobw a attack,the y did appear tob e associated with thehealin gproces s ofth etre e andparticularly ,periderm .Conse ­ quently attentionwa s focussed onth eperiderm s and their role inwoun d healing. Literature (Fahn,1967 ;Esau ,1977 )identifie s 2basi c typeso f 'na­ tural'perider m ingymnosperms :primary ,whic h replaces theepidermi s and secondary or sequent,whic h occurs indeepe rbar ktissue s and replacesth e primary.Thes e 2periderm s wereconsidere d todiffe ronl y inthei rtim eo f origin. Inaddition , itwa s recognized that anotherperiderm , woundo r pathological periderm, forms inrespons e tobioti c or abiotic injury. In many cases,woun dperiderm s havebee n associated with specific interactions withpathogen s (Hare,1966 ;Akai , 1959;Carter , 1962). Rhytidome,th edea d barktissue s external toth e last formedperiderm , (Srivastava, 1964)wa s suggested tob e causedb y impermeability ofth e suberizedperiderm s (Esau, 1977). Theperiderm swer e reported toaris eb ymeristemati c activity ofa layero fbar k cortical cells,th ephelloge n (corkcambium )whic hproduce d phellem (cork)toward s theoute rbar k surface andphelloder mtoward s the vascular cambium (Fig.2) .Th eperiderm swer e suggested topla y onlya passive role indefens ebecaus e theywer e distant from the siteo finterac ­ tionwit hth epes t andwer eofte nabsen t atth e timeo fpathoge ninactiva - tiono rdeliminatio n (Wood, 1967). Mullick (1971)foun dtha t adistinc tdifferenc e occurred inth epigmen -

95 m

«r m * % <è #

PERIDERMS

NECROPHYLACTfC (NP) EXOPHYLACTIC

USUAL SEQUENT PERIDERM FIRST (FEP) WOUND PERIDERM SEQUENT (SEP) PATHOLOGICAL PERIDERM ABSCISSION SCARS N*#*• ** * »

96 Figure 1. Cross section ofbalsa mwooll y aphid induced gout in Abies amabilis. Dark areaswithi n the swollenbar k tissue areth e reddish-purple feeding sites (fs).Severa l ofthes ehav ebee n isolated onth erigh t (x3) .

Figure 2. Structure ofperiderms .Th ecor kcambiu m (phellogen)produce s the cork (phellem)externall y towards theoute rbar k surface andphelloder m towards thevascula r cambium.Dea dphelle m cellsbecom e distorted duet o growthstresses .

Figure 3. The2 categorie s ofperiderm s inwood yplants .Th e necrophylactic category includesusua l sequent,wound ,pathologica l and abscission scar periderm, while theexophylacti c category includes the firstperider m and canoccu r as asequen tperiderm . (FEP= firstexophylacti c periderm,NI T= nonsuberize d impervious tissue,N P =necrophylacti c periderm, SEP =se ­ quentexophylacti c periderm, VC =vascula r cambium).

Figure 4. Cross sectiono fa nare ao f Abies grandis bark3 0day safte r mechanical wounding. Thebar k samplewa s F-F tested (seetext )throug hth e cambial surface.Th e dye failed topenetrat e theNI T and necrophylactic periderm (x 6).

Figure 5-8. Cryofixed sectionsphotomicrographe d using Carl Zeissfluor ­ escence exciterbarrie r filter combination 1/53. Figure 5.Radia l sectionthroug h theboundar y (arrows)o f aformin g rhytidome in T. heterophylla. Thecortica l and outer secondaryphloe m parenchyma arehypertrophie d below aregio nwher e some ofth ephelloge n cells ofFE Phav e developed fluorescentwalls .Compar e toth e outer tissues onth erigh t ofth e figure (x20) .

Figure 6. Radial section through thecentra l regiono f a1 4da yol dmechan ­ icalinjur y to Abies grandis. Theoute rnecroti c tissue (nt)ha scollapsed . A zone ofhypertrophie d cells internally abutsth enecroti c tissue.Th e innermost ofth ehypertrophie d cellshav e developed strongly fluorescent walls characteristic ofNIT .Th e zone internal tothes e cells,whic hha s little fluorescence, isth esit e ofN Pphelloge n formation (x20) .

Figure 7. Radial sectiono fmor e advancedhealin gtha ni nFig . 6.NI T formationha sbee ncomplete d includingmodification sproducin g fluorescence insiev e cellwall s (arrows).A non-fluorescen tphelloge n zone ispresent , producing phellem centrifugally andphelloder m centripetally. The outermost layer ofphelle m has developedwal l fluorescence (x20) . Figure 8. Late stageo fN P formation atrhytidom e in T. heterophylla. Tiers ofmaturin gphelle m with fluorescentwall s areclearl y seen (arrows). The outermostphelle m cells arebecomin g compressed andthei rcontent s are developing thetypica l reddishpurpl epigment .Th enecroti c tissuesexter ­ nal toN P have desiccated toth epoin twher e intactcryosta t sections could notb eobtaine d (x20) .

97 tationo fth eprimar y and sequentperiderm s in4 0differen tconife r spe­ cies.A technique called cryofixation was developedwhereb y thebar kwa s fixedb y freezing, sectioned ona cryosta t and observedwhil e frozenunde r amicroscop e using fluorescence andothe rmode s ofilluminatio n (Mullick, 1971). This cryofixation techniquepermitte d theobservatio n ofth epig ­ ments inthei rnatura l state.Detaile d analyseso f3 conife r species showed thatth eprimar yperider m was ofon etype ,wit h asoli dbrow nphelle m contentwherea s the sequentperider m consisted of2 differen t types.Th e mostcommo ntyp eo fsequen tperider mwa s reddishpurpl e incolo rwit h fluid contents init sphelle m while theothe rwa s identical inpigmentatio n and characteristics toth eprimar y periderm. Onth ebasi s of'1 5characteristic s revealedprimaril yb y cryofixation,Mullic k &Jense n (1973a)propose d that thebrow nprimar yperider m andbrow n sequentperider m were essentially the same andtha tth ereddish-purpl e sequentperider m comprised another dis­ tincttyp eo fperiderm .Mullic k& Jense n (1973b)subsequentl y verified this hypothesis and advocated ane wterminolog y andconcept s forperiderm s in woodyplants . The 2brow nperiderm s constituted asingl ecategor y ofperiderm s and wereterme d exophylactic (exo,external ; phylacta, aguard ) (Fig.3) .Thi s exophylactic periderm was considered tob eth enatura l 'skin'o fth etree s providingprotectio n of living tissues fromth e external environment (Mullick& Jensen,1973b).Tw oexophylacti cperiderm swer eproposed . The firstexophylacti cperider m replaced theepidermi s ofth e stem andwa sth e firstperider m formed,whil e the sequent'exophylacti cperider m wasassoci ­ atedwit hexfoliatio n or sloughing ofdea d anddisease d tissues anddevel ­ oped only rarely intree swit h adhering deadbark . Thereddis hpurpl eperider m constituted asecon d category andwa s termed necrophylactic (necrus, acorpse ; phylacta, aguard ) (Fig.3) . Mullick &Jense n (1973b)considere d thatth eprim e functiono fth e , necrophylactic peridermwa sth eprotectio n oflivin gtissu e fromth ead ­ verse effects associated withdeat ho fcells .Wheneve rphelloge n ceasedt o function,regardles s ofth e reason,necrophylacti c periderm would formt o maintainth e integrity ofth eoute r stemcovering .Formatio n ofthi s periderm was thus non-specifically induced, occurring throughwounding , disease,needl e scars,an dphysiologica l disorders allo fwhic hdamag e the phellogen.Damage d anddyin g cellstherefor e ledt operider m formation and notth e opposite as suggested inth e literature (Esau, 1977).Terminolog y inth eliteratur e referringt osequent ,woun d andpathologica l periderms was alsomisleadin g since according toMullick' s results theseperiderm s were allon e andth esame . The concepts ofexophylacti c andnecrophylacti c periderms wereprimar ­ ilybase d ondetaile d studies of3 Pinaceae species.T otes tth e general validity ofthi sconcep ti nrelatio n towood yplants ,So o (1977)observe d 15 species oftree s including representatives from allconife r familiesa s

98 well as afe w families of angiosperms.Withou texception , all species assessed contained only the 2classe s ofperiderms ,exophylacti c and necrophylactic.Also ,a s suggested byWullic k& Jense n (1973b), the necrophylacticperiderm s were always associated withwound s andrhytidom e while theexophylacti c was associated with thenorma l firstperiderm . Havingclarifie d the types and characteristics ofperiderm s intrees , investigations were initiated onth eproces s ofnecrophylacti c periderm formation inrespons e tomechanica l wounding and during rhytidome forma­ tion. Inth e latter case,Mullic k (1977)considere d rhytidome tob ea process ofnecrophylacti c periderm formation ata pathogen-free , 'non- induced' site.Earl y inthes e studies itwa s discovered thatprio rt o periderm formation, animperviou s layer oftissu e formed around theperiph ­ ery ofth ewounde d site (Mullick, 1975). This impervious tissuewa sde ­ tectedb y the failure of aferri c chloride-potassium ferricyanide (F-F) solution topenetrat e the impervious zone (Fig.4) . Histological examination showed theF- F solution stopped ata zon eo f hypertrophied parenchyma cellswhich , incryofixe d section,showe d achar ­ acteristic yellow greenwal l fluorescence. Sieve elements crossing thezon e also showedwal l fluorescence. Inlivin gbar k and inmos to fth enecroti c bark inth e injured area,parenchym a and sieveelement s showed no intrinsic fluorescence underblu e light stimulation.Necrophylacti c periderm occurred intissue s internally abuttingthes ehypertrophie d cells.Histochemica l test forsuberi n showed thewall s ofth ehypertrophie d cells tob enon - suberized and the layerwa s designated non-suberized impervious tissueo r NIT. Examination ofnecrophylacti c periderm atth evariou s siteso foccur ­ rence showed thatNI Twa s invariablypresen t externally abutting the phellem. Inaddition ,developmenta l studies ofmechanica l injuries showed thatNI Tprecede d formation ofphelle m anddevelope d specifically from tissues internally adjacentt oNIT .Thi s led toth e conclusion (Mullick, 1975)tha tNI T formationwa s anon-specifi c process thatprovide d the environment fornecrophylacti c periderm formation andwa s aprerequisit e for its formation. Itwa s suggested thatth eproces s ofNI Tproductio nwa s thephysiologica l basis ofhos trespons e topathogen s inbark . Byexaminin g theproces s ofrhytidom e formation in Tsuga heterophylla (Raf.)Sarg ,an dhealin g followingmechanica l injury in T. heterophylla and Abies spp.,th e stepb y step sequence ofcellula r and morphological changes that lead tonecrophylacti c periderm formationwa s delineated (Mullick, 1977). Initiation ofthi sproces s inrhytidom e formation appeared tob enon-functionin gphelloge n thatwa s distinguishable by its fluorescent walls (Fig.5) .Followin gmechanica l injuries,initiatio nwa s triggered by thephysica l destruction ofphellogen .Existin g cortical and secondary phloem cells adjacentt o the alteredphelloge n orinjure d surface,under ­ went structural andchemica l changes (asindicate db y fluorescentmodifi -

99 cations)becomin g hypertrophiée!(Fig .6) .Thes e cellsthe n dedifferentiated and someo fth e cells developed fluorescentreticulu m althoughn ofluor ­ escencewa s observable inth ecel lwalls. .Thi s reticulum eventually dis­ appeared and thecell s developedvarie d fluorescence including areas inth e cellwal l corners.A zone ofcell snea rth e internalboundar y of thehyper - trophied zone showed sporadic fluorescence inthei rwall s (Fig.7) .Thi s zone developed intoNIT .NI T formation, although encompassing adiversit y ofcel l types,di dno t involve anymeristemati c activity. Onth e cambial sideo fNIT ,th ecell s lostal l fluorescence,becam emeristemati c and developed intophellogen .Th ephelloge n divided,producin gphelle m cells towardsNI Twhic h developed reddishpurpl epigment s andproduce d phelloderm cells towardsth ecambiu m (Fig.8) . Theproces so fnecrophylacti cperider m formationwa sthu s foundt ob e very similar inrhytidom e formation and inmechanicall y induced injuries (Mullick, 1977). Inbot hcase scellula r reactionsoccurre d following damage toth ephelloge n followingwhic hNI Tdeveloped .NIT ,however ,di dno tfor m atth esam erat ethroughou tth eyea r andvarie d from about1 5day sfollow ­ ingmechanica l injuriesmad e inJun e andJul yt oove r 200day s for injuries made inNovembe r (Mullick& Jensen , 1976). Str-esscondition s also adversely affected thisnon-specifi c healingprocess .Drough tstres sdelaye d the formationo fNI T indirec tproportio n toth edegre eo fstres s (Puritch & Mullick, 1975). Thusnecrophylacti c periderm formationwa s adynami cpro ­ cess dependentupo nth ephysiologica l conditiono fth e tree andwa s im­ pairedunde r adverseconditions . Based onthes e findings,Mullic k (1977)presente d ane w concepto fth e process ofhost-pathoge n interaction inth e stemso ftrees .Sinc eth e process ofphelloge n restorationwa s autonomous andnon-specifi c hecon ­ cluded that itshoul dhav e acommo ngeneti cmechanis m inal lwood y plants (Mullick, 1977). Theprimar y step inestablishin g thenatur e ofth ehos t responsewa s the requirement for anunderstandin g ofth e interactiono fth e pathogenwit hth e firstlaye ro flivin g cells,th ephellogen .Th e reactions mightvar y from rapid death ton o response. Ifphelloge n restorationwa s triggered, resistance or susceptibility couldb e determined byho wsuccess ­ fully thehos tcomplete d the formation ofnecrophylacti c periderm while underth e influence ofth epathogen .Fo r instance,resistanc e could result from situationswher e thepathoge nwa sunabl e to successfully preventth e restorative process orwa s inactivatedprio r too rwithou t activating the process. Susceptibility could result from successful interference ofth e pathogenwit h theproces s or failure totrigge r it (Mullick, 1977). Once pathogens initiated theproces s ofphelloge n restoration theywoul db e interactingno tonl ywit h chemicalspresen t inth enorma lbar kbu tals o with ahos to fne w chemicalsproduce d during the cellular dedifferentia- tion. Inthi s regard,Mullic k felttha t sincephytoalexin s were non-specific

100 (Vanderplank, 1975), they were components of the phellogen restorative process, occurring in the dedifferentiating cells surrounding injury. Following this suggestion, Rahe & Arnold (1975) found that the bean phytoalexin, phaseollin, was located entirely in tissues abutting the injured zone. Mullick (1977) subsequently suggested that phaseollin as well as other non-specific compounds in wound healing, e.g. ethylene, cell-wall degrading enzymes, early RNase, phenolics and terpenoids, resulted from the processes of dedifferentiation leading to NIT and necrophylactic periderm formation rather than non-specific defense factors. He concluded that the response to injury was not one of defense but rather a response to the need for phellogen renewal. Mullick (1977) considered that defense, in the strict sense was only a fortuitous result of the restorative process. It was only through the understanding of this non-specific process that a true understanding of host-pathogen interactions could be obtained and the components of specific interactions isolated and identified. Mullick's research has provided major contributions to basic bark anatomy, periderm physiology and wound healing. Besides the phellogen restorative process described here he also discovered 2 other non-specific healing processes viz. vascular cambium restoration and sapwood tilockage (Mullick, 1977). These findings have given a solid foundation to the inves­ tigations of host-pathogen relationships and are a major contribution towards identifying the host component and genetic base of host-pathogen interactions in trees.

REFERENCES

Akai, S., 1959. Histology of defense in plants. In: J.G. Horsfall & A.E. Dimond (Eds): Plant pathology. Academic Press, New York. 674 p. Balch, R.E., 1952. Studies of the balsam woolly aphid, Adelges piceae (Ratz.) and its effects on balsam fir, Abies balsamea (L.) Mill. Canadian Department of Agriculture Publication 867. 76 p. Carter, W., 1962. Insects in relation to plant disease. Interscience Publishers, New York. 705 p. Esau, K., 1977. Anatomy of seed plants. 2nd edition. John Wiley and Sons, Toronto. 550 p. Fahn, A., 1967. Plant anatomy. Pergamon Press, New York. 534 p. Hare, R.C., 1966. Physiology of resistance to fungal diseases .in plants. Botanical Review 32: 95-137. Mullick, D.B., 1969a. Thin-layer chromatography of anthocyanidins, I. Journal of Chroma­ tography 39: 291-301. Mullick, D.B., 1969b. New tests for microscale identification of anthocyanidins on thin- layer chromatograms. Phytochemistry 8: 2003-2008. Mullick, D.B., 1969c. Reddish purple pigments in the secondary periderm tissues of wes­ tern North American conifers. Phytochemistry 8: 2205-2211. Mullick, D.B., 1971. Natural pigment differences distinguish first and seguent periderms through a cryofixation and chemical technigues. Canadian Journal of Botany 49: 1703-1711. Mullick, D.B., 1975. A new tissue essential to necrophylactic periderm formation in the bark of four conifers. Canadian Journal of Botany 53: 2443-2457. Mullick, D.B., 1977. The non-specific nature of defense in bark and wood during wounding, insect and pathogen attack. In: F.A. Loewus & V.C. Runeckles (Eds): Recent advances in phytochemistry. Vol. II. Plenum Publishing Co. New York. 527 p. Mullick, D.B. & G.D. Jensen, 1973a. Cryofixation reveals unigueness of reddish-purple

101 w

sequentperider m andequivalenc ebetwee nbrow nfirs tan dbrow nsequen tperiderm so f threeconifers .Canadia nJourna lo fBotan y51 :135-143 . Mullick,D.B .& G.D .Jensen ,1973b .Ne wconcept san dterminolog yo fconiferou speri ­ derms:necrophylacti can dexophylacti cperiderms .Canadia nJourna lo fBotan y 51:1459-1470 . Mullick,D.B .& G.D .Jensen ,1976 .Rate so fnonsuberize dimperviou stissu edevelopmen t afterwoundin ga tdifferen ttime so fth eyea ri nthre econife rspecies .Canadia n JournalO fBotan y 54:881-892 . Puritch,G.S .& D.B .Mullick ,1975 .Effect so fwate rstres so nth erat eo fnon-suberize d impervioustissu eformatio nfollowin gwoundin gi nAbie sgrandis .Journa lo fExperi ­ mentalBotan y26 :903-910 . Rahe,J.W .& R.M .Arnold ,1975 .Injury-relate dphaseolli naccumulatio ni nPhaseolu s vulgarisan dit simplication swit hregar dt ospecificit yo fhost-parasit einter ­ action.Canadia nJourna lo fBotan y53 :921-928 . Soo,B.V.L. ,1977 .Genera loccurrenc eo fexophylacti can dnecrophylacti cperiderm san d non-suberizedimperviou stissue si nwood yplants .Ph.D .Thesis ,Universit y ofBritis h Columbia.31 9p . Srivastava,L. ,1964 .Anatomy ,chemistr yan dphysiolog yo fbark .Internationa lRevie wo f ForestryResearc h1 :203-277 . Treshow,M. ,1970 .Environmen tan dplan tresponse .McGra wHill ,Ne wYork .42 2p . Vanderplank,J.E. ,1975 .Principle so fplan tinfection .Academi cPress ,Ne wYork .21 6p . Wood,R.K.S. ,1967 .Physiologica lplan tpathology .Blackwel lScientifi cPublications , Oxford.57 0p .

102 Trees resistant to spread of decay associated with wounds

AlexL .Shig o

ChiefScientist ,U.S .Departmen to fAgriculture ,Fores tService ,Northeaster nFores t ExperimentStation ,Durham ,Ne wHampshir e03824 ,US A

ABSTRACT

Trees arehigl ycompartmente dperennia l plants.Norma l growth isdependen t on shedding,wallin g off,an d regenerating newtissues .Tree scanno tmove .The yhav eevolve dunde rth e constant stress ofwound s and awid evariet y oforganism s that infectwounds .Tree s cannotpreven twound s and infection.Tree s survive after injury and infectionb y also shedding, orwallin g offth e infected tissues and thenb y regenerating similar tissues ina ne wposition .Tree s thathav eth e ability toshe d orwal l offrapidl y andeffectively , andt oregenerat ene w tissues rapidly,wil l survive fora lon gtim eunde rth e stress ofman y infections.Wallin g offprocesse s inth ebar k andxyle m arenon-specifi c totyp e ofinjur y or organism.Therefore , controlled wounding techniqueswher e therespons e ofth etre e canb emeasure d accurately,ma yb e aneffectiv ewa y todeter ­ mine atree' s genetic capacity tosurviv e after injury and infectionb y awid evariet y oforganisms .Preliminar y results fromwoundin g experiments onsevera l tree species indicate that thewallin g offprocesse s areunde r strong geneticcontrol . Some individuals ina specie swalle d offdecaye d wood tover y smallvolume swhil e other individuals stillwalle d offth e decayedwood ,bu t tomuc h largervolumes .A large scalepilo t projectusin gthi s information isno w inprogres s to select treeswit h strong resistance toth e spread ofdeca y associated withwounds .

INTRODUCTION

Decay is amajo r causeo f lowqualit y intree s throughout theworld . The economic loss isextremel yhigh .Deca y isals o amajo r cause ofhazar d trees.Whe n suchtree s fallthe ycaus e agrea t amounto fdamag e topowe r lines,property , andpeople .Wit h all this damage causedb y tree decay,wh y

103 has solittl ebee ndon et oproduc etree sresistan tt odecay ? Theliteratur eprovide sver y little informationo nth e subject.Th e textbookb yWrigh t (1976)o n forestgenetic s doesno tdiscus s it.Boyc e (1961), inhi s fine forestpatholog y text,give sonl y a fewcomment s on resistance totre e decay.Proceeding s frompas tconference s on forestgen ­ etics showtha ttre e decaywa sno tdiscussed . Several investigators have mentioned decay resistance,bu t inwoo dproducts . Itmus tb ekep t inmin d thatroo tdisease s aredifferen ti nman yway s fromthos einitiate db yme ­ chanicalwounds .Fo r acurren trevie w andexcellen tbackgroun d information onresistanc e toroo t andbut tro t inconifers ,se evo nWeissenber g (1980). The question remains,wh y solittl eprogress ? The answer istha tgen ­ eticistshav eno tbee ngive n anaccurat e accounto fth edeca yproces s and reliable simplemethod s towor kwit h tree decay.Geneticist s needt okno w how atre e isse t anatomically andbiochemicall y toresis t decay, andho w the decayproces s operates.Geneticist s need accuratemethod s forde ­ tecting,measuring , andworkin gwit htre edecay .Give nthes ebasic sthe y canprogres s rapidlywit hth eman y finetechnique s andbasi c information alreadywel l developed inth e field ofgenetics . Advances inscienc e aremad eno to nth epresentatio n ofne wobserva ­ tions,hypotheses ,an dmethod s alone,bu to nth e combination or connection ofthese ,whethe r they areol do rnew ,i nsuc h awa ytha ta ne wperspectiv e emerges. Iti swit h this rationale thatthi spape r ispresented : iti s hopedtha ta ne wperspectiv ewil lemerge .

BACKGROUND

Trees areperennia l plantswit hwel l developed systems for shedding parts afterthe yhav ebee nuse do r injured.Tree s shed fruits,leaves , twigs,branches ,bark , and roots.Th e openings leftb y thepart s shed are oftenwher emicroorganism s attack.T odefen d itself,th etre emus thav e systems toclos eo rt owal lof fth eopening .Timin g isextremel y important. Ifth esheddin g andwallin g offar eno twel l synchronized, thepathoge n willgai nth e advantage togro w into thetree .Thi sproces s is seenbes ti n the shedding ofbranches .Tree s alsowal l off injured and infected tissues inbar k (Mullick, 1977;se e alsoth eprecedin g chapter), trunkwoo d (Shigo, 1979b), androo twoo d (Tippett& Shigo, 1980). Iti sconjecture d thatth e sameprocesse s thatbecam ewel l developed through evolution for shedding parts,wer euse d forwallin g offinjure d and infected tissues thatb yna ­ tureo fthei rpositio n inth etre ecoul dno tb e shedeasily .Jus t asth e normal sheddingprocesse s aretime d tobes tbenefi tth e tree,ther emus tb e timeswhe nth ewallin g offprocesse s induced by injury and infectionfunc ­ tionmos trapidl y andeffectively , andtime swhe nthe y donot . Trees areconstructe d ashighl ycompartmente dplants .Afte rwoundin g theywal l offth e injured andinfecte dwoo d (Shigo, 1979b). Afterwounding ,

104 many typeso fmicroorganism s infectth ewoun d surface and exert aforc et o grow into thetree ,an dt odiges tth ewood .Th emicroorganism s may spread anddetermin e therat eo fdecay ,bu tth etre e defines the limits ofth e decayedwood .T ounderstan d thecompartmentalizatio nproces smor e easily, we developed asimpl emodel ,CODIT , anacrony m forCompartmentalizatio n Of Decay InTree s (Shigo& Marx , 1977). TheCODI Tmode lmake s itpossibl e tounderstan d the3-dimensiona lcon ­ figuration ofth edevelopin g columno fdiscolore d anddecaye dwood .Th e terms inth emode l -Wall s 1,2 ,3 an d4 -mus tno tb e confusedwit hth e realproces s of compartmentalization. Afterwounding , the cambiumbegin s to form cells thatdifferentiat e in­ to abarrie r zone,wal l 4o fCODI T (Sharon,1973 ;Moore ,1978 ;Mulher n et al., 1979). Once thebarrie r zone forms,th e continued spread ofth emicro ­ organisms isconfine d totissue s extant atwounding . Summaries ofwor k ondeca y resistance and adescriptio n of anon ­ destructive method using the Shigometer for selection of stronglycompart ­ mentalizing trees ina specie shav ebee npublishe d (Shigo,1979c ;Schmit t etal. ,1978 ).

RESEARCHRESULTS

Anatomical features Geneticists have showntha tman y anatomical features areunde r genetic control (fora revie w seeHattemer , 1963). Vessel featureswer e strongly related toth e degreeo fresistanc e to Dutchel m disease (Elgersma,1970 ,an dpag e 143 inthi sbook ;McNab b et al., 1970;Sinclai r et al., 1979). Vessel featureswer e also stronglyasso ­ ciatedwit hwound s inAmerica n elm (Shigo et al., 1980). Vessel grouping wasth e important feature.Tree s thatha d rapid lateral spread ofdiscol ­ oredwoo d hadvessel s closely grouped.Th e strongly compartmentalizing treesha d large singlevessel s surrounded byman y fibers.A similarsitua ­ tionwa s reportedb yEckstei n et al. (1979)fo rhybri dpoplar . Vessel groupingma yb eth emai n feature limitingth e lateral spread of infection inwood ,bu tvessel sma y alsob e important inlimitin g vertical spread (Bauche t al., 1980). Inre dmapl e (Acer rubrum L.)th e abilityt o plugvessel s rapidly afterwoundin g appeared tob e themai n featurelimit ­ ingvertica l spread. Forelm , seepag e 143 inthi sbook . Inth ewoun d studies,th ecolum n sizes ofdiscolore d wood associated with allth ewound swer e fairlyunifor m foreac htree ,suggestin g thati t was thetre e thatmainl y regulated thedimension s ofth e discolored wood.A similar resultwa s reportedb yEkma n& vo nWeissenber g (1979)fo r Picea abies (L.)Karst .Littl e isknow nabou tth epluggin g features inconifers . Inbirc h (Betula papyrifera Marsh., and B. alleghaniensis Britt.) lengths ofdiscolore d columns associated with drillwound swer e correlated

105 with lengths ofvessel s (Bauche t al., 1980).

Biochemical features Shortle (1979)give s anexcellen tdiscussio n ofth ebiochemica lmecha ­ nisms ofcompartmentalization .H e showsho wbiochemistr y relates toth e CODITmodel . Inothe r studies notye tpublished , heha s showntha twoo d from strongly compartmentalizing redmaple s andhybri dpoplar s doesno t supportrapi d growth ofselecte dwoo d decay fungi inlaborator yexperi ­ ments.Woo d fromweakl y compartmentalizing trees supportedmor e growtho f the fungi.

Compartmentalization in hybrids The original studies thatsuggeste dgeneti c controlo fcompartmentali ­ zationo fdecaye dwoo d associated withwound swer edon e onhybri d poplars (Garrette t al., 1976;1979 ;Shig oe t al., 1977). Santamour (1979)di dstu ­ dies oncompartmentalizatio n inre dmapl e (Acer rubrum L.), and silver maple (A. saccharinum L.) andhybrid s ofthese .Hi s conclusionwa s that compartmentalization ofdiscolore dwoo dwa sunde r geneticcontrol .

Roots Decayedwoo d associatedwit h Heterobasidion annosum (Fr.)Bref ,wa s compartmentalized inroot so fre dpin e (Pinus resinosa Ait.)(Shigo , 1979a). Compartmentalization mayhel p toexplai nwh y treestha t appear healthy and green canstil lhav e soman y decayed roots.Tree sma y remain alive afterman y infectionsbecaus e they constantly compartmentalize in­ fectedtissue s tosmal lvolumes .Recen t results suggest the samepatter n ofcompartmentalizatio n inroot so fsevera ltre especie s infectedwit h Armillaria mellea (Vahl.e xFr. )Quel . Greatcar emus tb e takenher ewit hth eroo tinfectin g fungith,a tar e also associatedwit hdecaye dwood .The yhav e the ability to acta spatho ­ gens first:the y kill cambialtissue s and alesio n forms.Th e tree responds by limitingth e size ofth elesion . Insom ecase s the fungi spread sorap ­ idly inth ebar ktha tth e rooto rtrun k iskilled .Bu t after the spreado f the lesion isstoppe db y thetree ,th e fungihav e the firstopportunit y to growrapidl y into thewoo dbeneat h thelesion .

Bark Itma yb etha tsom eo fth e informationgaine d from studieso fwallin g offi nwoo dwil lb e applicable tobark .Th ewor ko fMullic k (1977)doe s showtha tbar k forms aprotectiv e newperider m after injury. Iti s atyp e ofwallin g off.Hi swor kwil lhav e greatapplicatio nt oman ybar kdiseases , andma ywel l fitwit h H. annosum andA . mellea. Again,ho w fastan dho wef ­ fectively thetre ewall s off infections inth ebar k determines the sizeo f thelesion .

106 Wood decay Tothi spoin t2 type s ofdecay-causin g fungihav ebee n discussed. First,th e fungi thatente rwoo d throughwounds .The y stay inth ewood . This isth etyp etha tthi spape r addressesmainly . Second, thetyp etha t start inth ebar k andthe nmov e into thewood .Her e thetre emus t first limitthei r spread inth ebark , andthe n inth ewood .Whe nthe y grow into thewood , thenth e information givenher e isapplicable .A third type,no t discussed inthi spape r shouldb ementioned . These fungi start inth ewoo d afterwoundin g and latergro w intoth ebark .The y areth ecanke r rotfung i (Shigo, 1969).

Spread Itma yno tb eth enumbe r ofinfection s that injure orkil l atree ,bu t theexten to r spread ofeac h infection.A tre etha tcanno trespon d rapidly toth e spread of infection andhol d the infection inplac ewil l die. Iti s extremely important tonot etha t infectionspe r sed ono tkil l atree .I f they did,n otre ewoul d livemor e than afe wyears ,becaus e every treeha s small and largewound s andbranc h stubs that are infected. There ishardl y a tree thatdoe sno thav ehundred s ofinfection s atan yon e time.Tree s survive as longa sthe y are ablet owal l off the spread of infectionst o smallvolumes ,an dthe nregenerat ene whealth ytissues .

Heartwood compartmentalization Theheartro t concept states thatdecay-causin g fungi infectdead ,non - responsive heartwood exposedb ywounds ,an dtha t fungigro w throughth e heartwood until all isdigested , leaving ahollo w (Boyce, 1961). Itwa s alsobelieve d thatbecaus e thedeca y fungi infect dead,non-responsiv e heartwood, thatdeca ywa s nota disease .An dbecaus eheartwoo d was dead,i t wouldb e impossible to selecto rbree d for features indead , non-responsive tissue.Thes ebelief s havehel dbac kprogres s on selecting andbreedin g trees resistantt odecay . Heartwood mayb e dead according toou rdefinition ,bu t iti ssurel yno t non-responsive.Whe nhole swer e drilled intohealth yheartwood , acolum no f discolored heartwood formed andwa s compartmentalized (Shigo& Shortle , 1979). TheCODI Tmode l is applicable toheartwood ! Thispoin ti sver y important:investigator smus tno tthin k itimpossi ­ ble toselec t andbree d for strong compartmentalization ofdeca y inheart - wood. Themethod s described for selection of strongly compartmentalizing trees are also applicable forheartwood . Theheartwoo d confusion isa tth ever y core ofth ereaso n there isn o progress inth edevelopmen to fa decay-resistan ttree .Rennerfel t (1946) stated thatbecaus e decay goes oni ndea dwoo d of spruce itwoul db eimpos ­ sible tohav ephysiologicall y controlled resistance.

107 Pilot study for selecting decay-resistant trees The United States Forest Service is now conducting a large pilot study in natural forests in the north-eastern United States to select trees re­ sistant to the spread of decay. The trees in the study, over 200, were those already selected as superior on the basis of form and growth rate. The species are Acer saccharum Marsh., Betula alleghaniensis Britt., and B. papyrifera Marsh. Each tree received 4 large, deep drill wounds at 1.4 m aboveground. After 2 growing seasons the Shigometer method will be used to determine the length of the columns associated with the wounds. The trees with the smallest columns will be selected as superior, not only for growth rate and form, but for their ability to compartmentalize defects associated with wounds. Details on methodology have been published (Shigo et al., 1977).

REFERENCES

Bauch, J., A.L. Shigo &M . Starck, 1980. Wound effects in the xylem of Acer and Betula species. Holzforschung 34: 153-160. Boyce, J.A., 1961. Forest pathology. McGraw-Hill Book Cpmpany, NewYork/London . 572 p. Eckstein, D., W. Liese &A.L . Shigo, 1979. Relationship of wood structure to compart- mentalization of discolored wood in hybrid poplar. Canadian Journal of Forest Research 9: 205-210. Ekman, R. &K . von Weissenberg, 1979. Sapwood extractives in Norway spruce inoculated with Fomes annosus. Acta Academiae Aboensis, B. 39, No 7, 8 p. Elgersma, D.M., 1970. Length and diameter of xylem vessels as factors in resistance of elms to Ceratocystis ulmi. Netherlands Journal of Plant Pathology 76: 179-182. Garrett, P.W., A.L. Shigo &J . Carter, 1976. Variation in diameter of central columns of discoloration in six hybrid poplar clones. Canadian Journal of Forest Research 6: 475-477. Garrett, P.W., W.K. Randall, A.L. Shigo &W.C . Shortle, 1979. Inheritance of wound heal­ ing in sweetgum (Liquidambar styraciflua L.) and Eastern Cottonwood (Populus deltoides Bartr.). United States Forest Service Research Paper NE-443. 4 p. Hattemer, H.H., 1963. Estimates of heritability published in forest tree breeding research. Proceedings of the World Consultation on Forest Genetics and Tree Improve­ ment. Stockholm, Sweden. Vol 1, session 2a, paper 3. FA0/F0RGEN-63. 14 p. McNabb, H.S., H.M. Heybroek &W.L . MacDonald, 1970'. Anatomical factors in resistance to Dutch elm disease. Netherlands Journal of Plant Pathology 69: 1158-1159. Moore, K.E., 1978. Barrier zone formation in wounded stems of sweetgum. Canadian Journal of Forest Research 8: 389-397., Mulhern, J., W.C. Shortle &A.L . Shigo, 1979. Barrier zones in red maple: an optical and scanning electron microscope examination. Forest Science 25: 311-316. Mullick, D.B., 1977. The non-specific nature of defense in bark and wood during wounding, insect and pathogen attack. Recent Advances in Phytochemistry 11: 395-411. Rennerfelt, E., 1946. Om rotrotan (Polyporus annosus Fr.) i Sverige. Dess utbredning och satt att upptrada. Meddelanden fran Statens Skogsforskningsinstitut 35(8): 1-88. Santamour, F.S. Jr., 1979. Inheritance of wound compartmentalization in soft maples. Journal of Arboriculture 5: 220-225. Schmitt, D., P.W. Garrett &A.L . Shigo, 1978. Decay resistant hardwoods? You bet! North­ ern Logger and Timber Processor, September, p. 220-225. Sharon, E.M., 1973. Some histological features of Acer saccharum wood formed after wounding. Canadian Journal of Forest Research 3: 83-89. Shigo, A.L., 1969. How Poria obliqua and Polyporus glomeratus incite cankers. Phytopathology 59: 1164-1165. Shigo, A.L., 1979a. Compartmentalization of decay associated with Heterobasidion annosum in roots of Pinus resinosa. European Journal of Forest Pathology 9: 341-347. Shigo, A.L., 1979b. Tree decay: an expanded concept. United States Department of Agricul­ ture Information Bulletin 419. 73 p.

108 Shigo,A.L. ,1979c .Deca yresistan ttrees .North-easter nFores tTre eImprovemen t ConferenceProceedings .Schoo lo fFores tResources .Pennsylvani aStat eUniversity , p.64-69 . Shigo,A.L . &H.G .Marx ,1977 .Compartmentalizatio n ofdeca y intree s (CODIT).Unite d StatesDepartmen to fAgricultur eInformatio nBulleti n405 .7 3p . Shigo,A.L .& W.C .Shortle ,1979 .Compartmentalizatio no fdiscolore dwoo di nheartwoo do f redoak .Phytopatholog y69 :710-711 . Shigo,A.L. ,W.C .Shortl e& P.W .Garrett ,1977 .Geneti ccontro lsuggeste di ncompart ­ mentalizationo fdiscolore dwoo dassociate dwit htre ewounds .Fores tScienc e 23:179-182 . Shigo,A.L. ,R .Campana ,F .Hylan d& J .Anderson ,1980 .Anatom yo felm sinjecte dt o controlDutc hel mdisease .Journa lo fArboricultur e6 :96-100 . Shortle,W.C , 1979.Mechanism so fcompartmentalizatio no fdeca yi nlivin gtrees . Phytopathology69 :1147-1151 . Sinclair,W.A. ,J.P .Zahlan d& J.B .Melching ,1975 .Anatomica lmarke rfo rresistanc eo f Ulmusamerican at oCeratocysti sulmi .Phytopatholog y 65:349-352 . Tippett,J.T .& A.L .Shigo ,1980 .Barrie rzon eanatom yi nre dpin eroot sinvade db y Heterobasidionannosum .Canadia nJourna lo fFores tResearc h10 :224-232 . Weissenberg,K .von ,1980 .Resistanc eo fPice aabie st oHeterobasidio nannosum .Proceed ­ ingso fth eFift hInternationa lConferenc eo nProblem so fRoo tan dBut tRo ti n Conifers.Ed .L .Dimitri .Hessisch eForstlich eVersuchsanstalt ,Hann .Münden , p.67-74 . Wright,J.W. , 1976.Introductio nt ofores tgenetics .Academi cPress ,Ne wYork/London . 463p .

109 L Histopathology of anatomical mechanisms for resistance to fusiform rust in slashpin e

F.F. Jewell,Daphn e C.Jewel l& C.H .Walkinshaw 1

ABSTRACT

Anatomical expressions ofresistanc e to Cronartium quercuum f.sp. fusiforme, Southern fusiform rust,i ninoculate d slash pine seedlings (Pinus elliottii var. elliottii) were apparently initiated inth eprimar y tissue system ofth ehost .Artificia l wounding of slashpin e seedlings resulted inhos ttissu ereac ­ tions similar toreaction s from fusiform rust infection.Thi s intur n indicates thatth e initial tissue change inslas hpin e reported due to susceptibility or resistance to fusiform rust isno tuniqu e tothi sorganism ,bu t isa tfirs ta genera l host response to injury.A shos tgrowt h and stimulationb y the rust continues, differences inreactio no fth e rust-susceptible and resistantprogenie s areprobabl y duet othei r genetics.Th e resistantprogenie s were able tomaintai nth e differentiation oftissue s tocontai nth e rust.Thes e tissues apparently were incompatible togrowt h andexpansio n ofth erust . Susceptible progenies, although initially showing aresistanc e torus tin ­ fection,wer eunabl et operpetuat e this reactionthroug hdif ­ ferentiationo fincompatibl ehos ttissues .Instead ,hos ttis ­ sueswer eproduce d thatwer e compatiblewit hgrowt h and spread ofth erus t fungus.

INTRODUCTION

Certainearl y reports ofhistologica l studies ofgall-formin g pine rusts indicated sometyp eo fanatomica l reaction ofth ehos tt oth e

1.Respectively ,Professo ran dResearc hAssistant ,Schoo lo fForestry ,Louisian aTech . University,Ruston ,L A71272 ,an dPrincipa lPlan tPathologist ,Fores tDiseas eLab. ,USDA , Gulfport,M S39503 .Thi swor kwa ssupporte db yth eMclntire-Stenni sCooperativ eForestr y ResearchProgram ,an di ncooperatio nwit hth eSouther nFores tExperimen tStation ,USDA , ForestDiseas eLab. ,Gulfport , MS.

110 pathogen,whic hwa s concluded tob e afor m ofresistance .Hutchinso n (1935), True (1938), andMcKenzi e (1942)reporte d such findingsworkin g with Scotspine , Pinus sylvestris L., infectedwit h a Peridermium rust (Cronartium or Endocronartium). More recently, Jewell& Snow (1972), Jewell & Speirs (1976), andMille r et al. (1976)describe d resistantreac ­ tions in1 - and 2-year-old slashpin e (P. elliottii var. elliottii Engelm.) and loblollypin e (P. taeda L.)control-inoculate d with the Southern fusiform rust, Cronartium quercuum f. sp. fusiforme (Burdsall &Snow , 1977). Inslas hpine ,th ehos tarea sexpressin g resistancewer eterme d resistance-zones (Jewell &Speirs , 1976). Although the recentstudie s onresistance-reaction s inslas han d loblollypin ewer e rather intensive,th e initiationo fth ehos ttissue sas ­ sociated with theresistance-zone s wasno tdetermine d conclusively.How ­ ever,Jewel l & Snow (1972)an dJewel l& Speirs (1976)surmize d thatth e resistant-reactionswer e initiated inth eprimar y tissues ofth e slashpin e hosts studied. Thispape rwil l summarize aportio n ofth e research from our laboratory concerning the slashpine/fusifor m rusthost-parasit e relationship, theon - togeny ofanatomica l expressions ofhos t resistance toth e rustparasite , andth e reaction ofth ehos tt o artificialstimulus .

METHODS AND MATERIALS

Slashpin e tissue forexaminatio nwa s obtained fromhalf-si b progeny of knownparentag e (resistant)o rbul k (susceptible)see dcollections .Seed ­ lings4- 8 weeks of age from seedwer e control-inoculated withknow no rbul k cultures of fusiform rustb y thetechniqu e ofSno w& Kai s (1972). Sample collectionswer emad e atvariou speriod s following inoculation: 1an d2 years, including samples from seedlings classed asrecovere d orgalled ; and daily forth e first3 0 days.Uninoculate d samples from seedlings ofsimila r ages served aschec k samples. Six-week-old bulk slashpin e seedlingswer e artificiallywounde db y theinsertio no f sterile,stainles s steelpins . Thirty days afterwounding , sampleswer e collected fromth ewound-site . Unwounded seedlings ofth e same seed source andplan t age served aschecks . Tissue sampleswer eprocesse d aspreviousl y reported (Jewell et al.,1962 ; Jewell& Walker , 1967). Observationswer emad eb y lightmicroscopy .

RESULTS

Nine month and older infections Theearlie rwor k onth ehistopatholog y of fusiform rustgall s on 9-12 month old slashpin e revealed inpart ,th e commonpresenc e ofvertica l (wood)parenchyma ,terme d reactionparenchyma , ingal l tissue.Thi scellu ­ larconfiguration ,unusua l inpin e (Esau, 1953),wa so fparticula r interest

111 Figure 1. A. Macro-transverse section of a1-year-ol d slashpin ewit h internal necrotic symptoms (n)typica l ofresistance-zone s (6x) , B-C.Respectively,micro-transvers e view ofnecroti c resistance-zones in 1-an d 2-year rust infections.Not e separation ofpit h and cortical areasb ynorma lhos ttissu e inC an d inwardbendin g ofhos tcambiu m (c)(1 8 *). 112 asi tconsistentl y marked the innermostexten to fth emyceliu m ofth epara ­ site, andth ecorrespondin g host-tissue abnormality.A tthi s time,th eori ­ gin and importance ofthes ecell swer eno twel l understood. Subsequently, observations onrecovere d and gall samples from 1-an d 2-yearinfection s revealed thepresenc e ofreactio nparenchym a defining affected tissue areas.However , samples from recovered seedlings contained areas appearingnecroti cmacroscopicall y (Fig.1A )an dmicroscopicall y sim­ ilar tobu tmor e extensive thanth e reaction areas reported earlier for slashpin e (Jewell& Snow, 1972). Inth erecovere d seedlings,th enecrotic - appearing areas,terme d resistance-zones,varie d inamoun to f stem areain ­ volved.Resistance-zone s extended frompit h throughth e epidermis insom e samples (Fig. IB), orwer epresen t inth ecorte x and/orxyle m atvaryin g distances from thepith .Wher e cortical andxyle m zoneswer e radiallyoppo ­ site ina sample ,th e separating hosttissue swer e freeo fth e abnormal cells typical ofresistance-zone s (Fig.1C) .Th e latterwa s consistentwit h 2-yearol d infections.Th e resistance-zones,regardles s ofsampl eage , appeared tooriginat e inth eoute rpit h tissues,whic h comprised portions ofth emetaxyle m ofth eprimar yplan tbod ypresen t at inoculation (Fig. ID). ' The resistance-zones were sharply defined from thenorma l hostste m tissuesb ycel l areasconsidered , intransvers eview ,simila r to reaction parenchyma described forpine-gal l rusts (Jewell et al., 1962;Jewel l & Walker, 1967). However,longitudina l sections revealed notonl yvertica l parenchymabu tals o dark-staining, small,nearl y isodiametricparenchyma ­ likecell s (Fig. IE). Thecell scentra l toth ezone swer eheavil ytan - ninized and considered non-living,whil e others,alon gth eborder s ofth e zone, althoughdark-staining ,wer e considered tohav ebee n living atfix ­ ation (Fig. IF). Insom e instances,meristemati c activity ofcertai no f thesecell s appeared tohav eresulte d inproductio n of additionalparen ­ chyma orcallus-typ e cells.Thes e cells apparently wereproduce d to "heal" the areao f stem associated with the resistance-zone where tearingo fhos t tissue oftenresulte d duet ogrowt h stress (Fig. IG). The latter types of

D. Remnants ofprimar y needle tissue (p)burie d ina resistance-zone pushedb ynorma l growth into thecorte x (150x) . E. Longitudinal view ofmulti-typ e reactionparenchym a cells inxyle m (70 x). F. Tanninized non-functionalcell so fresistance-zon e (r)an ddark - stainingviabl eparenchym a (p)borderin g thezon e (150x) . G. Meristematic cells (callus) (c)differentiate d forclosin go fwoun d developing duet oresistance-zon e inth e innercorte x and cambial area (30 x). H. Tanninized non-functionalrus thypha e (h)i nresistance-zon e (150x) .

113 cellswer e absent intypica l galltissue . Establishment of fusiform rustha d occurred in all inoculated samples studied,bu tn o signo f infectionwa s observed inth echec k samples.Th e rustwas ,i nth e infected samples from resistantprogeny , confined toth e resistance-zonesan dwa s not foundi nth enorma lhos ttissu e surrounding thezones .Hypha ewer epresen t inth ereactio nparenchym a and,base d on staining reaction,considere d viable.Radia l andvertica l hyphal extension had occurred inth e zones.Haustoria ,typicall y abundantwit h fusiform rust,wer e difficult toobserve .Whe nobvious ,haustori awer e considered scanti nnumbe r related toth e amounto fhypha epresent .Hypha e andhaus ­ toriawer e abundanti ntanninize d areas ofth e zones,bu} ;degeneratio n and hypertrophy ofth erus twa s evidentan d itwa s considered non-functionali n this situation (Fig.1H) .Susceptibl eprogen y had developed the anatomy considered typical for fusiform rust (Jewell et al., 1962). Cambial cylinder interruptionb y the resistance-zones was common inth e 1-yearbu tno t inth e2-yea rinfection s (Fig. 1B,C). The latter consistenly had aninwar d curvature ofapparentl y normalhos tcambiu m oppositexyle m and/orcortica l resistance-zones (Fig.1C) .Thi s cambial abnormalitywa s considered indicative ofpreviou s injury or interruptiono ftypica l activ­ ityo fthi s tissue.Als o indicatedwa s thatth e resistant seedlings,i n time,woul dbur y thexylem-zon e innorma l stem tissue and slough-off the cortical zones and continue thenorma l growthprocess . Itwa s obvious that therewa s adegeneratio n orregressio n ofth e resistance-zones with an increase inhos tag e andgrowth , atleas t from 1yea rt o2 year s ofage ,a s observed.

30-day infection Susceptible and resistantplant s exhibited differences invisibl e rust-infectionsympto m appearance.Th e resistantprogen y exhibited symptoms aspurpl e spots onth ehos thypocoty l atth eprimar y needlebases .B y3 0 days, thehypocoty lwa s splita tth epurpl e area,bu tlittl eo rn o swelling wasevident .Th e susceptibleplant sexhibite d symptoms after inoculation primarily as spotso nth eprimar y needles.A t3 0 days, theseplant sexhib ­ itednoticeabl e swellingbu tn o splitting ofth ehypocotyl . Microscopically, themos tobviou s reaction torust-infectio n occurred inth eresistan t seedlings.Thi swa s evidencedb y localized areas ofheavi ­ lytanninize d cortical cellsradiall y opposite abnormalcellula rconfigura ­ tionextendin g fromth epith . Inth e affectedcortica l areas,th ecell s were conspicuously rounded with alos s of intercellular space (Fig.2A) . Hosttissue s adjacentt othes e abnormalitieswer e considerednorma l for pine andn o rusthypha e orhaustori awer e observed. Functional andnon ­ functional hyphaewer epresen ti nth e affected tissue areas,bu thaustori a wererare . The susceptibleplant sha d limited tanninization inaffecte dcortex ,

114 phloem, orxylem .Functiona l hyphaewer epresen t inth eparenchym a tissues ofth ehost .Typica l rusthaustori awer e abundant andwel l developed.Th e cellular characteristics developed appeared characteristic fortypica l fusiform rustgall s (Jewell et al., 1962).

Figure 2. A. Transverse view ofresistance-zon e (z)developin g 30day s afterinfec ­ tion, incontras tt oth enorma l tissue (n)(1 8x) . B Resistance-zone development 9day s following infection.Not ehypha e (h) and cellwal l thickening (w)i nzon e areacontraste d tounaffecte d tis­ sue (n) (70 x). Longitudinalvie w ofresistance-zon e 14day s following infection. Intra- and intercellular hyphaepresen t (h).Bas emeriste m (b)cell s are hypertrophied (150x) . D Longitudinal view of a30-day-ol dwound .Tanninizatio n (t)o fcell s presentwit hproliferatio n andmisalignmen t ofparenchym a (p)i nreac ­ tiont owound .Not e atypical development ofcell s (a)remove d fromth e wound (30x) .

115 Infection prior to 30 days Thehos treaction spresen t3 0day s after inoculation appeared, duet o their location inth e stem,t ohav ebee n initiated inprimar ytissue so f the stem and adjoining primary needle traces early inth e infectionpro ­ cess.Althoug hmacroscopi c symptom appearancevarie dbetwee n resistantan d susceptible plants,initia lmicroscopi c hostreaction swer e similar inbot h progeny types andoccurre d earlier inth e infectionprocess .Th e firsthos t responsewa s observed 8-9 days after inoculation immediately aboveth e primaryneedle/ste m junction or inth eprimar y needle traces.I nth e latter tissue,muc ho fth e reactionwa s inth ebas emeristem s andthei r cellular derivatives (Speirs& Jewell,1976 )associate dwit hthe,primar yneedl e traces.Th e affected zoneswer emos tobviou s inth eoute rprimar y cortex. Thiswa s evidencedb yhos tcel ltanninization ,cel lwal l thickening, cell contentdissolution , andth epresenc e ofinter -an d intracellular rust hyphae (Fig.2B) ,th e latterbein g atypical for fusiform rust.Haustori a were rare. With anincreas e intim e following inoculation,difference s inth e reactiont o infectiono fsusceptibl e andresistan tplant sbecam eevident . Atth etim eo fexterna l symptom appearence on'th esusceptibl eplant swel l developed inter-an dintracellula r rusthypha ewer epresen ti nan d among thebas emeriste mcells ,procambium , and adjacentparenchym a cells.Small , newly developed haustoriawer eofte nobserved . Cellso fth e affected areas exhibited limited tanninization, aswel l ashypertroph y inth eprimar y cortex andth epith .Conversely ,th eresistan tplant sexhibite d tanninized cells inth e affected areas,conspicuou s cellhypertroph y inth eoute r primary cortex,an d cellwal lthickenin g inthes e areas.Inter -an dintra ­ cellular rusthypha ewer epresent ,bu twer eusuall y larger thannorma l and frequently distorted, indicating thehypha ewer eunde r stress (Fig.2C) . Haustoria,norma l or atypicalwer erare .O fconsiderabl e interestwa s the appearance inth e affected areaso f abnormal hostcell s inadvanc e ofo r somewhatremove d from directcontac twit h therus thypha e and/orhaustoria . Frequently, asman y as3 cel l layers separated the rust from thehos tcel l abnormality.

Artificial wounding Thirty days afterwounding , allsample s exhibited alimite dbrow n discoloration around and alongth epat h ofth eneedle ,an d asligh tste m swelling.Thi s issomewha t similart o typical symptoms of fusiform rust infection on slashpine . Inaddition ,macroscopi c reactions often appeared tob e inconjunctio nwit hprimar y needlebases . Microscopically, cell destruction due toth e actual acto fwoundin g extended frompit h toepidermis .However ,th e effecto n adjacenthos t tissuewa swha twa s of interest.Simila r toth ehos trespons e inrust - resistantprogeny , cell tanninizationwit h limitednecrosi swa s frequenti n

116 thepith ,xylem ,phloem-cortex , andprimar y needle trace elements,an d needlebase s ofth ewounde d tissue.Littl e orn onecrosi swa s observed in the secondary vascular elements,althoug h tanninization,misalignment ,an d abnormalpittin gwer eobserve d (Fig.2D) .Norma l celldepositio n delimited thewoun d areas ofhos ttissue .However , atypical hostcell swer epresen t inarea s removed from thewound-site ,indicatin g thehos teffec twa s not limited to the actualwound-site . Atypicalparenchymatou s tissues appeared tohav e developed nearth e affected areas.Thes e atypical cells,whic h differentiated inth evascula r rays, resin ducts,an dprimar y cambium,possesse d contents,bu twer e tracheal-like inshape . Ingeneral ,thes e atypical parenchyma resembled the reactionparenchym a reported in fusiform rustgall s (Jewell et al., 1962). Inaddition ,peridermal-lik ecell swer eobserve d separating thewoun d areas from thenormal . Inth ewoun d area,light-staining , callus-like parenchyma developed between thenorma l andwoun d tissue.Thes ecell s appeared regenerative in nature indicating ameristemati cpotentia l forclosin g thewoun d area (Fig.2D) .Thi s cellular depositionwa s similar tobu t less extensive than thecallus-lik e cells associated with resistance-zones in rust-resistant slashpine .

DISCUSSION

Evidence indicates that anatomical expressions ofresistanc e to fusiform rust,characterize d asresistance-zones ,wer e consistently present incertai nprogen y ofrust-resistan t slashpin eparents .Th eresistance - zoneswer e quiteunifor m inth etype so f abnormal host-stem tissueproduce d inth e zone.Ther ewa s noevidenc e ofidentifiabl e type ofreactio nt o infection, although somevariatio n occurred duet o agedifferences . The initiation ofth e resistance-zones occurredver y early inth e hostparasite relationship.Observation s indicate thatth eresistance-zones , quite evident3 0day s after inoculation,wer e initiated inth eprimar y tissues ofth e seedlings studied andwer e adirec t.resul to fhos t reaction toth e fusiform rust fungususe d for inoculation.Th eresistan t reaction resulted inth eprimar y meristems (procambium, basemeristems , etc.) (Speirs& Jewell ,1976 )developin g alimite d but self-perpetuating abnormal parenchyma-like areao ftissu e inth ehos ttha tcontaine d the rustbu t prevented its spread orconcurren t growthwit h thehost .A s thehos tma ­ tured andproduce d secondary tissue systems,th ezone swit h ageregressed . Eventually, thenorma l tissueproductio n ofth ehos tovergre w thezon e resulting inburyin gportion s ofth ezon edee p inth exyle m andsloughing - offth eoute rportions .Thi swoul d result indevelopmen t of aseedling , recovered from rust infection,wit h thepossibilit y ofcontinue d normal growth.

117 In contrast, susceptible progeny, although initially showing a reaction to infection similar to the resistant progeny, exhibited no restraint on the rust fungus and by 30 days after inoculation a typical gall anatomy was developing. Artificial wounding of slash pine resulted in host reactions similar in nature to those initially expressed in rust-infected slash pine. The forma­ tion of periderm-like cells limiting the wound areas indicates the pin- wounded slash pine attempted to isolate the injury or stimulus in degener­ ating tissue. The pin-wounding appeared to stimulate parenchymatous tissue systems to become meristematic and differentiate callus-like cells to close the wound area. In time, normal host growth would continue until all signs of injury were hidden deep in the stem or sloughed off by radial growth of the host. Based on similarities between pin-wounded and fusiform rust-infected slash pine, it appears that the limited initial host response to non- or pathologic injury is basically the same.

REFERENCES

Burdsall, Jr., H.H. &G.A . Snow, 1977. Taxonomy of Cronartium quercuum and C. fusiforme. Mycologia 69: 503-508. Esau, K., 1953. Plant anatomy. John Wiley and Sons, New York. 735 p. Hutchinson, W.G., 1935. Resistance of Pinus sylvestris to a gall-forming Peridermium. Phytopathology 25: 819-843. Jewell, F.F. &G.A . Snow, 1972. Anatomical resistance to gall-rust infection in slash pine. Plant Disease Reporter 56: 531-534. Jewell, F.F. &D.C . Speirs, 1976. Histopathology of one- and two-year old resisted infections by Cronartium fusiforme in slash pine. Phytopathology 66: 741-748. Jewell, F.F., R.P. True &S.L . Mallett, 1962. Histology of Cronartium fusiforme in slash pine seedlings. Phytopathology 52: 850-858. Jewell, F.F. &N.M . Walker, 1967. Histology of Cronartium quercuum galls on shortleaf pine. Phytopathology 57: 545-550. McKenzie, M.A., 1942. Experimental autoecism and other biological studies of a gall- forming Peridermium on northern hard pines. Phytopathology 32: 785-798. Miller, T., E.B. Cowling, H.R. Powers, Jr. &T.E . Blalock, 1976. Types of resistance and compatibility in slash pine seedlings infected by Cronartium fusiforme. Phytopathology 66: 1229-1235. Snow, G.A. &A.G . Kais, 1972. Technique for inoculating pine seedlings with Cronartium fusiforme. In: R.T. Bingham, R.J. Hoff &G.I . McDonald (Eds): Biology of rust resistance in forest trees. Proceedings of the NATO-IUFRO advanced study institute. USDA Miscellaneous Publication 1221.p . 325-326. Speirs, D.C. &F.F . Jewell, 1976. Observations on the anatomy of the hypocotyl and epicotyl of slash pine seedlings. Bulletin No. 9, School of Forestry, Louisiana Tech University, Ruston, LA. 29 p. True, R.P., 1938. Gall development on Pinus sylvestris attacked by the Woodgate Peridermium and morphology of the parasite. Phytopathology 28: 24-49.

118 Mechanisms and inheritance of rust resistance in conifers

BohunB .Kinloc h

Geneticist,Pacifi cSouthwes tFores tan dRang eExperimen tStation ,Fores tService , U.S. Departmento fAgriculture ,Bo x245 ,Berkeley ,Californi a94701 ,US A

ABSTRACT

Relatively fewo fth eman y rustdisease s ofconifer s cause serious damage tothei r hostpopulations .Exception s include blister rusto fwhit epines ,a diseas e introduced toNort h America, and fusiform rust,a native .Bot hhav ebecom e epidemic but striking differences inth epatter no fgeneti cvariatio n in resistancebetwee nhost s ofthes e diseases areevident .Som e commonmechanism s ofresistanc e areshare db yhost s ofthes e andrelate dpin e stem rusts,includin g tolerance,hypersensi ­ tivity, andvariou s expressions ofincompatibility . Both oligogenic andpolygeni c modes of inheritance are indicated, but fewspecifi c geneshav ebee nidentified . More rapid pro­ gress ingen e identificationmigh tb emad eb y shifting the focus ofgeneti c analysis from hosts topathogens ,whic hca nb e genetically manipulated withmuc h lower investment intime , space, and cost.Becaus e ofth e complementarity thatexist s in gene-for-gene relationships,gene s for avirulence orvirulenc e infection type identified inpathogen s canb euse d todeduc e their complements forresistanc e and susceptibility inhosts .

INTRODUCTION

Of approximately 87know nrus tdisease s onNort hAmerica nconifers , less than aquarte r causemor e thanmoderat e or localized damage tothei r hostpopulations , andonl y about6 ar erecognize d asbein g actually or potentially epidemic (Hepting, 1971;Ziller , 1974).Mos to fthes eparasite s seemt ohav eevolve dway so fcoexistin gwit hthei rhost s thatavoi dsignif ­ icantbiologica l damage,eve nwhe nth e apparent amounto fdiseas e isenor ­ mous.Heptin g (1971,p .365 )vividl y describes acas e of Coleosporium needle rusto n loblollypine : 'Attacko nplante d stock issometime s so heavy thaton e cannotwal k through aninfecte dplantatio nwithou t choking onth e aeciospore load inth e air,ye t growth effectshav eusuall ybee n

119 temporary and light.'A mor egraphi c example ofhos ttoleranc e is difficult to imagine,ye ti tseem s likely thatman y conifers,coexis twit h theirmor e innocuous rustparasite s through thispoorl y understood.adaptation ,rathe r thanthroug hmor eobviou s anddramati cmechanism s ofresistance . Inan y case,ther eha sbee n little economicmotivatio n for studying the specific coadaptivemechanism s ofothe rtha n seriously destructive pathosystemso f commercial importance.Thi s isunfortunate , sinceproblem s indevelopin g genetic stability couldb emor e effectively approached throughbette r understanding ofparasit epopulation s thathav eevolve d suchbenig nrela ­ tionships. Inspit e ofth epromis e ofspectacula r gains inresistanc e overmor e than 3decade s of selection andprogen y testing (Bingham et al.,1973 ; Zobel,pag e 417o fthi s book), ourknowledg e ofgeneti c interactions even ofver y damagingpathosystem s remainsmeager .Fe w specific genes forresis ­ tancehav ebee n identified, and some ofthes e only tentatively. Documented mechanisms ofresistanc e -an dthei r inheritance,whe nknow n -ar e listed inTabl e 1 forsevera lpin erusts .Mos to fth edat apertai n to2 o fth e most serious and intensively studied ofthese :bliste r rusto fwhit epines , caused by Cronartium ribicola Fisch.,an d fusiform rusto f southernpines , caused by C. quercuum f. sp. fusiforme (Cumm.) Burds .e' tSnow .Thes e2 rusts, 1exoti c and the othernativ e inNort hAmerica ,offe r intriguing contrasts ingeneti c interactions withthei rhosts .

BLISTER RUST - WHITE PINES

Insuga rpin e (Pinus lambertiana Dougl.), ahypersensitiv e reactiont o invadingmyceliu m inneedl e tissues isconditione d by asingl e dominant gene (Kinloch& Littlefield , 1977). Seedlings carrying thisgen e develop small,brown ,necroti c flecks,whic h aremacroscopicall y distinct fromth e large, lustrous,yello w orre d lesions typical of susceptiblegenotypes . The gene isexpresse d intree so f all ages and insecondary ,primary , and evencotyledo n leaves (Kinloch& Comstock , 1980). Histologically, the hypersensitive reaction ischaracterize db y rapid degeneration of affected cells into granular cytoplasm devoid oforganelle s and filledwit h dense tannindeposits ,an d isessentiall y similar tohypersensitiv e reactions described inothe r rustpathosystems .Yet ,th edevelopmen t ofmyceliu m in tissues ofresistan t genotypesvarie s considerably, from novisibl e traces toextensiv e colonies thatoccasionall ypenetrat e intoth e stele,and ,les s frequently, grow downthroug hphloe m tissue into thebark .Whe nthi soc ­ curs, another hypersensitive reaction istriggere d inbar k tissue,furthe r development ofth epathoge n is arrested, and activehealin g ofth e lesion byperider m formation ensues (Kinloch& Littlefield , 1977). The occasional and limited expression ofpathogenicit y inneedl e tissuesmigh tb e duet o variation inaggressivenes s orvirulenc e inth epathoge n (Kinloch &

120 Comstock, 1980). Recently,w e found adistinc tvirulen t race causing typi­ calyello wneedl e lesions and subsequentbar kblister s onseedling s known tocarr y the dominantresistanc e gene (Kinloch& Comstock , 1981). Similarhypersensitiv e reactions occur inneedle s of P. armandii Franch.,a presume d nativehos to fth epathoge n (Bingham, 1977). Themod e ofinheritanc e could notb e determined,becaus e the seedlot studiedwa s from abul kcollectio n (Hoff& McDonald , 1975), butth e involvemento f major genes seems likely.Hypersensitiv e andyello w lesionswer emixe d on 3/4 ofth e seedlings,whil e onlyhypersensitiv e lesionswer e observed on theremainder .Thi s suggested thepossibilit y notonl y of differential races ofth epathogen ,bu t alsoo fmor e than 1majo r gene inth ehost . Needle infection inwhit epine s isonl y the firststag e indiseas e development, and isno tver yharmfu lb y itself.McDonal d &Hof f (1970) showed thatprematur e shedding ofinfecte d needles incertai nwester nwhit e pine (P. monticola Dougl.)familie swa s aneffectiv emechanis m inreducin g theprobabilit y ofth e fungus establishing inbar k tissue.Anothe rexpres ­ siono fresistanc e inth e 'needle spots only'syndrom ewa s the development ofa necroti c area ofhos tan d fungal cellsnea r thebas e ofth eneedl e fascicle (Hoff& McDonald , 1971). Mycelium developed normally inmesophyl l andvascula r tissues ofth eneedl e andwa s arrested only after contactwit h stem tissues ato r immediatelybelo w the small,dorman tmeriste m atth e baseo fth eneedl e fascicle.Segregatio n ratios insel f and full-sib fami­ lies ledMcDonal d &Hof f (1971)t ohypothesiz e thateac h ofthes emecha ­ nisms iscontrolle d by independently inherited recessive genes.However , consistent deficiencies innumber s ofresistan t seedlings inmos tfamilies , and discrepancies inperformanc e between selfed andoutcrosse d families of the sameparents ,leav e thishypothesi stentative . Two additional genes forresistanc e to specific races of C. ribicola werepropose db yMcDonal d &Hof f (1975). Identification ofthes e genes proceeded fromth eobservatio n that2 distinc t foliar lesiontype soccu r in westernwhit epin e following infection. Some seedlings developed only yel­ lowspots ,som eonl y red,whil e approximately aquarte r developed both types.Evidenc e for agene-for-gen e relationship wasbase d onth e additive property the color types exhibited: the average number of lesionspe r seedling on seedlings withbot h color typesnearl y equaled the combined average ofth e single types.Althoug h therelativ e frequencies ofth e yellow and redphenotypi c classeswer ehighl yvariable ,th e additiveprop ­ ertywa s consistent in2 experiment s indifferen tyears .Thi s ledMcDonal d &Hof f (1975)t oconclud e thaton ehos tgen eprevente d infectionb yth e 'redrace 'an d anotherprevente d infectionb y the 'yellowrace' ,bu tthei r hypothesis wasno tteste db y segregation ratios of seedlings inth e full- sib families used. Ina differen t approach,McDonal d (1978)analyze dsegre ­ gationratio s of24 5monoaeciospor e cultures derived from seedlings bearing yellow spotsonly , red spots only, andbot h spottypes .Unde r thehypothe -

121 sized gene-for-gene relationship,culture s from yellow-only and red-only spotted seedlings should have 'bredtrue 'whe nsusceptibl e test seedlings were inoculated withbasidiospore s resulting fromthes ecultures ,whil e cultures from seedlingswit hbot h spottype s should have segregated into 1:2:1 ratios ofyellow-only :mixe dyello w andred :red-onl y classes.Al ­ though the frequency of spottype s induced bybasidiospor eprogen y was strongly associated with the type ofth eparen tculture ,th eputativ e homocaryon cultures didno tbree d true,an dther ewa s alarg edeficienc y of mixed spottype s inth eputativ eheterocaryo n cultures.McDonal d (1978) proposed thatth e lacko f fitt oexpecte d ratios observedmigh thav ebee n due toirregularitie s in fertilization ofth epycni a orthall i ofth e original sources ofparen tcultures .Thus , the genetic control of lesion types inthi shost-pathoge n interaction remains unresolved. Other foliarmechanism s ofresistanc e havebee nreporte d butthei r inheritance iseve n less certain.Reduce dnumber s ofneedl e lesions among differentprogenie s havebee nnote d inwester nwhit epin e (Bingham et al., 1960), easternwhit epin e (P. strobus L.;Patton , 1967), and sugarpin e (Kinloch,unpublishe d data), and suggested tob eunde rpolygeni c control (Hoff& McDonald , 1980). Patton (1967)describe d amechanis m insecondar y needles ofgrafte d ramets from aresistan teaster nwhit epin e selection thatcompletel y inhibited the formation of substomatal vesicles ofth e fungus.Thi smechanis m was transmitted to seedlingprogenie s in different degrees. Bark reactions areth emos tcommo nmechanism s ofresistanc e inconifer s notonl y to stemrusts ,bu tt o injurious agents ingeneral .The y areex ­ pressed asnecrosi s andcollaps e oftissu e inrespons e totrauma , followed by formation ofperider m tissues thateffectivel y isolate the lesion (Hutchinson, 1935;True ,1938 ;Struckmeye r &Riker , 1951). Mullick (1977) andPuritc h& Jense n (page9 4i nthi sbook )hav e showntha tthe y arenon - specifically induced defense reactions toa variet y oftrauma s incitedb y mechanical injury or insecto rpathoge n attack.Thi s implies,then ,tha t normalpathogenesi s involvesspecifi c inhibition ofth e regulatory pro­ cesses (andgenes )tha ttrigge r thesereactions . Bark reactions havebee ndescribe d insevera l pine-rust systems (Table 1). Their salient features arethei rcommo nmod e of actionan d expression across differenthost-pathoge n combinations,an dth ewid e range inth ede ­ greeo f incompatibility expressed. They range continuously insiz e from microscopic (Hoff& McDonald , 1971)o rvisibl enecroti c flecks (Hutchinson, 1935; True, 1938), to sunken cankers several centimeters square.Bingha m et al. (1960)discriminate d betweenrelativel y small ('hypersensitive')an d large ('corked-out')bar k lesions,bu tther e isn o anatomical orphysiol ­ ogicalbasi s forthi s distinction.Often ,incompatibilit y isonl ypartial , aswhe nmyceliu m 'escapes'th ecollapsin g lesion and invadesperider m and healthy tissuebeyond ,whic h thencollapse s again (Hutchinson, 1935;True ,

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isB •P (B •H •H M O CL 0) ". D ft >i ra fi .fai a l race s m infectio n m fectio n s rH M -P fi OJ 'H •H fi X •rH o Ol 4-1 0 -H ^~M - IB (B rH ai ai •H o T3 V) •d fi P 0) •H •H 4-1 u -p si e 0 01 O) •-! rH M M X 18 c U 4-1 IB ai X U >, IB 0 IB (B -H iH id m ns q ra a) 4-1 OI-H ---4-1 X •p iH ft A! 14H-H fi ->W (B 4-1 fi ra ai i sra is >1 01 <44 > 3 raO) >lft S o us 4-1 rH -H -r4 MH >i P g •a-n Oi m •raH •Ç •H T> T3 4-1 > 4-1 o 01 -P fi ai c 0) ftMH •H ^- M q u .. iH IB S ->H 11)44 « O M rH fi •H 01 "rH O) -P X! a 4J fi O U 01 •H 0 4-1 S HH m o IB 0 M -H U fi U Ä-H •H id in fi 3-r4 (U H-I U fi M 0 3 •H in M HH Oi D-H iH 4-1 4-1 g is ra M O -H 4-1 P ra fi O fi HJ 01 fi «s S ufi 3-H T3 4-1 U ni ii U 3 OJ X) S fi 01 -P q m 4-1 M 01 -H 3 ft M m ai "o uo ai V4 ra <-H o -rH ai ura 10 3 0 XI U S ft U ra q U rH oi OJ O lH is bi u ËH3-H 4-1 0 X ai a) 3 3 ra o HH X oj 0 fiIB IB D O "O .C ü 1) ü 13 —-4J HH W ••H 0 X •M S M M! O) fi ra -v; c & OJ OJ fi •q13 0 <-H Œ HiMPiH »H M O 4-J ftj O M ns q O) 0 ns 'M Us 5T li. L ö co Kl 1938; Hoff& McDonald , 1972). Insuga rpine s lackingth emajo r gene for hypersensitivity described above,w ehav eobserve d. avariet y ofincompat ­ ible andcompatibl e reactions onth e same tree.Th e frequency ofbar k reactions, aswel l asthei r size,seem st ovar y amongdifferen tfamilies . These observations suggest additional complementary geneticvariabilit y in host andpathoge npopulations ,bu tproo f islacking .Th e only othertenta ­ tively identified gene forbar k reaction resistance isth e 'shortshoo t fungicidal reaction' described inwester nwhit epin eb yHof f& McDonal d (1971). Paired récessivesconfe rhypersensitivit y inbar kbu t -i ncon ­ trastt o sugarpin e -no tneedl etissues . A singlephenotypi c expressiontha tsimplifie s and.integrate scomplexl y inherited -an dperhap sunrecognize d -mechanism s ofresistanc e is 'slow rusting',whic h canb emeasure db y the apparent infection ratepe r unito f time (Vanderplank, 1963). Thisparamete r (r)i sa mor e sensitive indicator ofresistanc e than cumulative infection atan ysingl epoint .A mor e than threefold range inr value s among sugarpin e familieswa s demonstrated, comparedwit h differences incumulativ e infection of less than 20 % (Kinloch& Byler , 1981). Average rvalue s among families from slowx slo w rustingparent swer e onlyhal f aslarg e asthos e from slowx fastrustin g crosses, andonl y aquarte r of fastx fastcrosses .The'patter nstrongl y suggests additive inheritance,wit hth epotentia l forrapi d gains inresis ­ tance inearl ybreedin ggenerations . Adultplan tresistanc e iswel l known incerea l rustpathosystems ,bu t themagnitud e ofvariabilit y inthi s kind of age-related or ontogenetic resistanceha sno tbee n fully appreciated intre e rusts.Patto n (1961) showed thatgrafte d rametso fdifferen teaster nwhit epine s decreased in susceptibility tobliste r rusta sorte t ageincreased .Becaus ehi s choice ofortet swa s essentially random, the increase ofresistanc ewit h agewa s seena sa genera lpopulatio n characteristic. Insuga rpine ,however ,a n astonishing degreeo fvariabilit y insusceptibilit y was found amonggrafte d clones from sexuallymatur e ortetso froughl y the same age ina commo n garden (Kinloch &Byler , 1981). Different clonesvarie d from 0% to 100% infected, and from 0t o 50canker spe r ramet (Kinloch,unpublished) . Since allo fth eclone s inquestio nwer e from ortets highly selected forresis ­ tance, the true range of susceptibility isye tt ob e determined.Neverthe ­ less,th e amounto f infection among cloneswa s far lesstha ntha to fthei r seedling offspring.Thus , although resistance increaseswit h age generally, the degree towhic h iti sexpresse dvarie s greatly among genotypes.Th e specificmechanism s and inheritance ofontogeneti c resistance areno tknow n either,bu td ooperat e inbot hneedl e andbar ktissu e (Patton, 1961, 1967). Although oneo fth estronges t andperhap smos tvariabl e kinds ofresis ­ tance, ontogenetic resistance unfortunately mayb e the leastuseful ,sinc e iti sno t fully expressed until after atre ecro pha spasse d itsmos tvul ­ nerable growth stage.I nprogram sbase d onphenotypi c selection inwil d

124 Stands, itca n alsob e deceptive,sinc e itofte nmask s juvenile suscepti­ bility. Itnevertheles s hasvalu e instabilizin g resistance inlate r stages ofa rotatio n andreducin g levels of inoculum, especially ofrace s ofrus t virulentt oresistanc e genestha tar eeffectiv e inmor e juvenile stages.I n at least1 pine-ste m rust system (P. sylvestris - Peridermium pini), the ontogenetic relationship isreverse d and susceptibility increaseswit hag e (Vande rKamp , 1968). Slow rusting andontogeneti c resistance appeart ob e inherited indepen­ dentlyo f each other and ofmajo r gene resistance, since parent-offspring relationships forthes etrait s formn o discernible pattern (Kinloch & Byler, 1981).

FUSIFORM RUST - SOUTHERN PINES

Widevariatio n inresistanc e to fusiform rusto fsouther npine si s amply documented (seeZobel ,pag e41 7o fthi sbook) . Although themecha ­ nisms andexpressio no fresistanc ehav ebee nstudie d intensively, no spe­ cific geneshav eye tbee nidentified .Mille r et al. (1976)foun d thatmos t resistance ofyoun g slashpin e seedlingswa s expressed inbar ktissue s as hypersensitivity. Bothth e size and rateo f lesiondevelopmen tvarie dcon ­ siderably, from earlynecrosi s ofonl y afe wcel l layers inth ecorte xt o incipient formationo fgall swhic h subsequently aborted ina mas s ofne ­ crotic tissue surrounding the 'reactionzone' .Occasionally , the fungus escaped the reaction zone andresume d activeparasitism . These resistant reactionswer e observed onbot hrelativel y resistant and susceptiblewind - pollinated families,bu twer emor e frequento nth e former.Anothe r family- related expressiono fresistanc ewa s complete absence of incipient symptoms and anyevidenc eo ftissu epenetratio n orinfection .Sno we t al. (1975,an d page 243 ofthi sbook )measure d significantvariatio n amongdifferen t fami­ liesi nth eshap ean dgrowt hrat eo factiv egalls .Thus , acomplet espec ­ trumo fbot hqualitativ e andquantitativ e hostresponse s isapparen ti n thispathosystem . Equally dramatic variation inpathogenicit y of inoculum derived from different single gall isolates and evenmonoaeciospor e isolates froma single gallha sbee ndescribe d (Powerse t al., 1977;Powers ,1980 ;Sno we t al., 1975). Inmos t cases studied, family x isolatevarianc ewa shighl y significant,wit h frequentran k changes among either families orisolates . Differential interactions ofthi s kind arecharacteristi c of complementary gene-for-gene relationships,whic h almostcertainl y exist inthi spathosys ­ tem. Similar relationships apparently existbetwee n certain Mediterranean pines andC . flaccidum (Mittempergher& Raddi , 1977).

125 GENETIC VARIATION IN NATIVE AND EXOTIC PATHOSYSTEMS

Patterns ofvariatio n inresistanc e inhost s of fusiform rust andwhit e pinebliste r rustar e fundamentally different,an doffe rusefu lparadigm s ofth epotentia l genetic resources available inhos tpopulation s forcopin g withnativ e andexoti cpathogens .Th epathogen s areclosel y related taxonomically, andrelativel y minor differences distinguish thedisease s in their lifecycle ,etiology , andepidemiology . But C. q. f. sp. fusiforme coevolvedwit h its southernpin ehost swherea s C. ribicola was recently introduced (ca.1900 )fro mAsi avi aEurope . InAsia ,th elatte r isendemi c onwhit epine s relatedb y commonprogenitor s toNort hAmerica n species,bu t theAsiati c andAmerica n species have longbee ngeographicall y separated (Bingham, 1977). Inth e absence ofcoadaptiv e selectionpressures ,th e NorthAmerica nwhit epine sma y differ from either theirAsiati c or southern pine relatives inth e kinds ofresistanc e genes (majoreffect ,mino ref ­ fect), their frequencies,an deve nnumber s ofloci .Thi s is strongly sug­ gestedb y therelativ e lack ofvariabilit y inresistanc e tobliste r rusti n highly selected families ofwester nwhit epin e comparedwit h thehig h levels ofresistanc e found ina narbitraril y chosenbul k seed loto fArman d pine,whic h isnativ e toAsi a (Hoff& McDonald , 1975). Evenmor e striking aredifference s inpattern s of infectionbetwee n southernpine s andNort h Americanwhit epine s underepidemi c conditions forthei r respectiverusts . Anexampl e isgive n inFig .1 ,bu t itunderstate s thedifference s because the sugarpin eparen tpopulatio nwa s intensively selected for freedom from rust insevera l heavily infected stands,wherea s the loblollypin e parents were randomly chosen from asingl ewil d stand.Eve n so,muc hmor evariabil ­ ity inresistance ,distribute d nearly continuously, iseviden t inth e loblollypin e families.Th e relatively small degree ofresistanc e inth e sugarpin e families is largely duet o asingl e dominantgen e atlow ,fre ­ quency inth eparen tpopulation ,whic h isapparen ti nth e trimodal dis­ tribution ofoffspring .Th e situationwit h sugarpin e andothe rNort h Americanwhit epine s isi nsom eway s analogous toth e 'vertifolia' effect inth epotato-late-bligh tpathosyste m describedb yVanderplan k (1963), whereinhorizonta l resistance conditioned bypolygene swit h individual minor effects hasbee n lost incommercia l cultivars through selection primarily formajo r genes.Los s ofpolygene s reduces the apparentcomplex ­ ityo fhost-parasit e interactions,furthe rhighlightin g theeffect s of major genes.Thus , iti sperhap sno t surprising thatmajo r geneshav ebee n more clearlyperceive d inNort hAmerica nhost so fbliste r rust,whic har e relatively depauperate invariability , than innativ e hosts of fusiform rust,wher e theyma yb e obscured ina napparentl y largebackgroun d of quantitative variability.

126 Percent of families 30 C. fusiforme- P. taeda 20 54 families _,

10 — n 5 15 25 35 45 55 65 75 85 95 n 50 C. ribicola- P. lambertiana 40 124 families 30

20

10 n 5 15 25 35 45 55 65 n75 85 95 Percento ftree sinfecte d

Figure 1. Distribution of full-sib familymean s in infection ofloblolly - pineb y fusiform rust ando f sugarpin eb ybliste r rust,afte r 4year s in disease gardens inGeorgi a andCalifornia ,respectively . On each site alternate hostswer e interplanted among test families and infectionoc ­ curred naturally. (Data on loblollypin e fromKinloc h & Stonecypher,1969 ; on sugarpin e from Kinloch, unpublished.)

CONCLUSION

Since diversity ofresistanc e genes isth emos teffectiv emean s of stabilizingpathoge npopulation s (Browning et al.,pag e 371 ofthi sbook ) maximum protection ofhos tpopulation s willb epossibl e whenal l genes for resistance/virulence and their frequencies are identified. Themai n focus ofcurren t tree improvementprogram sha sbee no nprogen y testing forrela ­ tive levels ofresistance .Whil e effective forthi spurpose ,th eus e of open-pollinated families andheterogeneou s sources ofinoculu m isunlikel y toresul t inth emor eprecis e identification of resistance genesneeded . Because treebreedin gb y controlled pollination is soexpensiv e and time-consuming,mor e rapidprogres smigh tb emad eb y shifting emphasis from hostt opathogen . Inth e Cronartium rusts,fo rexample ,th e spore stage infective onpin e ishaploid , sotha tth ephenotyp e ofth e lesion (lowo r high infection type)als o denotes the genotype ofth e spore that induced it

127 for avirulence or virulence. Thus, an observed 1:1 segregation ratio of lesion types induced by basidiospores derived from mpnoaeciospore or mono- urediospore clones would identify a gene locus in the pathogen, and - by deduction from the gene-for-gene theory - a corresponding locus for resis­ tance/susceptibility in the host. The eventual goal would be to assign resistance/virulence formulae to parent trees selected for breeding pro­ grams, so that the genes identified could be deployed to maximum effect in commercial crops through appropriate matings. At the same time, important insights would be gained into the genetic structure of host-parasite inter­ actions in wild populations.

REFERENCES

Bingham, R.T., 1966. Breeding blister rust resistant western white pine. III. Comparative performance of clonal and seedling lines" from rust free selections. Silv. Genet. 15: 160-164. Bingham, R.T., 1977. Breeding white pines resistant to the white pine blister rust disease. In: R. Bogart (Ed.): Genetics lectures, Vol. 5, Oregon State Univ. Press, Corvallis. p. 33-44. Bingham, R.T., R.J. Hoff &G.I . McDonald, 1971. Disease resistance in forest trees. Ann. Rev. Phytopath. 9: 433-452. Bingham, R.T., R.J. Hoff &G.I . McDonald, 1973. Breeding blister rust resistant western white pine. VI. First results from field testing of resistant planting stock. USDA For. Serv., Res. Note INT-179. Ogden, UT. 12 p. Bingham, R.T., A.E. Squillace &J.W . Wright, 1960. Breeding blister rust resistant west­ ern white pine. II. First results of progeny tests including preliminary estimates of heritability and rate of improvement. Silv. Genet. 9: 33-41. Griggs, M.M. &R.J . Dinus, 1977. Patterns of fusiform rust increase their implications for selection and breeding. Proc. South. For. Tree Improv. Conf. 14: 43-52. Hepting, G.H., 1971. Diseases of forest and shade trees of the United States. USDA For. Serv. Handbook 386. Washington, D.C. 658 p. Hoff, R.J. &G.I . McDonald, 1971. Resistance to Cronartium ribicola in Pinus monticola: short shoot fungicidal reaction. Can. J. Bot. 49: 1235-1239. Hoff, R.J. &G.I . McDonald, 1972. Resistance in Pinus armandii to Cronartium ribicola. Can. J. Forest Res. 2: 303-307. Hoff, R.J. &G.I . McDonald, 1975. Hypersensitive reaction in Pinus armandii caused by Cronartium ribicola. Can. J. Forest Res. 5: 146-148. Hoff, R.J. &G.I . McDonald, 1980. Resistance to Cronartium ribicola in Pinus monticola: reduced needle spot frequency. Can. J. Bot. 58: 574-577. Hutchinson, W.G., 1935. Resistance of Pinus sylvestris to a gall-forming Peridermium. Phytopathology 25: 819-843. Jewell, F.F. &D.C . Speirs, 1976. Histopathology of one- and two-year-old resisted infections by Cronartium fusiforme in slash pine. Phytopathology 66: 741-748. Kinloch, B.B. &J.W . Byler, 1981. Relative effectiveness and stability of different resis­ tance mechanisms to white pine blister rust in sugar pine. Phytopathology 71: 386-391. Kinloch, B.B. &M . Comstock, 1980. Cotyledon test for major gene resistance to white pine blister rust in sugar pine. Can. J. Bot. 58: 1912-1914. Kinloch, B.B. &M . Comstock, 1981. Race of Cronartium ribicola virulent to major gene resistance in sugar pine. Plant Disease 65: 604-605 Kinloch, B.B., Jr. &J.L . Littlefield, 1977. White pine blister rust: hypersensitive resistance in sugar pine. Can. J. Bot. 55: 1148-1155. Kinloch, B.B., Jr. &R.W . Stonecypher, 1969. Genetic variation in susceptibility to fusiform rust in seedlings from a wild population of loblolly pine. Phytopathology 59: 1246-1255. Lundquist, J.E., 1979. The pre-gall symptoms of fusiform rust. Unpub. Ph.D. thesis. Univ. Georgia, Athens. 115 p. McDonald, G.I., 1978. Segregation of "red" and "yellow" needle lesion types among monoae- ciospore lines of Cronartium ribicola. Can. J. Genet. Cytol. 20: 313-324. McDonald, G.I. &R.J . Hoff, 1970. Resistance to Cronartium ribicola in Pinus monticola:

128 earlysheddin go finfecte dneedles .USD AFor .Serv .Res .Not eINT-124 . McDonald,G.I .& R.J .Hoff ,1971 .Resistanc e toCronartiu m ribicolai nPinu smonticola : geneticcontro lo fneedle-spots-onl y resistance factors.Can .J .Fores tRes .1 : 197-202. McDonald,G.I .& R.J .Hoff ,1975 .Resistanc e toCronartiu m ribicolai nPinu smonticola : ananalysi so fneedl espo ttype san dfrequencies .Can .J .Bot .53 :2497-2505 . Miller,T. ,E.B .Cowling ,H.R .Powers ,Jr . &T.E .Blalock ,1976 .Type so fresistanc ean d compatibility inslas hpin eseedling s infectedb yCronartiu mfusiforme . Phytopathology66 :1229-1235 . Mittempergher,L .& P .Raddi ,1977 .Variatio no fdivers esource so fCronartiu mflaccidum . Eur.J .For .Path .7 :93-98 . Mullick,D.B. , 1977.Th enon-specifi c natureo fdiseas ei nbar kan dwoo ddurin g wounding,insec tan dpathoge nattack .Rec .Adv .i nPhytochem .11 :395-440 . Patton,R.F. ,1961 .Th eeffec to fag eupo nsusceptibilit yo feaster nwhit epin et oinfec ­ tionb yCronartiu m ribicola.Phytopatholog y 51:429-434 . Patton,R.F. ,1967 .Factor si nwhit epin ebliste rrus tresistance .Proc .14t hIUFR OCon ­ gress,Sept .4-9 ,1967 .Munich ,Germany .Par t3 ,Sec .2 2an dIntersectiona lWorkin g Group22/24 .p .876-890 . Powers,H.R. ,Jr. ,1980 .Pathogeni cvariatio namon gsingl eaeciospor e isolateso fCronar ­ tiumquercuu mf .sp .fusiforme .For .Sei .26 :280-282 . Powers,H.R. ,Jr. ,F.R .Matthew s& L.D .Dwinell ,1977 .Evaluatio no fpathogeni cvariabili ­ tyo fCronartiu mfusiform e inlobloll ypin ei nth esouther nUSA .Phytopatholog y67 : 1403-1407. Quick,C.R. , 1966.Experimenta linoculatio no fponderos apin ewit hwester ngal lrust . PlantDis .Rep .50 :550-552 . Snow,G.A. ,R.I .Dinu s& A.G .Ross ,1975 .Variatio ni npathogenicit y ofdivers esource s ofCronartiu m fusiformeo nselecte dslas hpin efamilies .Phytopatholog y 65:170-175 . Struckmeyer,B.E .& A.J .Riker ,1951 .Woun dperider m formationi nwhit epin etree s resistant tobliste r rust.Phytopatholog y41 :276-281 . True,R.P. ,1938 .Gal ldevelopmen to nPinu ssylvestri sattacke db yth eWoodgat e Peridermiuman dmorpholog y ofth eparasite .Phytopatholog y28 :24-49 . Vande rKamp ,B.J. ,1968 .Peridermiu mpin i (Pers.)Lev .an dth eresin-to pdiseas eo f Scotspine .I .A revie wo fth eliterature .Forestr y41 :189-198 . Vanderplank,J.E. ,1963 .Plan tdiseases :Epidemic san dcontrol .Academi cPress ,Ne wYork . 349p . Ziller,W.G. ,1974 .Th etre erust so fwester nCanada .Can .For .Serv .Publ .1329 ,Dep .En ­ viron.,Victoria ,B.C .

129 'ÏÏT

Resistance mechanisms in interactions between poplars and rust1

2 2 3 4 R. Siwecki ,A .Werne r , Z. Krzan &F .Mlodzianowsk i

ABSTRACT

Predisposing factors on poplar leaves to infection by Melampsora larici-populina and resistance mechanisms involved in the penetration and colonization of poplar leaf tissues by the uredinial stage have been discovered. Disease incidence was estimated during 4 consecutive year.s, and the degree of resis­ tance of various poplar clones growing in a stool bed was de­ termined. Studies on germination of urediniospores and growth of germ tubes on poplar leaves showed that these processes are determinants of varietal resistance of hosts. Histological in­ vestigations of the penetration and pre-penetration growth of the pathogen revealed that in the incompatible association the host cells actively react against the pathogen. As an attempt to identify different resistance mechanisms against poplar rust a model is presented of the studies of host-pathogen interac­ tions which are in progress or are contemplated in the near future.

INTRODUCTION

Poplar plantations, regions of natural occurrence of the more important poplar species, and rich collections of poplar cultivars maintained by var­ ious research institutions are presently under constant biological study aimed at the determination and comparative analysis of variability in re­ sistance or susceptibility of poplars to infection by the common poplar rust fungus, Melampsora larici-populina Kleb.

1. This work has been supported partially by grant 11/15 from the Polish Academy of Sciences and partially by grants FG-po-298 and P-109 from the U.S. Department of Agriculture under PL-480. 2. Polish Academy of Sciences, Institute of Dendrology, 62-035 Kórnik, Poland. 3. Tatra National Park, 34-500 Zakopane, Poland. 4. A. Mickiewicz University, Institute of Biology, 61-713 Poznan, Poland.

130 The interdisciplinary studies involvingphytopathology ,genetics , physiology, andbiochemistr y create agoo dbas e forth e selection ofpopla r clones forresistanc e inorde rt orecommen dthe m forcultivatio n and further breeding.A tth e sametim eresult s ofstudie s onth einteractio nbetwee n both resistant and susceptible hosts andth epathoge nma y constitute aba ­ sis forth e identification ofsom ebiologica l resistancemechanisms . Various studies conductedb y agrou p ofscientist s atth e Institute of Dendrology, PolishAcadem y of Sciences inKórni kpermitte d adetaile dde ­ scription ofth e interactions betweenpoplar s and rust (Siwecki, 1978). Pre-infection factors existing onth epopla r leafan dresistanc e mechanisms involved inth epenetratio n andcolonizatio n ofpopla r leaftissue sb y M. larici-populina were discovered. Alsopermitte d was evaluation ofth e genetic resistance ofpoplar s toinfectio nb y therus t (Siwecki, 1981).

LIFE CYCLE OF THE PATHOGEN AND ESTIMATION OF DISEASE

The life cycleo f M. larici-populina on larch (Larix decidua) andpop ­ lars from sections Tacamahaca and Aigeiros and intra-o r inter-sectional hybridsha sbee ndescribe d withparticula r consideration givent o themos t pathogenic stage ofth eparasit e topoplars ,th euredinium . Characters of spore size,an dparticularl y thoseo furediniospores , cannotconstitut ea basis forth eidentificatio n ofrus t species onvariou spoplars .Ther e are probablynumerou sphysiologica l races ofth epathogen ,conditione d byth e response andvariabilit y ofth ehosts ,namel y thenumerou s poplar cultivars grown around theworl d (Krzan, 1981a). Optimalgerminatio n ofurediniospore s anddevelopmen to fger m tubeso n the lower surface ofpopla r leaves takesplac e at10 0 %relativ e humidity and anai rtemperatur e of+1 6 °C (Krzan, 1980). Insuc h conditions urediniospore germ tubes areabl e toinfec tth emesophyl lo fpoplar swithi n 2-4 hours.Successfu l infections,however , aredependen t onnumerou smech ­ anisms thathinde r orhaste n thisprocess . The studies conducted onultrastructur e permitted ust odescrib e in greater detail some ofth e stages ofth epathoge n and follow theultra - structural changes thatoccu r inpopla r clones differing inresistanc e (Miodzianowski& Siwecki , 1976,1978 ;Miodzianowsk ie t al., 1978). During 1975-1978 rust incidencewa s estimated andth e degree ofresis ­ tance torus twa s determined for26 8popla r clones growing ina stoo lbed , eachclon erepresente db y 5stump s (Krzan, 1981b). Observations included data on theoccurrenc e ofuredinia l and telial stages onpopla r leaves.Th e following six-point scale of infectionwa sused : 1.Al l leaves onth e stump completely freeo frus t sori -Immun e(I) . 2. Infrequent sori ofuredinia l ortelia l stages onon e oronl y afe w leavespe r stump -Ver yResistan t(VR) . 3.Mor e numerous sori onman y leaves.N onecrosi s -Resistan t(R) .

131 4. Sori commono n all or almostal l leaves.Localize d necrotic spots- MediumResistan t(MR) . 5.Sor icommo n on all leaves,numerou s necrotic spots orpatches , slight defoliation -Susceptibl e(S) . 6.Sor icommon ,almos tth eentir e leafblad enecrotic ,sever edefoliatio n- Very Susceptible(VS) . Observationswer econducte d for4 consecutive years atsevera ltime sdurin g theyea rwhe n theuredinia l andtelia l stageswer e occurring. From these observations foreac hclon e amea n infection index (Mil)wa s calculated, whichcharacterize d thedegre eo fresistanc e and susceptibility tonatura l rustinfection . Table 1 (fromdat ao fKrzan ,1981a ,b )show stha tther ewa sgrea tvari ­ ation inth e differentyear s inestimate s ofth edegre e ofrus tresistanc e ofth eclones .Durin g these4 year si twa sestablishe d thatth e finalex ­ tento frus tdevelopmen twa sdetermine db y thepatter no fprecedin g weather conditions.Fo r example the influence ofsom e climatic factors,especiall y relativehumidit y and airtemperatur e duringJun e andJuly ,wa s alsovari ­ ableove r thisperio d (compare Table 1wit h Fig.1) . Inhi s doctoral investigations Krzan (1980,1981a ,b )studie dresis ­ tancemechanism s andconfirme d theexistenc eo fecologica l andgeneti cfac ­ torswhic h influence resistance or susceptibility ofpopla r clones torus t andwhic hmak epossibl e selection forthi strait .

A 4 ' Mil RH % 3.00 •74 /^--—.-_ RH / ^. / Mil -72 2,50 / T 70 RH A' •68 N\ / / 2.00- "66 M"\ / ' / 64 1.50- -62 \ v' / -60 1,00- \^^^

0,50-

n nn 1975 1976 1977 1978

Figure 1. Therelationshi pbetwee nseverit y ofrus tinfectio no npopla r leaves (Mil), relative airhumidit y (RH),an d airtemperatur e (T)i nJun e andJul ydurin g 1975-1978 (FromKrzan , 1980).

132

Il co o rH in en o n co o CM G o co r- in o, en o 3 m O i-i o M CU co co iD t^ if CM

O •* O co CO CM O rH PO CO H O -aP *s H O) o en CM CM o en ^0 co m en (N en CO in ' 3 o CM UI 2 V t- in co H en a lß co o r- co o rH CM M U 00 i-H •* en r-H CM tO CU CU ra CM co in ^f l~- CM O >1 ,0 H co co O o S-S rH CU G 1-1 > •H O oo CM in co cn iD co •rH o m O en rH rH CO •P T3 3 CU 2 O CU 3 ra iû G ü o G o o 0) I i I I M •43 Cn o co en UI O G o o o CU 'M en UI S •H u •H UI -^ i> •P •P 3 CU 0) a e a w ra Ol i -rH o rB •Q ra >-i UI e >i 3 o 3 3 ja ui > C/3 oo 133 SOME PRE-INFECTION FACTORS ON THE POPLAR LEAF

Infectiono fpopla r leavesb y rustaeciospore s andurediniospore s in specificmicroclimati c conditions existing onth e leaf surface isdeter ­ minedb y the structure ofth e stomata, structure ofth e leaf surfaces,an d stomatal diffusion resistance andstomata lmovement .Th ecours e ofth e transpirationproces s insevera l resistanto r susceptible poplar cloneswa s studiedb y Siwecki& Przyby l (1981). Comparisonswer emad eo fdetache d and undetached non-infected poplar leavesunde r different experimentalcondi ­ tions.Th epopla r cloneswer echaracterize d byvariabl e frequency and size ofstomat ao nth e adaxial and abaxial leaf surfaces (Table2) . Therat eo ftranspiratio n ofdetache d leavesmeasure d ina win d tunnel and calculated onth ebasi s of leafdr yweigh twa s decidedly lower forth e resistantpopla r clones than forsusceptibl e ones (Table 3).Thi s ispar ­ ticularly emphasized by the rate oftranspiratio n inth e time intervals 0-15 min and 15-30min . Results ofmeasurin gtranspiratio no fundetache d leaves inth eporome - ter arepresente d onFig .2 .Th emos t interesting result istha t inth e morninghour s the 2susceptibl e clones P. x pet rowsky ana and P. tacamahaca demonstrated amor e rapid water loss inbot hwin d tunnel andporomete rex ­ periments than inth e afternoon orevening . Thisphenomeno n suggests alonge rperio do fstomata lopenin g inth e morning and analtere d cycleo fwate rmanagemen t inthes e clones compared totha ti nsom eresistan tclones ,fo rexampl e P. deltoïdes B-60.Th e entire

Figure 2. Water lossb yundetache d leaveso fresistan t ( )an dsuscep ­ tible ( )popla r clones at1 0a m (A),2 p m (B)an d6 p m (C). P. del­ toïdes x p. nigra 490-1 (DN), P. 'Serotina dePoitou ' (SP), P. deltoïdes B-60 (D), P. trichocarpa 404.(T) , P. x petrowskuana (P)an d P. tacamahaca (TA)(Fro m Siwecki &Przybyl , 1981).

134 Table 2. Mean stomatal frequency andmea n sizeo fstomat ao n adaxial and abaxial leafsurface s (From Siwecki &Przybi,1981) .

Clones Stomatal frequency Mean length and Mean (cm ) width ofstomat a (pm) Infection Index (Mil) Adaxial Adaxial Abaxial Abaxial

Resistant clones P. deltoïdes 1527 1± 36 1 27.30 x 16.94 B-60 (11892) 22 589± 43 7 28.07 x 17.50 0.00 P. deltoïdes ssp. angulata 13 664± 26 5 294 7x 18.48 (11621) 2726 3 ±51 4 28.91x 18.27 0.00 P. deltoïdes x P. nigra 1041 5± 21 5 491-1 (12299) 28 48x 1 6 12 0.11 30 088± 57 2 25 94x 1 5 50 P. trichocarpa 5 997± 11 2 32 24x 2 5 17 1200 (404) 10 764± 22 0 33.56x 17.55 0.96 P. 'Serotina de Poitou' (11309) 1104 6 ±34 2 27.37 x 16.17 1414 6± 34 1 32.55 x 20.09 1.05 P. deltoides x P. nigra 1051 3 ±21 7 490-1 (11888) 25.40 x 14.79 1488 3 ±32 7 26.50 x 16.23 1.17

Susceptible clones 8 552 ± 308 26 39 X 15 19 P. nigra PW3 21 444 ± 427 29 61 X 18 54 1.38 P. x petrows- 7 137 ± 252 27 85 X 16 73 kyana (5163) 20 673 ± 596 27 09 X 16 03 2.12 P. trichocarpa cl. 30 6 237 ± 309 32 05 X 19 52 19 033 ± 373 37 02 X 23 10 2.13 P. tacamahaca (3747) 6 880 ± 260 26 60 X 15 47 16 718± 34 7 27.58x 16.16 3.03

135 Table 3. Meantranspiratio n rateso fdetache d leaveso frust-resistan t and rust-susceptible poplar clones inth ewin d tunnel ascalculate d onth e basis of leaf area and dryweigh ti nth emornin g and afternoon (From Siwecki& Przybyl , 1981).

Clones Transpiration

mg •c m mg •m g dryw t per 15mi n per 15mi n

p.m. a.m. p.m.

Resistantclone s P. deltoïdes B-60 (11892) 0.1608 0.1117 0.0566 0.0398 P. deltoïdes ssp. angulata (11621) 0.2150 0.1622 0.0745 0.0574 P. deltoïdes x p. nigra 491-1 (12299) 0.2336 0.1013 0.0851 0.0400 P. 'Serotinad ePoitou ' (11309) 0.1599 0.Ö834 0.0573 0.0301 Mean 0.1923 0.1146 0.0684 0.0418

Susceptible clones P. nigra PW3 0.2279 0.1462 0.0742 0.0495 P. x petroviskyana (5163) 0.1835 0.0941 0.0765 0.0377 P. tacamahaca (3747) 0.2667 0.1539 0.0893 0.0534 P. trichocarpa (11651) 0.3469 0.2269 0.1118 0.0799

Mean 0.2562 0.1552 0.0879 0.0551

aspecto f leafwate r relations andwate rmanagemen t inth e2 susceptible poplar clones favors infectionb y germ tubes ofrus turediniospores .Also , thewate r relations ofthes epoplar s governpre-infectio nmechanism s that affectan d facilitate thenex tstep s incompatibl e host-pathogenrelation ­ ships. Werner (1981a), according tohi s studies on 59popla r clones,claime d thatth enumbe r of stomatape runi to f leaf surfacewa sno tcorrelate d with frequency of infection siteso n leaves affectedb y therust .However ,th e frequency of stomata on alea f surface could have some influence onth e number ofcontact sbetwee n hyphae and stomata. Inhi s opinionth ecours eo f thepre-penetratio n growth ofth epathoge n isdependen t on severalpre - infection factorswhic h limitmarkedl yth eintensit y ofinfection .Th edi ­ mensions ofth e stomata, sculpturing ofth e leafsurface ,anatomica lstruc -

136 Table 4. Meandimension s ofth esurfac e ofaréole so fresistan t andsuscep ­ tiblepopla r clones (FromWerner , 1981a).

Clones Surface ofaréole s (mm2)

Resistant clones P. deltoïdes B-60 (11892) 0.138 P. deltoïdes ssp. angulata (11621) 0.114 P. maximowiczii (4544) 1.116 P. 'Serotinad ePoitou ' (11309) 0.089

Susceptible clones P. nigra PW3 0.231 P. x petrowskyana (5163) 1.189 P. trichocarpa (11651) 1.277 P. balsamifera (8348) 1.975

tureo fth e guard cells and subsidiary cells all are factors determining thebehavio r ofth eger m tubeso nth e leaves and appressorial formation overth e stomata. Werner (1981a)indicate d thatth e structure of thevascula r bundles and thedimension s ofth e aréolesca ndetermin e thesiz eo fth e rustcolonie s developing onpopla r leaves (Table4) . Incompatibl e associations themesophyl l cells inhibitth e development ofth e fungus. Incompatibl e associations,however , astimulatio no f fungal growth may result.O nth ebasi s of ananalysi s ofcompactnes s ofpopla r leafmesophyl l we canconclud e thatth e development ofth epathoge n inth e tissue ofth ehos ti sdetermine d by areactio n ofth etissu e that isth e sum ofth e reactions of allth e individual cells.Inhibitio n ofgrowt ho f thepathoge nwa s stronger inresistan tpoplar swit h acompac tmesophyl l (e.g. P. deltoïdes B-60)tha n inthos ewit h ales s compactmesophyl l (P. maximowiczii). Onth e otherhand , insusceptibl epoplar swit h acompac t mesophyll theeffec t ofpathoge n growth stimulationwa s stronger thani n poplarswit h ales s compactmesophyll .Therefore , agreate r compactness of themesophyl l increases resistance inresistan tpoplar s and increases sus­ ceptibility insusceptibl e ones. Werner (1981a,b )showe d thatsevera l important resistance mechanisms ofpoplar s to rustbecom e apparentdurin g thepenetratio n andpost-penetra ­ tiongrowt h ofth epathogen . Someo fthos emechanism swer epresente d inth e paperb y Siwecki& Werne r (1980).

137 THE EPIPHYTIC PHASE OF PATHOGEN DEVELOPMENT

The germinationo furediniospore s of M. larici-populjna and thebeha ­ vior ofger m tubes aredifferen to nresistan t and susceptiblepoplars .I n the resistantpopla r clones (P. maximowiczii, P. deltoïdes B-60)th epro ­ cesses ofurediniospor e germination andger m tube developmentwer e dis­ tinctly inhibited during the firsthour s after inoculation. Some chemical factorspresen t onth e surface ofth epopla r leaveswer e responsible for this inhibition (Krawiarz &Werner , 1978). The structure ofth e stomatal surface andcertai n anatomical features ofth e guard and subsidiary cells constitute asyste m of factors that conditionth eappropriat e 'recognition'o fth estomat ab y thehyphae . Such features as:substantia l cuticular thickenings presento nth eex ­ ternalwall s ofth e subsidiary cells,depression s between theguar d cells and subsidiary cells,a swel l asth e small diameter ofstomata l pores and the large external ridges,al lhinde r the free growth ofhypha e over the stomata andthei rpenetration .Th ephysica l characteristicscondi ­ tioning recognition ofth e stomatab y thehypha e induceth e formationo f appressoria over the stomata. Insusceptibl e poplars thatwer echaracter ­ izedb y large stomata,smal lexterna l ridges andweakl ymarke d sculpturing ofth e leaf surface (e.g.i nP . trichocarpa), the germ tubes ofuredinio ­ spores accomplished penetration ofth e stomatawithou t forming appressoria over them.O nothe rpoplars ,bot h susceptible and resistant ones, theger m tubes formed appressoria overth e stomata.Thei rnumber ,an d asa conse ­ quenceth enumbe ro fpenetratio n sites,wa s decidedly greatero nsuscep ­ tiblepoplars .O nth e otherhand ,th e smallernumbe r ofpenetratio n sites inth e resistantpoplar swa s aconsequenc e ofth epoore r 'recognition'o f poplar stomatab y germ tubes.O nth e leaves ofthes epoplar s frequently growth ofth ehypha e near stomata orove r themwithou t appressoria forma­ tionwa s observed. Thisphenomeno n canb e explainedb y the facttha tth e small stomata ofth e resistantpoplar s couldno t induce appressoria forma­ tion andtherefor e inth emajorit y ofcase s remain 'unrecognized'.Whe n theywer e 'recognized', thehypha e alwaysproduce d appressoria overth e stomata.

PENETRATION OF THE PATHOGEN INTO THE LEAF

Withpoplar s resistant torus tdistinc treaction s ofstomat a toth e presence ofth epathoge n inth e stomatalpor ewer e observed.Thes ereac ­ tionswer emad evisibl eb y the strong fluorescence ofth eguar d cells and spongyparenchym a cells,afte rbein g labelledwit h afluorescen tbrightene r (Rohringer et al., 1977). This fluorescence indicated the formation ofcom ­ pounds ofphenoli c character intha tregion .Th e considerable speed ofthi s reaction allows us toclassif y ita s atypica l hypersensitive reaction.

138 Thisconfirme d the formation ofphenoli c compounds.Here ,th e appearance of phenolic compoundswa s associated with thedyin g ofpathoge nhypha e and hostcell s inth evicinit y ofth e stomata. Inspit eo fdifference s in opinionconcernin g therol e thathypersensitiv e reactionspla y inth eac ­ tive defense ofa plan t against apathogen ,th e rapidity ofthi s reaction inresistan tpoplar s (P. deltoïdes B-60)alway s resulted ina complet ein ­ hibition ofth epathoge nwithi n the substomatal cavity.Th e same reactions also tookplace ,bu twit h some delay,i nth eothe rpoplars .However ,i n these casesthe yneve r led toth ecomplet e cessation ofgrowt h ofth e pathogen inthi searl y stage ofit sdevelopment .Therefore ,w e assumetha t 'overcoming'o fth edefens e reaction ofth ehos tb y thepathoge nwithi n the substomatal cavity represents theke y factor inth eestablishmen t of a compatible relationbetwee n thehos t and thepathoge n and determines the subsequentprogres s ofinfection .

COLONIZATION OF HOST TISSUES

In P. deltoïdes B-60,an dt oa lesse rexten t inth eothe r 2resistan t poplars (P. deltoïdes ssp. angulata and P. maximowiczii), after theinfec ­ tionhypha e entered themesophyl l thegrowt h ofth epathoge nwa s stopped at the firstcontac t ofth e infectionhyph awit h themesophyl l cell, inth e phaseo fhaustoriu mmothe r cell formation, during thehaustoriu m formation, orjus tafte r that.Th e cessation ofgrowt h ofth epathoge nwa s alwaysas ­ sociatedwit h thedeat h ofth e fungus and ofth e adjacenthos tcells .Thes e were typicalnecroti c reactions,an dwhe n they appeared inlarg e areaso f themesophyll ,the ywer evisibl e asnecroti c spots atth epoin to finfec ­ tiono fth eleaf . InTabl e 5host-pathoge n interactions during the infectionproces s of poplar leavesb y the rust arepresente d descriptively.

NECESSITY FOR FUTURE STUDIES ON RESISTANCE MECHANISMS

Somepropose d trends inbiologica l research inorde r toexplai n there ­ sistance mechanisms inth econfrontatio nbetwee npoplar s and rustar epre ­ sented inFig .3 , according toth epatter n given inth epape r ofKaufman n & Wellendorf (1979). Thesekind so f studies shouldb eperforme d inth e field or laboratory, orboth ,o npopla r clones,hybri d families,an dprove ­ nances. Possible explanations ofth evariabilit y inresistanc e ofrust - infected poplars should arise from suchstudies . Clones selected forresistanc e or susceptibility shouldb e thebasi c material for investigations ofth e resistancemechanisms .Heritabilit y (h2) ofth e features characteristic ofresistanc e canb e estimated fromobserva ­ tions ofth e degree of infection ofparen ttree s andthei rhybri dprogen y (Siwecki, 1981).

139 I fi fi O 0) •H lP O O O O (U P 10 t0i Oi Oi o» fi -H ft 1) rH p p T3 >i >i >i >i Dl-H > > > > I 0) p 1 £1 1 , s fi fi fi rH O 01 u 10 & u o o I I 4-1 ï ID fi fi fi -H •H U) X! +J 4-1 +J 10 •P ü OOO 4-1 fi 0) « M M ui O O p P 4-1 fi 0) O O •H •H fi fi O 1-4 fi m DlXi P a) 4-1 10 o fi o P O 'M ft ID Dip M O rH P > en 4-1 4-1 P UftI G MHO fi Dl

a I 4-t I 10 •H fi O UI I P d) O) (0 10 (0 T3 U (1) p -H S fi p e H Dl ft P P O P oo ^ O CM rH 00 Ol o ft pu P <0 U) 4-1 p O U) in 00 co r~- •H >* o X! H W fi O en •H o P n id rH rH ro 4) +j M *—CM« ro — ^-v ai r-H w ro G co 3 lO .—» a. 1s UI O rH W w O rö W IH 11 O a U (D tu "H aj 3 10 »P te i •a ti S •fiH rH a, 0& 0 "M +J-M •'S g p XI !s o S fi 0 O S3 0 -H ro +J •q ns ro +J 4J •—• 'N p P 0) Ü W is P r-H rH rH X 0) ft O!•*l a, *H r-1 0) W U)

MELAMPSORA POPULUS GENOTYPES LARICI-POPULIMA

CONFRONTATION 1

HOST - PATHOGEN INTERACTIONS

RESISTANCE UREDINIUM STAGE IMMUNITY (urediniospores) LEAF CHEMICAL LEAF EPIPHYTIC ZL. STRUCTURE INHIBITORS MICROORGANISMS SELECTION Laboratory inoculation I j PhysiologicJ Axenic j after Size of rrn. by aeciospores Structure On Bacterial j t races , , CU|tures j natural infections aréoles & & urediniospores of surface jstomLaliJ^'"! ! * fu"9f' ' vascular TI tah b m surfaces bundles I of leaf | Ichamoers! ! ' " ! ! ™S \ i Degree of Resistance I Virulence l Selection clones I Movements | At infected I ' S aggressivness I | of stomata | j mesophyll t & their I I cells | 1 structure ' I i I Iactivitie si nprogres s 1" ~~ï contemplated activities

Figure 3. Some proposed trends in biological research in order to explain the resistance mechanisms in the confrontation between poplars and rust.

In such further studies 3 directions should be stressed: (1) leaf structure and its influence on the infection process by pathogen, (2) bio­ chemical inhibitors manifesting during infection process, and (3) the role of epiphytic microorganisms during the infection process. As shown in Fig. 3, and by the results discussed in this paper, only a few mechanisms of resistance active during the confrontation between pop­ lars and rust have been investigated. The other possible influences should be checked intensively in the near future. Our knowledge of the pathogen is still insufficient for the study of resistance from the standpoint of establishment of physiological races of the pathogen and their growth in axenic culture. Also, factors influencing the virulence and aggressiveness of the pathogen are still unknown. There is also a lack of information on other than the uredinial stage of develop­ ment of the pathogen (pycnium, aecium, telium and basidium). Those stages have a direct or indirect influence on the complex relationship between poplars and rust.

REFERENCES

Kaufmann, U. & H. Wellendorf, 1979. A study of mechanisms in Norway spruce having inhibitory effects on the growth of Forties annosus. Proceedings of the Conference on Biochemical Genetics of Forest Trees Umeâ, Sweden 1978. p. 50-59. Krawiarz, K. & A. Werner, 1978. Studies on the effect of surface chemistry of poplar leaves on urediniospore germination and growth of hyphae in development of Melampsora larici-populina Kleb. In: R. Siwecki: The mechanisms of poplar leaf resistance to fungal infection. Final Report, 1978. Mimeographed. 112 p.

141 Krzan,Z. ,1980 .Effec to fclimati c factorso ndevelopmen to fth ediseas ecause db y Melampsoralarici-populina .Foli aForestali a422 :10-13 . Krzan,Z. ,1981a .Cyk lrozwojow yi morfologi agrzyb aMelampsor a larici-populinaKleb . -sprawc yrdz ytopoli .Arboretu mKórnicki e2 6(i npress) . Krzan,Z. ,1981b .Popla rresistanc e toinfectio nb yth efungu sMelampsor alarici - populina infiel dconditions .Arboretu mKórnicki e 26 (inpress) . Mlodzianowski,F .& R .Siwecki ,1976 .Ultrastructur e ofpopla r leavesnaturall yinfecte d byrus tMelampsor a larici-populina.Arboretu mKórnicki e21 :375-400 . Mlodzianowski,F .& R .Siwecki ,1978 .Morpholog y andultrastructur e ofurediospore so f Melampsoralarici-populina .Europea nJourna lo fFores tPatholog y8 :43-48 . Mlodzianowski,F. ,A .Werne r& R .Siwecki ,1978 .Germinatio no fMelampsor a larici-populinauredospore so npopla rleaves .Europea nJourna lo fFores tPatholog y 8:119-125 . Rohringer,R. ,W.K .Kim ,D.J .Samborsk i& N.K .Howes ,1977 .Calcofluor :a noptica l brightenerfo rfluorescenc emicroscop yo ffunga lplan tparasite si nleaves .Phyto ­ pathology67 :808-810 . Siwecki,R. ,1978 .Th emechanis mo fpopla rlea fresistanc e tofungà linfection .Fina l Report,Jul y1 ,197 3- Octobe r31 ,1978 .Institut eo fDendrology ,Kórni k(Poland) . Mimeographed.11 2p . Siwecki,R. ,1981 .Characterizatio no fpopla rresistanc e tomor eimportan tdiseases . In:R .Siweck i& M .Rid é (Eds):Resistanc emechanism si npopla rdiseases . Proceedingsjoin tsymposiu mFA0/IUFR0 ,Kórni k 1980.p .41-49 . Siwecki,R .& K .Przybyl ,1981 .Wate rrelation si nth eleave so fpopla rclone sresistan t andsusceptibl e toMelampsor a larici-populina.Europea nJourna lo fFores tPatholog y 11:348-357 . Siwecki,R .& A .Werner ,1980 .Resistanc emechanis minvolve di nth epenetratio nan d colonizationpopla r leaftissue sb yMelampsor a rust.Phytopathologi aMediterrane a19 : 27-29. Werner,A. ,1981a .Histologiczn ebadani azaleznosc igospodarz-patoge nw przebieg u choroby liscitopol iwywolane jprze zMelampsor a larici-populina'Kleb.Arboretu m Kórnickie2 6(i npress) . Werner,A. ,1981b .Rozmieszczeni emetabolitó ww mezofil utopol iporazonyc hrdz aMelampsor a larici-populinaKleb .Arboretu mKórnicki e 26 (inpress) .

142 Resistance mechanisms of elms to Dutch elm disease

D.M. Elgersma

Phytopathological Laboratory 'Willie Commelin Schölten', Baarn, the Netherlands

ABSTRACT

Processes provoking symptoms inelm s after inoculation with theDutc hel m disease fungus arebriefl y discussed.A reviewo nresearc hconcernin gmechanism s ofresistanc e inelm s to Ophiostoma ulmi isgiven .Sprea d ofth e fungus throughout the tree ishampere d inresistan t trees compared to susceptible ones.Anatomica l factors,suc h asvesse l length and diameter and size ofvesse l groups,migh treduc e the speed of transport ofconidi a inresistan tplants .Tylosi s formation is fasteri n resistant trees than insusceptibl e ones, another factorwhic h might limitth e spread ofth e fungus.Th emeanin g ofphyto - alexins indiseas e resistance andth e role ofPhytotoxin s in pathogenesis are discussed.

INTRODUCTION

Dutch elm disease hasbecom e one ofth emos t severe treedisease s in NorthAmeric a andEurope .Recentl y anexcellen t review onDutc h elm disease hasbee ncompile d by Sinclair &Campan a (1978).Thi scompilatio n includesa paperb yMacHard y (1978)o nmechanism s ofresistance , andtherefor e there isn onee d for athoroug h anddetaile d review ofth e literature now.Th e purpose ofthi spape r ist odiscus s further research onphysiological ,bio ­ chemical ormorphologica l aspects ofth e expressiono fgene s forresistanc e toDutc h elm disease indifferen t species orclone s ofelms .A studyo f resistancemechanism s canb e fruitful only ifw e knowho w thenorma lpro ­ cesses ofpathogenesi s elapse.W ema yexpec ttha tproduct s ofgene s for resistancebloc k orcounterac t some,o r all,o fthos eprocesse s thatother ­ wiseultimatel y leadt o symptom expression and deatho fth ehost . Dutchel m disease iscause db y theAscomycet e Ophiostoma (Ceratocystis) ulmi (Buisman)Nannf. ,whic h after introduction intoth exyle mb ybar k beetles, grows andproduce s conidia inth evessels .Conidi a are transported with the transpiration stream throughout thetree .Durin g itsgrowt h inth e

143 vessels the funguscause schange s inp H ofth exyle m fluid, andproduce s metabolites such ascell-wal l degrading enzymes,growt h substances and toxins.Al l these factors inconcer tcaus ewiltin g andeventually , after1 or2 years ,deat ho f thewhol e tree. Iti su p toth eplan tpathologis t to determinewhic hmetabolite s orphysiologica l processes areth ecrucia l ones for disease expression.Processe s causedb yhost-parasit e interactions in thevesse l luminaar e socomple x and intermingled that astud y ofeac hfac ­ tor separately seems impossible. A cross-section of aninfecte d elmtwi g reveals adar k discoloration of thene w annual ring,cause db y oxydation andpolymerizatio n ofphenoli c compounds inth exyle m sap,xyle mparenchym a andra ycell s (Gagnon, 1967b). Gums andtylose s are formed inth e luminao fth exyle mvessels ,clearl y impedingth e sap flow (Gagnon, 1967a). Cell-wall degrading enzymesma y releasepartiall y degradedwal lpolymer s into thexyle m fluid.Thes e polymers aswel l ashigh-molecular-wéigh ttoxin s ascerato-ulmi n (Takai, 1974)an dglycopeptide s (Rebel,1969) , isolated from shakeculture ,ma y interferewit hwate r transport,probabl yb yphysica l plugging of thepi t membranes (VanAlfe n& Turner , 1975). Apart from the above-described parasitic stagew e know alsoa saprophytic stage ofth eDutc hel m disease fungus.Th epathoge n grows andproduce s coremia inbreedin g galleriesmad eb y elmbar kbeetle s in thebark .Fungu s spores adheret oemergin gbeetles ,an ddurin g feeding bybeetle s incrotche s oftwig so fhealth y elms, conidiama yb e introduced intoth exyle mvessel s and infection isestablished .Dyin g orweakene d elms areattractiv e forbeetle s asbroo d trees,thu s amplifying theepidemic . Justafte r the firstWorl dWa rth eDutc hel m disease appeared inEurop e and sweptthroug h theOl dWorl d (Gibbs, 1978). Inth e late 1920'sth efun ­ guswa s introduced intoth eUnite d Statesb y theimpor t ofvenee rwoo d from France. Atth e 'Willie CommelinScholten 'Laborator y atBaar nresearc h started onth ecaus e ofth e disease andth ediseas e cyclewa sunravele d (Buisman, 1928; Fransen& Buisman , 1935). Since allnativ e elm species inHollan d were susceptible, abreedin gprogra mwa s started.Elm s werecollecte d from all over theworl d and selected for resistance toth e disease,a progra m resulting inth ereleas e ofsevera l resistant elm clones (Heybroek,1957 , 1976). Itbecam e cleartha tgrea tcar e should be takent o avoidnon - heritablevariation s insymptom-expression , since trees are lesssuscep ­ tiblebecaus e of slow growtho rafte r transplanting (Heybroek, 1957;Went , 1954). Itals obecam e obvious thatresistanc e waspolygeni c (Heybroek, 1970), andn o substantial evidenceha sbee n found for ahost-parasit e dif­ ferential interaction (Gibbse t al., 1975). Inth emi d 1960'sresearc h onmechanism s ofresistanc e toDutc hel m diseasewa s started atth e 'WillieCommeli n Scholten' Laboratory atBaarn . By thengoo d resistant cloneswer e available and apropagatio nmetho dha d

144 beendevelope d (Tchernoff, 1963)tha tutilize d shoots cut from callused roots. Forphysiologica l experiments genetically uniformplan tmateria l is anecessity .Becaus eprobabl yman y genes areinvolve d inth emechanism s of resistance andn o race-specific effectha sbee ndiscovered , wema y expect to find effects ofa quantitativ e nature governedb ygene s thathav e anor ­ mal function inhealth yplant s (Vanderplank, 1978).

COMPOSITION OF XYLEM SAP

Instudyin g resistancemechanism sw e firststudie d the composition of thexyle m sap insusceptibl e andresistan t clones.Sinc e the fungusmus t grow inxyle m fluid,th enutrien t level ofsuc h fluidmigh thav e animpac t

)jmoles amino acids per ml

18 April Figure 1. A.Amin o acid contento fxyle m sapgathere d from ten 1-year-old callus cut­ tings growni npot s outdoors.o Amin o acid contento f susceptible elms. • Amino acidconten to fresistan t elms. B.Tota l sugarconten to fth e samexyle m sapdescribe d inA . oSuga r content of susceptible elms. •Suga r contento fresistan t elms.

145 onth e growtho fth epathogen .Amid e andamin o acidcontent s ofth exyle m sap ofsusceptibl e and resistant elms (Fig.1A )were ,hig h from thebegin ­ ning ofApri l until thebeginnin g ofMay ,decrease d rapidly inth emiddl e ofMay , andthe nremaine d rather constant from thebeginnin g ofJun eunti l August (Elgersma, 1969). During theperio d ofsusceptibility ,whic h inth e Netherlands is from aboutth emiddl e ofMa yunti l themiddl e ofJuly ,n o importantchange s occurred inamin o acid orsuga r content. In Ulmus americana L.,however , Singh& Smalley (1969b)foun d ahig h amide and alo w proline concentration inth exyle m sapdurin g theperio dwhe nth etree s were susceptible.The y found apositiv e correlationbetwee nprolin e content andresistanc e invariou s elm species (Singh& Smalley , 1969a).W e also found ahighe rprolin econten t inresistan t clones than in susceptible

ones. Bygrowin g elms ona nutrien t solutionwit hNH . instead ofN0 3, how­ ever,w ewer e able to increase theprolin e contento fth e sap ofa sus -

number of colonies

60.000

30.000

0

15.000

10.000

5.000

\A 10 Li days after inoculatio nv 1 2 3 A 15 30 Figure 2. Average number ofcolonie s grown from stempiece s thatwer e cut from susceptible (white)an dresistan t (blacks »1ms andhomogenize d onvar ­ iousday s after inoculation. A. stempieces, 25-3 0c m aboveth e site ofinoculation . B. stempieces, 1- 4 cm above thesit eo finoculation .

146 ceptible clonet o several timeshighe r than thato fresistan t elms grown onN0 ~ nutrient solutionwithou tobtainin g resistance (Elgersma, 1969). The relation ofprolin e and resistance isno twel l understood. Singh & Smalley (1969a)sugges ttha t ahig hconten to fprolin e reflects atoleranc e towaterstress . Data obtainedb y Singh& Smalley (1969a)an dElgersm a (1969)gav en o evidence thatgrowt h ofth e fungus inresistan t clones or species ofelm s compared totha t insusceptibl e oneswoul d be limitedb y shortage ofavail ­ able amino acids or sugars.Growt h ofth e fungus in2-year-ol d callus cut­ tingso fresistan t and susceptible cloneswa s comparedb y culturing the pathogen fromhomogenate s of stempiece s selected atvariou sheight s above the site of inoculation (Fig.2A) .Colon y counts showed that fungalmateri ­ alincrease d inresistan t and insusceptibl e trees inth e lower stempiece s atabou tth e same rate during the first4 days after inoculation.A t 7day s after inoculation thenumbe r ofpropagule s washighe r insusceptibl e than inresistan tplants ,becaus evessel sno tdirectl y infected atth e timeo f inoculationbecam e infected inth e susceptible plantsbu tno t inresistan t plants.Th enumbe r ofpropagule s isolated from stempiece s athighe rpart s ofresistan tplant swa smuc h lesstha n fromth e corresponding parts of sus­ ceptible elms (Fig.2B) .Th econclusio n istha tth epathoge n cangro wi n xylemvessel s ofresistan tplants ,bu t spread ofth e fungus through there ­ sistantplan t islimited .Thi s limited spread keeps thepathoge n localized and also its effecto nth etrees .Wate r conduction is interrupted onlypar ­ tially andthi s reductionha sn oo ronl yver y slighteffec to nth etre e as awhole .

ANATOMICAL DIFFERENCES

Thequestio nno w iswha t factors areresponsibl e for limiting the spread ofth e fungus. Iti sgenerall ybelieve d that spread ofth e fungus throughout thetre e isaccomplishe d bymovemen t ofth e conidia inth e transpiration stream (Banfield, 1968)an d therefore differences inanatomi ­ cal structure ofth evascula r systemmigh thav e animpac to ntranspor t of theconidia . Comparisons ofvesse l length andvesse l diameter of susceptible andre ­ sistantclone s revealed thatth eaverag e diameter ofth exyle mvessel s from a resistant clonewa s lower thantha to fvessel s ina susceptibl e clone (Elgersma, 1970). Smallvessel s area disadvantag e inth e spread ofth e fungusbecaus e transport canb eblocke d easierb y gums andtyloses .Thi s phenomenon alsoha sbee nobserve d inAmerica n elms (Sinclair et al., 1975). Another difference was thatth eeffectiv e length ofth evessel s appeared tob e shorter inth e resistant clonetha n inth e susceptible one.Thi sre ­ sulted in less spread ofconidi a inth eresistan ttree ,becaus e inshor t vessels thepathoge nha s topenetrat emor een dwall s to spread overth e

147 samedistanc e than inlon gvessels .Conductivit y forwate r and air through stempiece swa s lower inresistan ttha n insusceptibl e trees,whic h isi n agreementwit hth e observed differences in anatomical structure of theves ­ sels.Also ,th e decreased susceptibility oftree swhe n latewoo d is formed mayb e duet o the small diameter ofthes evessels .McNab b et al. (1970) found that anincreas e indiseas e susceptibility was correlated with anin ­ crease inaverag e sizeo fvesse l groups.Th e average size ofvesse l groups isdefine d asth eproduc t ofth e averagevesse l diameter andth e average number ofcontiguou s vessels.Al l theseobservation s indicate thatanatomi ­ cal factors are important inresistanc e toDutc h elmdisease .

TYLOSIS FORMATION

As alreadymentione d tylosis formation canbloc k the sap flow andi s considered ofparamoun t importance insympto m expression.O nth eothe rhan d tyloseswil l alsohinde r or arrestmovemen to fconidia , ahos t reaction thatmigh tb erathe r effective insmal l sizedvessels .Tylosi s formation hasbee ndescribe d asa possibl emechanis m ofresistanc e to Fusarium wilt inbanan aplants . Inth e incompatiblehost-parasit e combination rapidfor ­ mation oftylose s seals offth evessel s inadvanc e ofth epathogen ,thu s localizing the infection. Inth ecompatibl ehost-parasit e combination,how ­ ever, tylosis formation isdelayed , andth epathoge n can spread throughth e wholeplan t (Beekman& Halmos ,1962 ;Beekma ne t al., 1962). A significantdifferenc e intylosi s formation appearedbetwee n asus ­ ceptible and aresistan tel m clone after inoculationwit hth epathoge n (Fig. 3).Tylose s insusceptibl eplant s develop slowertha n inresistan t plants, andtherefor e theysupposedl ywer eno teffectiv e insealin gof fth e vessels inadvanc e ofth e infection (Elgersma, 1973). After discovery inEnglan d ofa highl y aggressive straino f O. ulmi (Gibbse t al., 1972)w e repeated theexperiment s and included this'aggres ­ sive strain aswel l asth evascula rparasit e Fusarium oxysporum f.sp . lycopersici, anon-pathoge n toelm s (Elgersma& Miller , 1976). Nosignifi ­ cant difference intylosi s formationwa s foundwithi n cloneBelgica , aver y susceptible clone,afte r inoculationwit h thenon-aggressiv e straino fO . ulmi, the aggressive strain,o r F. oxysporum f. sp. lycopersici. Similar resultswer e obtained inclon e390 ,a clon eresistan t toth e non-aggressive straino f O. ulmi. Tylosis formation inclon eBelgic a aswel l asi nclon e 390 isno t inhibited inindividual s of asusceptibl e reaction type,bu t seemst ob egeneticall y determined byth e elm clone alone andtherefor ew e consider tylosis formation anon-specifi c response ofelm st oinjur yo r microbial infestation. Sincetylosi s formation inclon e39 0 doesno tappea r tob e effective inarrestin g the transport ofth e aggressive straino fO . ulmi (Elgersma& Heybroek , 1979), wemus t assume thatbypassin g ofobsta ­ cles sucha stylose s isprobable .Thi s hasbee n suggestedpreviousl yb y

148 500 number of vessels with tyloses per 1000 vessels

400

300

200-

100

5 7 10 days after inoculation

Figure 3. Numbero fvessel s showingtylose spe r 1000vessel s incross - section,o susceptible ,• resistan tclone .

Miller& Elgersm a (1976)a sbein g duet omor e rapid growth andpenetratio n ofvesse lpit sb y the aggressive strain. Production ofcell-wal l degrading enzymesb y thepathoge nmigh tthere ­ foreb e of importance.A study ofcellulas e andendopolygalacturonas e pro­ ductionb y aggressive andnon-aggressiv e strains showed no differencebe ­ tweenthe m (Elgersma, 1976). Research onproductio n ofhemicellulas eb y these strains is inprogress .

PHYTOALEXINS AND TOXINS

Accumulation offungitoxi c compoundso rphytoalexin s after infection withmicro-organism s isa widesprea dphenomeno ni nangiosperms . Itappeare d alsotha tinfecte d elms accumulate fungitoxic compounds (Overeem & Elgersma, 1970). Thenaphthoquinone s mansononeE andF wer e isolated from susceptible aswel l asresistan t clones.Th eDutc h elmdiseas e fungus,how ­ ever,ca ntolerat e arelativel y high concentration ofthes ecompounds . Resultsobtaine d inlate r studies showed that infected resistant clones accumulated nomor e ofthes e compounds than susceptible plants (Elgersma & Overeem, 1971). The induction ofsynthesi s ofmansonone s appears tob e non-specific.Accumulatio n also occurs after chemical injury.Probabl yw e may consider theproductio n ofthes e fungitoxic compounds as awea kresis ­ tancemechanism . Suchcompound sma y slow downth e infectionproces s andth e

149 killing of the tree or inhibit other microbial infestation. 0. ulmi produces phytotoxic compounds in shake culture (Zentmeyer, 1942). Rebel (1969) isolated a toxic glycopeptide 'fraction, later purified by Strobel et al. (1978), that caused wilting of elm sprouts of susceptible as well as resistant trees. So it appeared to be non-specific. Van Alfen & Turner (1975) showed that even small amounts of this toxin could interfere with water conductivity in the xylem vessels. Probably the high molecular compounds plug the pit membranes of the vessels, thus hampering water transport. Recently we were able to detect this glycopeptide in infected trees by means of an advanced immunological technique (ELISA) (Scheffer & Elgersma, 1981). Important questions, such as can the toxin be neutralized by the resistant trees or does the aggressive strain produce more toxin in the tree than the non-aggressive one, still have to be answered.

CONCLUSIONS

Much evidence for the existence of mechanisms of resistance, as de­ scribed here, has been presented, but no absolute proof has been given because genetic crossings to determine if correlated phenomena segregate in absolute association have not been performed (Ellingboe, 1976). The polygenic and probably quantitative nature of resistance will make such experiments almost impossible. Elm breeding programs might use the accumulated information about the mechanisms of resistance, however. In the choice of alternative parent trees the nature of resistance should be taken into consideration. Crossing trees with different sets of genes for resistance would be advisable in­ stead of crossing parents with almost identical ones. In the future more mechanisms of resistance probably will be detected especially in the Asiatic elm species, which have not been studied very intensively till now.

REFERENCES

Banfield, W.M., 1968. Dutch elm disease recurrence and recovery in American elms. Phyto- pathologische Zeitschrift 62: 21-60. Beekman, C.H., 1971. The plasticizing of plant cell walls and tylose formation - a model. Physiological Plant Pathology 1: 1-10. Beekman, C.H. & S. Halmos, 1962. Relation of vascular occluding reactions in banana roots to pathogenicity of root invading fungi. Phytopathology 52: 893-897. Beekman, C.H., S. Halmos & M.E. Mace, 1962. The interactions of host, pathogen and soil temperature in relation to susceptibility to Fusarium wilt of bananas. Phytopathology 52: 134-140. Buisman, C.J., 1928. De oorzaak van de iepenziekte. Tijdschrift der Koninklijke Neder­ landse Heidemaatschappij 40: 338-345. Elgersma, D.M., 1969. Resistance mechanisms of elms to Ceratocystis ulmi. Mededelingen Phytopthologisch Laboratorium Willie Commelin Scholten, Baarn, No. 77. 84 p. Elgersma, D.M., 1970. Length and diameter of xylem vessels as factors in resistance of elms to Ceratocystis ulmi. Netherlands Journal of Plant Pathology 76: 179-182. Elgersma, D.M., 1973. Tylose formation in elms after inoculaton with Ceratocystis ulmi,

150 apossibl e resistancemechanism .Netherland sJourna lo fPlan tPatholog y79 :218-220 . Elgersma,D.M. , 1976.Productio no fpecti c andcellulolyti c enzymesb yaggressiv ean d non-aggressive strainso fOphiostom aulmi .Netherland sJourna lo fPlan tPatholog y 82:161-172 . Elgersma,D.M . &H.M .Heybroek ,1979 .Sprea dan dsurviva lo fa naggressiv ean da non - aggressive straino fOphiostom aulm ii nelms .Netherland sJourna lo fPlan tPatholog y 85:235-240 . Elgersma,D.M .& H.J .Miller ,1977 .Tylos e formationi nelm safte rinoculatio nwit ha n aggressiveo ra non-aggressiv e straino fOphiostom aulm io rwit ha non-pathoge nt o elms.Netherland sJourna lo fPlan tPatholog y83 :241-243 . Elgersma,D.M .& J.C .Overeem ,1971 .Th erelatio no fmansonone st oresistanc eagains t Dutchel mdiseas ean dthei raccumulation ,a sinduce db ysevera lagents .Netherland s Journalo fPlan tPatholog y77 :168-174 . Ellingboe,A.H. ,1976 .Genetic so fhost-parasit e interactions.In :R .Heitefus s& P.H.William s (Eds):Encyclopedi a ofplan tphysiology .Springer-Verlag ,Berlin / Heidelberg/NewYork .p .761-778 . Fransen,J.J . &C.H .Buisman ,1935 .Infectieproeve no pverschillend e iepensoortenme t behulpva niepenspintkevers .Tijdschrif tove rPlantenziekte n41 :221-239 . Gagnon,C. ,1967a .Histochemica lstudie so nth ealteratio no fligni nan dpecti csub ­ stancesi nwhit eel minfecte db yCeratocysti sulmi .Canadia nJourna lo fBotan y 45:1619-1623 . Gagnon,C. ,1967b .Polyphenol san ddiscoloratio ni nth eel mdiseas e investigatedb y histochemicaltechniques .Canadia nJourna lo fBotan y45 :2119-2124 . Gibbs,J.N. , 1978.Intercontinenta lepidemiolog yo fDutc hel mdisease .Annua lRevie wo f Phytopathology 16:287-307 . Gibbs,J.N. ,H.M .Heybroe k& F.W .Holmes ,1972 .Aggressiv e straino fCeratocysti sulm ii n Britain.Nature ,Londo n236 :121-122 . Gibbs,J.N. ,CM . Brasier,H.S .McNab bJr .& H.M .Heybroek ,1975 .Furthe rstudie so n pathogenicity inCeratocysti sulmi .Europea nJourna lo fFores tPatholog y5 :161-174 . Heybroek,H.M. , 1957.El mbreedin gi nth eNetherlands .Silva eGenetic a6 :112-117 . Heybroek,H.M. , 1970.Thre easpect so fbreedin gtree sfo rdiseas eresistance .2n dWorl d Consultationo nFores tTre eBreedin gProceeding sVol .I .FAO ,Rome .p .519-535 . Heybroek,H.M. , 1976.Dri enieuw eiepeklonen .Nederland sBosbou wTijdschrif t48 :117-123 . MacHardy,W.E. ,1978 .Mechanism so fresistance .In :W.A .Sinclai r& R.J .Campan a (Eds): Dutchel mdisease .Perspective safte r6 0years .Cornel lUniversity .Agricultura l ExperimentStation .Searc h (Agr.)Vol .8 :25-26 . McNabb,H.S. ,Jr. ,H.M .Heybroe k& W.L .MacDonald ,1970 .Anatomica lfactor si nresistanc e toDutc hel mdisease .Netherland sJourna lo fPlan tPatholog y76 :196-204 . Miller,H.J .& D.M .Elgersma ,1976 .Th egrowt ho faggressiv ean dnon-aggressiv e strains ofOphiostom aulm ii nsusceptibl ean dresistan telms ,a scannin gelectro nmicrosco ­ picalstudy .Netherland sJourna lo fPlan tPatholog y82 :51-65 . Overeem,J.C .& D.M .Elgersma ,1970 .Accumulatio no fmansonone sE an dF i nUlmu shollan - dicainfecte dwit hCeratocysti sulmi .Phytochemistr y 9:1949-1952 . Rebel,H. ,1969 .Phytotoxin so fCeratocysti sulmi .Thesis .Stat eUniversit yo fUtrecht , theNetherlands . Scheffer,R.J .& D.M .Elgersma ,1981 .Detectio no fa phytotoxi cglycopeptid eproduce d byOphiostom aulm ii nel mb yEnzyme-Linke dImmunospecifi cAssa y (ELISA).Physiolo ­ gicalPlan tPatholog y18 :27-32 . Sinclair,W.A .& R.J .Campan a (Eds),1978 .Dutc hel mdisease .Perspective safte r6 0 years.Cornel lUniversity .Agricultura lExperimen tStation .Searc h (Agr.)Vol .8 . 52p . Sinclair,W.A. ,J.P .Zahan d& J.B .Melching ,1975 .Localizatio no finfectio ni nAmerica n elmsresistan t toCeratocysti sulmi .Phytopatholog y 65:129-133 . Singh,D .& E.B .Smalley ,1969a .Nitrogenou san dcarbohydrat e compounds inth exyle msa p ofUlmacea e speciesvaryin gi nresistanc e toDutc hel mdisease .Canadia nJourna lo f Botany47 :335-339 . Singh,D .& E.B .Smalley ,1969b .Nitrogenou scompound si nth exyle msa po fUlmu sameri - cana;seasona lvariation si nrelatio nt oDutc hel mdiseas esusceptibility .Fores t Science 15:299-304 . Strobel,G. ,N .va nAlfen ,K.D .Hapner ,M .McNei l& P .Albersheim ,1978 .Som ephyto ­ toxicglycopeptide s fromCeratocysti sulmi ,th eDutc hel mdiseas epathogen .Bioche - micae tBiophysic aAct a538 :60-75 . Takai,S. ,1974 .Pathogenicit yan dcerato-ulmi nproductio n inCeratocysti sulmi .Nature , London252 :124-126 . Tchernoff,V. ,1963 .Vegetativ epropagatio no felm sb ymean so fshoot scu tfro mcalluse d

151 roots.Act aBotanic aNeerlandic a12 :40-50 . VanAlfen ,N .& N.C .Turner ,1975 .Influenc eo fCeratocysti sulm itoxi no nwate rrela ­ tionso fel m (Ulmusamericana) .Plan tPhysiolog y 55:312-316 . Vanderplank,J.E. ,1978 .Geneti can dmolecula rbasi so fplan tpathogenesis .(Advance d seriesi nagricultura lscience s6. )Springer-Verla gBerlin/Heid'êlberg/Ne wYork . 16Tp . Went,J.C. ,1954 .Th eDutc hel mdisease .Tijdschrif tove rPlantenziekte n60 :109-127 . Zentmeyer,G.A. , 1942.Toxi nformatio nb yCeratocysti sulmi .Scienc e95 :512-513 .

152 Pine wilt and pine wood nematode: histopathological aspects of disease development

Yasuharu Mamiya

Forestry and Forest Products Research Institute, Tsukuba Norin Kenkyu Danchi-nai, Ibaraki, Japan

ABSTRACT

Pinewil t involving thepin ewoo dnematode , Bursaphelenchus lignicolus, andth epin e sawyer, Monochamus alternatus, isth e mostseriou spes to f foresttree s inJapan . In197 9 itcause d the deatho f8 millio npin e trees (Pinus densiflora and P. thunbergii). The chronological development ofpin ewil tcoin - cideswit hth e lifecycl e ofth evector ,pin e sawyer.Nematod e inoculated pine seedlingswer e examined histopathologically to revealpathologica l responses ofpin e tonematod e infectiones ­ pecially during early stages ofpathogenesis .Deat h ofra yan d axialparenchym a cells occurred asth e firstvisibl epathologi ­ cal response.Thi scellula r response developed inrelatio nt o time after infection occurred. Cell deathwa s observed inwoo d tissues asearl y as2 4hour s after inoculation.Whe n aninocu ­ lated seedling ceased toexud eoleoresi n atth e 6tht o9t hda y after inoculation,deat ho faxia lparenchym a cellsha d pre­ vailedmarkedl y init swood .

INTRODUCTION

Theprimar y objectives ofthi spresentatio n aret oprovid e asummar y ofpin ewil t disease inJapa n andt opoin tou tth ehistopathologica l as­ pects ofdiseas e development emphasizing thosedurin gearl y stages of pathogenesis.Th e study onhistopathologica l changes ofpin e tissuesin ­ duced frompathologica l responses ofpin e tonematod e infectionwil lpro ­ vide abasi c step torevea l resistancemechanism s topin ewilt .

HISTORICAL REVIEW OF PINE WILT

Pinewil t firstoccurre d inJapa nearl y inthi s century.Yan o (1913) reported thedeat h ofpin e trees inNagasak i ofKyush u asepidemic .Th e damageha d spread since 1905 andth e causal agentha dno tbee n determined

153 attha ttime .Accordin g tohi sreport ,however ,diseas e symptomso fpin e trees corresponded well to symptoms currently inducedb y thepin ewoo d nematode. Since thenthi s dreadful diseaseha s spread inepidemi cpropor ­ tionthroughou t central to southwestern Japan,an dno w thediseas e covers thenorther n regiono fJapan . In197 8 itcause d the loss of2 millio nm 3 of pinewood , and deatho f8 millio npin e trees (Pinus densiflora Sieb.& Zucc, and P. thunbergii Pari.). Thisvolum e represented almost1 % o fth e growing stock ofpin e inJapan .Th epin ewoo d nematode isth emos t serious pesto f foresttrees .Th eJapanes eGovernmen t appropriated US $3 5millio n in fiscalyea r 1980t o address theproblem . Numerous investigations hadbee n conducted mainly on insects associated with deteriorated pine trees as suspected causal agents oftre emortality . Kiyohara& Tokushig e (1971)firs tsuggeste d that Bursaphelenchus lignicolus Mamiya& Kiyohara , thepin ewoo dnematode ,wa sth e causal organism ofth e pinewil t disease.The y found thisnematod e acommo n inhabitant inwoo do f deadpin e trees (Tokushige &Kiyohara , 1969), anddemonstrate d the drastic effecto fthi snematod e onpin e trees asth eresul to f inoculationtests . Sincethen ,man y investigators havepresente d experimental evidencewhic h showed astron g correlationbetwee nnematod e inoculation and death ofpin e trees (Mamiya, 1972). Amongman ybar k andwoo dborer s examined forthei r associationwit h B. lignicolus, Monochamus alternatus Hops, thepin e sawyer,wa s found tob eth emos t importantvecto r ofth enematod e (Mamiya & Enda,1972 ;Morimot o& Iwasaki, 1972).

DISEASE CYCLE

Affected trees areconspicuou s because ofquic k death after thesymp ­ toms appear.Pin e trees observed tob e healthy inearl y summer die inlat e summer, showing yellowish foliage.Th ever y same symptoms occurring innat ­ urally infected trees areproduce db y introducing nematode suspensions in healthy trees.Deat h oftree s follows in4 0t o 60day s after inoculation whenexperiment s areconducte d insummer . Thechronologica l developmento fpin ewil tunde rnatura l conditions coincideswit hth e lifecycl e of M. alternatus (Mamiya, 1972). Newly emerged adultpin e sawyers from deadpin e trees carrydauerlarva e ofÔ . lignicolus inthei rtracheae .Durin gmaturatio n feedingo fadul tbeetle s on freshbranche s ofhealth ypin e trees,nematode s leave thebeetl ebod y and enterpin e tissues through injuries.Nematod e infection occurs from early June to late July,th eperio d ofmaturatio n feeding of M. alternatus. The firstsympto m ofdiseas e is the failure ofa tre et oexud e oleoresin fromwounds .Adul tbeetle s oviposit successfully indisease d treeswhic h still appearhealth y attha ttime ,durin g Julyt o lateAugust .Th e rapid developmento fdiseas e results indeat h ofth e tree inlat eAugust .Hug e populations ofnematode s build throughout the diseased tree. Inwood ,

154 nematodes inhabitmostl y axial andradia l resincanal swher e they feedo n epithelial cells,mos t likely the sourceo fthei r food (Tamura &Mamiya , 1979).Hatche d larvae of M. alternatus boreunde rbar k and then through wood tissues.Larva e overwinter inth etunnel s andpupat e the following spring During February toMa ynematodes ,th edispersa l third stagelarvae , gather around thepupa l chambers ofbeetles . InMa y aggregated nematodes moltt oth edauerlarvae .Pupatio n ofbeetle sbegin s atmid-Ma y and adult beetles emerge frompupa e during lateMa y toJuly . Justafte remerging , adultbeetle sbecom e contaminated withdauerlarvae . Closely coordinated life cycles ofth epin ewoo dnematod e and thepin e sawyer causepin ewilt .

HISTOPATHOLOGICAL RESPONSE OF PINE TISSUES TO NEMATODE INFECTION

Histopathological studieshav ebee ncarrie d outt odemonstrat e thena ­ tureo fhos tresponse s inrelatio nt o feeding and reproduction ofnematode s inwood , especially inearl y stages ofpathogenesis .

Materials and methods , Three-year-old pine seedlings (Pinus densiflora and P. thunbergii) were inoculated with B. lignicolus cultured on Botrytis cinerea Pers. Inocula­ tionwa smad eb y injecting awate r suspensiono fnematodes , 500 0nematode s per seedling, into arubbe r tubewhic hwa s attached tocove r theen d ofa branchcu ta t5- 7 cm distant from thestem . Seedlingswer e kepta t27-3 0°C . Several inoculated anduninoculate d seedlingswer e collected 24hour s after inoculation and then atinterval s of3 days . Thenematod epopulatio n of eachseedlin gwa s estimated by extracting nematodes from thewhol e seedling without leaves using theBaerman n funnel technique.Fo r ahistopathologica l studywoo d cylinders 1c m inlengt hwer e collected from each inoculated branch, the stem justbelo w the inoculated branch, thebasa lpar t ofth e stem, and amiddl epar to fth e shoot,an dwer e fixed inFPA .Fixe dspeci ­ menswer edehydrate d and embedded in 10% celloidine.Celloidin e sections, 12ur nthick ,wer e stained with safranin and fastgreen .

Results Populations of B. lignicolus inwoo d ofcollecte d seedlings are shown inTabl e 1.Despit e the rapid spread ofnematode s from the inoculation site, almost allnematode s extracted from aseedlin gwer e found inth e inoculated branchunti l the 9thda y after inoculation.Result s ofobserva ­ tions atinterval s after inoculationwer e asfollows . 24 hours: Atth ecu ten d ofinoculate d branchesnematode s entered most axial resin canalswhos e epithelial cellswer e destroyed. Eggswer e also observed inthos e axial resincanals .Beyon d the inoculated branch afe w nematodeswer e found inresi ncanal s inth e cortex ofth e shoot and apar t

155 Table 1. Disease development and nematode population of 3 seedlings at each sampling date.

24 h 3rd day

a Oleoresin exudation + + + + + No. of nematodes per 2866 1297 1721 1638 908 756 seedling (406)b (237) (321) (18) (8) (346)

6th day 9th day

Oleoresin exudation + + - - No. of nematodes per 1343 850 1691 1582 5717 1122 seedling (303) (530) (291) (282) (4157) (322)

12th day 15th day

Oleoresin exudation - - - - No. of nematodes per 5800 12750 18.05 68980 104250 20770 seedling a. +: normal exudation, -: no exudation b. Number of nematodes except those from inoculated branch

of the stem. Cell death signaled by a granulation of the cytoplasm, de­ formed nucleus which was stained red with safranin, and browned cell con­ tents, was observed in axial parenchyma cells surrounding epithelial cells of an axial resin canal (Fig. 1) and in ray parenchyma cells at the basal part of the stem. At that time, however, it occurred separately oniy in a few cells. 3rd day. Destruction of epithelial cells of axial and radial resin ca­ nals in the inoculated branch became more advanced. Death of ray cells was commonly observed but sporadically distributed all over the anatomical cross sections or radial sections of the inoculated branch. Only a few nematodes were located in resin canals of the stem. Throughout the stem, death of axial parenchyma cells and ray parenchyma cells occurred spora­ dically, while no effect was observed on epithelial cells of resin canals which were associated with such dead axial parenchyma cells. 6th day: Almost all resin canals of the inoculated branch were de­ stroyed. One of the collected seedlings showed marked reduction of oleoresin exudation from a cross section of the stem cut at the basal part. In this seedling death of axial parenchyma cells was noticeable, especially at the basal part of the stem (Fig. 2),whil e no damage of epithelial cells

156 ofresi ncanal swa s observed.Pathologica l changes ofparenchym a cells did notoccu r inwoo d tissues ofth eshoot . 9th day: All3 seedlings collected atthi stim ecease d toexud e oleo- resino nth e surface ofcros s sections cut atth ebasa lpar to fstems .A nematodepopulatio n exceeding inoculum level (500 0pe r seedling)wa s found in1 o fthes e seedlingswhil e inth e other 2seedling s therewa sn opopula ­ tiongrowth .Dea d axialparenchym a cellsprevaile d throughout the stemo f the latter 2seedlings .Mos t axial resincanal swhos e epithelial cellswer e not affected atal lha d suchdea d axialparenchym a cells,a tleas ton eo f them.Deat h ofra ycell s alsobecam emor enoticeabl e than atth e 6thda y (Fig.3A) .A fewnematode swer e observed instems ,bu tn odestructio n of epithelial cells.Tylosoid s ofepithelia l cellswer e seen ina fe waxia l resincanals .Discoloratio n appeared sporadically alongth e cambialzone . Nematode distribution ata lo wleve l occurred inresi ncanal s ofth eshoot , butn o signo fcel l deathwa s detected there. Inth e stem ofth e seedling havingmor enematode s throughoutwoo d tissues thanth e inoculum level,a group of axialresi ncanal swhos eepithelia l cellswer e completelyde ­ stroyedb ynematode s occupiednearl y 1/4 ofth e cross section ofth e stem. Axialparenchym a cells ofthos e resincanal swer e also destroyed.Nematode s were observed commonly insuc hresi ncanal s (Fig.3B) .Number s ofdea d axialparenchym a cells andra yparenchym a cells increasedmarkedl y andthe y werewidel y distributed throughout thexylem .Discoloratio n ofcambiu m and phloem cellsbecam e advanced and crackswer e seen alongth ecambia lzone . Manyegg s found inresi ncanal s indicated thatreproductio n ofnematode s progressed rapidly. Smaller larvae,mostl y second stage larvae,wer eob ­ served passing throughwindow-lik e pits ofra ycell s and locating in tracheids. 12th day: The extento fdestructio n ofresi ncanal s andparenchym a cell death inwoo d ofthos e 2seedling s havingnematod epopulation s exceeding 5 000wer e similar toeffect so nseedling so fth e 9thda y inwhic hnematod e populations exceeded the inoculum level.However , itwa s clear thatdamage s became more advanced asnematod epopulation s became larger. Inaddition , discoloration ofcambiu m andcavit y formation along thecambia l zoneoc ­ curredmor e frequently thanbefore .Ther ewer e littlevisibl e pathological responses ofparenchym a cells inth e shoots ofthes e seedlings.Onl y aver y fewnematode swer e found inresi ncanal s ofth ecortex ,bu tno t inthos eo f thexylem .Th e other seedlingwhic h showed the symptom ofceasin g toexud e oleoresinha d asmalle rpopulatio n ofnematode s init swoo d thanth einocu ­ lum level.Jus ta sha dbee nobserve d atth e 6tho r9t hday ,deat h ofaxia l parenchyma cells and rayparenchym a cellsprevaile d throughoutth e stem while only afe w resincanal swer e affected and aver y fewnematode s were observed insuc hresi ncanals . 15th day: Rapid growtho fnematod epopulation s ininoculate d seedlings was found atth e 15thda y after inoculation. Itreache dmor e than1 0time s

157 Figure 1. 24hour s after inoculation. Celldeat h of axialparenchym a cells atth ebasa lpar to f stem.Cros ssection .

Figure 2. 6day s after inoculation.Cel l death ofra y and axialparenchym a cells atth ebasa lpar to f stem.Radia l section.

158 Figure 3. 9day s after inoculation. Celldeat h ofra y and axialparenchym a cells atth ebasa lpar to f stem.A .Radia l section.B .Radia l section. Nematodes in anaxia l resincanal .

the inoculum level. Inalmos t allresi ncanals ,epithelia l cells and axial parenchyma cellswer e destroyed throughout the shootan d the stem.Al l parenchyma cells ofth ecortex ,phloem , cambium, andray s seemed tob e dead judgingb y total shrinkage,discoloratio n andgranulatio n of cytoplasm, anddisappearin g orre d stained nuclei.Man y cavities that resulted from nematode attackswer e located along thecambia lzone . Therewer eman ynematode s andegg s inresi ncanal s and suchcavities .

Discussion Death ofra y and axialparenchym a cells occurred asth e firstvisibl e pathological response ofpin e tonematod e infectiondurin g early stages of pathogenesis.Anatomica l observations demonstrated thatthi s cellularre ­ sponse developed inrelatio n totim e after infection.Thus , itmigh tb e concluded thata stead y increase innumber s and extento fdea d parenchyma cellsreflecte d the disease development of seedlings.Cel l death occurred earlier than reduction orcessatio n ofoleoresi n exudation.Whe na n inoculated seedling ceased toexud e oleoresin,dea d axialparenchym a cells surrounding epithelial cellso f axial resincanal sprevaile d markedly in itswood . Thisprovide s aninterestin g subjectregardin g the supposedre -

159 lationship between cell death and reduction of oleoresin exudation. In addition, at this time no death of epithelial cells similar to that of axial parenchyma cells and ray parenchyma cells was observed. More developed pathological responses were commonly shown in the basal part of the stem than in the upper part, just below the inoculated branch, during early stages of pathogenesis. This indicates that disease develop­ ment began at the lower part of a seedling, except in the inoculated branch. At the inoculation site, the cross section of a branch, nematodes directly invaded resin canals just after inoculation and then gradually invaded areas extending deep inside the branch. In the inoculated branch, parenchyma cell death and destruction of epithelial cells directly caused by nematode attacks preceeded those of any other parts throughout a seed­ ling. Cell death occurring prior to nematode population growth and distribu­ tion through wood tissues indicates that pathological reactions of pine tissues to some chemicals might be involved. Oku et al. (1979, 1980) re­ ported that some metabolic substances of the pine wood nematode or bacteria associated with the pine wood nematode had toxic effects on a pine tree. Cell death occurring at initial stages of pathogenesis was detected as one of the most remarkable pathological responses of living cells to nematode infection through this study. Histopathological study of cell death will provide a useful measure to investigate differences of resis­ tance to the pine wood nematode within or among species of pine.

REFERENCES

Kiyohara, T. & Y. Tokushige, 1971. Inoculation experiments of a nematode, Bursaphelenchus sp., onto pine trees. Journal of Japanese Forestry Society 53: 210-218. (In Japanese with English summary). Mamiya, Y., 1972. Pine wood nematode, Bursaphelenchus lignicolus Mamiya and Kiyohara, as a causal agent of pine wilting disease. Review of Plant Protection Research 5: 46-60. Mamiya, Y. & N. Enda, 1972. Transmission of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae) by Monochamus alternatus (Coleoptera: Cerambycidae). Nematologica 18: 159-162. Morimoto, K. & A. Iwasaki, 1972. Role of Monochamus alternatus (Coleoptera: Cerambycidae) as a vector of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae). Journal of Japanese Forestry Society 54: 177-183. (In Japanese with English summary). Oku, H., T. Shiraishi & S. Kurozumi, 1979. Participation of toxin in wilting of Japanese pines caused by a nematode. Naturwissenschaften 66: 210. Oku, H., T. Shiraishi, S. Ouchi, S. Kurozumi & H. Ohta, 1980. Pine wilt toxin, the metabolite of a bacterium associated with a nematode. Naturwissenschaften 67: 198-199. Tamura, H. & Y. Mamiya, 1979. Reproduction of Bursaphelenchus lignicolus on pine callus tissues. Nematologica 25: 149-152. Tokushige, Y. & T. Kiyohara, 1969. Bursaphelenchus sp. in the wood of dead pine trees. Journal of Japanese Forestry Society 51: 193-195. (In Japanese). Yano, S., 1913. Reports of investigation on the death of pine trees. Sanrin Koho 4 suppl.: 1-22 (In Japanese).

160 Indirect selection for pest resistance using terpenoid compounds

Michael R.Bridge n& JamesW .Hanove r

ABSTRACT

Oneproble m indevelopin g genetic resistance intree st o insecto rdiseas epest s istha to ftre e longevity. Itma yb e overcomewit h theus e of anindirec t selectionprogram .An y secondary traituse d inindirec t selectionmus tb e highly correlated with theprimar y trait,bu ti tdoe sno tnee dt o be causally related toresistance . The identification ofsuitabl emarke r traits isdifficul t and expensive andma yb e theon e factormos t limiting theus e ofindirec t selection. Inoleoresin-producin g conifers,th e monoterpenes and resin acidsma yb eusefu l groups ofbiochemi ­ calmarker s for such aprogram .The y areunde r genetic control and insom e species arecorrelate d withresistanc e to insecto r diseasepests . Theresi n acid composition of Scotchpin e oleoresinma yb e involved inmechanism s ofresistanc e tosom e insectpests . Parallel seasonalvariatio n and larval feedingbehavio r andth e strongnegativ e correlations betweengeographi c variations in resin acids and insect susceptibility showtha t insectresis ­ tance ishighes twhe noleoresi n composition ishighest .Thes e compounds are strongly correlated with resistance and are rel­ atively easy and inexpensive tomeasure .The yma yb ever yuse ­ fulmarker s inindirec t selectionprogram s forpes t resistance inpines .

The forest industry inth eUnite d States isplacin g increasing emphasis onth e importance ofbasi c research in forestregeneration .Unde r pressures from aninflationar y economy, escalating fuelcosts ,governmen t and envi­ ronmental restrictions,companie s haveno wrecognize d thedesirabilit y of

1.Th eauthor sar erespectively :Researc hScientist ,Westvac oCorporation ,Rupert , West Virginia,USA ,an dProfessor ,Michiga nStat eUniversity ,Eas tLansing ,Michigan ,USA .

161 having themos t superior seedlings available toreplac e stands afterhar ­ vesting.Althoug h improved seedlings areexpensive,.hig hsite-preparation , planting, and sitemaintenanc e costsovershado w the initia.1investmen to f improvedplantin g stock.No tonl y shouldplante d seedlings grow quickly and possess desirable form andwoo d quality characteristics,the y should also be able towithstan d various insect and diseasepest swhic hma y attackthe m atan ytim e during their life,i norde r thatth ecost s ofth e seedlingsma y becompletel y recovered in futuregrowth . Although research intre e improvement and genetics hasproduce d rap­ idly growing genotypes,littl ewor kha sbee ndon e todevelo p insectan d disease resistance inth e samematerial .Ther e are several reasons forthi s (Hanover, 1980): forestgenetic s is arelativel y new field andther e are few genetic researchers;th e simplicity ofnatura l regeneration makes it'swid e usemor e attractive thanplanting ; andth e lacko fbasi c knowledge ofhost - pestphysiolog y andth e longperiod s oftim ebetwee n generations intree s discourage researchers fromworkin g inth e field. Concurrentwit h thelon g time intervalsbetwee n generations and the longevity oftree s isth epossi ­ bility thattree sma y notexhibi tresistanc e to insecto rdiseas epest s at anearl y stage asthe yma yno tb e attacked by thesepest s forman y years. Thismake s traditional selection schemesver y difficult and their results oflimite dvalue . Theus e ofindirec t selectionma yb e onemetho d ofovercomin g theprob ­ lem oftre e longevity. Indirect selection isth eearl y identification of superior individuals orpopulation s for atrai tothe r than the one for which improvement is sought.Th e traitbein guse d forselection , thesec ­ ondary trait,mus tb ehighl y correlated with thedesire d orprimar ytrait , whether orno t iti scausall y related. Indirect selection isespeciall y useful ifgreate r ormor e rapid success canb eha db y selecting for acor ­ related trait instead of aprimar y character. The theoretical background forindirect'selectio nha sbee noutline db y Falconer (1960). Weissenberg (1976)ha spreviousl y described thetheory , advantages, andus e ofindirec tselectio n inbreedin g forvariou s traits in foresttre especies . Directselectio n has asimpl emathematica l basis.Wit h directselec ­ tion, anumbe r of individuals from apopulatio n arechose n tob e thepar ­ ental stock for futuregenerations .Th e intensity ofselectio n isdefine d asth e ratio ofth emea nphenotypi cvalu e ofth e individuals chosen,S ,t o thephenotypi c standard deviation, a .

i =S/a p

Heritability, h2, isdefine d asth erati o ofth emea nphenotypi c value ofth eprogen y of selectedparents ,R , toth emea nphenotypi c value ofth e parents.Heritabilit y isals oth erati o of additive geneticvarianc e toto -

162 talphenotypi c variance ofth epopulation .

h2 =R/ S =a 2/!?2 a' p

Theexpecte d gain orchang e from themea no fth eorigina lpopulation , R, isth eproduc t ofth e intensity ofselectio n times the square rooto f theheritabilit y ofth e traittime s the square rooto fth e additive genetic variance ofth e selectedtrait .

R =ih a a

By using indirect selection, asecondar y trait ischose nwhic h is highly correlated with theprimar y trait.Th egai n forth eprimar ytrait , then, isth eproduc to fth e intensity of selection forth e secondarytrait , the additive genetic correlationcoefficient , andth e square rooto fth e heritability ofth esecondar y traittime s theadditiv e geneticvarianc e of theprimar ytrait .

Gx = ih ry ay aa x

The relative efficiency ofusin g indirect instead ofdirec t selection is shownb y arati o ofth eexpecte d gaino f indirect selection totha t expected with the directmethod .Thi s ratio isth e correlation coefficient times the selection intensity and square rooto fheritabilit y ofth esec ­ ondary traitdivide d by the selection intensity and square rooto fherita ­ bility ofth eprimar ytrait .

G /R =r (i v h )/(i h ) x' x a y Y xx '

Indirect selection canb e asuperio r method todirec t selectionif : - ahig h genetic correlation existsbetwee nth e2 traits ; - the secondary traitha s ahighe rheritabilit y thanth e desired character; - ahighe r selection intensity canb e applied onth e secondary traittha n onth eprimar ytrait . The terpenesma yb e anidea l group ofbiochemical st ous e asmarker s in anindirec tselectio nprogram , especially forpines ,spruce s andothe r resinproducin g conifers.Monoterpene s andditerpenoi d resin acids comprise thegreates tportio n oftota loleoresin .Th emonoterpene s areunde r strong genetical control (Baradate t al., 1975;Hanover , 1966a;Squillace , 1971) and showminimu m influenceb y environmental variation (Hanover, 1966b). Geneticvariatio n inmonoterpene s between seed sourcesha sbee ndemon ­ strated forman ypin e and spruce species (Arbeze t al., 1974;Bridge n et al., 1979;Ganse l& Squillace,1976 ;Tobolsk i &Hanover , 1971;Wilkinso n & Hanover, 1972). Thegenetic s ofresi nacid shav eno tbee n studiedver y

163 widely.Ther e appearst ob en ogeneti cvariatio n inth eresi nacid so fth e oleoresin ofwhit epin e (Bridgene t al., 1979)bu t seedlot and regional variationha sbee n found inScotc hpin e (Bridgen, 1979). Further studies should identify geneticvariatio n and control ofresi n acid inothe r spe­ cies. There are4 characteristic s ofterpene s thatmak e these compounds use­ ful inindirec t selection forpes tresistance : 1.Severa l terpenes havebee n identified as attractants or repellents to insects and so aredirectl y involved inresistanc emechanism s (All & Anderson, 1974;Bordasc h &Berryman , 1977;Hanover , 1975). Similarly, they areprobabl y involved inresistanc e to some fungal diseases (Bailey et al., 1975; Cobb et al., 1968). 2. Oleoresinma yb e sampled from tree seedlingswhil eman y insect anddis ­ easepest sma yno tb e observed until thetre e isman yyear s old.Althoug h there issom evariatio n inoleoresi n causedb y the ageo f thetree ,cor ­ relations of juvenile traitswit h resistancepattern s should be equally useful andvali d ascorrelation s traitsmeasure d atth e same time asre ­ sistance. 3.Measurin g errors canb eminimize d and large*number s oftree s canb ean ­ alyzedusin g theterpenes .Th equantitativ e accuracy ofchemica lmeasure ­ mentsusin g gaschromatograph y greatlyovershadow s thequalitative ,ofte n ocular,measurement s which areusuall y applied toward insect and disease infection rates.Man y samplesca neasil yb ehandle d aslittl e storage space isnecessar y forth e small quantities ofoleoresi n used and automatic sam­ pling devices allow acontinua l analysis,eve nwhe n labpersonne l areno t available. 4. Finally,man y terpenes are simultaneously analyzed.A typicalpin eo r spruce analysiswil lyiel d4-1 5 ormor emonoterpene san d 16o rmor e resin acids.A seac h compound ispresumabl y under independent genetic control and insects and disease reactdifferentl y tovariou s chemicals,th eprobabilit y of finding acorrelate d trait ismuc hhighe r thani fonl y 1o r afe wtrait s orchemical swer ebein g studied. Anexampl eo fth eus eo fterpene s asmarker s forresistanc e to adis ­ ease on foresttree s istha to f fusiform rust (Cronartium fusiforme Hedge. & Hunte xCumm. )i nlobloll y (Pinus taeda L.) and slash (Pinus elliottii Engelm.)pines .Rockwoo d (1973)studie d thephenotypi c andgeneti ccorre ­ lationsbetwee n fusiform rust infestation andth e chemical compositiono f both stemxyle m andbranc hcorte x oleoresin oflobloll ypine .H e collected oleoresin from about35 0 treesrepresentin g 36 full-sib families.Th e trees were 5year s old.Non e ofth e3 primar ymonoterpene s ofste mxylem ,a - pinene, ß-pinene, ormyrcene , showed anyrelationshi p with resistance,bu t branch ß-phellandrenewa sphenotypicall y correlated with susceptibility.A s ß-phellandrenecompositio n increased,percen t infection also increased. Rust infection isdirectl y incontac twit h thebranc h cortex tissues,an d

164 iti sreasonabl e tobeliev e thatcorte x oleoresinwoul d bemor e involved in any resistancemechanis m than stem xylemoleoresin . To expand his studies onth erol e ofmonoterpene s and rustresistance , Rockwood (1974)examine d anotherpin e specieswhic h also showsvariatio n in resistance toth erust ,slas hpine .H ecollecte d branch oleoresin inabou t 300 trees fromclones ,win dpollinate d families, full-sib families,an d existing rusttest s inFlorida ,Mississipp i andGeorgia .Ther e areonl y 4 majormonoterpene s inslas hpine ,a-pinene , ß-pinene,myrcen e andß - phellandrene.A s inlobloll ypine ,ß-phellandren ewa sth e onlymonoterpen e showing arelationshi p with fusiform rust,bu ti nthi s case,resistanc ewa s associated withhig h levels ofth eterpen e rather than low.Th e monoterpene may notb e thecausa l agento fresistanc e orsusceptibility ,bu trathe rb e genetically linked toth econtrollin g traits.Therefore ,i tma yb esuccess ­ fullyuse d ina nindirec t selectionprogram . Several years agow ebega nt o study thephysiolog y and genetics ofth e oleoresin system ofScotc hpin e (Pinus sylvestris L.). Totes tth ehypothe ­ sis thatditerpene sar eusefu l indicators of insectresistance , comparisons weremad ebetwee n data collected inth ephysiolog y andgeneti c studieswit h the knownpattern s ofresistanc e to several insectpest s common'onScotc h pine.Al l ofth e insectresistanc e informationha dbee npreviousl y collect­ ed fromprovenanc e testswithi nMichiga n (Steiner, 1974;Wrigh t& Wilson , 1972; Wright et al., 1967,1975 ,1976) . Results from some ofou robserva ­ tions indicate thata relationshi p may existbetwee n the diterpenoid resin acids and insectresistanc e inthi sspecies . Beginning 1Apri l 1978,oleoresi nwa s collected from 14Scotc hpin e treesever y 15day s throughout the growing season.Th e treeswer e 18year s old,originatin g inSwede no fth evariet y septentrionalis. Oleoresinwa s collected incapillar y tubeswhic hwer e tapped in2-year-ol d tissue and theresi n acidswer e analyzed using gas-liquid chromatography (Bridgene t al., 1979). Twelve resin acids orpair s ofunseparabl e resinacid swer emeasured . Fourmajo r resin acids,sandaracopimarate , levopimarate +palustrate , strobate +dehydroabietat e andneoa'bietate ,2 mino r acids andtota l resin acids showed significant seasonal variation.Th e combined peak of strobic + dehydroabietic acids showed anincreas e inconcentration s between 15Ma y and 15June ,afte rwhic h itdecrease d to levels close tothos eprio rt o 15May .Al l ofth eothe r resinacids ,an d total acids,ha d anoticeabl e decrease during this sametim eperiod , extending to 1July .Thi s drop in total resin acid concentrations coincideswit h the time ofmos trapi d shoot elongation. Itals oparallel s thedevelopmenta l physiology of severalin ­ sectswhic h attack Scotchpine .Th e larvaeo fth eEuropea npin e sawfly (Neodiprion serti fer Geoff.) feedo nyear-ol d foliagebetwee n earlyMa y andmid-June .Als o during thisperiod , larvaeo f theeaster n pineshoot borer (Eucosma gloriola Heinrich)bor e into lateral and terminalshoots .

165 Table 1. Simple correlations ofresi n acidswit h susceptibility patterns of several Scotchpin e insectpests .

European Pine Zimmerman Pine root pine shoot pinemot h collarweevi l sawfly borer

Unknown2 8 0.23 0.07 0.12 0.23 Unknown3 0 -0.06 -0.05 -0.26 -0.15 Unknown3 1 -0.23 0.14 -0.16 -0.21 Pimarate -0.46** -0.27 -0.37** -0.34* Sandaracopimarate -0.29 -0.33* -0.23 -0.21 Levopimarate + palustrate -0.44** -0.21 -0.36* -0.36* Isopimarate -0.43** -0.51** -0.30* -0.19 Unknown3 6 -0.28 * -0.32* -0.20 -0.16 Strobate +dehydroab : etate -0.15 -0.14 -0.20 -0.14 Unknown3 8 -0.01 0.08 -0.14 -0.13 Abietate -0.42** -0.40* -0.35* -0.31* Neoabietate -0.04 0.11. -0.15 -0.12 Unknown4 1 -0.04 0.11 -0.15 -0.12 Total resin acids -0.46** -0.27 -0.37** -0.36*

Significant atp £0.0 5 andp <_0.0 1 respectively.

The timing ofthes e events corresponds very closely toth edecreas e of total resinacid s inth e oleoresin ofbranches . Itma yb e only coincidental thatth edevelopmenta l physiology ofth e insectparallel s thechang e of chemical composition inth e oleoresin,bu t iti s alsopossibl e thatth e insectha s adapted its developmental stages genetically to feed ata tim e whenundesirabl e compounds inth ehos t area ta lo wconcentration .A simi­ lar situationha sbee ndescribe dwit h thewinte rmot h (Operophtera brumata L.). The tannin concentration ofoa k leavesma yhav ecause d evolutiono f thewinte rmot h sotha t its feeding ceasesbefor e tannins increase incon ­ centration inth e leaves (Feeny, 1970). Alongwit h seasonalvariation ,variatio nbetwee n seedlots of Scotch pine exists forth e resin acids.I nth elatte rpar to fJun e 1978,oleoresi n was collected fromth ebole s of30 0 trees representing 50 seedlots and1 8 varieties of Scotchpine .Tota l resin acids ranged inconcentratio n from 8.5 %t o 54% ofoleoresin .Th e Scandinavianvarietie s had thehighes tcon ­ centrations oftota l resin acidswhil e thecentra l Europeanvarietie s had the lowestconcentration s ofpimaric , abietic,neoabieti c andtota l resin acids. Scandinavianvarietie s alsohav e thehighes t resistance to European pine sawfly (Wrighte t al., 1967)an d theeaster npineshoo tbore r (Steiner,

166 1974). Correlation analysis between insect resistance and resin acids for all seedlots show a significant negative relationship between these traits (Table1) .

REFERENCES

All, J.N.& R.F .Anderson ,1974 .Th einfluenc eo fvariou sodor si nhos tselectio no f pioneerbeetle so fIp sgrandicollis .Journa lo fth eGeorgi aEntomologica lSociet y 9:223-228 . Arbez,M. ,C .Bernard-Daga n& C .Fillon ,1974 .Variabilit é intraspécifiquede s monoterpènes dePinu snigr aArn. :de spremier srésultats .Annale sde sScience s Forestières31 :57-70 . Bailey,J.A. ,G.A .Carter ,R.S .Burde n& R.L .Wain ,1975 .Contro lo frus tdisease sb y diterpeneso fNicotian aglutinosa .Natur e255 :328-329 . Baradat,Ph. ,C .Bernard-Dagan ,G .Paul y& C .Zimmerma nFillon ,1975 .Le sterpène sd u pinmaritime .Aspect sbiologique se tgénétiques .III .Hérédit é del ateneu re n myrcène.Annale sde sScience sForestière s32 :29-54 . Bordasch,R.P .& A.A .Berryman ,1977 .Hos tresistanc e toth efi rengrave rbeetl e Scolytusventrali s (Coleoptera:Scolytidae) .2 .Repellanc yo fAbie sgrandi sresin s andsom emonoterpènes .Canadia nEntomologis t102 :95-100 . Bridgen,M.R. ,1979 .Geneti cvariatio no fScotc hpin eoleoresi nphysiology .Ph.D . DissertationMichiga nStat eUniversity ,Eas tLansing ,Michigan .7 8p . Bridgen,M.R. ,J.W .Hanove r& R.C .Wilkinson ,1979 .Oleoresi ncharacteristic s ofeaster n whitepin esee dsource san drelationshi p toweevi lresistance .Fores tScienc e 25: 175-183. ' Cobb,F.W. ,Jr. ,M .Krstic ,E .Zavari n& H.W .Barber ,Jr. ,1968 .Inhibitor yeffect so f volatileoleoresi ncomponent so nFome sannosu san dfou rCeratocysti sspecies . Phytopathology 58:1327-1337 . Falconer,D.S. ,1960 .Introductio n toquantitativ egenetics .Th eRonal dPres sCompany , NewYork .36 5p . Feeny,P.P. , 1970.Seasona lchange si noa klea ftannin san dnutrient sa sa caus eo f springfeedin gb ywinte rmot hcaterpillars .Ecolog y 51:565-581 . Gansel,C.R .& A.E .Squillace ,1976 .Geographi cvariatio no fmonoterpen ei ncortica l oleoresino fslas hpine .Silva eGenetic a25 :150-154 . Hanover,J.W. , 1966a.Genetic so fterpenes .I .Gen econtro lo fmonoterpen e levelsi n Pinusmonticol aDougl .Heredit y21 :73-84 . Hanover,J.W. , 1966b.Environmenta lvariatio ni nth emonoterpène so fPinu smonticol a Dougl.Phytochemistr y 5:713-717 . Hanover,J.W. , 1975.Physiolog yo ftre eresistanc e toinsects .Annua lRevie wo f Entomology20 :75-95 . Hanover,J.W. , 1980.Breedin gfores ttree sresistan t toinsects .In :F.G .Maxwel l& P.R. Jennings (Eds): Breedingplant sresistan tt oinsects .Wile y& Sons ,Inc . p.487-511 . Rockwood,D.L. ,1973 .Monoterpene-fusifor mrus trelationship s inlobloll ypine . Phytopathology63 :551-553 . Rockwood,D.L. ,1974 .Cortica lmonoterpen e andfusifor m rustresistanc e relationshipsi n slashpine .Phytopatholog y64 :976-979 . Squillace,A.E. ,1971 .Inheritanc e ofmonoterpen e composition incortica loleoresi no f slashpine .Fores tScienc e17 :381-387 . Steiner,K. ,1974 .Geneti cdifference si nresistanc eo fScotc hpin e toeaster npineshoo t borer.Grea tLake sEntomologis t7 :103-107 . Tobolski,J.J .& J.W .Hanover ,1971 .Geneti cvariatio ni nth emonoterpène so fScotc h pine.Fores tScienc e17 :293-299 . Weissenberg,K .von ,1976 .Indirec tselectio nfo rimprovemen to fdesire dtraits .In : J.P.Miksch e (Ed.):Moder nmethod si nfores tgenetics .Springer-Verlag .28 8p . Wilkinson,R.C . &J.W .Hanover ,1972 .Geographica lvariatio ni nth emonoterpen e compositiono fre dspruce .Phytochemistr y11 :2007-2010 . Wright,J.W. ,W.A .Lemmien ,J.N .Bright ,M.W .Da y& R.L .Sajdak ,1976 .Scotc hpin e varietiesfo rChristma s treean dfores tplantin gi nMichigan .Michiga nStat e UniversityAgricultura lExperimen tStatio nResearc hRepor t293 .1 6p . Wright,J.W . &L.F .Wilson ,1972 .Geneti cdifference si nScotc hpin e resistance topin e rootcolla rweevil .Michiga nStat eUniversit yAgricultura lExperimen tStatio n

167 ResearchRepor t159 .6 p.. Wright,J.W. ,L.F .Wilso n& J.M .Bright ,1975 .Geneti cvariatio ni nresistanc eo f Scotchpin e toZimmerma npin emoth .Grea tLake sEntomolo.gis t8 :231-236 . Wright,J.W. ,L.F .Wilso n& W.K .Randall ,1967 .Difference samon gScotc hpin evarietie s insusceptibilit y toEuropea npin esawfly .Fores tScienc e 13:175-181 .

168 Monoterpenes and resistance of conifers to fungi

H.J. Schuck

Lehrstuhl für Forstbotanik, Universität München, D-8000 München 40, Amalienstr. 52, BRD

ABSTRACT

Monoterpenes usually inhibitth edevelopmen t ofpathogeni c fungi.Thi s effectvarie s over awid e range. Ina fe wcase sth e composition ofth eterpen e fractioncoul db e correlatedwit h theresistanc e of atre e againstpathogens .Afte rbein g wounded or infected aconiferou s tree canexhibi t achang e inth e composition ofth eterpen e fraction.Thi s reactionperhap s determines atleas tth e actual resistance againstpathogens .

Monoterpenes arewidel y distributed inth eplan tkingdom .No tonl yth e conifers can synthesize them,bu tothe r systematically unrelated families such as the Lauraceae, Poaceae, Liliaceae, Rutaceae, Compositae and Lamia- ceae containmonoterpenes ,to o (Fretz,1978) . Compared with theseplan t families theconifer sproduc e thegreates t amounts ofterpenes ,whic h are constituents ofresi n thatca nmak eu p to amaximu m of2 5% (in Pinus elliottii Engelm.)o fth edr yweigh to fheartwoo d (Parham, 1976). In Pinus sylvestris L. theheartwoo d contains 8% resin consisting of2 5 %monoter ­ penes. Certainly thesevalue s cannotb e generalized for all conifers,an d furthermore greatvariatio noccur s amongindividuals . Iti scharacteristi c inconifer s thatth eresi n islocate d ina specia l resin duct system, usually existing inal lpart s of atree .Onl y some genera, like Abies, normally lackresi nducts ,bu t asa reactio n toinju ­ ries they candevelo p such systems,calle d traumatic resin ducts.Othe r conifers react inth e samewa yupo nwounding . The development of traumatic resinduct s shows thatth e resin andth e monoterpenes play apar t inth edefens emechanism s againstpathogeni c organisms. Surely oneo fth emai n functions ofresin s isclosin g wounds quickly.The y establishmechanica l andchemica lbarrier s topreven tinfec ­ tionsb y fungi (Rishbeth, 1972)an d insects (Smith, 1977). Themechanica l barrier depends onth eresi nacids ,whic h aremai nconstituent s ofth e resin,wherea s themonoterpene s ofth eresi n act astoxi c agents.Generall y thesebarrier s arebuil tu pb y resin thatcome s from ducts inth ebark .

169 Thewoo d can also form abarrie r zone:injurie s induce the development ofne wresi nduct s inth ewoo db ycambia l cellso fth etree ,an d somor e resinca nb emobilize d topreven t aninfection . Inolder ,xyle mn one w ducts canb e differentiated. This traumatic reaction signals the significance ofresin s inhost-para ­ site interactions. Iti sno t surprising that for several years the effects ofmonoterpene s onpathogeni c fungihav ebee n investigated (Table 1).Her e only theexperiment s withmonoterpen ehydrocarbon s arecatalogued ; other derivatives, inliteratur e oftenname d asmonoterpenes , areno tconsidered . Mostly the influence onmycelia l growth of fungiwa s tested;bu t insom e cases also spore germination, drymatte rproduction , andth eperoxidas e activity of fungi growing in aterpen e atmospherewer e studied. Inmos t cases (Table 1)th emycelia l growthwa s inhibitedb ymonoter ­ penes. Stimulating effectswer e rarely observed (Fries,1973 ;Väisälä , 1974). Mosto fth e fungi testedwer ewood-decomposin g or wood-inhabiting organisms.Th e fungi growing inconife rwoo d seem tob emor e tolerant againsthig h levels ofterpene s than the species growing inwoo d ofdecidu ­ ous trees (Hintikka, 1970). Fries (1973)recorde d that,althoug h linear mycelial growthwa s reduced, the fungus could'increas eproductio n ofit s drymatte r whenexpose d to aterpen e atmosphere. The effectswer e always correlated with theconcentratio n ofth eap ­ plied terpenes,an d the terpenes themselves were alsodifferen t inthei r effects.Difference s may also exist inth eeffect s ofa monoterpen e on differentpathogens .Fo rexampl eA-3-caren e seems tob e relatively harmless against Fomes annosus (Fr.)Cook e (Schuck, 1977;Cob be t al., 1968), but very aggressive against Diplodia pinea (Desm.)Kick x (Chou& Zabkiewicz , 1976). Finally, different effects were also related toth etes tmethods . Fig. 1show sth e influence ofa-pinen e onth egerminatio n rate,th e linear mycelial growth, and thewood-decomposin g activity of Fomes annosus. Inal l 3 experiments the inhibition increasedwit h theconcentratio n ofth eter ­ pene.Th e germination ratewa sno treduce d as fasta sth emycelia l growth. Thewood-decomposin g activity,measure d asweigh t loss, cannotb e compared with theothe r test-methods,becaus e greater quantities ofterpen ewer e applied. Thistes ttoo k 31/ 2 months.Abou t every 10day sth e quantities mentioned inth e figurewer e added toth eculture s of Fomes annosus. Because ofth e greatvariatio n inthei r effects,i ti sno tpossibl et o postulate agradatio n inth e aggression ofterpenes . Inaddition ,th e results reported inth e literature differ sometimes,eve nthoug h the same funguswa s examined.Hintikka(1970 )foun dtha tA-3-caren e and limonene reduced themycelia l growth of Fomes annosus very stronglywherea s a-pinene had only asligh t reducing effect. Incontrast ,i nm y investigations (Schuck, 1977)caren ewa smor eharmles s anda-pinen e themos t aggressiveterpene . This difference cannotb e explained with certainty.Perhap s itdepend s on differences inmetho d oro nth epurit y ofterpene s appliedb y the2 investi -

170 ^-v N co O 10 -—• •H en O -—CN- S tn H o r- 0) ^co rH. r- en en rH en (8 -—' cn H fim rH fc. rH 18 4-1 H •— N — N—- 4J Ä 0 —• vO :I0 ^ ^ (1) A! 0 M <« r> m rH G •— G •H n u> (1) en O :(d •H C0 -H fi 4-1 Ü 1) fi P rH 3 m T3 t> T3 fi G •1-1 fi 0 ~ s: •H 0 en 0 0 •H 4) U 3 fi 0 :(0 rH rH rH U œ T3 In a u l/î > Ju — tu

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171 % Contr. 100-

90

80

70-

60- germination rate

50

40

30-

20

10 wood-decomposing activity -i—=t ->Cone, (ppm) oooooooo o UI o U1 o IT) o C\( (\|^ Ol to

Figure 1. Inhibitory effects (asa percentag e ofth econtrol )o fa-pinen e onth edevelopmen t of Fomes annosus in3 differen ttests .

gators.Nevertheless ,al lth epaper s describe the inhibitory effecto f terpenes onth evegetativ e developmento f fungi.Therefor e attempts have beenmad e to distinguishbetwee ntree s that areresistan t and susceptible to aparticula r fungus according toth ecompositio n ofthei r terpenefrac ­ tions. Such investigations arever y difficult,becaus eth ecompositio ni s highlyvariable . Iti s influenced by season (vonRudloff , 1975;Powel l & Adams, 1973;Hrutfior d et al., 1974), waterbalanc e (Hodges& Lorio ,1975 ; Gilmore1977 )an deve ndiurna lvariation s (Adams,1979) . Verygrea tinter - and intra-individualvariation s complicate such investigations (Schuck & Schutt, 1975;vo nRudlof f& Hunt , 1977). Nevertheless,recen t results show thatterpen e composition canb e related tosusceptibilit y topathogeni c fungi insom e cases.Rockwoo d (1974)demonstrate d acorrelatio nbetwee nth e percentage of ß-phellandrenei nth e resino f Pinus elliottii and resistance against Cronartium fusiforme Hedge. &Hunt .Cho u& Zabkiewicz (1976)foun d

172 higherpercentage s ofA-3-caren e intree s resistant to Diplodia pinea than insusceptibl e ones.Ther e aren o differences inth eterpen e fractions of severalwester nwhit e pines (Pinus monticola Dougl.)resistan t against Cronartium ribicola J.C.Fishe r (Hunt& vo nRudloff , 1977). Allthi s information couldb eusefu l inbreedin g forresistance ,bu t only ifth ecompositio n ofth eterpen e fraction isunde r geneticcontrol . Estebane tal.(1976 )an dHanove r (1966)postulate d genetic control systems forth eheredit y ofmonoterpenes . Many ofth ewood-decomposin g fungi areno tabl et openetrat e the intact

%

A-3- CARENE

3-

1-

~~~-~.-"' Distance to 1 1 1 1 1 1 1 7 ,, . -20 -10 -5 0 +5 +10 +20 wound(cm)

45-

40-

35-

ß- PI'NENE 30

Distance to 1 i i i i i woun crr -20 -10 -5 0 +5 +10 +20 ' d ( >) Figure 2. Effecto fwoundin g oncompositio n ofth emonoterpen e fractiono f Picea abies sapwood.A :wounded ;B :control .

173 bark.The y infect atre eonl yb ywounds ,wher eno tonl y the amounto f terpenes isincreased ,bu t alsoth ecompositio n ofth e terpene fractionha s beenchanged . These alterations areo fgrea t importance forth e defense againstpathogens ,becaus e they atleas tdetermin e the actual resistancet o fungal attack.Th e significance of increasing amounts ofresi n inwounde d parts of atre eha sbee n recognized by some authors.Cob b etal.(1968 ) postulated thatth e accumulation ofterpene s canb e afacto r inresistanc e topathogeni c fungi,an dRishbet h (1951)suppose d thatresi nproductio n in rootsca ninhibi t Fomes annosus infections.S oth e resistance against pathogens which infect through wounds couldb e correlated with the ability oftree s tomobiliz e resin (Gibbs,1968 )i nth ewounde d region.Thi strau ­ matic resinha s adifferen t chemical composition.Rüsse l& Berryma n (1976) found ahig hmyrcen e andA-3-caren e content in fungus-infected wounds of Abies grandis Lindl. Recently suchchange s inth e terpene fraction of Picea abies (L.) Karst,wer e found.Tw o remarkable chemical alterations inth e terpene fraction of sapwood were demonstrated 2month s after artificially wounding with abore r (Fig.2) .Lin eA indicates thepercentag e of adistinc tter ­ pene inwounde dwoo d (atpositio n 0)an d inwoo d located 5, 10,o r 20c m below (-) or above (+)th ewound . Forcontro l (B)w e took intactwoo d samples from comparable positions not influenced by thewound .Th e ß-pinene contentdecrease d inwounde dparts ,an dthi s effectwa s strongly limited to thewounde d regionwherea s theA-3-caren e content increased andwa sno t strongly limited towounds . Inth e samples taken atdistance s up to 10c mbelo w and aboveth e wound, the altered resincoul db e found.Bu t theeffec tdecrease d with growingdistanc e from thewound .No tonl y sapwood but also theheartwoo d canreac tb y synthesizing traumaticterpenes . Theseresult s cannotb egeneralize d because theywer eno tuniversal . Not allo fou r tested trees showed suchtraumati c reactions,o rthe'reac ­ tionswer eno tsimilar .Thi s is ane w aspect ofth e resistance mechanism duet omonoterpenes . Iti snecessar y to investigate whether these different reactions intree s ofth e same species arecorrelate d with their resistance against fungal attack. Iti spossibl e thatsuc hchange s takeplac e alsoi n infected butno t injuredwood .N o investigations of thisphenomeno n have been started atou r laboratory.

REFERENCES

Adams,R.P. ,1979 .Diurna lvariatio ni nth eterpenoid so fJuniperu sscopuloru m (Cupressa- ceae)- Summe rversu swinter .America nJourna lo fBotan y66 :986-988 . Chou,C.R.S .& J.A .Zabkiewicz ,1976 .Toxicit yo fmonoterpene s fromPinu sradiat acortica l oleoresint oDiplodi apine aspores .Europea nJourna lo fFores tPatholog y6 :354-359 . Cobb,F.W. ,M .Krstic ,E .Zavari n& H.W .Barber ,1968 .Inhibitor yeffect so fvolatil e oleoresincomponent so nFome sannosu san dfou rCeratocysti s species.Phytopatholog y 58: 1327-1335.

174 Esteban,I. ,F .Bergmann ,H.R .Gregoriu s& 0 .Huhtinen ,1976 .Compositio nan dgenetic so f monoterpenesfro mcortica loleoresi no fNorwa ySpruc ean dthei rsignificanc efo r cloneidentification .Silva eGenetic a25 :59-66 . Flodin,K. ,1979 .Effect so fmonoterpen eo fFome sannosu s (Fr.)Cook ean dit spheno l oxidaseactivity .Europea nJourna lo fFores tPatholog y9 :1-6 . Flodin,K .& N .Fries ,1978 .Studie so nvolatil e compoundsfro mPinu ssilvestri san dthei r effecto nwood-decomposin g fungi.II .Effect so fsom evolatil ecompound so nfunga l growth.Europea nJourna lo fFores tPatholog y8 :300-310 . Fretz,T.A. , 1978.Chemica lidentificatio no fwood yornamenta lplants .Ohi oRepor t63 : 23-25. Fries,N. ,1973 .Th egrowth-promotin gactivit yo fterpenoid so nwood-decomposin gfungi . EuropeanJourna lo fFores tPatholog y3 :169-180 . Gibbs,J.N. , 1968.Resi nan dth eresistanc eo fconifer st oFome sannosus .Annal so f Botany32 :649-665 . Gilmore,A.R. ,1977 .Effect so fsoi lmoistur e stresso nmonoterpene s inLobloll yPine . Journalo fChemica lEcolog y3 :667-676 . Groot,R.C .de ,1972 .Growt ho fwood-inhabitin gfung ii nsaturate datmosphere so fmonoter - penoids.Mycologi a64 :863-870 . Hanover,J.W. ,1966 .Genetic si nterpenes .I .Gen econtro lo fmonoterpen e levelsi nPinu s monticolaDougl .Heredit y21 :73-84 . Hintikka,V. ,1970 .Selectiv eeffect so fterpene so nwood-decomposin gHymenomycetes . Karstenia11 :28-32 . Hodges,J.D .& P.L .Lorio ,1975 .Moistur e stressan dcompositio no fxyle moleoresi ni n loblollypine .Fores tScienc e22 :283-290 . Hrutfiord,B.F. ,S.M .Hople y& R.J .Gara ,1974 .Monoterpene s inSitk aspruce :withi ntre e andseasona lvariation .Phytochemistr y 13:2167-2170 . Hubbes,M. ,1975 .Terpene san dunsaturate d fattyacid strigge rcoremi a formationb y Ceratocystisulmi .Europea nJourna lo fFores tPatholog y5 :129-137 . ' Hunt,R.S .& E .v .Rudloff ,1977 .Leaf-oil-terpen evariatio ni nwester nwhit epin epopu ­ lationso fth ePacifi cNorthwest .Fores tScienc e23 :507-516 . Parham,M.R. ,1976 .Stimulatio no foleoresi nyiel di nconifers .Outloo ko nAgricultur e 9:76-81 . Powell,R.A .& R.P .Adams ,1973 .Seasona lvariatio ni nth evolatil e terpenoidso fJuni - perusscopuloru m (Cupressaceae).America nJourna lo fBotan y60 :1041-1050 . Rishbeth,J. , 1951.Observation so nth ebiolog yo fFome sannosu swit hparticula rrefe ­ rencet oEas tAnglia npin eplantations .III .Natura lan dexperimenta linfection so f pines,an dsom efactor saffectin gseverit yo fth edisease .Annal so fBotan y (N.S.) 15:221-246 . Rishbeth,J. , 1972.Resistanc e tofunga lpathogen so ftre eroots .Proceeding so fth e RoyalSociety ,Londe nB 181 :333-351 . Rockwood,D.L. , 1974.Cortica lmonoterpen ean dfusifor m rustresistanc e relationships inslas hpine .Phytopatholog y 64:976-979 . Rudloff,E .von ,1975 .Seasona lvariatio ni nth eterpene so fth efoliag eo fblac k spruce.Phytochemistr y 14:1695-1699 . Rudloff,E .vo n &R.S .Hunt ,1977 .Chemosystemati c studiesi nth egenu sAbies .III .Lea f andtwi goi lanalysi so famabili sfir .Canadia nJourna lo fBotan y55 :3087-3092 . Rüssel,CE . &A.A .Berryman ,1976 .Hos tresistanc e toth efi rengrave rbeetle .1 .Mono ­ terpenecompositio no fAbie sgrandi spitc hblister san dfungus-infecte dwounds . CanadianJourna lo fBotan y54 :14-18 . Schuck,H.J. , 1977.Di eWirkun gvo nMonoterpene nau fda sMycelwachstu mvo nFome sannosu s (Fr.)Cke .Europea nJourna lo fFores tPatholog y7 :374-384 . Schuck,H.J .& P .Schutt ,1975 .Di eVerteilun gflüchtige rTerpen ei mFichtenstam mun d ihreBedeutun gfü rdi eAusbreitungsresisten z gegenüberFome sannosus .Biochemi eun d Physiologiede rPflanze n167 :65-77 . Smith,R.H. ,1977 .Monoterpene so fponderos apin exyle mresi ni nwester nUnite dStates . US.Fores tService .Technica lBulleti n1532 . Väisälä,L. ,1974 .Effect so fterpen ecompound so nth egrowt ho fwood-decomposin gfungi . AnnalesBotanic iFennic i11 :275-278 . Väisälä,L. ,1978 .Effect so fterpen ecompound so nth egrowt han dperoxidas e activityo f Phellinuspini .Annale sBotanic iFennic i15 :131-137 .

175 Buffering of pH in plant organs and resistance against fungi

F. Scholz& B.R . Stephan

Bundesforschungsanstaltfü rForst -un dHolzwirtschaf tHamburg-Reinbek ,Institu tfü r Forstgenetikun dForstpflanzenzüchtun gSchmalenbeck ,Siekerlandstrass e2 ,D-207 0Gross ­ hansdorf,BR D

ABSTRACT

Thepresen t knowledge ofrelation sbetwee n resistancet o fungalparasite s andbufferin g capacity invulnerabl e plant organs isreviewed .Result s arediscusse dwit hrespec tt othei r importance forou runderstandin g interactions inhost-parasit e systems.

INTRODUCTION

Resistance orsusceptibilit y isth e resulto fa n interaction between host andparasite .Physiologica l andbiochemica l processes areinvolve d in this interaction.T o agrea texten tthe y are influenced by hydrogen ion concentration and its regulation. Inth e followingpape rpH-relation swil l bediscusse d with regard to theirpossibl e importance forresistanc e of trees against fungalparasites .

IMPORTANCE OF PH FOR REGULATION OF METABOLIC PROCESSES

When after infectionhos tplan t andpathoge n arei ncontac tth einterac ­ tionbetwee nmetaboli c mechanisms ofvirulenc e andresistanc e determines the course ofpathogenesis .No tonl yth eprocesse s themselvesbu t alsoth e medium inwhic h they interact are important.Thi smediu m is characterized, forexample ,b y itspH . Thep H isver y important forth eoptima l functioning ofmetaboli c processes.Deviation s from optimal hydrogen ionconcentratio n influence biomembranes,affectin g their structure and function sotha t transport processes between cells andwithi n cells aredisturbed .Additionall y the biochemical reactions themselves canb e influenced, aswel l asth e activity ofenzyme s involved.A n example (Fig.1 )i sth eequilibriu m between3 molecular states of ahypothetica l enzyme inrelatio n top H and enzyme activity.Thu s regulation ofintracellula r pH is afundamenta l biological process (Raven& Smith , 1973).

176 inactive active inactive

R + R R H-C-NH3 -H^ H-C-NH3 -H^ H-C-NH2 H-C-COOH ^ H-C-COO" 'H* H-C-COO" 1 1 1 R' R' R'

pH Figure 1. Equilibrium between3 molecula r stateso fa hypothetica l enzyme inrelatio nt op Han dvelocit yo freactio n (v)(fro mSchol z& Knabe , 1976).

Within eachorganism ,varyin gwit hth erespectiv e organan dth einvol ­ ved cells,a noptima lp Hi smaintaine db yregulation .Th eregulatio ni sdu e to internalmetaboli c processes consumingo rproducin gH of0H~ ,an dp Hi s stablei na centai n range againstexterna l influences (Smith& Raven , 1976).Th eobjectiv eo fth eparasiti c fungusi st ometaboliz e substratesi n the internalmilie uo fth ehost ,whic h sumultaneouslyi sth eexterna l milieufo rth efungus .Thi smean s thatfo rth efungu s alsoth ehydrogen-io n concentration inth ehos tplan tcell si so fimportance . Inman yhost-pathoge n systemsth eexterna l optimump Hfo rfunga lsub ­ stratemetabolis m ishighe r thanth ep Hi nth einterna lmilie uo fth ehos t plant. Therefore2 differen t regulationmechanism s interactwit h each other.Thi s interaction isillustrate db ya cyberneti c model (Fig.2 )i n whichth erelationshi p isshow nbetwee nth eintracellula rpH-milie uo fth e hostan dth eextracellula r pH-milieuo fth eparasit e underth eassumptio n thatth eintracellula rp Ho fth ehos ti ssuboptima l forth eparasite . Measurementso fp Hinteraction s inlivin gplan tcell sar edifficult . Hence, forcharacterizin gp Hrelations ,titratio no faqueou splan t extracts canb ecarrie dou twit hH or0H~ .Th eresistanc e againstp Hchang e during titrant additionca nb econsidere d asa measur eo fth estabilit yo fth epH ,

177 reference input HOST: intracellular pH-optimum

controlle reference input PARASITE: « 5 extracellular pH-optimum Ü sensor,. I final control element finalcontro l element controlleUJMLI r I HOST H+x pH /OH" PARASITE i disturbancefo rPARASI T disturbance forHOS T «

controlled variable: i extracellular pH •valueFttRASITE ) ^"sensor

controlled variable: intracellular pH-value HOST

Figure 2. Acyberneti c model ofth e interaction ofhos t andparasit ewit h respectt oth e intracellular pH-milieuo fth ehos t and the extracellular pH-milieuo fth eparasit ewhe nth ep Ho f thehos t is lower thanth e optimum pH forth eparasit e (after Scholz& Stephan, 1975).

expressed asbufferin g capacity. Thehypothesi s is thatth ehighe r the buffering capacity, themor e resistant isth ehos tplant . These interactions were tested firsti nth ehost-parasit e system Pinus sylvestris L./Lophodermium pinastri (Schrad. exHook. )Chev . (Scholz & Stephan, 1974).

THE HOST-PARASITE SYSTEM PINUS, SYLVESTRIS/LOPHODERHIUM PINASTRI

Themos t interestingperio d for studying thishost-parasit e interaction is soon after infection inautum n and during the latentphas e inwinter , which isdecisiv e forpathogenesis . With reference toth e cybernetic model (Fig.2) ,2 subject sma yb e investigated: thecontributio n ofth epH-regulatio nb y thehos tplan tt o itsresistance , and the contribution ofpH-regulatio nb y the fungust oit s pathogenicity. Firstw eca ntes twhethe r the fungus canregulat ep H toth e level for itsoptimu m growth,whic h is assumed tob e approximately pH5. 0 top H 5.5, depending onth e fungus strain. Intrial swit h a L. pinastri straino naga rmedi a containingvariou spin eneedl e extractsw e found that the fungus can shiftth ep H toward itsexterna l optimump H (Fig.3) .

178 pH 5.5-

5.0

45-

40-

3.5- fungus strains on R"and R* clones SIL A 5 A A SILA 20 D • SIL 156 O • 2 3 4 5 measuring points on the radius of fungus colony

Figure 3. ThepH-changin g influence of3 strains of Lophodermium pinastri onneedl e decoction agar.p Hwa smeasure d alongth eradiu s ofculture s growing inPetr i dishes.Curve s aremean s of4 resistant (R )an d4 suscep ­ tible (R~)non-infecte d Pinus sylvestris clones,respectivel y (from Scholz & Stephan, 1981).

Inthi s connection Schutt (1964a)foun d thatth e colony diameter of L. pinastri decreased with increasing concentrations inth e culturemediu m of needle extracts ofresistan t Scotspin e clones.Th ediamete r increased, however,whe n increasing concentrations ofneedl e extracts of susceptible cloneswer e added. Schutt (1964a)suppose dbiochemica l components aspossi ­ ble influencing factors.Highe rbufferin g capacity inresistan t clones also could have contributed tothi seffect . Corresponding effects shouldb e expected inth e livinghos tplan tcell . Thehos tplan tshoul dmaintai np H atit s own intracellular lower optimumb y its inherentbufferin g capacity orb y additionally synthesizedbufferin g substances.

179 pH

Bruns 6 / - Schlesw 77/1 / susceptible / resistant /

pH-optimum of L.pinastri / /

! I l 1

n NaOH CmI)

Figure 4. Buffering capacity inneedl e extracts fromnon-infecte d Pinus sylvestris cloneswit hdifferen t degreeso fresistanc e to Lophodermium pinastri (after Scholz& Stephan, 1974).

Forprocedura l reasonsw e cantes tth e inherentbufferin g capacity only invitro ,tha t is,i naqueou s extracts ofth eparticula rplan torga n that the fungus infects,i nth epresen tcas e theneedles .Sinc e inth ecours eo f pathogenesisnucle i of infectedplan tcell s degenerate andcompartmentatio n withinth e singleplan tcel l isdisintegrated , this state ispresumabl y similar totha t foundb y the fungus inth e invitr oextracts . Inaqueou sneedl e extracts from 8clone s of Pinus sylvestris ofdiffer ­ entdegree s ofresistanc e thep Hwa s artificially influenced by adding 0H~ This titration resulted incurve s (Fig.4 )whic hshowe d that asusceptibl e cloneha d lowerbufferin g capacity than aresistan t one.Th ebufferin g capacity ofresistan t cloneswa s significantly highertha n thato fsuscep ­ tibleclones .A n analysiso fvarianc ewa sbase d onclone s (Table 1).Vari ­ ancebetwee n ramets inclone swa s small.Bufferin g capacity and resistance werehighl y correlated (r= 0.84 ) (Scholz& Stephan, 1974). These summarized investigations support thehypothesi s givenabove :

EXAMPLES OF RELATION OF BUFFERING CAPACITY AND RESISTANCE

Therelatio nbetwee nbufferin g capacity and resistancewa s tested repeatedly forth e Pinus sylvestris/Lophodermium pinastri-systemwit h differentpin epopulations ,an d investigated also forothe r host-parasite

180 Table 1. Analysis ofvarianc e ofbufferin g capacity ofdifferen t resistant clones of Pinus sylvestris.

Sourceo f df F-value Variance Relative variation component variance a- 2 component (%)

between clones 7 22.01*** 18953.7 83.4 betweemramet s 16 4.84*** 2146.8 9.4 error 48 - 1675.3 7.4

***significan t atp S0.001 .

systems.Th e results ofth evariou s authors aresummarize d inTabl e 2.Mos t ofth e investigations confirm thepreviou s results.Bu tther e areals o deviating results.The y canb ecause d byvariou s reasons several ofwhic h will be discussed inmor e detail,becaus e they lead toa mor e thorough understanding ofhost-parasit e interaction: / 1. Procedural reasons related to statistical aspects of sampling,handlin g ofplan torga n samplesbefor e titration, and titrationmethod s allca n influence theresults .Difference s caused by thesereason s canb e excluded by standardization ofmethod s andwil l notb e discussed further. 2. Physiological processes areinfluence d bydiurna l or seasonalvaria ­ tion,o rboth , andma y changewit h the ageo fth ehos tplant .Thus , results ofinvestigation s onth erelatio nbetwee n resistance andbufferin g capacity inth erespectiv e plantorga n also canb e influenced. 3. The resistance inth e investigated hostpopulation s mayb ebase d on different or interacting resistancemechanisms . 4. Finally, thewell-know n greatvariabilit y ofparasit epopulation s may cause differences invirulenc e indifferen tbiotype s orraces .Henc eresis ­ tancemechanism swhic h areeffectiv e againsta certai n funguspopulatio n mayb e less effective against another funguspopulation . Sucheffect s are knownt ooccu r insom ehost-parasit e systems,bu thav eno tbee ninvesti ­ gated extensively in foresttrees .

EFFECTS ON THE RELATION BETWEEN BUFFERING CAPACITY AND RESISTANCE

Influence of age of the plant organ Buffering capacity andp H inaqueou s extracts ofScot spin e needles vary during the ontogeny ofth eneedle s as shownb yDupat e (1977),Martins - son (1979), Wind (1979)an d Scholz& Stephan (1981). Buffering capacitywa s highest inyoun g needles afterneedl e elongation, decreased during summer, autumn andwinte r ofth e firstvegetatio nperiod , and stayed atlo wvalue s

181 .H CO <# H r-- Ol ci r-- oo IX ^ r- Ol Ol Ol CD r- - f- o Ol O! Ol H rH f-H Ol Ol fi r^ Ol fi rH rH O <« ^ Oi H rd fi Ol H -fi e .fi .fi o> .fi Ü rd Ol M d 01 U P U P 0 +J p 3 en P U u m rH rH o CO s a CO co CO 02 CO CO co 10 en Q s S w lu s CO « H

fi O •tf o «Ji 01 •H oo fi o P fi •H ^^ fi rd o -a tf CJ H >i >i >- >H a) U >i — >H 0 >i fi fi C M O rH P P O Oi P (H l Ü O m fi fi 01 0 •H ai rd p M O ai O o u rH •H rH rQ M w co rd N. rd ja 01 \ 0) P (1) ai ai 3 3 >i •0 ft o rH o « p P Oi fi fi >i ai p e s > rd 01 rH •H rd ai rd 3 3 3 u o 1-) 1-5 rH 01 EH CO O ra •a t-j fi o H <

CH O P m •H fi rH T3 TS r* T3 U rd P •o T3 •a •0 xs •O ai ai 01 01 01 Ol Oi CO ai ai 01 u m 01 ai ai 0) 01 0) rd O) U 0 ai ai fi rQ rd o rfi fi fi fi fi fi fi fi fi Ü

Cn co co C Ol •H co ai 0) rH 01 01 01 u CH co ü P p ra ra rfid •H CH rd 01 ai 0) CO 3 •H co •H •H •H 01 0) o rH rH rH 0fi) rH 01 rH rQ H 0) ai ru •H •H •H > Oi c en p fi S g 0 0 C o s fi rH -fiH O rd rd rd rd H •H £ rH CH OH CM ft CO U ra ra H S u M O Jfi W r-, 01 & >*H Ol Ss •H W C0 co co Is •Ci <*H fi lu 13 HJ 01 MS 0) 01 l-i ns m m tj a, O fi O O Oi •a +J C! q R CI co •p co *«N "M 'rn 'M Is Is 0 > > r>n > r>H &i i-H •-H bi Öl is •H rH u •H ft 3 01 S» Si "M 'M +J 3l Öl 3l 3i US ft W is CO CO CO rH W co q co co co Is q ft CO CO CO US C0 w CO co CO co co W co CO 3 01 3 3 3 3 3 3 3 3 3 3 3 O •o « q q q q q q q q q q "M "rH •H •rn '"H -H •H ro C O a •H a< û. n. EH rd m a. ft. ft. ft. ft.

182 inth ebeginnin g ofth e second year.Durin g thesam eperio d pH increased. Parallel tothos e changes the lifecycl eo fth eneedl e cast fungus Lophodermium pinastri takesplace .Th ehighes tlevel s ofascospor eproduc ­ tion and release in late summer and autumncoincide ,a tleas tunde r central European conditions,wit h the low-value period ofbufferin g capacity in currentyea rpin eneedles . During this infectionperio d and the following latentphas e ofth e pathogen resistancemechanism s ofth ehos tplan t shouldb emos teffective . Results of several investigations showed clearly thatonl y inlat esummer , autumn andwinte r did the resistant Scotspin eclon e group havesignifi ­ cantlyhighe rbufferin g capacity thanth e susceptible group (Scholz & Stephan,1974 ,1981) . Iti simportant ,therefore ,t otim e investigations of traits likebufferin g capacity toth eperio d when thedecisiv ephysiolo ­ gical interactionbetwee nhos t andparasit e takesplace . Resistancemechanism s varywit h further ontogeny ofa plan t organ.Fo r example, Scotspin eneedle s oldertha n 1yea r areresistan t toth eparasit e Lophodermium pinastri. Also, differences inag eo fth ehos tplan t itselfcoul d influence the relative importance ofdifferen tmechanism s inhost-parasit e systems. Dupate (1977)foun d arelatio nbetwee nbufferin g capacity and resistance in young Scotspin eplants ,bu tMartinsso n (1979), who investigated other young Scotspin e families,di dnot .Studie s described abovewer e conducted on25-yea r old Scotspin e clones (Scholz& Stephan, 1974,1981) . Further­ more, Franich &Well s (1977)foun d norelatio nbetwee nbufferin g capacity of Pinus radiata D.Do nneedle s andresistanc e against Dothistroma pini Hulb. in5-yea rol d trees,bu t in40-yea r old treeshig hbufferin g capacity and resistance toth eparasit e generallyexist .

Influence of host population For investigations onth erelatio nbetwee nbufferin g capacity and resistance,host-plan tmaterial s ofquit e different character and origin havebee nuse db y different authors.I nvariou s regions ofth enatura l distribution areao f afores ttre e species differentcoevolutio nwit hth e respectiveparasit ema y have resulted indifferen t resistancemechanisms . Thus relationsbetwee n traits aton e location canb edifferen t from those atanothe rone . A fewexample sma y supportthi s concept forth e Pinus sylvestris/Lopho­ dermium pinastri-system. Relative resistance ofprovenance s may changewhe n they arecultivate d ina distan t location incompariso nt o thato fprove ­ nances from other regions ortha to f localprovenances .Whe n Scotspin e provenances aregrow nunde rcentra l European conditions,thos e fromScandi ­ navian countries often showhighe r resistance toth eneedl e cast fungus thanthos e from southern France (Schutt, 1964b). Ina Dutc h trialGerma n Scotspin eprovenance s werekille db y Lophodermium pinastri whereas some

183 localprovenance s remained largelyunattacke d (Kriek& Bikker , 1973). Ina south-westGerma ntria l ofth eForstlich eVersuchs -un d Forschungsanstalt Baden-Württemberg theprogenie s ofnon-loca l Scotspin eplu s trees selected forneedl e castresistanc ewer eheavil y attacked by L. pinastri butth e localprovenanc e remained unattacked (dataunpublished) . These reactions wereth eresul to f interactionswit henvironmenta l factors,bu t alsodiffe ­ rences inresistanc emechanism smigh thav ebee n involved. Perhaps such differences could explainth e results ofMartinsso n (1979)wh o didno tfin d a relationbetwee nbufferin g capacity of Swedish Scotspin e families and resistance. Inou r own investigations onScot spin e full-sib families from crosses inan dbetwee nAlpin e andMazuria nprovenance s theAlpin e crosses showed highbufferin gcapacit y and susceptibility to L. pinastri, whereas Mazurian crosses showed lowvalue s forbot h traits (Stephan& Scholz , 1981). Withinth eprovenances ,however ,thi s relationwa s notgeneral . Resistance of P. sylvestris to L. pinastri, which isassume d tob e polygenic, iscertainl y governedno tb y only onemechanism . Thismigh t explain differences betweenpin epopulation s investigated foron eresis ­ tance traitonly .

Influence by parasite population Finally the interactionbetwee nhos t anddifferen tpopulation s from different locations shouldb e considered. Lophodermium pinastri, forin ­ stance, showsgrea tvariabilit y with respect tomorphological ,physiologi ­ cal andbiochemica l traits (Millar& Watson , 1971;Stephan ,1973a ,b) , which recently lead toth e separation ofdifferen t species (Minter et al., 1978)wit h apparently different life cycles andbiolog y (Minter& Millar , 1980). Obviouslyvariatio n inth evirulenc e ofth epathoge n ist ob eexpec ­ ted.Therefor e invariou shost-parasit epopulation s different interactions andbalance sbetwee n resistancemechanism s andvirulenc emechanism s are probable. Inconsiderin gp H regulation as apossibl emechanis m ofhost-parasit e interaction Stephan (1975)reporte dvariatio nbetwee n L. pinastri strains isolated fromdifferen tpin e species.Th e ability ofth e fungal strains to changep H aswel l asth e reaction of thehos t species showed remarkable variation invitro .Suc hvariatio n alsoha sbee n foundbetwee n fungal strains isolated only from Scotspin e (Scholz& Stephan, 1981). These strains differ inth e amounto fthei rpH-changin g influence onneedl e agar andeve n inth edirectio no fthi s influence (Fig.3) . The results of L. pinastri strains growing onneedle-decoctio n agar were largely confirmed with cultures inliqui dneedl e extracts of resistant and susceptible Scotspin e clones (dataunpublished) .Th ep Hnormall y changed either tohighe r orlowe rvalues ,dependin g onth e fungal strain used.Ther ewer enegativ e correlationsbetwee nth edegre eo fresistanc e of respective Scotspin e clones andth eexten to fp H change invitr o after

184 14day s ofculture .Th e importance ofth evariabilit y ofth eparasit e in therelatio nbetwee n resistance andbufferin g capacity isunderline d by the facttha t 1strai n showed adifferen tbehaviou r inculture . Inthi s case the amounto fp Hchang ewa spositivel y correlated with degree of resistance ofth epin e clones.Th ebehaviou r ofth epathoge npopulatio n interacting withth erespectiv e hostpopulatio nwil l depend on its inherent requirement for anoptimu m pH and its ability toregulat epH .

INHERITANCE OF BUFFERING CAPACITY

Inheritance ofbufferin g capacity isno tye twel l known.Preliminar y results of investigations ofcrosse sbetwee n andwithi n 2Scot spin eprove ­ nances showed significant differences betweenprogenie s (Stephan& Scholz , 1981).Alpin e crosses showed thehighest , andMazuria ncrosse s the lowest means. Interprovenance crosseswer e intermediate forthi strait . Estimations ofgeneti cparameter s in families ofNorwa y spruce (Picea abies (L.)Karst. )showe d thatth ephenotypi c variance ofbufferin gcapaci ­ ty isgoverne d mainlyb y genetic factors (Scholz& Reck , 1977).Heritabili - 2 ' tywa s estimated roughly ath =0.78.

CONSEQUENCES AND CONCLUSIONS

1. Forsuitabl e comparability ofresult s sampling criteria andbufferin g capacity evaluationmethod s shouldb e standardized. With the necessary phytosanitary restriction,th eexchang e ofhos tplan tmateria l and cultures ofparasite s for suchhost-parasit e interaction studies is also suggested. 2. Abette r understanding ofvirulenc e and resistancemechanism s isneces ­ sary includingmor e detailed knowledge ofp H regulation inhost-parasit e interaction and its relationt oothe r resistancemechanisms .Thi s shouldb e investigated invariou s host-parasite systems. 3. Up tono wbufferin g capacity cannotb e recommended generally asa trai t forus e inindirec t selection.Befor e generalizing aboutth e importance of a resistancemechanism , one should test iti ndifferen thos tpopulations . 4. Investigations onp H regulation canhel p inunderstandin g theinterac ­ tionbetwee n host andparasit e atth e level ofbiochemistr y aswel l asa t the level ofth epopulation s involved.

REFERENCES

Dupate,A.I. ,1977 .Investigation so nresistanc eo fPinu ssylvestri sL .t oLophodermiu m pinastri (Schrad.)Chev .In :Ökolog :Besonderheite nde rniedere nPflanze nde ssow ­ jetischenOstseeraumes .Materia lzu m8 .Symposiu m derbalt .un dweissruss .Mykologe n undLichenologen .Wiln a1977 .p .74-75 . Franich,R.A . &L.G .Wells ,1977 .Infectio no fPinu sradiat ab yDothistrom apini :effec t ofbuffe rcapacit yo fneedl ehomogenates .Ne wZealan dJourna lo fFores tScienc e7 : 35-39.

185 Kriek,W .& G .Bikker ,1973 .Germa nan dDutc hprovenance so fScot spin ei nth eNether ­ lands.Nederland sBosbou wTijdschrif t45 :154-161 . Kumi,J .& K.J .Lang ,1979 .Th esusceptibilit yo fvariou sspruc especie st oRhizosphaer a kalkhoffiian dsom ecultura lcharacteristic so fth efungu si nvitro .Europea nJourna l ofFores tPatholog y9 :35-46 . Martinsson,0. ,1979 .Testin gScot spin e forresistanc e toLophodermiu mneedl ecast . StudiaForestali aSuecic a 150:1-63 . Matschke,J. &E .Scholz ,1980 .Biologisch eGrundlage nde rForstpflanzenzüchtung .Bei ­ trägefü rdi eForstwirtschaf t 14:116-121 . Miliar,CS .& A.R .Watson ,1971 .Tw obiotype so fLophodermiu mpinastr ii nScotland . EuropeanJourna lo fFores tPatholog y 1:87-93 . Minter,D.W .& C.S .Millar ,1980 .Ecolog yan dbiolog yo fthre eLophodermiu m specieso n secondaryneedle so fPinu ssylvestris .Europea nJourna lo fFores tPatholog y10 : 169-181. Minter,D.W. , J.M. Staley& C.S .Millar ,1978 .Fou rspecie so fLophodermiu m onPinu ssyl ­ vestris.Transaction so fth eBritis hMycologica lSociet y71 :295-301 . Raven,J.A .& F.A .Smith ,1973 .Th eregulatio no fintracellula rp Ha sa fundamenta lbio ­ logicalprocess .In :W.P .Anderso n (Ed.): Iontranspor ti nplants .Academi cPress , London/NewYork ,p .271-278 . Scholz,F. ,1973 .Biochemisch eMethode ni nde rForstgenetik .Forstarchi v44 :171-174 . Scholz,F .& W .Knabe ,1976 .Investigation s onbufferin gcapacit y inspruc eclone so f differentresistanc e toai rpollution.'XVI .IUFR OWorl dCongress ,Osl oJun e1976 . WorkingPart yS 2.09.0 4- Physiologica leffects .Mimeograph .1 0p . Scholz,F .& S .Reck ,1977 .Effect so facid so nfores ttree sa smeasure db ytitratio ni n vitro,inheritanc eo fbufferin gcapacit y inPice aabies .Water ,Ai ran dSoi lPollu ­ tion8 :41-45 . Scholz,F .& B.R .Stephan ,1974 .Physiologisch eUntersuchunge nübe rdi eunterschiedlich e Resistenzvo nPinu ssylvestri sgege nLophodermiu mpinastri .I .Di ePufferkapazitä ti n Nadeln.Europea nJourna lo fFores tPatholog y4 :118-126 . Scholz,F .& B.R .Stephan ,1975 .Zu rpH-Regulatio nal sFakto rde rResisten zvo nPinu s spp.gege nLophodermiu m pinastri.Angewandt eBotani k49 :55-63 . Scholz,F .& B.R .Stephan ,1981 .Physiologica l studieso nvariatio ni nresistanc eo f Pinussylvestri st oLophodermiu mpinastri .II .Variatio no fbufferin gcapacit yi n needleso fdifferen tag ewit hrelatio nt oresistance .Europea nJourna lo fFores t Pathology 11(i npress) . Schutt,P. ,1964a .Ein emykologisch eMethod ede rResistenzprüfung ,entwickel tfü rde n Lophodermium-Befallde rKiefer .Phytopathologisch eZeitschrif t51 :62-72 . Schutt,P. , 1964b.De rSchüttebefal lde rKiefe r inAbhängigkei tvo nHerkunf tun dAnbau ­ ort.Forstwissenschaftliche s Çentralblatt83 :129-192 . Smith,F.A .& J.A .Raven ,1976 .H transportan dregulatio no fcel lpH .In :U .Lüttg e& M.G.Pitma n (Eds):Encyclopaedi a ofplan tphysiology ,Ne wSeries ,2 .Transpor ti n plantsII ,Par tA ,Cells .Springer ,Berlin/Heidelberg/Ne wYork .p .317-346 . Stephan,B.R. , 1973a.Untersuchunge nzu rVariabilitä tvo nLophodermiu mpinastri .I .Kul ­ turvarianten.Europea nJourna lo fFores tPatholog y3 :6-12 . Stephan,B.R. , 1973b.Untersuchunge nzu rVariabilitä tvo nLophodermiu mpinastri .II .Un ­ terschiede imphysiologische nVerhalten .Europea nJourna lo fFores tPatholog y3 : 112-120. Stephan,B.R. , 1975.Variabilit yo fLophodermiu mpinastr ii nculture .Mitteilunge nde r Bundesforschungsanstalt fürForst -un dHolzwirtschaf tHamburg-Reinbe k108 :69-78 . Stephan,B.R .& F .Scholz ,1979 .Weiter eUntersuchunge nzu runterschiedliche nAnfällig ­ keitvo nPinu snigra-Klone ngegenübe rScleroderri slagerbergii .Europea nJourna lo f ForestPatholog y9 :46-51 . Stephan,B.R .& F .Scholz ,1981 .Preliminar y resultso nLophodermiu mneedl ecas tresis ­ tancean dbufferin go fp Hi nScot spin ecrosses .Proceedin go fth emeetin go fth e IUFROWorkin gPart yS 2.06.0 4- Needl ediseases .Sarajevo ,Yugoslavia ,Sept .1980 . 6p . (inpress) . Wind,E. ,1979 .Pufferkapazitä t inKoniferennadeln .Phyto n19 :197-215 .

186 Histoenzymological approach for detecting host-parasite interactions in forest trees caused by heart-rot diseases

Zuei-ching Chen & Paul D. Manion

Departmento fBotany ,Nationa lTaiwa nUniversity ,Taipei ,Taiwan ,Republi co fChina ,an d Departmento fFores tBotan yan dPathology ,Stat eUniversit yo fNe wYork ,Colleg eo fEnvi ­ ronmentalScienc ean dForestry ,Syracuse ,Ne wYork ,US A1321 0

ABSTRACT

Aprogressiv e change ofcell-wal l degrading enzymeactivi ­ ties inlivin g aspen (Populus tremuloides) sapwood inoculated withbasidiospore s of Phellinus tremulae were investigated bya routine enzymological method and amodifie d histoenzymological method.Peroxidas e activity was inhibitedmoderatel y inth e wounded tissue andseverel y inth e inoculated tissue/duringth e earlyperio d ofdiscoloration .Th e activity of laccase inth e inoculated tissuewa s stimulated immediately after treatment and increased 4t o6 time sove r that inth e sound tissuean d abouttwic e over thati nth ewounde d tissue.Mor e cellulase activity wasnote d inth ewounde d than inth e inoculatedtissue . A clearerpictur e ofhost-parasit e interactions was obtained by thehistoenzymologica l method.Th e sensitivity of thehistoen ­ zymological methodwa s about12-101 8 timeshighe r thano fth e routine extractionmethod .

INTRODUCTION

Basidiospores of Phellinus tremulae (Bond.)Bond .& Boriso v germinate only on fresh aspen (Populus tremuloides Michx.)sapwoo d wounds duringth e summer (Manion& French , 1968;Chen , 1971). Recently Shigo's succession theory (Shigo,1963 ,1967 )cas tdoub to nth epathogenicit y ofprincipa l decayHymenomycete s inth e initial stage ofdecay . Phellinus tremulae, the white-rotter of aspen,possesse s anenzym e systemwhic h can attack all components ofth ewoo d cellwal l including lignins,resultin g in agenera l thinning ofth ecel lwall , even inth eearl y stage ofdeca y (Basham,1958 ; Cowling, 1961;Wilcox , 1968). Cellulases ofth ewhite-ro t fungi attackth e crystalline and amorphous cellulose ofth ehos tcel lwal l ata simila r rate, and act from the surface ofth ewall . Lignin-depolymerizingenzyme s ofth ewhite-ro t fungi are ablet openetrat e rapidly the secondarywal l of thewoo d cells and acto nth e compound middle lamella.Thi s results inth e

187 rapid utilization of ligninb y the fungus inth eearlies t stage ofdecay . Enzymatic degradation ofth ecel lwal lb ywhite-ro t fungi,therefore , involvesbot h alignin - and acellulose-degradatio n system (Cowling, 1961). Lignin-degradationenzyme s involvepolypheno l oxidases ofth e laccase type (Aufsess et al., 1968;Fâhraeus ,1952 ;Kir k et al., 1968a,1968b )o rth e peroxidase type (Ishikawa &Oki ,1964 ;Ly r& Ziegler, 1959). Thepresen t investigation attempts todetermin e the decay capability of P. tremvlae in fresh sapwoodwound s of living aspentree s aftergerminatio n ofbasidiospo - res.Histoenzymolog y is introduced into this study ina compariso n withth e routine enzyme extractmetho d forth eevaluatio n ofth emagnitud e ofhost - parasite interactions inthi sheart-ro t system.

MATERIALS AND METHODS

Basidiospore inoculumwa s collected ona clea nglas s slideplace d ina plasticbo x attached to abigtoot h aspen (Populus grandidentata Michx.) bearing sporulating fruitingbodies .Spore swer e collected during a16 -t o 18-hour-period starting at3- 5 p.m. Spores from asingl e fruitingbod ywer e washed from the slideswit h sterilized distill'edwate rprio r toinoculat ­ ion. The concentration of sporeswa s approximately 2-6 millionpe rm l of solution.O nJun e 1, 1969,wound s 2c m deep and 0.5 cm indiamete r were made ata heigh to f 1.4 m onopposit e sides oftremblin g aspentree s 10-15 cm indiamete r located inth eHeiber gMemoria l Forest,Tully ,Ne w York,USA .Afte r thewound swer emade ,on ebor ehol ewa s inoculated with 1 mlo fth ebasidiospor e suspension of P. tremulae andth eothe rhol ei n theopposit e sidewa s injected with 1m l of sterilized distilledwater . Collections from treated aspentree swer emad emonthl y after inoculation until October.Eac hmont h 15-20tree swer e cut and1-mete rsection swer e removed and stored at-2 0 °C.On ehal fo feac hcollectio nwa sused_fo r investigating symptom development, fungal flora,cytologica l changes,an d enzyme assays.Th e restwa suse d for investigating pH change,as han d mineral content.Th e stem sectionswer ecu tverticall y through thebore ­ hole intohalve s to observe thedevelopmen t ofdiscoloratio n and the change inmorphology .Piece s ofwoo d about1 x lx 2-3 mm insiz ewer e removed aseptically from differentpart s ofth e discolored column andculture d on 2 %mal t agarmediu m at2 5 °Ci norde r torecove r P. tremulae. Forenzym epreparatio n extracted fromwoo d meal, frozensapwoo d blocks 2.5 cmx l. Oc mx 0.5 cmwer e removed near thewoun d andgroun d ina Wile y Mill over a10 0mes h screen.On e gram ofwoo dmea lwa s then suspended in5 m lo f0.0 5 Mphosphat ebuffe r containing 0.25 M sucrose and 0.03 M ascorbic acid atp H 7.0.Th emixtur ewa s ground in amorta r for 2minute s at5 °C.Th emixtur e was thencentrifuge d at4 °C for3 0minute s at 1500 0x g .T o the supernatant extract ammonium sulfatewa s added to 0.8 saturation and the resultant enzymeprecipitat e wascollecte db ycentrifu -

188 gationa t4 °Cfo r1 0minute s at200 0x g. Theprecipitate d enzymewa s dissolved in5 m lo fdistille dwate r anddialyse d incol dwate r for1 2h . Thedialyse d enzyme solutionwa suse d formeasurin gperoxidase ,laccase , catalase,an dcellulases . Forenzym epreparatio n from thin sections ofsapwoo d tissue, frozen woodblock s about0. 5c mx 0. 5c mx 0. 5c mi nsiz ewer e sectioned radially toa thicknes so f2 0 ymwit h acryostat .Abou t2 0section s were addedt o eachenzym e reaction solution.Afte rth ereactio nwa smeasured , thesec ­ tionswer e collectedo nfilte rpaper s anddrie d at10 5° Covernigh tbefor e theirdr yweigh twa sobtained . Peroxidase activitywa sdetermine db yth emetho d describedb yJenning s etal.(1969) .Th eenzym e solutionwa sdilute d 1:5wit hwate r and0. 5m lwa s used todetermin e peroxidase activity.Th ereactio nmixtur e consistedo f 1.5m lo f0.02 5M citrate-phosphat e buffer atp H5.0 ,0. 5m lo f0.0 2M guiacol,an d0. 5m lo f0.0 6M hydroge nperoxide .Activit ywa srecorde da s thechang e inabsorbanc epe rminut e at48 3nm .Th ereactio nwa sinitiate d byth eadditio no fhydroge nperoxide .Fo rth equantitativ e histochemical method, thetissu e sectionswer e added directly toth ereaction/Solution . Themixtur ewa sincubate d for3 0minute s at3 0°C .A preliminar y testwit h various incubation times from 1t o24 0minute s revealed that3 0minute swa s thebes tchoic e forobtainin gth elinea r relationbetwee n enzyme activity and incubation time.Th ereactio nwa sterminate db yswif ttransfe r ofth e reaction solution into ice.Th ereactio n solutionwa sfiltere d through WhatmanNo .1 filte rpape r andfiltrate s transfered tocuvettes .Fo rth e control,tissu e sections killedb yautoclavin g at12 0° Cfo r2 0minutes ,o r living tissue sections incubated inth ereactio n solutionwithou thydroge n peroxide,wer eused . Laccase activitywa sdetermine db yth emetho d describedb yJenning se t al.(1969).Th ereactio n solution contained 1.5m lo f0.0 2M citrate-phos ­ phatebuffe r atp H6.0 ,0. 5m lprolin e (5mg/ml) , and0. 5m lcatecho l (2mg/ml) . Themixtur ewa saerate d through aglas s capillary for2 minute s before addition ofcatechol ,whic h initiated thereaction .Fo reac h assay 0.5m lo fundilute d enzyme solutionwa sused . Catalase activitywa sassaye db yth emetho d ofBee r& Size r (1952)a s modifiedb yth eWorthingto n Biochemical Corp.(1963).Th edecompositio no f hydrogenperoxid ewa sdetermine d in3 m lo freactio nmixtur e containing 0.1m lo fenzym epreparation , 1m lo f0.05 9M hydroge nperoxid e in0.0 5M phosphate buffer atp H7.0 ,an ddistille dwater .Th echang e inabsorbanc e at24 0n mwa srecorde d every 10second s for7 0seconds .A uni to fcatalas e activitywa sdefine d astha t amountwhic h catalyzesth edecompositio n of1 pmol eo fhydroge nperoxid epe rminut e underth ereactio n condition described above. Cellulases activitywa sdetermine db yth emetho d ofMandel s& Rees e (1964)a smodifie db yth eWorthingto n Biochemical Corp.(1963).Thi s activ-

189 itywa s demonstrated by adecreas e inviscosit y orb y the formationo f glucose incellulos epowde r andcarboxymethy l cellulose (CMC).

RESULTS

The amounto fenzym e activity detectedb y the extracted enzymeprepara ­ tionmetho d was so lowtha tonl y the activity atth e thirdmont h oftreat ­ ment ispresente d (Table 1).Th e inoculated tissue showed thehighes t catalase activity and thewounde d the lowest. Inth econtrary , thewounde d tissue had thehighes tperoxidas e activity. Its activity was2 an d 7.5 timeshighe r thantha to fth e inoculated and sound tissu,e,respectively . Laccase activity inth e inoculated tissuewa s thehighes t amongth e3 treatments. Cellulase activitywa snegligibl e inth e sound tissue but higher activities were found inbot h the inoculated andth ewounde d tis­ sues. Ingeneral ,al l enzyme activitiesmeasure db y thismetho d tendedt o indicate low activity inaspe nsapwood , evenafte rth ewoun d inoculation. Thismetho d alsowa s unable tosho wth e low level ofenzym e activity taking place atth e site ofcell-wal l decay inth ehos tplants . Thehistochemica lmetho d showedprogressiv echange s ofenzym eactivi ­ ties inth ehos ttissues .Th eperoxidas e activity inbot h the inoculated and thewounde d tissueswa s inhibited during the first2 month s (Table1) . Inth e thirdmonth , the activity increased sharply andexceede d theactivi ­ tyi nth e sound tissue.Althoug h the activity inth e inoculated tissuewa s lowest inth e firstmonth , itbecam e thehighes t inth ethir dmonth . Inocu­ lationwit h P. tremulae basidiospores caused astron g inhibition ofperoxi ­ dase activity inth ehos ttissu e inth eearl y stage of infection.Activit y oflaccas e inth e sound tissuewa s inth erang e ofAA=0.1 5 to 0.2. Contrary toth e influence ofhos ttissu e onperoxidas e activity, laccase activity in both the inoculated and thewounde d tissueswa shighe r than inth e sound tissue. Incomparison sbetwee n the inoculated and thewounde dtissues , activity inth e inoculated was slightly higher (AA=0.80)tha n inth ewoun ­ dedtissu e (AA=0.62)on emont h after inoculation and reached AA=1.14,whic h was twice asmuc h astha t inth ewounde d tissue (AA=0.56)i nth ethir d month.A slightcellulas e activity was detected inth e sound tissue.Th e activity innorma l aspen sapwood ranged from the lowest inSeptembe r (0.08) toth ehighes t inmid-summe r (0.52). Thewounde d tissue showed the highest activity ofth e 3types .Almos t alinea r increase of 'C.' enzyme activity inth ewounde d tissue wasnotice d through the second month.Activit y inth e inoculated tissue increased inth e secondmont hbu twa s only 2/7 ofth e activity inth ewounded .A steady activity wasobserve d inbot h theinocu ­ lated and thewounde d tissues during the second and thethir dmonth swhil e therewa s ashar p drop ofcellulas e activity inth enorma l aspen sapwood.

190 4-1 +J W c 1 m •H •H 4-1 o 1 o 73 rH U) M ra H G r- CM r~- 01 t~ ie -3 § ^ 73 •O o in 0 o rH Ü -p 10 10 10 rH (0 o ja ja G G 10 •o 3 CM co O >1 •ri G o O 3 r- rH L/1 m CO m o m ^p O lO -H ja 10 o o 1 co rH *£> o rH in co iD H co c- 4-1 •P s G • 10 73 G w a o o r-\ « 0) 10 nj 3 s iH s ra sQ U o 0 0 rH 1 0 ifl o e 0 rH O» +J G 4-\ 1 O 3 •P Xi G I« O •H 4-1 rH G U m •H Oi G 10 10 Id 10 •p D Q U w «s > ra .H 3 *j" in ID r~- O O G O o G M S rH CM M dl n n n n n -H •P MH 0

Cl a) CI Oi m m 10 10 10 73 M m rH rH (NI 0 0 Q) 3 3 0) O 1 m rH rH S (>0 m 10 CI rH rH 1ra0 N m T) C) CJ CI 71 ft10 0 w• S m -rH m 1) 0 •H ra •p 10 tj U 0 G 0 •ri •H M 0 rH rO ft M T) ft = ~ ft ja 1ra0 73 O 0 • ^ O I4H Dl m o Ifl 0 OS 1 0 rH Oi S o •H 0 •P •r| N. i > ft •H •H id 4-1 CI 10 (H -H « S S O u ü M xi Q •P r4 \ \ O 10 10 u O < < o, 1-1 73 < CI <

191 DISCUSSION AND CONCLUSION

There are 2theorie s onth eenzym e system responsible-.for lignindegra ­ dation.On e theory stresses therol e of laccase (Aufsesse t al.,1968 ; Fâhraeus, 1952;Kir k et al., 1968a, 1968b). Theothe r stressesperoxidas e asmor e suitable forth edegradatio n of lignin inth e semi-anaerobic en­ vironment ofwoo d tissue (Ishikawa& Oki , 1964;Ly r& Ziegler , 1959). In vivo,peroxidas e activity was inhibitedmoderatel y inth ewounde d tissue and severely inth e inoculated tissue during the first2 months .A t theen d ofth e thirdmonth , the activity inth e inoculated tissue recovered and finally exceeded the levels inth ewounde d andth e sound tissues.Th e mechanism(s)operatin g inth eearl y inhibition and late reactivation of peroxidase inth e inoculated tissue isno tunderstood . An increase of laccase activitywa s noted fromth ever ybeginnin g ofwounding . Laccase activity inth e inoculated tissuewa s 5.7 timeshighe r than inth e sound tissue and 2time shighe r than inth ewounde d tissue 3month s afterth e inoculation. The impacto fhig h activity ofpolypheno l oxidases could affect lignin catabolism andpheno lmetabolis m inth ehost .Pigmentatio n ofth ehos t tissue afterwoundin g isconsidere d tob e the result ofoxidatio n ofpheno l compounds catalyzed byman y oxidative enzymes (Kellin& Mann , 1939)includ ­ ingth eperoxidas e system ofcontaminatin g bacteria (Shortle et al., 1978). The ability toutiliz e these oxidativeproduct s couldb e acrucia l advan­ tage forthos eorganism s thatgro w and survive in thewoun d tissue (Lyr, 1962). Organismswit hhig hpolypheno l oxidase activitywoul db ebette r able todominat e and survive inth ephenol-ric h tissue.Hig hpheno l oxidase activity inth ediscolore d tissuemigh tcontribut e to thedegradatio no f aromatic inhibitors ofperoxidas e inth e tissue andpermi t reactivation in the late stage ofdiscoloration . Itseem s thatperoxidas e is less important than laccase inth e initial stage ofdiscoloratio n anddeca y ofth ewounde d aspen sapwood by P. tremulae, because ofth eevidenc e ofchange s that occurred inth e inoculatedtissue . Infiel d inoculations,cellulas e activity inth e inoculated tissuewa s significantly higher than inth e sound tissuebu t lower than inth ewounde d tissue. Invivo ,cellulas e activity inth e sound sapwood decreaseddrasti ­ cally after the summer growth season. Inoculationwit hbasidiospore s of P. tremulae altered cellulase activity inth e inoculated tissue and activity was lower than inth enorma l sound sapwood inth e firstmonth , thengradu ­ ally increased from the second month.Th ecaus e ofthi s decrease inth e early stage ofwoun d inoculation isno tknown .Shortl e etal.(1978 )repor ­ ted thatbacteri a found indiscolore d woodmigh t alter the cellulase acti­ vity ofwhite-ro t fungi to somedegree . Inth epresen t investigation, bacteriawer e isolated inabou tth e same frequency from thewounde d andth e inoculated tissues.Therefore ,th ecaus e of inhibition ofcellulas eactivi -

192 ty in the inoculated tissue probably is related to P. tremulae itself. Only slight enzyme activities in the host tissue 3 months after wound inoculation were detected by the enzyme extraction method. In contrast, the histoenzymological method was 12-1018 times as sensitive. The results indi­ cated that the majority of enzymes exuded from living hyphae in the wood decay ecosystem were tightly bound with their substrates such as lignin and cellulose components of the wood cell wall. The method modified in this way resulted in a very sensitive enzyme reaction and the outcome of the experi­ ment was very satisfactory. A clearer picture of host-parasite interaction in terms of the progressive change of enzyme activity was obtained by this method. Histoenzymology, therefore, proved to be a dependable research technique.

REFERENCES

Aufsess, H. von, H. von Pechmann &E . Graessle, 1968. Fluoreszenzmikroskopische Beobach­ tungen an pilzbefallenem Holz. Holz als Roh- und Werkstoff 26: 50-61. Basham, J.T., 1958. Decay of trembling aspen. Canadian Journal of Botany 36: 491-505. Beer Jr, R.F. &I.W . Sizer, 1952. A spectrophotometry method for measuring the break­ down of hydrogen peroxide by catalase. Journal of Biological Chemistry 195:133 . Chen, Z.C., 1971. Host response of aspen after wound inoculation with Fomes igniarius basidiospores. Ph.D. Thesis. SUNY College of Forestry at Syracuse, New York. 100 p. Cowling, E.B., 1961. Comparative biochemistry of the decay of sweetgum sapwood by white- rot and brown-rot fungi. U.S. Department of Agriculture, Technical Bulletin 1258. Fahraeus, G., 1952. Formation of laccase by Polyporus versicolor in different culture media. Physiologia Plantarum 5: 284-291. Ishikawa, H. &T . Oki, 1964. The oxidative decomposition of lignin I. The enzymatic de­ gradation of softwood lignin and related aromatic compounds by peroxidase. Journal of Wood Society of Japan 10: 207-213. Jennings, P.H., B.L. Brannaman &F.P . Zscheile Jr., 1969. Peroxidase and polyphenol oxi­ dase activity associated with Helminthosporium leaf spot of maize. Phytopathology 59: 963-967. Kellin, D. &T . Mann, 1939. Laccase, a blue copper-protein oxidase from the latex of Rhus succedanea. Nature 143: 23-24. Kirk, T.K., J.M. Harkin &E.B . Cowling, 1968a. Oxidation of guaiacyl-and veratryl- glycerol-B-guaiacyl ether by Polyporus versicolor and Stereum frustulatum. Biochemica et Biophysica Acta 165: 134-144. Kirk, T.K., J.M. Harkin &E.B . Cowling, 1968b. Degradation of the lignin model compound syringyl-glycol-B-quaiacyl ether by Polyporus versicolor and Stereum frustulatum. Biochemica et Biophysica Acta 165: 145-163. Lyr, H. &H . Ziegler, 1959. Enzymausscheidung und Holzabbau durch Phellinus igniarius Quel, und Collybia velutipes Curt. Phytopathologische Zeitschrift 35: 173-200. Lyr, H., 1962. Detoxification of heartwood toxins and chlorophenols by higher fungi. Nature 195: 289-290. Mandels, M. &E.T . Reese, 1964. Fungal cellulases and the microbial decomposition of cellulosic fabric. Developments in Industrial Microbiology 5: 5. Manion, P.D. &. D.W. French, 1968. Inoculation of living aspen trees with basidiospores of Fomes igniarius var. populinus. Phytopathology 58: 1302-1304. Shigo, A.L., 1963. Fungi associated with the discolorations around rot columns caused by Fomes igniarius. Plant Disease Reporter 47: 820-823. Shigo, A.L., 1967. Successions of organisms in discoloration and decay of wood. Interna­ tional Review of Forest Research 2: 237-299. Shortle, W.C., J.A. Menge &E.B . Cowling, 1978. Interaction of bacteria, decay fungi, and live sapwood in discoloration and decay of trees. European Journal of Forest Pathology 8: 293-300. Wilcox, W.W., 1968. Changes in wood microstructure through progressive stages of decay. U.S. Forest Service Research Paper, FPL 70. 46 p. Worthington manual, 1963. Enzymes. Worthington Biochem. Corp., Freehold, New Jersey, USA.

193 Preliminary work on the relation between resistance to Fomes annosus and the monoterpene composition of Sitka spruce resin

G.I. Forrest

ForestryCommission ,Norther nResearc hStation ,Roslin ,Midlothian ,Scotland ,U K

ABSTRACT

Apreliminar y studywa s carried out ina Sitk a sprucepopu ­ lationheavil y infectedb y Fomes annosus. Significantdiffer ­ ences inmonoterpen e compositiono fth eroo tcortica l oleoresin were foundbetwee n infected anduninfecte d trees,a sjudge db y thepresenc e or absence of Fomes inradia l cores taken fromth e lower stem. Inparticular , Fomes wa'sabsen t fromnearl y all those trees inwhic ha-pinen e formed atleas t3 7% ofth eroo t oleoresinmonoterpen e fraction.

INTRODUCTION

Several ofth emonoterpene s which commonly occur inth eoleoresi n of conifers havebee n shown toposses s fungitoxicpropertie s (e.g.Hintikka , 1970; Gibbs,1972 ;d eGroot , 1972), andth edegre e ofresistanc e ofcertai n species to attackb y Fomes annosus (Fr.)Cook eha s insom e instancesbee n connectedwit h themonoterpen e composition ofthei roleoresi n (e.g.fo r Pinus sylvestris L.b yChudny i et al., 1972;Fedoro v &Manukov , 1972). Sitka spruce (Picea sitchensis (Bong.)Carr. )i sth emai n forestry species planted commercially inBritain , and is frequently subjected to serious attacks from Fomes annosus. Ina preliminar y attemptt oelucidat e anycon ­ nectionbetwee n its degree ofresistanc e toinfectio n and thechemica l com­ position of its oleoresin, astud ywa s carried outi na heavil y infected stand growing incentra l Scotland.

EXPERIMENTAL METHODS

The area sampled formedpar t ofCompartmen t 86A ofDrummon d Hill for­ est, Tayside Region,plante d in1943 .A 4 m m diameterwoo d corewa s taken from the lower trunk ofeac ho f10 2trees .A sample ofbar k andwoo dwa s taken fromth emai nroo t system ofeac htre e forresi n analysis.Thi swa s usually taken from theuppe r surface ofon eo fth emai nroots ,abou t3 0c m

194 fromth e trunkbase .Th e samplewa s square,an dmeasure d about5 c m square and 1c mdeep . Eachwoo d corewa s examined forth epresenc e of Fomes annosus, bothb y directmicroscopi c examination ofth ecor e surface after suitable incuba­ tion, andb y culturing thin slices from thecor e onaga rmediu m atroo m temperature. Cortical oleoresinwa s extracted fromth e rootbar k samples eitherb y direct suction into glass capillary tubing from resincanal swher epossi ­ ble, or alternatively bymaceratin g small quantities ofth e rootcorte x in n-pentane andevaporatin g the solution downt oa smal lvolume .Oleoresi n was analysed by gas chromatography.Monoterpene swer equantifie d asth e percentage contribution ofeac hpea k toth e totalmonoterpene spresent .

RESULTS

Thepresenc e of Fomes was confirmed in4 1ou to fth e 102core s either by fruiting onth ecor e surface orb y growth inaga rculture .Th emono - terpene compositions ofth e4 0% ofth epopulatio n inwhic h infectionha d beenconfirme d and the 60% showingn o evidence ofinfectio n couldthe nb e compared.Th emea nmonoterpen ecompositio n ofeac hdivision ,togethe rwit h standard errors,i s showni nTabl e 1. Ofth ethre emonoterpene s showinghighl y significant differencesbe ­ tweenth e twogroups ,th emos toutstandin gwa sa-pinene ; ascatter-diagra m ofth edat a showed thatther ewa s aquit eclearl y defined cut-offpoin ta t about3 7% a-pinene , abovewhic hver y fewinfecte d samples occurred.Th e distributionswer e quite different inth etw o groups:o fth e 28tree s in

Table 1. Meanmonoterpen epercentag e composition ofroo tcortica l oleoresin related to incidence of Fomes inwoo d cores frommai ntrun k ofSitk a sprucetrees .

Monoterpene Fomes present Fomes absent tvalu e Significance of difference a-pinene 25.6 ± 1.6 33.4± 1.3 3.67 *** camphene 0.1 ± 0.0 0.2 ± 0.0 2.89 ** ß-pinene 20.1 ± 0.7 18.7± 0.4 1.70 - 3-carene 2.1 ± 0.7 0.6 ± 0.2 2.28 * limonene 4.3 ± 0.7 1.8 ± 0.4 3.34 ** ß-phel1andrene 46.9 ± 1.2 44.5 ± 1.0 1.50 - terpinolene 1.0 ± 0.2 0.8 ± 0.1 0.81 -

Significance levels:** *= P i 0.001; **= P S 0.01; *= P S 0.05.

195 Table 2. Percentage representation ofmonoterpen epatter n types ininfec ­ tedan d inuninfecte d Sitkaspruc e trees.Th epredominanc eo fa-pinene - dominated types inuninfecte d trees iswel l shown,whil e infected trees were characterised by apredominanc e of ß-phellandrene.

Dominant Sub-dominant Percentage representation monoterpenes monoterpenes Forne s Fome s pre sent absent

ß-phellandrene a- pmen é 24 16 ß-phel1andrene ß-pinene 10 2 ß-phe11andrene a-pinene, ß-pinene 10 3 ß-pinene ß-phellandrene, - a-pinene 2 a-pinene ß-phellandrene 29 67 a-pinene ß-phellandrene, ß-pinene 2 - ß-phellandrene, a-pinene ß-pinene 20 10 ß-phellandrene, ß-pinene a-pinene 2 2

whicha-pinen e exceeded 37% ofth etota lmonoterpen e component, Fomes was detected inonl y three ofthem .Th e frequency distributions ofth etw o groups were alsodifferen tbelo w the4 0% mark ,wit h infected treeshavin g generally lower levels ofa-pinene . Camphene, although forming only aver y smallpercentag e ofth etota l monoterpenes,showe d arelativel y striking differencebetwee n infected and uninfected trees:it s frequency distributionwa sbimodal ,an dth e higher mode, at0. 4 %, was represented only inuninfecte dtrees . Each treema yb e characterised genotypically by itsga s chromatogram pattern-type,whic htake s into accountth equantitiv erelationship s between the differentmonoterpenes .A system for allocating eachtre e to adefinit e typeb yvisua l inspection ofit schromatogra m hasbee n devised forSitk a spruce (Forrest, 1980), andth e results ofclassifyin g the infected and uninfected groups inthi swa y are shown inTabl e 2.

CONCLUSIONS

Themai n conclusion tob e drawn from thispreliminar y studywa s that Fomes was absent fromnearl y allthos e trees inwhic ha-pinen e formed at least3 7% ofth e rootcortica loleoresi nmonoterpen e fraction.Ther e are twopossibl e reasons forthi s effect.First ,a hig ha-pinen e contentma y

196 confer a genuine resistance to attack by Fomes by virtue of the known fungitoxic properties of this monoterpene. Secondly, the invasion of the root system by Fomes may induce a decrease in the percentage representation of a-pinene in the cortical oleoresin. In the latter case, the relative increases in the synthesis of 3-carene and limonene following infection may be of biological significance. Further work is necessary to decide between these two possibilities. The main aim will be to measure the resin composition both before and at various periods after artificial inoculation of tree roots with Fomes. If the composition is found to be unaltered by infection, then the possibility exists that a-pinene is the main fungitoxic monoterpene in Sitka spruce root cortical oleoresin. Breeding programmes could then be modified to incorporate a screening procedure favouring the preferential selection of high-a-pinene trees, in order to build up a breeding population with high resistance to Fomes attack.

ACKNOWLEDGEMENTS

I am grateful to Dr Redfern and his staff for assistance with sampling and culturing.

REFERENCES

Chudnyi, A.V., R.A. Krangauz & E.I. Gundaeva, 1972. Effect of oleoresin composition on the resistance of Scots pine to Fomes annosus. Lesnoe Khozyaistovo 7: 60-61. (In: Forestry Abstracts 34, No. 4654.) Fedorov, N.I. & E.N. Manukov, 1972. Composition of gum turpentine and wood turpentine of Scots pine trees infected by Fomes annosus. Lesovedenie i Les. Kh.-vo, Minsk, USSR no. 5: 128-133. (In: Forestry Abstracts 34, no. 690.) Forrest, G.I., 1980. Geographic variation in the monoterpene composition of Sitka spruce cortical oleoresin. Canadian Journal of Forest Research 10: 458-463. Gibbs, J.N., 1972. Tolerance of Fomes annosus isolates to pine oleoresins and pinosylvins. European Journal of Forest Pathology 2: 147-151. de Groot, R.C., 1972. Growth of wood-inhabiting fungi in saturated atmospheres of monoterpenoids. Mycologia 64: 863-870. Hintikka, V., 1970. Selective effects of terpenes on wood-decomposing hymenomycetes. Karstenia 11: 28-32.

197 Biochemical aspects of the resistance of Pinus sylvestris to Fomes annosus

E.I. Ladejtschikova & G.M. Pasternak

Ukrainian Scientific Research Institute of Forestry and Ägroforestry Amelioration, Kharkow, USSR

ABSTRACT

Diagnostic traitswer e studied inorde r to select resistant trees of Scotchpin e indiseas e centres.Th e difference between resistanttree s andtree swit h lowresistanc e (bothinfecte d andnon-infected )wa s established forth e followingparameters : resistance ofwoo d todeca y through thepathoge n invitro ; thickness ofth ewall s oftracheids ;conten to f hemicelluloses inwoo d inrelatio n toon euni to fcellulose ;qualitativ e com­ position ofextractive s fromphloe m tissue andthei r inhibitory effecto ngrowt h ofth epathoge n inpur eculture ;qualitativ e composition ofextractive s fromwood ,bein g extracted by ace­ tone, and their contribution toth eresistanc e ofwoo d todeca y through thepathoge n invitro ;conten to ftrac e elements (e.g., Mn, B, Cu)i nth ephloe m tissue ofth e stem.N o relationwa s foundbetwee n thecompositio n ofmonoterpene s and theresis ­ tance of Scotchpin et o Fomes annosus.

INTRODUCTION

The range of Fomes annosus (Fr.)Cook e indicates thatthi sproble mha s evolvedprimaril y duet oman-mad e disturbance ofecologica l conditions. There ismuc h evidence ofth erelatio nbetwee n the resistance ofconiferou s stands andecologica l conditions (Laatsche t al., 1969;Ladejtschikov a et al., 1975). Alongwit h resistance of stands,individua l resistanceha sbee nestab ­ lished,bu t itsnatur e hasno tye tbee ndetermined . There isevidenc e of the formation of areactio n zone inScotc hpin e (Shain,1967 )a s amanifes ­ tationo f activeprotectiv ereactions . Some individuals of Scotchpin emaintai n theirviabilit y indiseas e centres (=opening s due to F. annosus -attac k instands )durin g 20-30 years ormore .The yma yb ecompletel y non-infected (these cases areex ­ tremely rare)o r theyma y have local,mino r infection ofroots .Researc h

198 resultsbea rwitnes s toa stabilit y ofthei rphysiologica l vigour,whic h is typical for Scotchpin e ingive nconditions :provisio nwit h the inorganic nutrients,nuclei n and carbohydrate metabolism, tissuemoisture ,growt h and thevalu e ofelectrophysiologica l parameters (Ladejtschikova et al.,1975 ; Pasternak, 1975).

MATERIALS AND METHODS

The sample treeswer e selected in40-6 0year s old stands,i ndiseas e centres infectedb y F. annosus 20-30year s ago.The ywer e selected onth e basis ofthei r location ina n infested stand,thei rexterna l features,an d thevalu e of anelectrophysiologica l parameter (impedance)o fth e stem phloem tissue complex incontac twit h the cambium (measured with aninstru ­ mentdesigne d byA.G .Korobtschenko) ,whe nhorizonta l rootswer e carefully dugup .Upo n completion ofth ebiochemica l tests,al lth eroot swer ecom ­ pletely dugup .Thi smad e itpossibl e tocompar e thevalue s ofbiochemica l and biophysical traits with the actualvigou r ofth eroo tsystem . The experimental model consisted oftree swit h 3differen t categories ofvigour :categor y I-Ia,consistin g oftree swit hhig h andmediu mresis ­ tance growing inth ever y disease centre;categor y II,consistin g of healthy trees growingbeyon d theboundarie s ofa diseas e centre serving as anexampl e ofa stan dwit h lowresistanc e -sometime s thesetree s could have infection ofth e initialphas e (category IIa);categor y III,con ­ sisting ofdisease d treesgrowin g inth eperipher y of adiseas ecentre . Thiscategor y oftree sha s features oflo wresistanc e and, inaddition , theeffec t ofth epathologica lproces s isreflecte d init . Resistant treeswer e selected inth ever y disease centre among trees withwell-developed , pointed, dense crowns,wit h dark-green needleso f normal length, andwit hnorma l shootgrowth .Th evalu e ofth e stem impedance ofsuc htree s should exceed the averagevalu e ofth e impedance ofthos e treeswit h equal diameter, growing ina health ypar to fth e stand. Forthi spurpos e thevalu e ofth e impedance of 15tree shavin g equal diam­ eterwit h theresistan t treewa smeasured .A health y treewit h impedance valueequa l toth e average of alltree s inth e standwa s selected forth e experiment (category II).Disease d treeswer e selected along theperipher y ofth ediseas e centre.The ywer e characterized by reduced growth and thin crownswit h lightgree nneedles ,usuall y shortened. Their impedancevalu e was 60% ofth evalu e ofth e sound tree or less. Acharacteristi c feature ofth e applied technique istha tonl y healthy roots areanalyse d irrespective ofth ecategor y ofvigou r ofth etree ,an d thatno tonl yphloe m tissue andwoo d ofth eroots ,bu t also those ofth e stem areuse d asdiagnosti c tissues.Phloe m tissue andwoo d ofth e stem chosen for analysiswer eno tdirectl y affected by themyceliu m ofth e pathogen.

199 Furthermore, itha s tob etake n into accounttha twoo d ofth e stempro ­ videsmor e comparable samples inrespec tt ometholog y thandoe swoo d ofth e root. Samples ofth e latter arecharacterize d byuneve nstructur e ofth e wood andb y eccentricity. Forth etest swit h the decay ofwoo d this iso f methological importance.Relate d conclusions ofth ediagnosti c value ofth e phloem tissue of the stemwer emad eb y other scientists,to o (Alcubilla et al., 1971). Theconten to f structural substances inth ewoo d ofroot sha sbee n studied (Melikyan, 1959), aswel l as theresistanc e ofwoo d todeca y in vitro (Naumov, 1932), the contento fextractive s soluble inchlorofor m and acetone (Ivanov,1946 )an d their importance forth eresistanc e ofwoo dt o decay (Shain, 1967), thecompositio n ofextractive s inth ephloe m tissueo f the stem (Ivanov, 1946), and thecompositio n ofturpentin e oils. Biotests withphloe m tissue or extractives fromphloe m tissue andpur e cultures of F. annosus were also conducted (Laatsche t al., 1969).

RESULTS AND DISCUSSION

Researchha s showntha tth e stemwoo d ofresistan t50-yea r old treesi s decayed less thanth ewoo d ofhealth y treesbeyon d thediseas e centre (by 14% less)an d also less than thewoo d ofdisease d trees (by2 3% less). Earlier thereha d already been stated anopinio n thatth e resistance ofa living tree towood-attackin g fungi covers thewood , too (Rypacek, 1957). A question arises:wha tar eth e reasons forth egreate r resistance of thewoo d ofresistan t trees? Inthi s respect information aboutth e anatomic structure andcompositio n ofth e structural substances ofth ecel l membrane ofwoo d iso finterest ,a swel l asinformatio n aboutth econten t ofdiffer ­ entgroup s ofextractives . Itturne d outtha twoo d ofth e roots andth e stemso f resistant treesha s agrea tquantit y of late tracheids anda thicker structure ofcel lwall s (Ladejtschikovae t al., 1973). The followingphas e inresearc hwa st o find out,wha t kind ofstruc ­ tural substances determine the formation ofthic k cellwall s inth ewoo d ofresistan ttrees . Whenth e investigations wereoutline d itwa s supposed thatthi sphenom ­ enon isconnecte d with lignin. Itturne d out,however , thatth e contento f lignin inth ehealth y rootso fdisease d treesma y evenb ehighe r (Table1) . A correlationwa s foundbetwee n thevigou r ofth etre e and theconten to f hemicellulosesbot h inroot s and instem : treeswit h low resistance havea very lowcontent .Thi s kindo fregularit y isquit e evidentwhe nth e content isrelate d todr yweigh t ofwoo d and, especially, to auni to fcellulose . Inth eroot s and stem of Scotchpin eth econten t ofth e analysed substances insapwoo d isno tdifferen t from theconten t inth e central parto fth e root. Itma yb e assumed thatresult s ofth e chemical analysis ofwoo d ofth e

200 Table 1. The contento fstructura l substances incell so f thewoo d of roots and stemso ftree s indifferen tvigou rcategories .

Category Treeorga n Sapwood Central part oftre e vigour cellu- hemi- lignin cellu- hemi- lignin lose cellu- lose cellu­ lose lose

In % of the dry weight of wood I healthy roots 46.3 22.0 28.3 45.9 22.0 29.5 II healthy roots 47.4 23.7 26.1 47.7 23.4 26.4 III healthy roots 50.0 18.9 27.6 50.0 17.9 29.0 I stem 47.6 23.9 26.0 44.7 23.8 28.3 II stem 46.7 24.3 25.6 43.1 25.2 27.9 III stem 47.6 21.3 27.1 44.7 21.7 29.0

Per unit of cellulose I healthy roots 0 .47 0 61 0 50 0 64 II healthy roots 0 50 0 55 0 50 0 55 III healthy roots 0 38 0 55 0 36 0 58 I stem 0 50 0 55 0 53 0 63 II stem 0 52 0 55 0 58 0 65 III stem 0 45 0 57 0 49 0 65

centralpar to froo t and stem characterize the content of structural sub­ stances inth eroot s of 10-15yea r old Scotchpine ,and , furthermore,tha t wood ofth eroo tan d stemwa s formed atthi s agebefor e the infectiono f thetre eb y F. annosus. Apparently lowconten to fhemicellulose s insom e trees ison eo f thereason s forthei r low resistance to F. annosus. Correlationbetwee n the resistance ofwoo d to decay andth e composition ofextractive s isconfirme d by theresult s ofexperiment s invitr owit h wood,whic hwa sconsistentl y treated with different solvents (Shain, 1967). Aswa s alreadymentione d aboveth ewoo d ofresistan t trees isdecaye d less thanth ewoo d ofbot h healthy anddisease d trees.Whe na grou p ofextrac ­ tives soluble inchlorofor m was separated fromwood , the degree ofdeca yo f thewoo d inal l categories ofvigou rwa s increased by 20-24% ,bu tth e formerregularit y isstil lmaintained : thewoo d ofresistan ttree swa s decayed less. Further treatment ofwoo dwit h acetone increased thedegre e ofdeca y only ofth eresistan t trees (by 6 %). Consequently, thegrea t resistance of thewoo d of resistant trees isevidentl y related to agrou p ofextractive s

201 soluble in acetone.Extractive s soluble inchlorofor m produce acertai n level ofresistanc e inth ewoo d ofScotc hpin e irrespective ofth evigou r ofth etree .Th egreate r degree ofdeca y ofth ewoo d ofdisease d treeswit h low resistance is also related toa lowe rconten to fhem'icellulos ean d lignin. Composition ofextractive s inphloe m tissue andthei r inhibitory effect may alsob e acriterio n forresistance ,a sth eresult s of astud y on F. annosus showed (Alcubilla et al., 1971). However,th ephloe m tissue of Scotchpine ,unlik e thato fspruce ,di dno thav ea ninhibitor y effecto n thegrowt h of F. annosus inpur e culture (Table 2).Thi s isdu et o the fact thatth econten to fth emai ngrou p ofextractive s having an inhibitory effecto nth egrowt h of F. annosus -pheno l compounds -i sapproximatel y 5 times smaller inth ephloe m tissue of Scotchpin e than inspruce ,whil eth e contento fcarbohydrate s ispracticall y equal.Now ,th ebiologica l effect ofth ephloe m tissue ofth e stemso fresistan t trees isth e same astha to f healthy trees,bu t thephloe m tissue ofdisease d trees is ales s favourable substrate forth e growth ofmyceliu m ofth epathogen .

Table 2. Theconten to fextractive s (M1 m )i nphloe m tissue ofth estem s of Scotchpin e and their effecto nth egrowt h of F. annosus depending on thevigou r oftree s (n=10). LSD:Leas t significantdifference .

Index Indiseas e centre Ininterme - LSD diatezon e

resistant diseased healthy

The contento fextractive s solublein : chloroform (%) 6.5010.40 8.610.72 5.110.40 1.41 acetone (%) 3.010.58 4.310.42 2.810.33 1.24

Thebiomas s ofmycelium : a. inexperiment s withextracts : chloroform extracts (mg) 71.9110.1 44.9±4.8 91.6±8.2 21.71 acetone extracts (mg) 63.018.6 67.517.7 87.8±8.5 20.09

b. inexperiment s with phloem tissue (mg) 162.0110.0 128.019.7 151.019.4 27.33

Inhibitory effect ofextractive s fromphloe m tissue solublein : chloroform (%) 51.6 65.0 39.4 acetone (%) 61.1 47.3 41.9

202 Chloroform and acetone extracts ofth ephloe m tissue of Scotchpin e contain substanceswhic h inhibitth e growth ofth epathogen .Th e inhibitory effecto fchlorofor m extracts increases directly with the quantitative content;wherea s the effecto f acetone extracts depends onthei rqualita ­ tivecomposition .Whil e the contents ofextractive s soluble inaceton e are equal inth ephloe m tissue ofresistan t andhealth y trees (3.0% and 2.8 % respectively), the inhibitory effecto nth e growth ofth emyceliu m of F. annosus wasgreate r inth ecas e ofresistan t trees (61.1% versu s 41.9 %). Thepathologica l process leads toa remarkabl e increase inth e content ofth e group ofextractive s soluble inaceton e (4.3 %), but iti s not followedb y an increase ofthei r inhibitory effect,whic h remained on the level ofhealth y trees andwa smuc h lowercompare d withresistan t trees (47.3 %). Thechromotographi c (solvents:H-buty l alcohol:aceti c acid:wate r 4:1:5)divisio n of acetone extracts fromphloe m tissuemad e itpossibl e to detect thedifferenc e inth e composition depending onth e category oftre e vigour: inresistan t treesunidentifie d extractives were discovered with Rfvalue s 0- 0.4 0 and 0.55 whichwer e lacking inhealth y and diseased trees. ' Experiments inou r laboratory have established thatanalyse s ofphloe m tissue give us information fordrawin g conclusions aboutth erelatio nbe ­ tweenprovisio nwit h inorganic nutrients andvigou r oftree s ina n infected stand (Pobegailo &Ladejtschikova , 1975). This regularity is alsovali d for theprovisio n of Scotchpin ewit h trace elements.Resistan t trees aredis -

Table 3. The average content ofbasi cmonoterpene s and itsvariabilit y in treeswit h differentvigour .

Category oftre evigou r a-pinene ß-pinene Ä-3-carene Indiseas e centre Ihig hdegre e ofresistanc e (n=l) 52.7 1.7 22.0 laresistan t (n=15) 53.1 6.3 20.5 36.2-77.6 1.0-26.2 2.6-38.6 III diseased (n=7) 50.3 4.7 28.5 28.2-62.8 0.9-26.6 16.5-40.5 Beyond thediseas e centre IIhealth y (n=4) 45.4 6.2 31.8 34.6-56.5 0.7-21.8 21.7-51.5 III initially diseased (n=5) 58.1 1.6 24.9 33.4-76.7 0.1-2.7 5.0-55.9

203 tinguished by a markedly higher content of trace elements, such as Mn, B, and Cu (respectively 34 %, 32 %, and 33 %), whereas diseased trees have a lower content (respectively 52 %, 43 %, and 53 %) compared with healthy trees. In genetic research with coniferous species - including that where resistance of Scotch pine to F. annosus is selected - great importance is attached to composition of turpentine oils (Table 3). Studies of the tur­ pentine of 32 trees with different degrees of resistance and a complete digging up of roots made it possible to compare the composition of monoterpenes with the degree of disease (Ladejtschikova et al., 1975). Among 15 resistant trees there was only 1 which was. totally free of F. annosus (an example of a high level of resistance). The content of A-3-carene (a monoterpene to which resistance to the pathogen is related) in such a tree turned out to be lower (22 %) than what was observed in re­ sistant trees having local signs of disease (38.6 %) or in diseased trees (40.5 %). Beyond the disease centre among a group of healthy trees, trees with signs of initial disease were discovered in which the content of A-3-carene reached its maximum value: 55.9 %.

CONCLUSIONS

Resistant trees, which are a product of natural selection in heavily infectious surroundings, are characterized by the following features dis­ tinguishing them from trees with low resistance (both diseased and not diseased): higher resistance of wood to decay in vitro; greater thickness of the walls of thracheids; higher content of hemicelluloses in wood per unit of cellulose; specific composition of extractives soluble in acetone, which are found in phloem tissue and wood, with a higher inhibitory effect on the growth of F. annosus in pure culture; and higher content of trace elements (e.g., Mn, B, Cu) in the phloem tissue of stems. Obviously part of these traits are genetically predetermined. Investi­ gations have been started in ,the field of genetic resistance.

REFERENCES

Alcubilla, M, M.P. Diaz-Palacio, K. Kreutzer, W. Laatsch, K.E. Rehfuess & G. Wenzel, 1971. Beziehungen zwischen dem Ernährungszustand der Fichte (Picea abies Karst.), ihrem Kernfäulebefall und der Pilzhemmwirkung des Basts. Eur. J. For. Path. 1: 100-114. Ivanov, N.N., 1946. Methods of physiology and biochemistry of plants. State Publishing House of Agricultural Literature, Moscow-Leningrad. 494 p.*. Laatsch, W., M. Alcubilla, G. Wenzel & H. von Aufsess, 1969. Beziehungen zwischen dem Standort und der Kernfäule-Disposition der Fichte (Picea abies Karst.). Forstwiss. Cbl. 87: 193-203.

* Titles translated to English by the authors.

204 Ladejtschikova,E.I. ,A.G .Tschernyc h& A.I .Pobegailo ,1973 .Anatomica lan dchemica l characteristics ofpin eroot swoo dconditione db yvariou sdegre eo fFome sannosu s injury.In :Silvicultur ean dAfforestatio nAmelioration ,33 ,Urozhay ,Kiev . p.137-142* . Ladejtschikova,E.I. ,A.I .Pobegailo ,G.D .Byely ,I.A .Alekseyev ,A.G .Tschernych , G.M.Pasternak ,A.G .Shekhovtso v& A.G .Korobchenko ,1975a .Reason so fpredispositio n ofpinet ao nol darabl eland st odiseases .In :Prapor ,Kharkov ,p .22-30* . Ladejtschikova,E.I. ,A.I .Pobegailo ,G.M .Pasternak ,A.G .Korobchenk o& L.F .Ladnych , 1975b.Estimatio no fPinu ssilvestri smorphologica lan dphysiological-biochemica l indicesfo rplant-breedin go nresistanc e toFome sannosus .In :Silvicultur ean d AfforestationAmelioration ,40 ,Urozhay ,Kiev .p .3-15* . Melikyan,N. ,1959 .Structura lchange san daccumulatio no fligni ndependin go nth e conditionso fth eenvironment .Publishin gHous eo fYereva nUniversity ,Yerevan . 200p.* . Naumov,N.A. ,1936 .Method so fmicroscopica l researchesi nphytopathology .Stat e PublishingHous eo fAgricultura lLiterature ,Moscow-Leningrad ,22 4p.* . Pasternak,G.M. , 1975.Effec to fFome sannosu sinjur yo nnuclei cacid sconten ti npin e phloem tissue.In :Silvicultur ean dAfforestatio nAmelioration ,40 ,Urozhay ,Kiev . p.84-90* . Pobegailo,A.I .& E.I .Ladejtschikova ,1975 .A wa yo fdiagnostic so finorgani cnutrient s ofstands ,injure db yFome sannosus .Author' sCertificat eN 475546 .USS RCounci lo f MinistersStat eCommitte eOfficia lBulleti ni nDiscoverie san dInventions ,N 24 . p.90* . Rypacek,V. ,1957 .Biologi edreokaznyc hhub .Nakladatelstv iCeskoslovensk eAkademi evëd . Praha.27 5p . Shain,L. ,1967 .Resistanc eo fsapwoo di nstem so flobloll ypin et oinfectio nb yFome s annosus.Phytopatholog y 57:1034-1045 . /

205 Susceptibility of European black pine (Pinus nigra) to the European pine shoot moth (Rhyacionia buoliana):variation s of susceptibility at the provenance and individual level of the pine and effect of terpene composition

112 2 P.J. Charles , A. Delplanque , Anne Marpeau , Colette Bernard-Dagan & M. Arbez3

ABSTRACT

The number of attacks of shoot moth (Rhyacionia buoliana) on individual trees were noted during 5 consecutive years in a provenance trial of European black pine (Pinus nigra). Results show that great differences exist among provenances in the lev­ el of attacks. Undamaged branch samples were taken from 5 heavily-attacked and 5 non-attacked trees within each of 4 provenances. Two provenances of the subspecies nigricans were heavily attacked, and the other 2, from the Corsican group of the subspecies laricio and from the subspecies pallasiana, were slightly attacked. The cortical tissues of these branch samples were peeled, macerated in pentane, and the terpene concentra­ tions measured by gas chromatography. The results show that it is difficult to distinguish between terpene profiles of heavily-attacked trees and non-attacked trees within very sus­ ceptible provenances, but that a clear difference seems to ex­ ist between heavily-attacked trees and non-attacked trees when the provenances are partially resistant. Judged only from a statistical relationship, it seems that phellandrene, 3-carene and camphene could play a role in the biochemical mechanism of resistance. If confirmed, such a result indicates the promising possibility of making indirect selections of black pines which would be less susceptible to the European shoot moth and conse­ quently developing fewer forks.

1. INRA, Station de Recherches de Zoologie et de Biocoenotique forestière, Ardon, 45160 Olivet, France 2. Université de Bordeaux I, Laboratoire de Physiologie cellulaire végétale, 33405 Talence, France 3. INRA, Laboratoire d'Amélioration des Arbres forestiers, Pierroton 33610 Cestas, France

206 INTRODUCTION

Inspit e of largegeographi cvariation ,excellen t straightness ofth e trunk is atypica l characteristic ofth eEuropea nblac kpine .Nevertheless , and depending onth e environmental conditions,thi s average growthhabi t canchang e and acertai n amounto f forking canappear .Fro m results ofsom e exploratory investigations, itseem s that atleas t2 mai n factors couldex ­ plainth e incidence of forked trees:th eoccasiona l occurrence of ashor t second terminal shootwhe nth e location iswel lprovide dwit hwater ,an d damage toth etermina lbu d caused by Rhyacionia buoliana Schiff. Provisionally disregarding environmental effects,w e shall consider the susceptibility ofth eblac kpin e toth eEuropea n shootmoth . In1 planta ­ tion,th e infestation rateb y Rhyacionia varied amongprovenance s (geo­ graphic origins)an d among individualswithi nprovenance s (Charles,1974 ; Mallet, 1975).Malle tha s already noted significant differences amongprov ­ enances of Pinus nigra Arn. fromvariou s geographical origins.Thes eobser ­ vationswer emad e in aprovenanc e trial located inth eFrenc h southern Piedmonto f theAlps . For agive nyea r ofobservation s (1975)th e totalnumbe r ofattack spe r tree decreased from the subspecies nigricans (Laragne, aFrenc h introduced population, andTzarvaritza , anatura l Bulgarian one)t oth e Corsican branch ofth e subspecies laricio (Vizzavona, from the centralpar to f Corsica)an dth e subspecies pallasiana (Mugla, aprovenanc e fromsouth ­ western Turkey). Suchconclusion s remained truewhe nth e incidence of damaged terminal shootswa s considered inplac e ofth etota l numbero f attackspe rtree . Besides this,w ehav e alsonote d clear differences in Rhyacionia- susceptibility among individuals withinprovenances . Thesepreliminar y observations werepromisin g enought oencourag e ust o continuemor e intensive studies onth e sameprovenanc e experiment during5 consecutiveyears . Previous research (Arbez et al., 1974)als o has showntha t largevaria ­ tion inth emonoterpen ecompositio n ofth ecortica l tissue exists amongth e different subspecies of P. nigra. Because ofsuc hcondition s itseeme d of interest to investigate whether arelationshi p between terpene composition and shootmot h susceptibility existed.O fcourse ,suc h arelationshi p could behighl y valuable inmakin g indirect selections forshoo tmot hresistance .

EXPERIMENTAL SCHEME

Observationswer e carried oni nth e sameprovenanc e trial.Nin ediffer ­ entprovenance s were involved inthi stes tplante d inth e spring of 1971,6 ofthe mbelongin g toth e subspecies nigricans, 2belongin g toth esubspe ­ cies laricio, and 1t oth esubspecie s pallasiana (Table 1).Th emai n design

207 Table 1. Description ofth epopulation s used inth eprovenanc etest .

Abréviations Provenances Country Subspecies

VIZZ Vizzavona France (Corsica) laricio COSE Cosenza Italy (Calabria) laricio LEDE Ledererkogel Austria nigricans STUD II Studenica II Yugoslavia (Serbia) nigricans VGRA II Visegrad II Yugoslavia (Bosnia) nigricans TZAR Tzarvaritza Bulgaria nigricans POLV Popova-Livada Yugoslavia (Macedonia) nigricans LARA Laragne France (artificial stand) nigricans MUGL Mugla Turkey pallasiana

was abalance d lattice with4 replication s and 56tree spe rplot .Th eex ­ periment is located inth ePellen q State Forest*nea rMontmeya n (VarPrefec ­ ture); theelevatio n is 550m , the aspect is flat,an dth e soil overliesa hard limestone.Th eplantin g issurrounde d by anatura l oak forest {Quercus pubescens Willd.). Trees hadbee nattacke d by theEuropea n shootmot h since 1972.Damag ewa s important from 1973 to 1975bu tdecrease d everyyea r since 1973. Thenumbe r of attackspe r treewer ecounte d aswel l as attacko fth e terminal shoot.Finall y these observations were combined forth e first5 consecutive years. With avie w toward eventually establishing moreprecisel y arelation ­ shipbetwee n terpene composition and shootmot h resistance, in 1978w e decided tochoos e asampl e including 4provenances :2 heavil y and regularly attacked (Tzarvaritza (TZAR)an dLaragn e (LARA))an d2 wit h lightdamag e (Vizzavona (VIZZ)an dMugl a (MUGL)). Ineac hprovenance ,w e chose 10indi ­ viduals: 5heavil y and regularly attacked during the last5 year s and 5no t oronl y slightly attacked during the sameperiod . Unattacked 1-year-old branch sampleswer e taken from each selected tree.Afte rmaceratio n and extraction inpentan e ofcortica l tissues of these individual samples,th eextract swer e submitted to gas chromatography analysis. The relative concentrations of9 terpene s (expressed inpercen to f totalvolatil eterpen e hydrocarbons)wer ethe n compared by ahierarchica l desing for analysis ofvarianc e (provenances and individuals withinprov ­ enances)an d aprincipa l component analysis.Th e 9terpene s involved in thega schromatograph y studywere :a - and ß-pinene, camphene,myrcene , 3-carene, limonene, ß-phellandrene,terpinolen e andcaryophyllene .Th e first8 aremonoterpenes , the laston e isa sesquiterpene .

208 RESULTS

Provenance and individual variation in shoot moth susceptibility (5 years' performance) Observations made during 5consecutiv e years show that some trees sus­ tained repeated attacks,wherea s otherswer eneve r attackedb y the European shootmoth .Th epercentage s oftree swithou t attacks andthos e oftree s heavily attacked aregive n inTabl e2 . Itappear s that largevariatio n in susceptibility occurred, depending ongeographi c origin ofth epine .Difference s among trees,a swel l asamon g provenances were large immediately afterplanting ,whe ntree swer e still small.Th e 'silhouette effect',ofte nuse dwhe nmoth s are concerned toex ­ plaindifference s in female attraction during the egg layingproces s does not apply inou rcase .Suc hdifference s insusceptibilit y to R. buoliana havebee n observed previously inothe rpin e species such as P. contorta (Esjberg& Feilberg , 1971).

Relationship between intensity of attack and terpene composition of the cortical tissues Inever yprovenance ,non-attacke d trees andheavily-attacke d trees (4 to 5time s during 5consecutiv e years)wer e analysed separately. Subpopula­ tionswit h anod dnumbe r correspond toheavil y attacked trees and those with aneve nnumbe r tonon-attacke d trees (Fig.1) . Terpene compositions ofcortica l tissues (percento ftota l volatile terpenes) forth e4 provenance s and the8 subpopulation s arepresente d in

Table 2. Percentages oftree sneve r attacked orheavil y attacked depending onprovenanc e level of susceptibility.

Provenances Treesneve r attacked Trees attacked 3 Trees attacked5 during a5-yea r period or4 times during times duringa a 5-year period 5-year period

MUGL 21 1 0 VIZZ 30 4 0.5 COSE 3 4 0.5 POLV 8 9 1 STUDI I 15 9 2 LEDE 10 17 2 VGRAI I 7 21 3 LARA 5 25 1.5 TZAR 4 25 2

209 Table 3. Terpene composition ofcortica l tissue of4 provenance s and8 subpopulations.A =heavil y attacked trees,N A =non-attacke d trees.

a-pin. camph. ß-pin.myrc . 3-car. limon,phell . terpi.cary . provenances LARA (I) 72 59 1 17 10 47 2 .17 0 20 13 05 0 24 0 20 3 77 TZAR (II) 63 81 1 19 5 82 2 98 0 43 25 50 0 15 0 23 3 86 MUGL (III) 72 15 1 35 15 10 2 95 0 36 7 05 0 63 0 27 7 90 VIZ2 (IV) 37 86 0 84 3 16 5 88 0 26 42 58 6 53 0 89 1 23

subpopulati ans LARA 1 (A) 78 49 1 18 3 80 2 24 0 25 13 91 0 12 0 16 5 37 LARA 2 (NA) 66 68 1 16 17 13 2 09 0 15 12 19 0 36 0 25 2 17 TZAR 3 (A) 66 99 1 17 7 50 2 87 0 40 20 86 0 10 0 22 2 26 TZAR 4 (NA) 59 04 1 23 3 31 3 14 0 46 32 46 0 23 0 25 6 25 MUGL 5 (A) 66 47 1 05 19 64 2 85 0 62 8 59 0 56 0 25 9 17 MUGL 6 (NA) 77 83 1 64 10 57 3 06 0 11 5 50 0 70 0 28 6 62 VIZZ 7 (A) 26 45 0 59 3 19 5 69 0 34 58 30 3 40 0 80 0 97 VIZZ 8 (NA) 49 27 1 10 3 13 6 07 0 18 26 85 9 66 0 97 1 49

Table3 . Ata provenanc e level,th eresult s are ingoo d agreementwit hth e preliminary conclusions ofArbe ze t al. (1974).Provenance s belonging to the subspecies nigricans and pallasiana were similar,wit h arelativel y high levelo f a- and ß-pinene, asmal l amounto fmyrcen e and arelativel y scant amounto flimonene .Th eCorsica npin ebelongin g toth e subspecies laricio wascharacterize d by alo wleve l ofpinenes ,a typicall y high amounto f limonene andphellandren e and amoderatel y high level ofmyrcene . Inconsideratio n ofthes eresult s and thequit edifferen tterpen ecom ­ positions whichoccurre d forth e 2highl y resistantprovenances ,Vizzavon a andMugla , itappear s thatth e singlepoin t ofsimilarit y interpen ecompo ­ sitionwa s thehighe r amounto fphellandrene .Tha twa s also thecas e for terpinolene concentration butth edifference s between resistant andsuscep ­ tibleprovenance s were smaller. Inan ycase ,onl y theprovenanc e Vizzavona

PROV I PROV II PROV III PROV IV LARA TZAR MUGL VIZZ / \ / \ / \ / \ Subpopulation: 1(A) 2(NA) 3(A) 4(NA) 5(A) 6(NA) 7(A) 8(NA)

Figure 1. Experimental design.A = heavily-attacke d trees,N A =non-attacke d trees.

210 was significantly different from theothers . Atth e subpopulation level,i tappear s thatwithi n the sameprovenanc e thenon-attacke d individuals had thegreate r amounto fphellandrene ;al ­ though the differences inconcentration s betweenheavily-attacke d individ­ uals andnon-attacke d individuals were sometimes slight,thi s relationship wasconsistent . Analysis ofth ecorrelatio nmatri xwit hprovenanc e means ofterpen e concentrations shows thatmyrcene , limonene,phellandren e and terpinolene concentrations werehighl y correlated. Aprincipa l component analysis wasperforme d withth emean s ofth e8 subpopulations. The first2 principa l components explain 78% ofth e totalvariance .

EV1 H

1.95 *MUGL(NA) *MUGL(A )

* LARA (A) * LARA(NA')

* TZAR (A) * TZAR(NA ;

0.

•kviZZ (NA;

4,00. *VIZZ(A ) -, ^ EV2 1,68 0 1,89

Figure 2. Projections ofpoint s of attacked (A)an dnon-attacke d (NA) groups of individuals from the4 provenances :eige nvector s EV-.e nEV_ .

211 Except for 3-carene, almost all the other terpenes involved in this study were correlated with the first principal component, but mainly ct-pinene positively, and myrcene, limonene and terpinolene negatively. The third principal component is mainly correlated with ß-pinene and phellandrene concentrations. The graphical representation of the 8 subpopu­ lations in the first 2 plans of the eigen vector EV-, and EV„ is shown in Fig. 2. The most striking fact is that the differences between attacked and non-attacked individuals are clear mainly for the slightly-attacked prove­ nances Vizzavona and Mugla. In Fig. 2, such differences can be seen only on the second eigen vector EV2 (3-carene+, camphene-). This is also the case with the third eigen vector EV, (ß-pinene+, phellandrene+). If we disregard the details, it seems that attacked and non-attacked individuals have similar terpene profiles within highly-susceptible prove­ nances, but within slightly-attacked provenances their terpene compositions are clearly different. This could mean that, in explanation of differences between individ­ uals, terpenes (directly or through some biochemical precursors) can be a sufficient barrier against Rhyacionia attacks only when the resistance level of the population is already high. This leads us to suppose that terpenes act probably in association with more important factors of resis­ tance . If confirmed with different provenances, in different environments, such results could make possible indirect selection for Corsican pine that would be less sensitive to the European shoot moth, with a subsequent decrease in the incidence of forking.

REFERENCES

Arbez, M., C. Bernard-Dagan &C . Fillon, 1974. Variabilité intraspécifique des monoterpènes de Pinus nigra Arn. Bilan des premiers résultats. Annales des Sciences forestières 31: 57-70. Charles, P.J., 1974. Incidence des attaques de la tordeuse des pousses Rhyacionia (Evetria) buoliana Schiff, sur 'le choix des espèces de pins suceptibles d'être plantées en région méditerranéenne. Comte rendu IVèmes journées Phytiatrie- Phytopharmacie Circum-méditerranéennes, Montpellier, p. 468-471. Esbjerg, P. &L . Feilberg, 1971. Infestation level of the European pine shoot moth (Rhyacionia buoliana Schiff.) on some provenances of lodgepole pine (Pinus contorta Loud.). Danish Forest Experiment Station Report no. 254, 32 (4): 345-358. Mallet, B-, 1975. Contribution à l'étude de la variabilité infraspécifique de la sensibilité à Rhyacionia buoliana Schiff, chez Pinus ponderosa et Pinus nigra. Rapport D.E.A. (limited distribution).

212 Genetic basis for variation in fungi

CE. Caten

Departmento fGenetics ,Universit yo fBirmingham ,Birmingha mB1 52TT ,U K

ABSTRACT

Fungal species arehighl yvariabl e inrespec to fbot h qualitative andquantitativ e characters.Mos t ofthi s phenotypic variation isgeneticall y determined and originates fromnuclear-gen e mutations and from therepeate d shuffling of these intone w combinations byheterokaryosi s and sexual and parasexual hybridization. Changes inth emitochondria l genome andth e effects ofmycoviruse sma y alsoproduc evariation .Al l theseprocesse s occur innatur ebu tth econtributio n ofeac ht o theobserve d variation isdifficul t to assess andprobabl yvari ­ able.Amon g the factors involved, incompatibility systems act­ ing atsexua l reproduction-orbetwee nvegetativ e cells arepar ­ ticularly important incontrollin g the flowo fgeneti cdetermi ­ nantsbetwee n strains.Th evariabilit y of fungal speciesex ­ tends tothei rpathogenicity , which is acomple x characterwit h qualitative andquantitativ e components.Th equalitativ ecompo ­ nent (virulence)i sprimaril y involved inhos t specificity and iscontrolle d bymajo r genes,whil e thequantitativ e component (aggressiveness) ispolygenicall y determined.

INTRODUCTION

The genetic basis forvariatio n in fungi encompasses much of fungal genetics and cannotb e comprehensively treated in the spaceavailable . Thispape r concentrates on3 topics : (i)th evariabilit y of fungalpopu ­ lations, (ii)th e origino fvariatio n and (iii)th egeneti c control of pathogenicity. Forbasi c information onth e genetics of fungi and for dis­ cussion ofothe r aspects of fungalvariatio n andpathogenicit y the reader isreferre d toth etext sb yDa y (1974), Burnett (1975)an dFincha m et al. (1979).

213 VARIABILITY OF FUNGAL POPULATIONS

Variationma yb e conveniently divided into qualitative andquantitat ­ ive. With the former,th edifference s arediscret e and any individual can beunambiguousl y assigned to aparticula r class,whil ewit hth e latter there is acontinuou s gradiento ftype s and discrete classes cannotb erec ­ ognised.Physiologi c specializationprovide s thebes tdocumente d example of qualitative variation in fungi. Isolates areclassifie d intodiscret e races onth ebasi s ofthei rreactio nwit hhos tcultivar s carrying different sources ofresistanc e andpathoge npopulation s mayb epolymorphi c withre ­ spectt othei r racial composition.Likewise ,Spiet h (1975)showe d thatpop ­ ulations of Neurospora intermedia Tai are frequentlypolymorphi c forelec - trophoretically-distinct formso findividua l proteins.Th epresenc e of quantitative variation infunga lpopulation s isals owidel y appreciated. Brasier (1970)foun d thatth egrowt h rateo f 77dikaryon s of SchizophyHum commune FT. sampled from asingl e localityvarie d continuously and thatth e isolates could notb eplace d intodiscret e classes onthi sbasis .Extensiv e quantitative variationoccur snaturall y for arang eo fothe r biologically important characters including fruiting (Brasjer, 1970), fungicideresis ­ tance (Hollomon, 1978)an dpathogenit y (Clifford &Clothier , 1974). These comparative studies ofisolate s reveal only thephenotypi c variation pres­ ent innatura lpopulations .However ,man y caseso fqualitativ e andquanti ­ tativephenotypi c variationhav ebee n shownt ohav e ageneti cbasi s (e.g., Flor, 1956;Crof t& Jinks ,1977 ;Caten , 1979)an d iti sreasonabl e tocon ­ cludetha tthi s isgenerall y thecase .

ORIGIN OF VARIATION

Geneticvariatio n isdu et oth epresenc e of2 o rmor e different forms (alleles)o fa gen ei na population . Since,eve nsuc hsimpl eorganism sa s fungicontai n sufficientnuclea rDN A formor e than 10 000gene s iti sclea r that alarg enumbe r ofdifferen t genotypes arepossible ,eve n allowing for the facttha tno t allgene s arepolymorphic .Electrophoreti c surveys ofa rangeo forganism s have showntha ta high ,althoug h somewhatvariable ,pro ­ portion ofgene s isnaturall ypolymorphi c (Nevo, 1978). These different alleles ariseb ymutatio nwhic h istherefor e theultimat ebasi s forth e geneticvariatio n in fungi and all other organisms.However ,mutatio n isa rareeven t and thecontributio n ofne wmutant s toth evariabilit y of apop ­ ulation inan yon egeneratio n isver y small.Mos to fth eobserve dvariatio n results fromprio rmutation s and from therepeate d shuffling ofthes e into new combinations.Laborator y studies have showntha ti n fungi reassociation and recombination ofnuclea r genes occurs throughheterokaryosis ,sexua l hybridization andparasexua l hybridization. Themechanism s underlying these processes arewel l understood ina fe w species (Burnett,1975 ,1976 ;

214 Fincharae t al., 1979)bu tthei r contribution toth evariabilit y of fungal populations is far from clear. Inadditio n toth enuclea r system, fungi,i n commonwit h other eukaryotes,posses s asecon d genetic system located in thecytoplasm . This cytoplasmic system doesno tconfor m toth e lawso f Mendeliangenetic s thatgover nth enuclea r system.Nevertheless , cytoplasmic genes aresubjec tt omutatio n andma yb e anadditiona l source ofvariation . Ihav e identified mutation,heterokaryosis , sexual andparasexua lhy ­ bridization and cytoplasmic inheritance aspotentia l sources ofgeneti c variation in fungi.T owha texten td othes eprocesse s occur innatur e and what arethei r contributions toth eobserve dvariabilit y of fungalpopula ­ tions?N o definitive answers tothes equestion s arepossibl ebu t Iai mt o identify some ofth emajo r factors involved, topoin tou tsom eo fth egap s inou rknowledg e and todra w afe wtentativ econclusions .

Mutation Several factors suggest thatcurren tmutatio n iso fmor e significance forth evariatio n and evolution of fungi than iti s forhighe rorganisms . Firstly, the dominantnuclea r phase ofmos t fungi ishaploi d andhenc ene w mutations areexpresse d oncethe y are segregated into aspor e orhypha . Secondly, although spontaneous mutation rates arever y lowth e large size ofman y fungalpopulation s ensures thatmutan t alleles arise continuously. Slootmaker (citedb yDay , 1974)calculate d that ahectar e ofbarle yin - fectedwit hpowder ymilde wma yproduc e 10 conidi13a pe r day.Eve nwit ha conservative estimate ofth emutatio n rate tovirulenc e to aparticula r —8 5 resistance geneo f 10 ,1 0 virulentconidi ama yb eproduce d per hectare per day and doublemutant svirulen t to2 resistanc e genes are area lpossi ­ bility (Persone t al., 1976). The frequency ofthes evirulen t alleles in thepopulatio nma yb ever y low (toolo w tob e detected)bu tthei rpractica l significance isenormou sbecaus e ofth e large selective advantage theyhav e onresistan t hosts.Thirdly , thehig h asexualmultiplicatio n rates ofman y fungi ensures thata ninitiall y rare alleleca nunde r selection increase in number and frequencyver y rapidly.

Heterokaryosis The role ofheterokaryosi s inth evariabilit y of fungiha sbee nth e subjecto fconsiderabl e speculation (e.g.,Snyder , 1961). However,knowl ­ edgeo fheterokaryosi s derives largely from laboratory studies utilizing rather artificial techniques and confirmed examples ofnatura lhetero - karyons arerar e (Caten& Jinks , 1966). (Thedikaryon s ofheterothalli c Basidiomycetes areinvariabl y heterokaryotic butconstitut e aspecia l case aspar to fth e sexual cycle ofthes e fungi.)Th enatura l significance of heterokaryosis will depend notonl yupo n itsoccurrenc ebu t alsoupo nth e diversity ofth e associated genomes.Attempt s to form heterokaryons between independent isolates revealed thepresenc e ofheterokaryo n (vegetative)

215 incompatibility systems inman y species (Caten& Jinks ,1966 ;Esse r & Blaich, 1973). Strains carrying different alleles aton eo rmor e specific heterokaryon-incompatibilitygene s (het) arerestricte d orblocke d in heterokaryon formation (Esser& Blaich , 1973;Crof t& Jinks , 1977). Consequently heterokaryons form readily onlybetwee n strains ofsimila r genotype.Rayne r &Tod d (1979)hav erecentl y showntha tnuclea r exchange between dikaryons ofwood-decayin g Basidiomycetes isblocke db y asimila r incompatibility system whichthe y term intraspecific antagonism. These considerations ofth e incidence ofheterokaryosi s and itsgenetica l control suggesttha t itsnatura l significance is less thanha s frequently been assumed inth epast .

Sexual hybridization Where itoccur s sexual hybridization isundoubtedl y themajo r sourceo f variability in fungalpopulations ."I nrusts ,man y different racesma yb e produced by crossing 2contrastin g parentrace s (Flor, 1955). Withquanti ­ tative characters evenhybridizatio n ofphenotypicall y similar strainsma y give awid e spectrum ofvariatio n (e.g.Fig .1 i nCate n& Jinks , 1976). Despite the obvious effecto f sexual hybridization inthes e examples it shouldno tb e forgotten thatman y fungal species are asexual and further­ more thatth esexua l stageo fcertai nperfec t species occursrarel y orno t atal l innature . The amounto fvariatio n released byhybridizatio n dependsupo nth e diversity ofth e interacting genomeswhic h isregulate d by 2 independent and opposing sexual incompatibility systems,terme d homogenic andhetero ­ genic incompatibility (Esser, 1971). Homogenic incompatibility isth e control exertedb y themating-typ egene s and encourages outbreedingb y blockingmating sbetwee n individuals of likegenotype .Th eoccurrence , genetic control andbiologica l significance ofhomogeni c incompatibility in fungi iswidel y appreciated (Raper,1966 ;Burnett ,1976 ;Fincha m et al., 1979)an dwil lno tb econsidere d furtherhere .Heterogeni c incompatibili­ ty, incontrast ,stop smating sbetwee n strains ofdifferen t compatibility genotype, thereby reducing outbreeding and thereleas e ofgeneti cvaria ­ tion.Knowledg e ofheterogeni c incompatibility derives largely frominves ­ tigationsb yEsse r and associates intomatin gbetwee n races of Podospora anserina (Ces.)Niessl .where , in1 cas e analysed indetail ,compatibi ­ lity/incompatibility was determined by complex interactionsbetwee n4 unlinked genes (Esser& Blaich , 1973). Thehomothalli cAscomycete , Aspergillus amstelodami (Mangin)Thor n& Church ,provide s asecon d example (Caten,unpublished) . Inhomothalli c species thegeneti c significance of sex dependsupo nth e frequency ofhybridizatio n asoppose d toselfing .Gen ­ etic control ofthi s characterwa s soughtb y comparing theproportio n of hybrid asci (outcrossing index)i nmixe d cultures ofrelate d und unrelated strains.Althoug h all the strains hybridized, related strains did somor e

216 Tabel 1. Heterokaryon formation andoutcrossin g between related andunrelate d strains of Aspergillus amstelo- dami. + =heterokaryo n compatible, -= heterokaryo n incom­ patible.Th e figures indicate theproportio no fhybri d asci among total asci. Strainswit h thesam enumbe r are related, strain13 w isrelate d to9b .

White-spored strains

9w 37w 115w 164w 13w

+ - - - + in 9b a 0.24 0.03 0.01 0.01 0.22 •H a u •p m 37b •a

readily thanunrelate d giving ahighe r outcrossing index (Table 1).Th e related strains were allheterokaryon-compatibl e while theunrelate d strainswer e incompatible, implying thatlo whybridizatio n isrelate d to heterokaryon incompatibility. Thiswa s investigated by determining the outcrossing index forpair s ofstrain s differing atknow n het genes.O f5 het genes tested, 1 (hetB) gave a5-fol dreductio n inoutcrossin g when heterozygous while theother s hadn o effect.Clearl y in A. amstelodami some,bu tno tall , het genes also acta sheterogeni c sexual incompatibil­ ity factors.

Parasexual hybridization The occurrence in fungi ofrecombinatio n byprocesse s other than sexual reproduction was firstclearl y demonstrated in Aspergillus nidulans (Eidam) Wint.b y Pontecorvo and associates (Pontecorvo, 1956). The sequence of eventswa s termed theparasexua l cycle and isno w extensively documented andwidel y appreciated (forrevie w seeRoper ,1966 ;Caten , 1981). More recently itha sbee n shown thatparasexua l recombination indikaryon s of heterothallic Basidiomycetes canoccu r throughprocesse s other thanth e

217 parasexual cycle (Leonard et al., 1978;Day , 1978;Frankel , 1979). At least oneo fthes eprocesses ,meiotic-lik erecombination ,produce sbot hinter - and intra-chromosomal recombinants ata frequenc ymuc h higher than expected from theparasexua l cycle (Frankel, 1979). Evidence thatparasexualit y occurs outside the laboratory hasaccumu ­ lated over recentyears .Somati c diploid strains havebee n isolated from nature insevera l habitually-haploid species (Caten& Day ,1977 ;Caten , 1981)an dne w genotypes ofrust s and smutshav ebee n shownt oaris e in mixtures ofdikaryon s undernear-natura l conditions (Bartose t al.,1969 ; Megginson& Person , 1974). Theorigi no fthes evariant s isunclear ,al ­ though diploid clones of Puccinica graminis Pers f. sp.- tritici Eriks.& E. Henn arise inaxeni c cultures (Macleane t al., 1974)an dproduc evari ­ ants (Green et al., 1978). Eventhoug hparasexua l processes occur naturally theircontributio n tovariatio nma yb e reducedb y heterokaryonincompati ­ bility sincethi s restricts nuclear association to strains ofsimila r genotype. Iti ssignifican t inthi s contexttha tman y naturally-occurring somatic diploids appearhomozygou s (Caten, 1981). Furtherwor k is required toestablis h how common somatic diploids are innatur e andth eexten tan d nature ofth egeneti cvariatio n theycarry .

Cytoplasmic inheritance The cytoplasm isimportan t forth evariabilit y of fungi in2 respects : (i)th epresenc e of ageneti c system inth emitochondri a and (ii)th eoc ­ currence of stable double-stranded RNA (dsRNA )molecule s with geneticac ­ tivity. Eukaryotic mitochondria carryDN A genomeswhic h canunderg omutation , recombination and segregation inmanner s analogous tothei rnuclea rcoun ­ terparts (Gi,lham, 1978). Thenumbe ro fgene s inth emitochondria l genome islimite d (approximately 50i nyeast )bu tthe y areessentia l forth efunc ­ tioning ofth emitochondria . Themitochondria l genetic diversity present innatura lpopulation s remains tob e established but analyseswit hrestric ­ tionenzyme s have revealed sequence heterogeneity within species (Sanders et al., 1977). Iti sparticularl y interesting that2 long-recognise d degen­ erative conditions in fungi,senescenc e andvegetativ e death,hav e recently beenshow nt oresul t from gross changes inth emitochondria l genome (Cummingse t al., 1979;Lazaru s et al., 1980). Double-stranded RNAmolecule s havebee ndetecte d inman y fungal species and they arebelieve d toreflec tmycoviru s infections, althoughvirus-lik e particles areno talway svisibl e (Ghabrial, 1980). Notal l strainswithi n a species carry anyo r the same dsRN Amolecule s andther ema y orma y not bephenotypi c differences associated with thevariou s complements.Double - strandedRN Adetermine s theproductio n of,an d sensitivity to,th e toxins responsible forth e 'killer'phenotyp e in Ustilago maydis (DC.)Cda .an d is almost certainly responsible forth ehypovirulen tphenotyp e in Endothia

218 parasitica (Murr.)P.J . &H.W . And. (Day& Dodds , 1979). These phenotypic effects raise thepossibilit y ofusin gmycoviruse s tocontro l fungalpatho ­ gens. Sincecell-fre e mycoviruspreparation s areunabl e to infectintac t fungal cells thenorma l route oftransmissio n isvi ahypha l anastomosis and willb e affectedb y those factorswhic h control hyphal fusionan dhetero - karyon formation (Ghabrial, 1980). Forexample ,transmissio n ofhypoviru - lencei n Endothia isrestricte db yheterokaryo nincompatibilit ybu tsuffi ­ cient infection occurs togiv e curing ofcanker s following inoculationwit h hypovirulent strains (Day& Dodds , 1979).

GENETIC CONTROL OF PATHOGENICITY

Whena singl ehos tgenotyp e is inoculatedwit hindependen t isolates of aphytopathogeni c fungus andth e level ofdiseas e is assessed quantitat­ ively iti stypicall y found thatth e isolates differ inpathogenicit y and thatthi svariatio n iscontinuou s ranging from isolateswhic h given oobvi ­ ous symptoms toother swhic hproduc e severe disease (Schwarzbach &Wolfe , 1976; Fig.1) .O nman yhos tgenotype s there isa nexces s of isolateswhic h give only low levels ofdiseas e implying thatth eoveral lpictur e reflects 2 overlapping distributions,on equalitativ e and theothe r quantitative. In considering thegeneti c control ofpathogenicit y iti sconventiona l to distinguishbetwee n thesequalitativ e andquantitativ e components, although thisdistinctio nma yno t alwaysb e clear inpractice .Th e low-disease pro­ ducing isolates aregenerall y classified as avirulentt otha thos t genotype incontras t toth evirulen t isolateswhic hproduc e significant levelso f disease (Fig.1) .Thi sclassificatio nprovide s thequalitativ e componento f

quantitative qualitative assessment assessment 10( 100i

80 0) o (/) 0) a m V) ;/> 60- •o virulent T» o «•- o 40 "c o D Ê o 20- } avirulent Figure 1. Assessmento fth evariatio n inpathogenicit y shownb y independent isolates inoculated onto asingl ehos tcultivar .Eac hhorizonta l barrepre ­ sents the amounto fdiseas eproduce db y anindividua l isolate.

219 pathogenic variation andwil lb e referred to asvirulence . Iti sparticu ­ larly important indenotin g thehos trang e ofpathoge n isolates.However , asFig .1 shows ,no t allvirulen t isolatesproduc e the same level ofdis ­ ease and thisrepresent s thequantitativ e componento fpathogeni c vari­ ationwhic hwil lb e referred to asaggressiveness .

Virulence Theclassica l studies ofFlo r (1942, 1956)wit h flaxrus t established that avirulence/virulence toa particula r resistance genewa s controlled by alternate alleles ata correspondin g major gene inth epathogen .Thi sone - for-one relationship applied to several resistance geneg, suchtha tfo r every gene determining resistance inth ehos tther ewa s aspecifi ccomple ­ mentary gene determiningvirulenc e inth epathogen . Itha s subsequently beendemonstrate d insevera l otherhost-parasit e systems (Person& Sidhu , 1971;Day , 1974)an d is formalized asth egene-for-gen ehypothesis . However, Lawrence et al. (1981a)hav e recently found thatpathogenicit y of flaxrus t toeac h ofth eM ,L ,L ,L andL resistance allelesi s determined by 2interactin gpathoge n genes,indicatin g that,althoug ho f widevalidity , thegene-for-gen e relationshiptloe sno t fully account for allqualitativ e host-parasite interactions.Th e avirulent and resistant alleles inpathoge n andhos trespectivel y aregenerall y dominant over their alternate alleles (Person& Sidhu,1971 )an d arethough t tob eth ephysio ­ logically-active forms ofthes e genes,i.e . host-pathogen incompatibility isth e active response (Ellingboe, 1978). Ina numbe r ofcases ,resistanc e factors againstth e samepathoge n areclosel y linked andma yb e allelic (Shepherd &Mayo , 1972). The little information available onth echromo ­ somal location ofth epathoge n genes indicates that they areno tclustered , 12 3 although in flax rustth e avirulencedeterminant s toth eP ,P andP resistance alleles aretightl y linked andma yb e allelic (Lawrence et al., 1981b).

Aggressiveness The genetic control of aggressiveness has rarelybee nexamine d despite the facttha tthi s componentma y determine the severity of anepidemi c or whether anewly-arise n race spreads ornot .A s aquantitativ e characterag ­ gressiveness wouldb e expected tob epolygenicall y determined and thefe w analysed examples indicate thatthi s isth ecase .Th e enhancedpathogenici ­ tyo fth e aggressive straino f Ceratocystis ulmi (Buism.)C .Morea u isde ­ terminedb y anunknow nnumbe r ofgene swhic h interact in acomplementar y fashion (Brasier, 1977). Strains of Ustilago hordei (Pers.)Lagerh . differ inth e% infection theyproduc e oncompatibl ebarle y cultivars.Thi svari ­ ation inaggressivenes s isdetermine d by apolygeni c system (Emara& Sidhu , 1974)and , atleas t inon e strain,th egene s involved show additive,domi ­ nance andepistati c effects (Caten,Groth ,Perso n andDhahi ,unpublished) .

220 CONCLUDING REMARKS

Fungi possess a variety of mechanisms which can generate, store and re­ lease genetic variation and it is not surprising that natural populations are highly variable. The relative importance in nature of these different mechanisms is not clear and is likely to vary between species, between lo­ calities and over time, precluding any general conclusions. The mechanisms are themselves subject to genetic control and may evolve to higher or lower efficiency in response to selection pressures and we might expect related species inhabiting contrasting environments to utilize different mechanisms and to exhibit different patterns of variation. Many agricultural chemicals have genetic effects on fungi (Kappas et al., 1974; Bignami, 1977) and they may enhance the activity of the genetic mechanisms and thereby increase the general variability of fungal populations. Just as important as the genetic mechanisms are the selection pressures operating in nature. These will mark­ edly influence the fate of new mutations and the rate of change of gene and genotype frequencies. Clearly the host population is the major selective force affecting pathogen populations, but many detailed questions remain to be answered. For example, are virulent and avirulent alleles equally fit m the absence of the matching host resistance and what will be the response of pathogens to heterogeneous host populations? It is hoped that study of the population genetics of pathogens will lead both to a better understand­ ing of host-parasite interactions and to the development of practical con­ trol measures (Person et al., 1976; Wolfe & Barrett, 1980).

REFERENCES

Bartos, P., G. Fleischmann, D.J. Samborski & W.A. Shipton, 1969. Studies on asexual variation in the virulence of oat rust, Puccinia coronata f. sp. avanae and wheat leaf rust, Puccinia recondita. Canadian Journal of Botany 47: 1383-1387. Bignami, M., 1977. Mutagenic and recombinogenic action of pesticides in Aspergillus nidulans. Mutation Research 46: 395-402. Brasier, CM., 1970. Variation in a natural population of Schizophyllum commune. American Naturalist 104: 191-204. Brasier, CM., 1977. Inheritance of pathogenicity and cultural characters in Ceratocystis ulmi; hybridization of protoperithecial and non-aggressive strains. Transactions of the British Mycological Society 68: 45-52. Burnett, J.H., 1975. Mycogenetics. Wiley, London. 375 p. Burnett, J.H., 1976. Fundamentals of mycology. 2nd ed. Edward Arnold, London. 673 p. Caten, CE., 1979. Quantitative genetic variation in fungi. In: J.N. Thompson, Jr. & J.M. Thoday (Eds): Quantitative genetic variation. Academic Press, New York. p. 35-59. Caten, CE., 1981. Parasexual processes in fungi. In: K. Gull & S. Oliver (Eds): The fungal nucleus. Cambridge University Press, Cambridge (in press). Caten, CE. & A.W. Day, 1977. Diploidy in plant pathogenic fungi. Annual Review of Phytopathology 15: 295-318. Caten, CE. & J.L. Jinks, 1966. Heterokaryosis: Its significance in wild homothallic Ascomycetes and Fungi imperfecti. Transactions of the British Mycological Society 49: 81-93. Caten, CE. & J.L. Jinks, 1976. Quantitative genetics. In: K.D. Macdonald (Ed.): Second international symposium on the genetics of industrial micro-organisms. Academic Press, London, p. 93-111.

221 Clifford,B.C .& R.B .Clothier ,1974 .Physiologi c specializationo fPuccini ahorde io n barleyhost swit hnon-hypersensitiv e resistance.Transaction so fth eBritis h MycologicalSociet y63 :421-430 . Croft,J.H .& J.L .Jinks ,1977 .Aspect so fth epopulatio ngenetic so fAspergillu s nidulans.In :J.E .Smit h& J.A .Patema n (Eds):Genetic san dphysiolog yo f Aspergillus.Academi cPress ,London ,p .339-360 . Cummings,D.J. ,L .Belcou r& C .Grandchamp ,1979 .Mitochondria lDN Afro mPodospor a anserinaII .Propertie so fmutan tDN Aan dmultimeri ccircula rDN Afro msenescen t cultures.Molecula ran dGenera lGenetic s171 :239-250 . Day,A.W. ,1978 .Chromosom e transfer indikaryon so fa smu tfungus .Natur e273 :753-755 . Day,P.R. ,1974 .Genetic so fhost-parasit einteraction .Freeman ,Sa nFrancisco .23 8p . Day,P.R . &J.A .Dodds ,1979 .Viruse so fplan tpathogeni cfungi .In :P.A .Lemk e (Ed.): Virusesan dplasmid si nfungi .Marce lDekker ,Ne wYork .p .201-238 . Ellingboe,A.H. ,1978 .A geneti canalysi so fhost-parasit e interactions.In :D.M .Spence r (Ed.):Th epowder ymildews .Academi cPress ,London ,p .159-181 . Emara,Y.A .& G .Sidhu ,1974 .Polygeni c inheritanceo faggressivenes s inUstilag ohordei . Heredity32 :219-224 . Esser,K. ,1971 .Breedin gsystem si nfung ian dthei rsignificanc e forgeneti c recombination.Molecula r andGenera lGenetic s110 :86-100 . Esser,K .& R .Blaich ,1973 .Heterogeni c incompatibility inplant san danimals .Advance s inGenetic s17 :107-152 . Fincham,J.R.S. ,P.R .Da y& A .Radford ,1979 .Funga lgenetics .4t hed .Blackwells , Oxford.63 6p . Flor,H.H. ,1942 .Inheritanc eo fpathogenicit y inMelampsor a lini.Phytopatholog y 32:653-669 . Flor,H.H. ,1955 .Host-parasit e interactioni nfla xrus t- it sgenetic san dothe r implications.Phytopatholog y45 :680-685 . Flor,H.H. ,1956 .Th ecomplementar y geniesystem si nfla xan dfla xrust .Advance si n Genetics8 :29-54 . Frankel,C , 1979.Meiotic-lik erecombinatio ni nvegetativ edikaryon so fSchizophyllu m commune.Genetic s92 :1121-1126 . Ghabrial,S.A. , 1980.Effect so ffunga lviruse so nthei rhosts .Annua lRevie wo f Phytopathology 18:441-461 . Gillham,N.W. ,1978 .Organell eheredity .Rave nPress ,Ne wYork .60 2p . Green,G.J. ,P.G .William s& D.J .Maclean ,1978 .Variabilit y ofuredia lculture so f Pucciniagramini stritlc iderive dfro mvegetativ eaxeni cmycelium .Canadia nJourna l ofBotan y56 :855-861 . Hollomon,D.W. , 1978.Competitiv eabilit yan dethirimo lsensitivit y instrain so fbarle y powderymildew .Annal so fApplie dBiolog y90 :195-204 . Kappas,A. ,S.G .Georgopoulo s& A.C .Hastie ,1974 .O nth egeneti cactivit yo f benzimi-dazolean dthiophanat efungicide so ndiploi dAspergillu snidulans .Mutatio n Research26 :17-27 . Lawrence,G.J. ,G.M.E .May o& K.W .Shepherd ,1981a .Interaction sbetwee ngene scontrol ­ lingpathogenicit y inth efla xrus tfungus .Phytopatholog y71 :12-19 . Lawrence,G.J. ,K.W .Shepher d& G.M.E .Mayo ,1981b .Fin estructur eo fgene scontrollin g pathogenicity infla xrust ,Melampsor a lini.Heredit y4 6 (inpress) . Lazarus,CM. , A.J.Earl ,G .Turne r& H .Kiintzel ,1980 .Amplificatio no fa mitochondria l DNAsequenc ei nth ecytoplasmicall yinherite d 'ragged'mutan to fAspergillu s amstelodami.Europea nJourna lo fBiochemistr y106 :633-641 . Leonard,T.J. ,R.F .Gabe r& S .Dick ,1978 .Internuclea r genetic transferi ndikaryon s ofSchizophyllu m commune.II .Direc trecover yan danalysi so frecombinan tnuclei . Genetics89 :685-693 . Maclean,D.J. , I.e.Tommeru p& K.J .Scott ,1974 .Geneti cstatu so fmonokaryoti cvariant s ofth ewhea tste mrus tfungu sisolate dfro maxeni cculture .Journa lo fGenera l Microbiology84 :364-378 . Megginson,F.G.A .& CO . Person,1974 .Somati crecombinatio n inUstilag ohorde idurin g theparasiti cphas eo nbarley .Canadia nJourna lo fGenetic san dCytolog y16 :851-855 . Nevo,E. ,1978 .Geneti cvariatio n innatura lpopulations- ,pattern san dtheory . TheoreticalPopulatio nBiolog y13 :121-177 . Person,C .& G .Sidhu ,1971 .Genetic so fhost-parasit e interactions.In :Mutatio n breedingfo rdiseas eresistance .Internationa lAtomi cEnerg yAgency ,Vienna . p.31-38 . Person,C , J.V.Grot h& O.M .Mylyk ,1976 .Geneti cchang ea tth epopulationa l leveli n host-parasite systems.Annua lRevie wo fPhytopatholog y 14:177-188 . Pontecorvo,G. ,1956 .Th eparasexua lcycle .Annua lRevie wo fMicrobiolog y 10:393-400 .

222 Râper,J.R. , 1966.Lif ecycles ,basi cpattern so fsexualit yan dsexua lmechanisms .In : G.C.Ainswort h& A.S .Sussma n (Eds):Th efungi ,volum eII .Academi cPress ,Ne wYork . p.473-511 . Rayner,A.D.M .& N.K .Todd ,1979 .Populatio nan dcommunit ystructur ean ddynamic so f fungii ndecayin gwood .Advance si nBotanica lResearc h7 :333-420 . Roper,J.A .1966 .Th eparasexua lcycle .In :G.C .Ainswort h& A.S .Sussma n (Eds):Th e fungi,volum eII .Academi cPress ,Ne wYork .p .589-617 . Sanders,J.P.M. ,C .Heyting ,M.P .Verbeet ,F.C.P.W .Meylin k& P .Borst ,1977 .Th e organisationo fgene si nyeas tmitochondria lDNA .III .Compariso no fth ephysica l mapso fmitochondria lDNA sfro mthre ewild-typ eSaccharomyce s strains.Molecula r andGenera lGenetic s157 :239-261 . Schwarzbach,E .& M.S .Wolfe ,1976 .Consideratio no fth erelationshi pbetwee nErysiph e graminisf .sp .horde ian dit shost ,barley ,i nth eexploitatio no fhos tresistance . In:H .Gau l (Ed.):Barle ygenetic sIII .Kar lThiemig ,Munich ,p .426-432 . Shepherd,K.W .& G.M.E .Mayo ,1972 .Gene sconferrin gspecifi cplan tdiseas eresistance . Science175 :375-380 . Snyder,W.C. ,1961 .Heterokaryosi sa sa natura lphenomenon .In :Recen tadvance si n botany,vol .I .Proceeding so fth e9t hinternationa lbotanica lcongress ,Montreal , 1959. Universityo fToront oPress ,Toronto ,p .371-374 . Spieth,P.T. ,1975 .Populatio ngenetic so fallozym evariatio ni nNeurospor aintermedia . Genetics80 :785-805 . Wolfe,M.S .& J.A .Barrett ,1980 .Ca nw elea dth epathoge nastray ?Plan tDiseas e 64:148-155 .

223 Genetics of pathogenicity in Ceratocystis ulmi and its significance for elm breeding

CM. Brasier

Forest Research Station. Alice Holt Lodge, Farnham, Surrey, UK

ABSTRACT

The relationship between research onvariatio n in Ceratocystis ulmi andel mbreedin g isdiscussed . C. ulmi falls into2 majo r sub-groups,a highl ypathogeni c 'aggressive strain' and amor eweakl y pathogenic 'non-aggressive strain'. The 2strain s also differ inmos t othermajo r characteristics including colonymorphology , growth rate,optimu m temperature forgrowt h andmatin g system.Eac h strainshow s awid e range ofvariation .Th e 2strain s showgeneti c isolation andprobabl y dono t freely interbreed. Laboratory forced crossesbetwee nth e aggressive andnon-aggressiv e strains indicate thatth e genetic systems governing theirpathogeni c abilities arecomplex , polygenic andqualitativel y different.Th e aggressive strain divides into 2 further sub-groups,th eEurasia n andNort h American races,whic h differ ina numbe r of characteristics including colonymorpholog y andpathogenicity . They showa degree ofreproductiv e isolation,an d afairl ydiscret e geo­ graphical distribution.Th e terms 'strain'an d 'race'a suse d in C. ulmi arediscusse d anddefined .

INTRODUCTION

Theel m ison eo fth emos timportan t shelter andamenit y trees inth e NorthernHemisphere , comprising some3 0tax a extending fromNort hAmeric a throughEurop e andWester nAsi aeastward svi a theHimalaya s and thecentra l Asianupland s toChin a andJapan .Broadl y speaking,Nort hAmerica n elms tendt o include themos t susceptible species toDutc hel m disease,th e Asiatic elms themos tresistan t species,an dman y ofth eEuropea n elms lie somewhere inbetween . Currently, 3mai ncentre s atWisconsi nUSA ,Wageninge n theNetherland s andVolgogra d USSR arecarryin g outth ebreedin g and selection ofelms . Dutch elmdiseas e isb y farth ebigges tthrea tt oth eproduct s ofthes e

224 breedingprogrammes ,an d all3 centre s are attempting to incorporate disease resistance fromhighl y resistantAsiati c elms such as Ulmus pumila L.int o theirbreedin gmaterial . Treebreedin g isobviousl y along-ter mproces s and iti s consequently important tohav e clear aims. Inbreedin g forresistanc e todiseas e iti s importantt okno wwha ton e isbreedin g forresistanc e to i.e. the extent ofan dth epotentia l forvariatio n inth epathogen .Fo r anumbe r ofrea ­ sons, knowledge ofpathogeni cvariatio n in forestpathogen s hasbee n laggingbehin d that inpathogen s of arable crops. Indeed,unti l quite recently elmbreeder swer ever ymuc h inignoranc e ofwha tthe ywer ebreed ­ ing for resistance to. Itwa s only inth eearl y 1970s that the existence of ahighl ypathogeni c 'aggressive'strai no fth eDutc h elmdiseas efungus , Ceratocustis ulmi (Buism.)C .Moreau ,wa s recognised, and inconsequenc e the elmbreedin g programmes inth eUSS R and inth eNetherland swer e severly shaken (Kryukova, 1972;Gibb s& Brasier , 1973;Heybroek , 1976). Thebreed ­ ingprogramm e atWisconsi n survived the trauma largely through geographical accident,sinc e they had,unknowingly , alreadybee nusin g the aggressive strain inthei r resistance testingprocedures . Clearly we as forestpathologist s and fungal geneticists haVe adut yt o the treebreeder s to attemptt okee p knowledge ofvariatio n in forest pathogens atleas tone ,an dhopefull y several steps ahead ofwha t isgoin g on inthei rbreedin g programmes.Ou r failure torecognis e the existence of several pathogenic formswithi n C. ulmi hasha d anumbe r ofrepercussions . Firstly, asjus tmentioned , itha sundermine d thebasi s uponwhic hth e selection ofresistan t elmmateria l wascarrie d out.Secondly , itha s resulted in alac ko fdat a on interactions between theproduct s ofth eel m breeding programmes -th edifferen t elm species and theirprogenie s -an d thevariou s strains and races ofth e fungus.Thi s intur nmus thav eresult ­ ed inth e loss ofvaluabl e information onth egeneti c nature ofhos tresis ­ tance ando f fungalpathogenicity . Thirdly, itha s allowed 2ne w epidemics ofDutc h elm disease toprogres svirtuall y uncharted and unrecognised acrossmuc h ofEurop e and SouthWes tAsi a during thepas t 10-20year s (Brasier, 1979). Evidently continued negligence ofvariatio n inou r forestpathogen s is a short sightedpolicy . Itshoul d thereforeb e our constant aim tomaximis e information onth epathogeni c potential ofthes e organisms,an d to attempt tounderstan d thegeneti c systems controlling theirpathogenicit y bysur ­ veying andmonitorin g theexten t ofvariatio n inth e field andb yexperi ­ menting onpotentia lvariatio n inth e laboratory. Inth ecas e of C. ulmi, anattemp tt oidentif y thegeographica l centre oforigi n ofDutc hel m disease,stil lrathe rvaguel y thoughtt ob e 'some­ where inEaster nAsia' , is athir d andvita l requirement.

225 PRESENT KNOWLEDGE OF STRAINS AND RACES WITHIN CERATOCYSTIS ULMI

C. ulmi falls into 3geneticall y distinct sub-groupings:a highl y pathogenic aggressive strainwhic h itselfdivide s into 2distinc traces , and amor eweakl ypathogeni c non-aggressive strain (Gibbs& Brasier ,1973 ; Brasier, 1979). Thenon-aggressiv e straini s found fromNort hAmeric a throughEurop e to Iran.O fth e2 race s ofth e aggressive strain,terme d the NorthAmerica n orNA N and Eurasiano rEA Nraces ,th eNA N is distributed throughoutNort hAmerica . Inth e late 1960s itwa s introduced intoBritai n and itha s since spread to adjoiningpart s ofnorth-wes tEurop e as fareas t as Italy andnorther nYugoslavia .Th eEA Nrac e occursmainl y toth e easto f a line fromDenmar k to Italy,extendin g toVolgogra d and theCaspia n Sea areao f Iran.Th epresen t situation inEurop e israpidl y changing asth e NAN raceo fth e aggressive strain spreads eastwards and southwards andth e EAN race spreadswestwards . The aggressive andnon-aggressiv e strainshav eman ydifferin gproper ­ ties (Table 1). Inaga rcultur e they differ ingrowt h rate,optimu m tem­ perature forgrowth ,uppe r temperature limit forgrowt h and incolon y morphology. They alsodiffe r inth e frequency ofthei rmatin g types in nature, and abarrie r tohybridizatio n existsbetwee n them.Moreover ,a s shownbelow ,crosse sprovid e evidence ofconsiderabl e genetic isolation between them. The 2race s ofth e aggressive strain also differ incultura l charac­ teristics and some other features (Table 1).Barrier s tothei rhybrid ­ ization alsoexis tbu tthes e areles smarke d thanthos eoccurrin gbetwee n the aggressive andnon-aggressiv e strains.

DEFINITION OF 'STRAIN' AND 'RACE' IN C. ULMI

The terminology applied to sub-unitsbelo wth e species level in'funga l populations is ina rathe r confused state,reflectin gperhap s theyouthfu l stateo fth e art!Th e field of fungalpathogeni c polymorphism isparticu ­ larlydifficult , and there certainly appears tob en oestablishe dterminol ­ ogy inth e literature appropriate tomajo r sub-groups ofth e sortencoun ­ tered in C. ulmi. Inth eeven tth e somewhatneutra l term 'strain'wa s chosent o apply toth e aggressive andnon-aggressiv e sub-groups of C. ulmi (Gibbs& Brasier , 1973)despit e the facttha tth e samewor d is sometimes used rather looselyb ymycologist swhe nthe yma yreall ymea n 'isolate' (the differencebetwee n 'strain'an d 'isolate'ha sbee nclearl y definedb y Ainsworth (1961)). Subsequently itha sbee nnecessar y to find asuitabl e word to apply to sub-groups of lower rank than strain in C. ulmi, andi n thiscas eth ewor d 'race' sensu Stebbins (1966)ha sbee nchose n (Brasier, 1979). Following Stebbins'hierarch y 'strain'a suse d in C. ulmi should probably assume aran k roughly equivalent tohi s 'sub-species',an d indeed

226 •O >D

rfiO firö rH 0) 0) t) rO 2 O 0) 4J •H O 3 4-1 rd ,v M rH -H fi ra 2 p 4-1 X) rfl 4-1 «C ra 0fi fi0 ^~. m fi fi ra •rH 0) Ol •H 0) 0 0) ra •Ö H u CD O in T3 M ft 01 ü rrad rH 4-1 0) 0) M ra 0 (0 XI^ - m M >i O 0ra1 Lfl 01 >TO fi ft HOfiM >1 r> 0 *}• rH -H • o rH «fi rH Oi M •H CTi rd rH fi 0 fi •H 01 rö Oi Oi O Ol 'S H U •* CM 0 rH 0) +J •H • fi rö o > rH i O CM g rd > rd & 4-1 V O 1 rH 0 3 V •-***a . *z rH 1 1 g P o> 4-1 MUM 1 u rd 0) O CM 0 01 fi ofi 1 ro ro 4-1 oc O Ol IT) X! W«f i co» O CM CO U ft 3 N M4 01 & u •rra4 r> m m fi 4-1 ai -H 1 M rH r-l 1 >i XI fi <ü wra o ^^ 3 •H M 0) o O. 4-1 ra ro u •P XraI 1 • 0) M >i Di rO XI 3fi -rfiH ra "0 rfiO U 4-1 rH Ol M •H ro 3-H rd ra Oi rd CJ — M O 0 rH •fiH U fi 1 u u 4-1 M H rü S 01 0 fi <3 oi ra XI rrj fi o X! > U o 1 0 M ra 01 MH 0> 3 01 o ra t/2 fi M ro ft-H U XI to 01 Ol H > 0) . ra •H u •H a) >i T3 fi ft X! rö fi ra m ra 0 co in rH (1) o m u >i4-i M >0 •H m m rO fi 4J Ol-r4 01 4-1 DlrH 4-1 O 0 (0 M • •. 0) U <#• o • CM O ro fi 4-1 ft DlrH ra ra O 01 M ca M i I CM g-H O rd ft 01 ld rd 4-1 rH M p ^ai^ Di *z CM rH 1 g U U fi 4-1 ü 3 u 0) 1 / ra Di «fi O CO 0 4-1 4J 0 1 O fH D< 0) o o •- »ai «C m• O CM CO u ra t/3 N ca H co fi 01 .—01» rH a r- • •>H ai 1 1 tl) H rü-H >i •—' rH p e (H -H 4-1 ft ro id ft Ü M ro 01 u i ra ro U 01 G o m X) •H ra i N ro fi SH -o io ra ra *—•> 4-> X rH-H U & O fi M u <« ro ra rO 0) 4J 3 01 P ro P 01 0) M l'­ S 4-1 fi H ft ra 0) ai u ro 0) T) ra 0) 0) o> •>H •H rH X! M U 0) 0 > 01 u ra ra •— •>H 4l«llÄ O-H > Ol ra JO ra O "O M-l ro ft rö ra -H SH ai M (H O O 0ra> g g'H S 1 i-H 0) ra ix Oi •H SH ra T) m rö M 01 rH Di ra Oi rH CO >H fi ra Oi M rH 10 .< ro Oi 10 o 3 g •o M Ol Ol rö ai rO CO CM 3 oi ro oi in u aj r> 1 1 I g fi >i-H rfiö > i rd CO -H X! 01 m O H O SHH •H fi •P rH u < 13 fi 0 rH 4-1 afi> oo^ XraI CJ XI -—U • !-01- •H 0) o s -—s 4-1 rH U Oi fi fi 0> fi u — -H 0) fi ro XI ro 0 X! rfi O Is r» ^ 4-1 Jx fi -H 0) ra r> rH S Ü 0 -M O 1 OX! O rH S5, 0fi —' —• g 4-1 Oi gl -H 4-1 ü o M M 0 rö u 0 01 4J 0 ^1*4 W 01 0 u M 0 rH (H rj ~- 0) 3 l*H •H rH 0) i m iX'O S m ra ro •—' ft X! 0) XI 01 4-1 M •H (0 01 S 4-1 0) M U •H ^SC> Q U 4J P 0) *—>U . •H uft T3 CO 0 a) ro *—>1* 4-1 0 S o & >i 0) u •H SH rH m s™* U iDUU •H g 4-1 4-1 W \ fi 0 Ifl ^H» MH M T"J o 0 S r-< •H-H ra 01 "H fi o a) XÎX 3X3 >1 Di O rH rH fi oi o 4) •H 4-1 S O ro g 4J (H fi fi M -H -H -H O O) rH >-H m m s g CM ro •H S O 0 •H 3 4-1 4-1 rO X! Ol u •Cl 4-1 ro o P 0 a rH 4-1 4-1 M M M •p fi ra (0 M u fi a SH 0. 0 ro CO 01 Ol 4-J ro ro fi E-. Tl m o H o Oi D u S fi fa ^ ra P4 SH O

227 this seems appropriate forth e aggressive andnon-aggressiv e strains. The following definitions of 'strain' and 'race'a s applied to C. ulmi areoffered : Strain. (Aggressive andnon-aggressiv e strains).Majo r sub-groupswithi n the C. ulmi populationwhic hd ono t freely interbreed andwhic hdiffe r in almost all importantphysiologica l andpathologica l characteristics (see Table 1). Each strain shows awid e rangeo fvariatio n comparable totha t often found ina singl e species.Inter-strai nhybrid shav eye tt ob e found innature . Race. (NorthAmerica n andEurasia n races). Sub-groupswithi nth eaggress ­ ive strainpopulatio nwhic h dono t freely interbreed andwhic h differ in certain importantphysiologica l andpathologica l characteristics,bu ti n which their similaritiesbroadl y outweigh theirdifference s (seeTabl e1) . Thepopulatio n ofeac h race shows awid e range ofvariation .N o inter-race hybridshav eye tbee n foundi nnature .

DIFFERENCES IN PATHOGENICITY

The strains and raceso fC . ulmi arebes t"differentiate do nelm so f moderate disease resistance sucha s Ulmus procera Salisbury orcertai n clones of U. japonica (Rehd.)Sarg .Highl y susceptible orhighl y resistant elms such as V. americana L.o r U. pumila are, forobviou s reasons,unsuit ­ able. Fig. 1show sth e differences inpathogenicit y between population samples ofth enon-aggressiv e strainan do fth eEA N andNA Nrace so fth e aggressive strainwhe n inoculated intoclona l U. procera. Fig.l a isth e resulto f apathogenicit y testcarrie d outi n197 7comparin g 13non - aggressive isolates originating from 7countrie s from theUS At oIran . After 10weeks ,al l the isolatesha d caused only relatively low levelso f defoliation ranging from 14t o3 9% wit h amea no f28. 0% .Thi s isa typi ­ cal result forth enon-aggressiv e strain. The existence ofth eNort hAmerica n andEurasia n raceso fth eaggress ­ ive strainwa s only recognised asrecentl y as 1978,whe n itwa s supposed thatthe ybot hha d comparably high levels ofpathogenicit y (Brasier, 1979). However, apathogenicit y testcarrie d outi n197 9 showed otherwise.Th e test (Fig.lb ,c )comprise d asampl e of1 4EA N race isolates originating from 7countrie s for Ireland to Iran,an d 14NA Nrac e isolates originating from 7countrie sbetwee n theUS A andAustria .Tw onon-aggressiv e control isolates (arrow)gav e 16% mea ndefoliation .A s inpreviou s tests theNA N race isolates causedver yhig h levels ofdefoliatio n ranging from8 1t o 100% .Wit hth eEA N aggressive,however , defoliation ranged from 38t o 100 %, with amea no f8 2% , significantly below theNA Nmea no f9 5% . Thus theEA N aggressive showed anunexpecte d range ofvariatio n in pathogenicity,wit h some isolates significantly lesspathogeni c thanth e

228 No of Isolates

10- A Non Aggressive strain 8- 6- h 4-

2-

0- i • i ii i i i 0 50 100 6- EAN race 4-

2

0- T r" r

14

12 NAN race 8

6

4

2

50 100 Pathogenicity(/6 defoliation) Figure 1. Pathogenicity ofth enon-aggressiv e and ofth eEA N andNA N races ofth e aggressive straino fC . ulmi. A, sample of1 3 non-aggressive isolates inoculated into 3-year-old clonal V. procera in1977 ,showin g % defoliation after 10weeks . B, C, samples of 14EA N and 14NA N aggressive isolates inoculated into 5-year-old clonal U. procera in1979 ,showin g % defoliation after 13weeks .Th e arrow indicates themea n of2 non-aggress ­ ivecontro l isolates.Heav ybar s represent samplemeans . Therewer e 3rep ­ licatetree s foreac hisolate .

NAN race,thoug hstil lmor epathogeni c thanth enon-aggressiv e strain. Further important differences between the strains arereveale d in diseaseprogres s curves.Fig .2 show sth ediseas eprogres s curves forth e abovepathogenicit y test.Typically ,wit hth enon-aggressiv e straino n U. procera aburs to fdiseas e occurs inth e first fewweek s after inoculation,

229 Mean % defoliation 1U0

50-

100 Time(days ) Figure 2. Diseaseprogres s curves (mean% defoliation againsttim e from inoculation)fo rth enon-aggressiv e strain and theEA N andNA N races ofth e aggressive straino f C. ulmi on V. procera. Note finaldifference s inmea n pathogenicity levels ofth eEA N andNA Nraces ,an dth erecover y oftree s inoculated with thenon-aggressiv e strain.Th e testswer e carried outo n 5-year-old clonal U. procera in1979 .Th ecurve s areth emean s of 14EAN , 14NA N and2 contro lnon-aggressiv e isolates.Ther ewer e 3replicat e trees foreac hisolate . butthereafte r diseaseprogres s ishalte d'an dth etree s go intoa recover y phasewit h the diseased shootsbudding-u p andeventuall y flushing.Ther e is normally nore-occurrenc e ofth ediseas e inth e following season. Withbot h theEA N andNA N raceso fth e aggressive strainther e is a greater initialburs to fdiseas e activity.Wit hth eNA N race disease usuallyprogresse s until defoliation levels of90-10 0% havebee n reached andmos ttree s diewithi nth e same season. Ifthe y donot ,diseas e re­ occurrence and deathusuall y follow inth e second season. Withth eEA N raceth emos thighl ypathogeni c isolatesbehav e similarly toth eNA N isolates,bu twit hth e lesspathogeni c isolates some recovery may takeplace .A full recoveryma y occur inth e following seasonwhe n dis­ ease re-occurrence appears tob e arelativel y uncommonphenomenon . TheNA N race isth e form ofth e aggressive strain firstdescribe d in 1973b yGibb s& Brasier .Fro mit spathogeni cbehaviou r theEA Nrac ei s clearly amor e distinctgeneti c entity thanwa s originally supposed. Its

230 presentstatu s as arac ewithi n the aggressive strain (alongside theNA N race)rathe rtha ntha to f athir d straini nit sow nrigh tma yye tb ecalle d into question. Itsbiologica l characteristics,distributio n andhistor y of spread are,togethe rwit h those ofth eNA N race and the non-aggressive strain, still undergoing detailed survey and investigation.

GENETIC CONTROL OF PATHOGENICITY: CROSSES BETWEEN THE AGGRESSIVE AND NON- AGGRESSIVE STRAINS

Although the aggressive andnon-aggressiv e strainsma yno thybridis e freely innature ,crosse s canb e forcedbetwee n them inth e laboratory.Nu ­ merous crosseshav eno wbee nundertake nusin g aggressive andnon-aggressiv e isolates from 3continents ,an dth eresult shav ebee nbroadl y thesame . Interm s ofth e inheritance ofgrowt hrate ,th eprogen y of aggressive x non-aggressive crosses showconsiderabl e variation,man y growingsignifi ­ cantly faster thanth e aggressiveparen to r slowertha nth e non-aggressive parent.Fig .3 show sth eresul to fon esuc hcros sbetwee n non-aggressive

No of Isolates ' 15i „,, . -

10-

1.5 2.5 3.5 4 4.5 5 Growth rate mm-day-1

Figure 3. Growth rate distribution ofF ,progen y from acros sbetwee n aNA N aggressive (S30)an d anon-aggressiv e (273A)isolat e ofC . ulmi. Theheav y bar shows theprogen ymean ,an dth e arrowsth eparenta l growth ratesan d theparen tmid-poin tvalue .Th etes twa s carried outa t1 8 °Can dther e were2 replicate spe risolate .

231 parent273 A and aggressiveparen t S30.Th eprogen y distribution indicates that alarg e component ofth evariatio n is simply additive,bu t adegre eo f negative interactionbetwee n theparent s canoften 'b e detected, ashere , with themea no fth eprogen y (heavybar )lyin g significantly below themid ­ pointo fth e 2parent s (middle arrow) (seeBrasier , 1977). Incolon ymorphology ,progen y of aggressive xnon-aggressiv e crosses are always,extraordinar y variable (seeBrasie r& Gibbs ,1976 ,Plat e1) . Usually very fewo fth eprogen y canb e characterised interm s ofth e striate-petaloid colonymorpholog y ofth e aggressiveparent ,o rth e flat relatively undifferentiated morphology ofth enon-aggressiv eparent ,givin g a further indication of segregation ofextensiv e genetic differencesbe ­ tween the2 parents . Inthei rpathogeni c behaviour theprogen y of aggressive xnon-aggress ­ ivecrosse s areparticularl y interesting. Fig.4 shows theresult s of2 suchcrosses ,on ebetwee nnon-aggressiv e isolateM3 5 and aggressive isolate 027 (left)th eothe rbetwee nnon-aggressiv e isolateW1 0 and aggressive iso­ lateG3 6 (right).Abov e (Fig.4a ,c )i sth eresul twhe n asampl e ofthei r progenies were inoculated into themoderatel y resistant U. procera. Points tonot e are asfollows : -Th e aggressiveparent s gave characteristically high and thenon-aggress ­ iveparent s characteristically lowlevel s ofdefoliation . - Themajorit y ofth eprogen y arewea kpathogens .Wit hth eexceptio n of1 isolate (inth ecros sW1 0x G36 ,Fig .4c )non e approached the aggressive strain inpathogenicity . Statistically, only afe wprogen ywer esignifi ­ cantlymor epathogeni c thanthei rnon-aggressiv eparent . -Ther e is astron gnegativ e interaction: inbot hcrosse s theprogen ymea n (heavybar )fel l significantly below themid-poin t ofth eparent s (middle arrow). Inoculation ofth eprogen y ofW1 0x G3 6 into themor e susceptible U. glabra Hudson (Fig.4d )produce d agreate r spread inthei rpathogeni c distribution thusprovidin g acleare rpictur e ofthei rpathogeni c behav­ iour.Again ,non e ofth eprogen y approached the aggressiveparen t inpatho ­ genicity, andth eprogen ymea n (heavybar )la ysignificantl y below thepar ­ entmid-poin tvalu e (middle arrow). Thebehaviou r ofth eprogen yM3 5x 02 7o nth eeve nmor e susceptible U. laevis Pallas (Fig.4b )amplifie s thepictur e even further.O nthi s species eventh enon-aggressiv e parent (leftarrow )cause d 57% defoliation,wit h the aggressiveparen t (rightarrow )causin g 97% defoliation.Th eprogen y showed abroa d distribution.No tonl ywa s theprogen ymea n (heavybar )sig ­ nificantly below theparen tmid-poin t (middle arrow)bu t statistically many progenywer e significantly lesspathogeni c thanth enon-aggressiv eparent , 1progen y isolate causing only 0.5 % defoliation.A fewprogen y approached thepathogenicit y level ofth e aggressiveparent ,th ehighes tgivin g 90% meandefoliatio n (comparedwit h the aggressiveparen tvalu e of9 7 %).

232 Thepictur e givenb y theprogen y istherefor e oneo f alarg enegativ e interactionbetwee n thegeneti c systems ofth e aggressive andnon-aggress ­ ivestrain s (i.e.a dro p inpathogenicit y ofth eprogen y relative totha t ofth eparents) , together (asindicate db y the V. laevis data)wit h some additive component.Th e interaction cannotb e accounted forb y cytoplasmic

No of Isolates

M35 X 0 27 1 \ \ 48-

I W10 X G 36 15- Uli

10- Ln 5- A

=LT 1 n

15 M35 X 0 27 I I I

10- G 36 ruv I I I 5-

q (W\ 50 100 50 100 Pathogenicity (% defoliation) Figure 4. Pathogenicity ofF ,progen y from crossesbetwee n theNA Naggress ­ ive andnon-aggressiv e strains ofC . ulmi. A, B, progeny sample ofnon - aggressive x aggressive crossM3 5x 02 7o n 5-year-old clonal U. procera and 3-year-old seedling V. laevis respectively. The inoculationswer e carried outi n197 3 anddat a are for 10week s after inoculation.C , D, progeny sampleo fnon-aggressiv e xaggressiv ecros sW1 0x G3 6 on3-year-ol d clonal U. procera and 3-year-old seedling V. glabra respectively. The inoculations were carried outi n1974 ,an ddat a are for1 2week s after inoculation. Therewer e 3replicat e treespe r isolate.Th eheav ybar s represent the progeny means and the arrows thepathogenicit y levels ofth eparent s and theparen tmid-poin tvalues .

233 effects:reciproca l aggressivex non-aggressiv e crosses giveth e samere ­ sults (seeBrasie r &Gibbs ,1976 ;Brasier , 1977). The following explanation ispropose d forth enegativ e interaction: -Th e genetic systems governingpathogenicit y inth eaggressiv e andnon - aggressive strains arecomplex ,polygenic , andqualitativel y different. -Th ehig h levelo fpathogenicit y inth e aggressive strain isconferre d by special genecombination s which onhybridizatio n with the non-aggressive strain aredispersed , resulting ina larg enumbe r ofweakl y pathogenic genotypes (Fig.4) . - Iti s suggested thatth e special genecombination s may involve genese ­ quences acting as functional units,suc h asa serie s ofstructura l genes governedb y anoperato r gene.Thu s thephysiolog y ofth e host-parasite relationship inthi svascula rwil tdiseas e ishighl y complex, and several highly specialised enzyme systems arelikel y tob e involved.Thei r control byoperon-typ egeneti c systems is a'distinc tpossibility . Substitution of anoperato r allele inth e aggressive strainwit h theequivalen t locus in thenon-aggressiv e strainmigh t lead tohal fo fth eprogen ybein gnon ­ functional atth e operon concerned. Substitution of alleles atth estruc ­ tural locimigh t also impair theefficienc y ofth emetaboli c pathwayin ­ volved (seeBrasier , 1977).

CONCLUSIONS

To summarize,i ti sclea r thatther e areextensiv e genetic differences betweenth e aggressive andnon-aggressiv e strains of C. ulmi, These are exemplified firstlyb y their differingpropertie s ofgrowth-rate ,thei r differing colonymorphologies ,an d inthei r remarkably differenttempera ­ ture-growth*response s. Secondly ,the y areshow ni nthei rdifferen tmatin g systems, and inth e fertilitybarrie r thatoperate sbetwee n them.Thirdly , they arehighlighte d by the complexpattern s of inheritance thatoccu r among theirprogeny , andb yth e facttha t although thousands ofwil d iso­ lates of C. ulmi havebee nexamined ,n o indication ofhybridizatio n between the 2strain s innatur e hasbee n found. Many ofthes e factorspoin tt o aconsiderabl e degree ofgeneti c iso­ lationo revolutionar y divergence. Indeed,w e appear tohav eher e 2dis ­ cretevirtuall y non-hybridizing populations which approximate toth e level ofsub-species .Whethe r the 2strain shav e evolved as aconsequenc e ofsym - patric or allopatric evolutionha sbee ninvestigated . Experimental evidence points to asympatri c divergence,wit h the aggressive strainhavin g evolved fromth enon-aggressiv e strain (Brasier,i npreparation) . Turning toth e 2race s ofth e aggressive strain,the yhav e anumbe ro f features incommo n indicating common ancestry,bu t iti s alsotru e that ever greater genetic differences arebein g foundbetwee n them asshow ni n thepathogenicit y datapresente d here.Th epresen t status ofth eEA Nag -

234 gressive as a race rather than a separate strain could readily be called into question as mentioned above. Further light may be thrown on this prob­ lem when crosses between the EAN and NAN races, now being carried out, have been analysed in detail. Thus it is clear that there are 3 distinct genetic groupings within the wild population of C. ulmi. With the geographical centre of origin of Dutch elm disease still to be determined, the possible existence of further dis­ crete pathogenic forms of the fungus cannot be ruled out. This possibility represents a continuing threat to the products of our expensive elm breed­ ing programmes. Such a threat can only be alleviated by further detailed mycological surveys, in particular in the several remaining uncharted areas of elm diversity such as the Himalayas and parts of China.

REFERENCES

Ainsworth, G.C., 1961. Dictionary of the fungi. Commonwealth Mycological Institute, London. 547 p. Brasier, CM., 1977. Inheritance of pathogenicity and cultural characters in Ceratocystis ulmi: hybridization of protoperithecial and non-aggressive strains. Transactions of the British Mycological Society 68: 45-52. / Brasier, CM., 1978. Mites and reproduction in Ceratocystis ulmi and other fungi. Transactions of the British Mycological Society 70: 81-89. Brasier, CM., 1979. Dual origin of recent Dutch elm disease outbreaks in Europe. Nature 281: 78-80. Brasier, CM. &F . Afsharpour, 1979. The aggressive and non-aggressive strains of Ceratocystis ulmi in Iran. European Journal of Forest Pathology 9: 113-122. Brasier, CM. &J.N . Gibbs, 1975a. A highly fertile form of the aggressive strain of Ceratocystis ulmi. Nature 257: 128-131. Brasier, CM. &J.N . Gibbs, 1975b. Variation in Ceratocystis ulmi. Significance of the aggressive and non-aggressive strains. In: D.A. Burdekin &H.M . Heybroek (Eds): Dutch elm disease. Proceedings of IUFRO Conference, Minneapolis - St. Paul, 1973.USD A Northeastern Forest Experiment Station, Upper Darby, Pennsylvania, p. 53-66. Brasier, CM. &J.N . Gibbs, 1976. Hybridization of the aggressive and non-aggressive strains of Ceratocystis ulmi. Annals of Applied Biology 83: 31-37. Brasier, CM., J. Lea &M.K . Rawlings, 1981. Aggressive and non-aggressive strains of Ceratocystis ulmi have different temperature optima for growth. Transactions of the British Mycological Society 76: 213-218. Gibbs, J.N. &CM . Brasier, 1973. Correlation between cultural characters and pathogenicity in Ceratocystis ulmi isolates from Britain, Europe and North America. Nature 241: 381-383. Gibbs, J.N., D.R. Houston &E.B . Smalley, 1979. Aggressive and non-aggressive strains of Ceratocystis ulmi in North America. Phytopathology 69: 1215-1219. Heybroek, H.M., 1976. Chapters on the genetic improvement of elms. In: Better trees for metropolitan landscapes. USDA Forest Service General Technical Report NE22 . p. 203-214. Kryukova, E.A., 1972. The Dutch elm disease pathogen of elms in the droughty zone of South East European Russia. In: Protection of Forests against Pests and Diseases. Kolos, Moscow, p. 153-161. Stebbins, G.L., 1966. Processes of organic evolution. Prentice Hall, Englewood Cliffs, New Jersey. 179 p.

235 Pathogenic variability within the genus Cronartium

H.R. Powers, Jr.

ChiefPlan tPathologist ,Southeaster nFores tExperimen tStation ,USDA ,Forestr yScience s Laboratory,Athens ,Georgi a 30602,US A

ABSTRACT

Inth egenu s Cronartium, eachpathoge ntha tha sbee n investigated has shown ahig h degree ofpathogeni cvariabil ­ ityo nbot hpin ehost s and alternate host.Thus , constant monitoring ofth epathoge nwil lb erequire d todetec tshift s inpathogenicity , andt odetermin e themos teffectiv e placement ofresistan tmaterials . Inaddition , anextremel y broad genetic base ofresistanc e willb erequire d ineac ho fth ehos t species iftre e rusts aret ob e controlled by the development ofresis ­ tantstrain s ofpines .

INTRODUCTION

Therus t fungihav eearne d areputatio n forvariability . Thischarac ­ teristicwa s firstdemonstrate d inrust so fcereals ,particularl y theblac k stem rustosganis m (Puccinia graminis Pers.)whic hha s several hundred physiologic races.Researc h onpathogeni c variability ofrus t fungi on foresttree sbega nmuc h later.On e reasoni stha tth euredial ,o r repeating stage,o fcerea l rusts occurs onth eeconomicall y importanthost .Wit hth e treerusts ,thi s stage isusuall y onth e less importanthosts .

WHITE PINE BLISTER RUST

Whitepin ebliste r rust,cause db y Cronartium ribicola J.C. Fisch,e x Rabenh.,wa s the firsttre e rustt ob e closely studied.Widesprea d inAsi a andEurope , itwa s introduced into theUnite d States early inthi s century. The first5 0year s ofresearc h onwhit epin ebliste r rustyielde d little evidence ofpathogeni c strains forthi s fungus.Th e fungus attacksman y softpines ,rangin g from thever y susceptible sugar (Pinus lambertiana Dougl.)an dwester nwhit e (P. monticola Dougl.)pine s toth ehighl yresis ­ tant Swiss stonepin e (P. cembra L.)o fEurope .Al l species of Ribes can serve asalternat e hosts,bu tthe y alsovar ywidel y in susceptibility to

236 infection.Mielk ee tal . (1937)showe dvariatio nbot hbetwee n andwithi n 4 species of Ribes. Somehighl y resistant individuals were found inth egen ­ erally susceptible R. petiolare Dougl.Anderso n& French (1955)reporte d variation inrespons e toinfectio no n R. hirtellum Michx.whe n collections of C. ribicola from 3differen tpin e specieswer euse d asinocula . Yokota et al. (1975)reporte d afurthe r step invariability— ,a straino fth eorganis m fromJapa ntha twa s able toutiliz ebot h Pedicularis and Ribes asalternat e hosts.Subsequently , Yokota &Uozum i (1976)mad ea tentative revision of Cronartium rusts onwhit epin e (P. strobus L.)i n Japan: C. ribicola f.sp . ribicola on P. strobus and Ribes sp.; C. ribicola f. sp. pedicularis on P. strobus and P. pumila, withbot h Pedicularis and Ribes as alternate hosts. InCanada ,Hiratsuk a &Maruyam a (1976)foun da straino f C. ribicola thatwa s able toinfec tbot h Ribes and Castilleja miniata Douglas.Thi swa s the firstrepor to f C. ribicola infecting a scrophulariaceous host inNort hAmerica . Inaddition , Saho& Takahash i (1976)reporte d a Peridermium species on P. pumila Regel inJapa nwhic h theyconside r tob e anautoeciou s form of C. ribicola. That is,a pine-to-pin e rustwit h anintermediat e hostno t being required tocomplet e thelif e cycle.Thi swoul db epathogeni cvari ­ ability ofth e firstmagnitude . Some ofth emos textensiv ewor k indicatingvariatio nwithi n C. ribicola hasbee ndon e onwester nwhit epin eb yMcDonal d &Hof f (1975). Theyhav e shownextensiv evariatio n inpigmentatio n of spots developing on inoculated pine seedlings.Thei rresult s demonstrate theexistenc e of4 seedling types: thosewit h only red spots,thos ewit h onlyyello w spots,thos ewit h both red andyello w spots,an d thosewit hn o spots.The yhypothesiz e that these reactions indicate raceso f C. ribicola, andtha tth epin ehost s are differentiallyresistant .Further ,the y feel resistance tothes e races is simply inherited. Seedlingswit hre dneedl e spots areresistan t to therus t shown toproduc eyello w spots.Conversely , the seedlingswit hyello w spots areresistan t toth e strainproducin g red spots.Seedling swit h spotso f both colors are,therefore , susceptible tobot h strains,an d seedlingswit h nospot s areresistan t tobot hstrains . Recently,McDonal d &Andrew s (1980)foun dhighl y significantvariatio n indevelopmen t ofth epathoge no n Ribes. They found differences indevelop ­ mento furedia l infections after single aeciospore inoculations onlea f disks, and inth epercen t ofthes e infections thatproduce d teliospores within 35 days.Thes evariation s could influencepathogeni c capabilities of the fungus. Two recentinvestigation s provide some ofth e strongest evidence found todat e onpathogeni c variability ofC . ribicola onpine .Kinloc h & Comstock (1981)foun d thatsuga rpine swit h singlemajo r generesistanc e to blister rust suddenlybecam e susceptible to infection.Thes etree sha dbee n free ofdiseas e under extremely hazardous conditions foru p to1 4year s in

237 fieldprogen y tests.Thei r sudden susceptibility demonstrates ane w raceo f C. ribicola thatovercome s thisgen e forresistanc e insuga rpine .Thes e authors indicate thatnecroti c flecks on inoculated seedlings indicate resistancewhil e yellow oroccasiona l red lesions indicate susceptibility. G.I. McDonald (personal communication)als oha s found anew ,mor evirulen t straino f C. ribicola on P. monticola. Compared to standard isolates ofC . ribicola, thene w strain (ChampionMine )mor ereadil y infected previously tested seedlings fromon e specific areao fth ePacifi cNorthwest .

HARD PINE RUSTS

Red andyello w needle spots also occur in Italy onseedling s of ahar d pine (P. pinaster Ait.)inoculate d with C. flaccidum (Alb.e tSchw. )Wint . These spots indicate 2race so fth epathoge n anddifferentia l resistance of maritimepin e seedlings to these 2race s (Raddi, 1976). The host-parasite interaction, inthi s case on ahar dpine ,closel y resembles thato f soft pineswit h C. ribicola. Studies onpathogeni c variability ofhar dpin e rusts inth eUnite d Stateshav eprimaril y dealtwit h the C. quercuum complex.Kai s& Snow (1972)an dPower s (1972)indicate d sharp differential responses between jack (P. banksiana Lamb.)an dVirgini a (P. virginiana Mill.)pine swhe n inoculated with cultures of C. quercuum derived from each species.Eac h pine species was susceptible to its isolate ofth eorganism ,bu t resistant toth e isolate from the other species.Thi swor k demonstrates arelation ­ ship forthi shost-parasit e complex comparable totha twit h thewhea trust . That is,th e isolates ofC . quercuum from agive npin e specieswer e similar toth e formae speciales onth evariou s cereals.Thes e relationships within the C. quercuum complexwer e clarified subsequently byBurdsal l &Sno w (1977). Four formae speciales (banksianae, virginianae, echinatae, and fusiforme) were established toaccommodat e the rusto njack ,Virginia , shortleaf (P. echinata Mill.), and thecombinatio n of loblolly (P. taeda L.) and slash (P. elliottii var. elliottii Engelm.)pines . Snow& Kai s (1970)provide d evidence ofpathogeni cvariabilit y within the fusiform rustorganis m (C. q. f. sp. fusiforme). They founddifferen ­ tial reactionsbetwee n resistant slashpin eprogenie s and rust isolates from 5states .Later ,Sno w et al. (1975)foun d asignifican t inoculum sourcex family interaction amongrus t isolates collected ona east-wes t transect (Florida-Texas)an d2 north-sout h transects (Mississippi and Florida-Georgia).Amon g 8individua l galls and3 resistan tslas hpin e families asignifican t inoculum x family interactionwa s also found, aswa s substantial pathogenic variability among collections from individualgalls . An absence ofactiv egall so n1 resistan t familywhe n inoculatedwit h 2o f the rust isolateswa s the firstobservatio n ofa nimmun ereactio nbetwee n thispathoge n and slashpine .I naddition ,2 o fth e 10 familieswer eresis -

238 tantt o allo fth e inocula from 5states .Th e authors hypothesized that thisphenomeno nmigh tb ecomparabl e tohorizonta l resistance invariou s agronomic crops. Pathogenic variability of C. quercuum f.sp . fusiforme onlobloll ypin e was studiedb y extensively sampling thepathoge n across the SouthernUnite d States (Powers et al., 1978). Isolates from 56 individual rustgall s from7 states from Louisiana throughNort h Carolinawer e used to inoculate3 loblollypin e families rated asresistant , intermediate, and susceptible to fusiform rust.Th e results ofthes e inoculations showed highlysignifi ­ cantvariabilit y atever y level examined.Th eproportio n of seedlingsin ­ fectedwa s influenced by thehos t family,th e state inwhic h therus twa s collected, interactions betweenhos t family and stateo frus tcollection , the individual rustcollectio nwithi n states,an d interactionsbetwee nhos t families and individual collections within states.Severa l interactions between specific rustcollection s within states and families demonstrate thenee d to labelhighl yvirulen t strains andmak e treebreeder s aware of the facttha tthes e isolates cancaus eheav y damage on someo fth emos t resistant clones or selections available.Extensiv eplantin gbase d ononl y 1 sourceo fresistanc e could lead to arapi dbuild-u p of suchvirulen t strains ofrust .Fo r example,1 o fth e isolates from SouthCarolin awa s highlyvirulen t onresistan t family 11-20,whic hwa s selected inSout h Carolina. Infact ,i tproduce d ahighe rpercentag e of infectiono nth e resistant family thano nth e susceptible family.Thi s study alsoproduce d evidence ofth eclassi c reversal of infection typesbetwee n families and rustisolates ,simila r todifferentia l reactions onwhea thos t lines that result inth eclassificatio n ofphysiologica l raceso fth ewhea tste m rust fungus. Inbot h slash and loblollypine ,difference s among infection levels causedb y individual rustcollection s fromwithi n astat ewer e sometimes greater thanth edifference s amongcollection s fromdifferen t states.Thus , abroa d geneticbas e ofresistanc e isneede d forplanting , andtest s for resistancemus tinclud e rangeo frus t isolates from different geographic areas. Arecen t study included 7lobloll ypin e families rated asresistan tt o intermediate inresistance ,2 susceptibl e families,an d 5rus t isolates (Powers& Matthews , 1979). Again,ther ewer ehighl y significant differences inresistanc e amonghos t families andi ninfectio n levelsproduce d byth e various rust isolates.Al l resistant families had relatively low levels of infection,bu tonl y 1wa s resistant toal l rust isolates.Highl y virulent rustisolate s causedparticularl y heavy infectiono npin e familiesorig ­ inating inth e same geographic area.Th e SouthCarolin a rust collection againcause d heavy infection on family 11-20. Families fromTexa s and Louisianawer emos t susceptible torus tisolate s fromthei row nstates . Snowe t al. (1976)demonstrate d increasingvirulenc e ofrus t isolates developing onresistan tpin e selections.The y found astrikin g increase in

239 virulenceproduce d by collections of C. quercuum f. sp. fusiforme gathered from one specific resistant slashpin e family.Becaus e such increases chal­ lengeprogram s ofselectin g andbreedin g forresistanc e toth erus tpatho ­ gen, additional studieswit h loblollypin ewer e carried out (Powers et al., 1977). Theyuse d 3 families:a resistan t family fromwhic h the rustcollec ­ tionswer e obtained, anadditional ,non-relate d resistant family, anda susceptible check. Collections of individual rustgall s from fieldplant ­ ings ofth e resistant familywer ecompare d withgall s representing thegen ­ eral rustpopulation s fromnearb y trees.Th e rust isolates from theresis ­ tant familyproduce d 9% mor e infection onseedling s oftha t family than did isolates from thegenera lpopulation .Thi s increase-mayb e biologically significant,eve nthoug h itwa sno t statistically significant.Ther ewa sn o increase invirulenc e onth eunrelate d rustresistan t family.Th erelative ­ lymodes t increase invirulenc e ofthes e family-specific rustisolate s andmaintenanc e of resistance to all isolatesb y theunrelate d resistant loblolly familywer eencouraging . Todetermin e ifa tren d for increasing virulence hasbee n developing inrecen tyears ,isolate s ofth e fusiform rustorganis m originating inap ­ proximately 1945wer e comparedwit h those originating around 1970 (Powers & Dwinell, 1978). No differences invirulenc ewer e foundbetwee n the2 groups,bu tth emor e recent isolateswer emuc hmor evariable . Pathogenic variability has alsobee n found among 10 single-spore iso­ lates originating from 1isolate d rustgal l (Powers, 1980). Three half-sib families of loblollypin epreviousl y determined tob e resistant,inter ­ mediate, and susceptible to fusiform rustwer e inoculated with eacho fth e isolates. Isolates differed significantly inpercentag e of seedlingsin ­ fected; therange ,4 2% to5 6% ,wa s similar totha tproduce d by unrelated gallswithi n anindividua l county orplantation . Inpopulation s ofslas h and loblollypines ,resistanc e to C. quercuum f. sp. fusiforme hasbee n found insingle ,rando m trees. Inlobloll ypin e there are also specific geographic zones,primaril y around theperipher y of itsnatura l range,wher ebulk.collection s ofsee d aremoderatel y resistant (Wells& Wakeley , 1966). To compare seeds from 6o fthes e geographic areas and rustcollection s from eacharea ,a stud ywa s designed tocross-inocu ­ late seedlings from each seed sourcewit h eachrus tcollectio n (Powers & Matthews, 1980). Seed andrus tspore swer e collected fromArkansas ,eas t Texas; Livingstonparish , Louisiana;th eeaster n shore ofMaryland ; and Marion county,Florida . Seeds fromcentra l Georgiawer e included asa susceptible control.Th eMarylan d seed sourcewa s significantly morere ­ sistanttha nan yothe r source.Virulenc e ofrus tisolate svarie db y state ofcollection ,bu tth erang e inpercentag e of seedlings infected (<1 0% ) was too small tob ebiologicall y meaningful. Interactionsbetwee n seed sources and rustcollection s from different geographic areaswer esignifi ­ cant.Th e range ofresponse swa s from 48% infection onth eArkansa s seed

240 Table 1. Incidence of infection on loblollypin e seedlings from 6geo ­ graphic sources after inoculationwit h rustcollection s from each geo­ graphic area.

Seed sources Seedlings (%) with galls 9mo .afte r inocu- Hostmea n lationwit h spores from:

Ga. Md. Ark. Fla. Tex. La.

Maryland 53 69 49 52 56 55 56a Arkansas 54 48 72 61 68 61 61b Florida 69 74 49 76 61 73 67c Texas 58 53 80 68 84 83 71c Louisiana 78 73 81 81 81 82 79d Georgia control 88 89 81 82 80 87 84e

Mean 66a 68ab 69abc 70abc 72bc 73c

1. Infection percentages followed by the same letter do not differ signifi cantly (P -è 0.01 for host means and P s 0.05 for spore means) as determine by Duncan's multiple range test.

sources produced by the Maryland rust, to 89 % infection on the Georgia check lot by the Maryland rust. In all cases except the control, the re­ sulting infection level on a specific seed source was highest when inocu­ lated with the rust isolates from the same geographic area (Table 1). This and earlier results indicate that genes for virulence tend to develop where the complementary genes for resistance are found, and that while this viru­ lence is relatively common in these areas, such genes are not common throughout the ranges of the host and its parasite.

REFERENCES

Anderson, R.L. &D.W . French, 1955. Evidence of races of Cronartium ribicola on Ribes. Forest Science 1: 38-39. Burdsall, H.H., Jr. &G.A . Snow, 1977. Taxonomy of Cronartium quercuum and C. fusiforme. Mycologia 69: 503-508. Hiratsuka, Y. &P.J . Maruyama, 1976. Castilleja miniata, a new alternate host of Cronartium ribicola. Plant Disease Reporter 60: 241. Kais, A.G. &G.A . Snow, 1972. Host response of pines to various isolates of Cronartium quercuum and Cronartium fusiforme. In: R.T. Bingham, R.J. Hoff &G.I . McDonald (Eds): Biology of Rust Resistance in Forest Trees, USDA Miscellaneous Publication No. 1221. p. 495-503. Kinloch, B.B. &M . Comstock, 1981. Virulent race of Cronartium ribicola to major gene resistance in sugar pine. Plant Disease 65: 604-605. McDonald, G.I. &D.S . Andrews, 1980. Influence of temperature and spore stage on production of teliospores by single aeciospore lines of Cronartium ribicola. USDA

241 1 ForestServic eResearc hPape r256 .9 p . McDonald,G.I .& R.J .Hoff ,1975 .Resistanc e toCronartiu mribicol ai nPinu smonticola : ananalysi so fneedle-spo ttype san dfrequencies .Canadia nJourna lo fBotan y 53:2497-2505 . Mielke,J.L. ,T.W .Child s& H.G .Lachmund ,1937 .Susceptibilit y toCronartiu m ribicolao f thefou rprincipa lRibe sspecie sfoun dwithi nth ecommercia lrang eo fPinu s monticola.Journa lo fAgricultur eResearc h55 :317-346 . Powers,H.R. ,Jr. ,1972 .Testin gfo rpathogeni cvariabilit ywithi nCronartiu mfusiform e andC .quercuum .p .505-509 .In :R.T .Bingham ,R.J .Hof f& G.I .McDonal d (Eds): Biologyo frus tresistanc e infores ttrees .USD AFores tServic eMiscellaneou s PublicationNo .1221 . Powers,H.R. ,Jr. ,1980 .Pathogeni cvariatio namon gsingle-aeciospor e isolateso f Cronartiumquercuu m f.sp .fusiforme .Fores tScienc e26 :280-282 . Powers,H.R. ,Jr .& L.D .Dwinell ,1978 .Virulenc eo fCronartiu m fusiformestabl eafte r 25years .Plan tDiseas eReporte r62 :877-879 . Powers,H.R. ,Jr .& F.R .Matthews ,1979 .Interaction sbetwee nvirulen tisolate so f Cronartiumquercuu m f.sp .fusiform ean dlobloll ypin efamilie so fvaryin gresis ­ tance.Phytopatholog y69 :720-722 . Powers,H.R. ,Jr .& F.R .Matthews ,1980 .Compariso no fsi xgeographi csource so flobloll y pinefo rfusifor m rustresistance .Phytopatholog y 70:1141-1143 . Powers,H.R. ,Jr. ,F.R .Matthew s& L.D .Dwinell ,1977 .Evaluatio no fpathogeni c variability ofCronartiu m fusiformeo nlobloll ypin ei nth eSouther nUSA . Phytopathology67 :1403-1407 . Powers,H.R. ,Jr. ,F.R .Matthew s& L.D .Dwinell ,1978 .Th epotentia lfo rincrease d virulenceo fCronartiu m fusiformeo nresistan tlobloll ypine .Phytopatholog y 68:808-810 . Raddi,P. ,1976 .Indagin esu itip id imacchi e fogliarie su llor osignificat oi n discendenzed iPinu spinaste rinfett ed aCronartiu mïlaccidum .Rivist ad i PatologiaVégétal e12 :91-98 . Saho,H .& I .Takahashi ,1976 .A preliminar y reporto na Peridermiu m speciesfoun do n Pinuspumil aRege li nJapan .Europea nJourna lo fFores tPatholog y6 :187-191 . Snow,G.A. ,R.J .Dinu s& A.G .Kais ,1975 .Variatio ni npathogenicit y ofdivers esource s ofCronartiu m fusiformeo nselecte dslas hpin efamilies .Phytopatholog y 65:170-175 . Snow,G.A. ,R.J .Dinu s& C.H .Walkinshaw ,1976 .Increas ei nvirulenc eo fCronartiu m fusiformeo nresistan tslas hpine .Phytopatholog y66 :511-513 . Snow,G.A .& A.G .Kais ,1970 .Pathogeni cvariabilit y inisolate so fCronartiu mfusiform e fromfiv esouther nstates .Phytopatholog y60 :1730-1731 . Wells,0.0 .& P.C .Wakeley ,1966 .Geographi cvariatio n insurvival ,growth ,an d fusiform-rustinfectio no fplante dlobloll ypine .Fores tScienc eMonograph sII .4 0p . Yakota,S .& T .Uozumi ,1976 .Ne wdevelopment si nwhit epin ebliste rrust si nJapan .XV I IUFROWorl dCongress .Proceeding sDivisio nII .Oslo ,Norway ,p .330-337 . Yokota,S. ,T .Uozumi ,K .End o& S .Matsuzaki ,1975 .A Cronartiu m rusto fstrob epin ei n easternHokkaido ,Japan .Plan tDiseas eReporte r59 :419-422 .

242 Relative virulence of Cronartium quercuum f. sp. fusiforme on loblolly pine from Livingston parish

G.A. Snow,W.L .Nanc e& E.B . Snyder

USDAFores tService ,Forestr yScience sLaboratory ,Gulfport ,Mississippi ,US A

ABSTRACT

Seedlings of4 3controlled-pollinate d pine familieswer e inoculated with 5source s of Cronartium quercuum f.sp . fusiforme. The seedlingswer egrow n fromsee dproduce d ina diallel crossing experimentwit h 10lobloll ypine s in LivingstonParish .Dat awer e assessed ast opercentag e oftree s with galls and the form ofgall s oninfecte d trees.Difference s inpin e families,inocula ,an d apin e familyx inocula interac­ tionwer e identified withbot hvariables .Th emos tprecis esep ­ aration ofinocul awa s achievedwit h gall-form. Thevariabl e response of full-sibpin e families todivers e inoculaempha ­ sizes thenee d toevaluat epotentia l breeding stockwit hsever ­ aldifferen t rust inocula.Progen y from someparen ttree swer e relatively stable toth evaryin g sources of inocula.Suc htree s mayb evaluabl e astester s in futurebreedin gprograms .

INTRODUCTION

Resistance to fusiform rust (Cronartium quercuum (Berk.)Miyab ee x Shirai f. sp. fusiforme) hasbee n found incertai n geographic seed sources oflobloll y (Pinus taeda L.)pin e (Wells& Wakeley , 1966;Well s &Switzer , 1971). Since large quantities ofthi s seed isreadil y available,i tha s beenuse d extensively. LivingstonParish ,La. ,i sa nimportan t source for loblolly seed.Tree s grown from this seedperfor mwel l ingrowt h andresis ­ tance to fusiform rust.Moreover ,th e seed hasperforme d well throughout much ofth e southernpin e region.A s aresult ,th eLivingsto nParis h area will likely remain important as asourc e ofresistan t trees forsee dor ­ chards. Powers et al. (1977)recentl y reported thatLivingsto nParis h (LP) seedlings grown frombul k seed collections varied insusceptibilit y to different sources ofrus t inocula.A tleas t 1o f1 0rus tcollection s from eacho f7 state swa s relativelyvirulen to nbul k stock. Inocula from

243 Mississippi were generallymor evirulen ttha ninocul a from otherstates . Insubsequen t tests,difference s invirulenc e ofth epathoge nwer edemon ­ stratedo na half-si b LP family (Powers& Matthews , 1979)^.Thes e findings indicate anee dt o evaluatepotentia l breeding stock from the Livingston Parish areawit h an array ofdifferen t inocula toidentif y andmaintai n adequate genetic resistance. This study seekst oevaluat e the interaction amongL Ppin e families withvaryin g levels ofresistanc e andrus tinocul awit hvaryin g levelso f virulence.Understandin g this interaction isnecessar y tomaximiz eresis ­ tanttrait s that are stablet oth edivers e rustpopulation .

METHODS

Hostmaterial :Forty-thre econtrol-pollinate d pine families from1 0 randomly selected loblolly pines inLivingsto nParish ,La. ,wer e tested. Thetree sha dbee n intercrossed inal l combinations without regardt o whichwa suse d asth emal e or femaleparen to f agive n cross.Th e result was ahal f diallelwit h4 5possibl e crosses.Tree s fromthi s experiment hadbee nplante d inreplicate d plots onth eHa'rriso nExperimenta l Forest inMississipp i in1968 .Extr a seedwa s available from allbu t2 o fth e crosses. Inthi s experiment,pin e seedlings from eachcros swer e growni n 2.5 cmx 12.7c mplasti c tubescontainin g a1: 1 mixture ofvermiculit e and peatmoss .The ywer etransplante d tonurser ybed s4- 6 weeks afterinocu ­ lation. Inocula:Fiv e different inocula of C.q. f. sp. fusiforme were used. Thesewer e chosen tob e diverse invirulenc e onth e stock.Eac h inoculum was derived from 1lobloll ypin e gall, the collectionpoint s for allinocu ­ lawer ewidel y separated, and2 inocul awer e from LPtrees .Th e inocula from LP treeswer e expected tob emor evirulen t thanth eothe r inocula (Snowe t al., 1975;Power s etal. , 1978). InoculumA wa s from atre egrow ­ ing inLivingsto nParish ; inoculaB ,C , andD wer e collected nearJasper , Texas; Kiln,Mississippi ; andBrewton ,Alabama ,respectively , andwer e taken fromtree sthough tt ob enativ et oth ecollectio npoints .Inoculu m E was collected from atre e inth edialle lplantin g onth eHarriso nExperi ­ mentalForest . Artificial inoculations:Te n5-week-ol d seedlings from eacho fth e4 3 pine familieswer e inoculated with eacho fth e inocula in 1978.Th e entire procedurewa srepeate d in197 9t oobtai n asecon d replication.Th e ordero f inoculation,wit h respectt obot h family and inoculum,wa s determinedran ­ domly toprovid e acomplet e factorial arrangement.A forced air inoculation system (Snow& Kais , 1972)wa suse d throughoutth e experimentwit hbasidio - spore densitymaintaine d at15-25/mm 2. Sixmonth s afterinoculation , the seedlingswer e scored forpresenc e of galls, gall length and diameter. Several derivedvariable swer e studied

244 graphically to identify thosemos trepeatabl e from replication torep ­ lication and thatbes t separated families and inocula.Th evariable sse ­ lectedwer epercentag e ofplant swit h galls andgall-for m (gall length/gall diameter).Gall-for m isa quantitativ emeasur e of shape;roun d gallspro ­ duce gall-form valuesnea r 1;elongated , or spindle-shaped gallsyiel d largervalues .T o interpret theresults ,i twa s assumed thatresistanc e of an individual pine family couldb eexpresse d as alo wpercentag e ofplant s with galls orb y lowgall-for m values.Lo wgall-for m valueswoul d indicate a reduction inlongitudina l growth ofth e fungus inth ehos ttissues . Analysis ofvarianc ewa suse d totes t for significance of differences among families and inocula and forth e familyx inocula interaction. Significance was appraised atth e 0.01 level.

RESULTS

Analyses ofpercentag e ofplant s galled and gall-form showedsignifi ­ cantdifference s amongbot h inocula andpin e families,an d significantpin e family x inoculainteractions :

Df Fvalue s

Percentgalle d Gall form

Replication 1 11.24 12.31 Inocula 4 3.75 21.69 Pine family 42 8.12 7.75 Inoculax fan n iy 168 1.70 1.54 Error 214 Total 429

TheF value s forpin e familyan dth e inoculab ypin e family interaction were slightly higher forpercen t galled thangall-form . However,th eF value for inoculawa smuc h larger forgall-form , indicating thatmor epre ­ cision inseparatio n of inoculawa s achieved withth egall-for mvariable . This resulted from (1)les sreplication-to-replicatio n variation inth e gall-form datatha noccurre d inth epercentag e data and (2)th e uniformly highpercentag e levels obtainedwit hmos to fth e familyx inoculacombina ­ tions. Moretha n 70percen t ofth etree s developed galls in4 5o fth e 50half - sib family x inoculum combinations (Fig.1) .Familie s 3, 5,an d 7ha dth e lowestpercentage s and families 3an d 7showe d themos tvariatio nwit hin ­ ocula source.Familie s 4, 5,8 , and 9showe dmuc h lessvariatio nwit hin ­ ocula than theothe r families. InoculumA ranked firsto r second highest on 5o f 6mor evariabl e families (nos.1 ,3 , 6, 7, and 10). The rankings

245 PERCENTGALLED

•A .A .E 90 •D - 5c A : .D .c •ÔC •D •B .B E •E •B C 'D •B .D •A •B •E •E •C 80 ~ E •C •E •E : c •A •C .A .A *S> •D •B 70 •D

INOCULUM SOURCES 60 A- LIVINGSTON,LA. • B B-JASPER.TEX. • B C-KILN,MISS. •C D-BREWTON,ALA. 50 E-SAUCIER,MISS .

•D /in i i , i i 1 1 . i 10 5 3 PINE FAMILY

Figure 1. Percentage oftree swit h galls for 10half-si b pine families6 months after exposure to 5differen t rust inocula.Th ehalf-si b family values are anaverag e of8 o r9 full-si b familieswit h acommo nparent ; familynumber s correspond with thecommo nparen tnumbers .Familie s are ranked indecreasin g ordero fsusceptibility . Eachentr y represents the meanpercentag e infection foral ltree s ina particula r familyx inocula combination.

ofth e other inocula changed onthes epin e families andnon eranke dcon ­ sistentlyhig h orlow . Rankings ofth e 5inocul awer emuc hmor e consistentwit h regardt o gall-formvalue s (Fig.2) .Inoculu mA ranked firsto rsecon d highesto nal l half-sib families. InoculumE also ranked highwit h theexceptio n of family 10. InoculaB andC ranke d lowesto nmos t families,indicatin g thatgall s causedb y these inoculawer e generally rounder thangall scause db y inocula A,E , andp . Gall-formwa s generally related topercen t galled intha t families3 , 5,an d 7wit hth e lowestpercentage s (Fig.1 )als oha d the lowestgall-for m values (Fig.2) .Conversely , families4 and 8,whic h ranked first andthir d forhighes tpercentages ,ha d the largest gall-form values.A noticeabl eex ­ ceptionwa s family 9whic hwa s second highest inpercen tgalle d and fourth lowest for gall-form.

246 GALL FORM 5

.A •A •D •E .A

•E •D .D .A .A •C •B •E •D •E •E •A .A •B •B •D •D •E •E •C •E .C,E .A •C •C •B •C •D •C •D •B - INOCULUM SOURCES •B A- -LIVINGSTON,LA . B- -JASPER.TEX. •B, D •C C-- K1LN.MI5S. D--BREWTON,ALA •B E- - SAUCIER,MISS . •B

• i i i i i , 8 10 2 16 9 3 7 5 PINEFAMIL Y Figure 2. Form (gall length/gall diameter)o fgall so n 10differen trus t inocula.Th ehalf-si b familyvalue s are anaverag e of8 o r 9full-si b familieswit h acommo nparent ; familynumber s correspond with the common parentnumbers .Familie s areranke d indecreasin g order foraverag e gall- formvalues .Eac hentr y represents themea ngall-for m for allgalle d trees ina particula r familyx inoculacombination .

The interactionbetwee n full-sib families and inoculawa s strong. Graphic analysis ofthi s interactionreveale d thatcrosse s fromparent s5 and8 showe d much lesstendenc y to interactwit h inoculum sourcetha nth e crosses from otherparents .Gall-for m values forth e full-sib crosseso f parent 5ar e shown inFig .3 .Cros s 7x5 ranked lowestwit h inoculaB ,D , andE ,an d intermediatewit h inoculaA andC ; families 8x5, and 4x5 were intermediate tohig h on all inocula. Incontrast ,th e rankingo f family 3x5 changed from lowt o intermediate tohigh ,dependin g onth e inocula employed. Similarchange s inran kwit h inoculaoccurre d forth e crosses ofparen t8 (Fig.4) .Famil y 9x8 was one ofth emos tvariabl e while 5x8 ranked uniformly low and4x 8 wasuniforml y highwit hmos t inocula.Th erang ebetwee n familieswit h the lowest andhighes t gall-form values oneac hinocul awa s greater forth e families ofparen t 8tha nth e families ofparen t5 .

247 GALL FORM 5|

•8 •4 «A •10 •1 •1 •6 •4 •7 "•3 •2 '3 .6 .1 •3 •8 .10 •6,9 •9 •7 -.10 •2 .2 .7 •7 •2,4 -»a •1,9 *6

•3 Figure 3. Form ofgall so n9 full-sib •7 pine familieswit hcommo nparen tNo .5 sixmonth s after inoculationwit h5 sources of e.g. f. sp. fusiforme.

.4 •4

•2 .10 • 4 •2 »10 • 9 .1 »2 _ •4 •3 .6 •3 .1 •9 Ï6 _». : 9 «9 •6 .1 . 5 .2,5 •1 »10 •10 4- -*• •6 •4 • 5 j 6,9 •10 .3 »3 »?

•5

•5 Figure 4. Form ofgall so n8 full-sib •3 pine familieswit hcommo nparen tNo .8 i i C B sixmonth s after inoculationwit h5 INOCULA sources of C.q. f. sp. fusiforme.

248 DISCUSSION

Infection levels of inoculated seedlingswer emuc hhighe r thanex ­ pected. Inth e fieldplantin g atth eHarriso nExperimenta l Forestonl y 5.2 % of allth etree sha dbranc h or stem galls atag e 10,whil e another seed source oflobloll y pine included inth eplantin g had 57% infection . Itappear s thatal l components ofth edialle l have ahig h level ofresis ­ tance.Ther e issom ereservatio n therefore aboutusin g thepercentag e data from the artificial inoculations tomak e interferences aboutth e fieldper ­ formance ofthes e trees.However ,th e resultswit h the gall-form dataar e encouraging and iti shope d thatmeaningfu l correlations canb emad ebe ­ tween field and greenhouse resultsusin g form data. Infection levelswer e too low inth e fieldplantin g tomak e these correlations.Th e diallelwa s re-established ina hig h rusthazar d area inJanuar y 1980,an dw ehop e sufficient infectionwil l occur formor evali d comparisons in2 o r3 years . Iti sno tknow nwhethe r themechanis m that limits longitudinal growth ofth erus t fungus inth ehos t isdifferen t from thatwhic h limits its establishment.Unti l thebiologica l relationship between thehos tresponse s isbette r understood,variable s thatquantif y bothevent s seem necessary to assess resistance.Th e gall-form variable appears tob e anadequat eex ­ pression oflongitudina l growth andha s atleas t3 advantage s overpercen ­ tagedata : (1)Dat a are from infectedplant s only andexperimenta l error associated with inoculationprocedure s isreduced ; (2)fewe rplant s are required toobtai n repeatable results,an d (3)ther e isn ouppe r limitt o gall-form as inpercentag e data.Th edisadvantag e ofgall-for m istha tin ­ fectedplant s thatd ono t develop galls areignored . Itwa sno t surprising to find thatinocul aA andE ,whic hwer e col­ lected from LPtrees ,wer eusuall y morevirulen t thanth e otherinocula . Although alarge r sampleo f inoculawoul db eneede d toestablis h thatL P stock selectvirulen t formso fth erus tpopulation ,th eresult s arei nlin e with those ofPower s &Matthew s (1980)wh o recently reported inocula from LivingstonParis h tend tob emor evirulen t onL P stocktha n inocula from other areas. The differences invirulenc e ofth e 5culture s of C.q. f. sp. fusiforme observed inthi s experiment aresimila r tothos e thathav ebee nobserve db y Powers et al. (1977), andPower s &Matthew s (1979)i nothe r experiments with loblolly pine.Us eo f full-sibpin e families andth e gall-form vari­ ablehav eprovide d adifferen t andperhap s closer look atth eeffect so f diverse inoculao npine s thanpreviou s researchha s indicated. With respect to selection andbreedin g rustresistan t trees,th ere ­ sults thus far are ingenera l agreementwit h otherexperiment s ofthi s nature (Powers et al., 1977;Sno we t al., 1975). That is,resistanc e inL P stock isrelativ e toth evirulenc e ofth epathoge n andeffort s shouldb e made toward finding resistance thati sstabl et o asman y forms ofth epath -

249 ogen as possible. The stability of progeny from 2 of the pine parents (8 and 5) is encouraging. These trees seem to impart stable response to vary­ ing sources of inocula when used in combinations with other parents in this study. Such parents could prove valuable as top-cross testers in breeding programs.

REFERENCES

Powers, H.R., Jr. &F.R . Matthews, 1979. Interactions between virulent isolates of Cronartium quercuum f. sp. fusiforme and loblolly pine families of varying resistance. Phytopathology 69: 720-722. Powers, H.R., Jr. &F.R . Matthews, 1980. Comparison of six geographic sources of loblolly pine for fusiform rust resistance. Phytopathology 70: 1141-1143. Powers, H.R., Jr., F.R. Matthews &L.D . Dwinell, 1977. Evaluation of pathogenic variability of Cronartium fusiforme on loblolly pine in the southern USA. Phytopathology 67: 1403-1407. Powers, H.R., Jr., F.R. Matthews &L.D . Dwinell, 1978. The potential for increased virulence of Cronartium fusiforme on resistant loblolly pine. Phytopathology 68: 808-810. Snow, G.A. &A.G . Kais, 1972. Technique for inoculating pine seedlings with Cronartium fusiforme. In: R.T. Bingham, R.J. Hoff &G.I . McDonald (Eds): Biology of rust resistance in forest trees. USDA Miscellaneous Publication 1221.p . 325-326. Snow, G.A., R.J. Dinus &A.G . Kais, 1975. Variation in pathogenicity of diverse sources of Cronartium fusiforme on selected slash pine families. Phytopathology 65: 170-175. Wells, 0.0. &P.C . Wakeley, 1966. Geographic variation in survival, growth, and fusiform- rust infection of planted loblolly pine. Forest Science Monograph II. 40 p. Wells, 0.0. &G.L . Switzer, 1971. Variation in rust resistance in Mississippi loblolly pine. Eleventh Southern Forest Tree Improvement Conference Proceedings, 1971. p. 25-30.

250 Variation in pathogenicity and virulence in Fomes annosus and Armillaria mellea

RobertD .Raab e

Departmento fPlan tPathology ,Universit yo fCalifornia ,Berkeley ,Californi a94720 ,US A

ABSTRACT

There arevaryin g reports regarding thevariabilit y in pathogenicity andvirulenc e of Fomes annosus. The facttha t there arereport s ofth e fungusdifferin g inthes echaracteris ­ tics suggests thatth e fungus is somewhatvariable . Itals o suggests thatthi s is anare awher emor e researchneed s tob e done. Reports thatcrosse shav ebee nmad e incultur e andtha t fruitingbodie shav ebee nproduce d incultur e should leadt o studies onth e inheritance ofman y characteristics ofth e fungus, including itspathogenicit y andvirulence . Incon ­ trast, Armillaria mellea isa nextremel yvariabl e fungus in many characteristics including itsgrowt h incultur emedia , pathogenicity andvirulence . Itals o isvariabl e inmatin g types and recentresearc hha s showntha tbase d onthi sphenom ­ enon, these are3 distinc t 'biologic species'i nFinlan d and 10i nth eUnite d States.Mating sbetwee n 5 fromEurop e andth e 10 fromth eUnite d Stateshav e shownther e tob e someinterfer - tilitybetwee n isolates from the2 continents .Th ecomplica ­ tionsresultin g from such 'biologic species' suggest additional researchneed s tob e done concerningvariou s aspects ofthi s fungus, andparticularl y inregar d topathogenicit y andviru ­ lence.Althoug h fruitingbodie shav ebee nproduce d inculture , a consistentmetho d forthi s isno t available.Whe n developed, itwil lb e ofgrea tvalu e inth e study ofman y inheritedchar ­ acteristics including those regarding pathogenicity andviru ­ lence.

INTRODUCTION

A review ofth e literature regarding Fomes annosus (Fr.)Cook e and Armillaria mellea (Vahle xFr. )Kumme r reveals anextensiv e amounto fre - .searchconcernin g these fungi andth e diseases resulting from infection

251 by them.Ver y little,however ,ha sbee npublishe d regarding the genetic variability ofthes e fungi,particularl y inregard.t opathogenicit y and virulence.On ecoul d speculate ast oth ereason s forthis, .Inoculation so f seedlings areeas ybu tinoculation s of larger trees aremor edifficult . Thismigh tb e complicated by the facttha ttree so fgive n speciesma yvar y insusceptibilit y dependingupo nth e agewhe nexpose d toth epathogen .Fo r example,peac h (Prunus persica Batsch)i ssusceptibl e atan yag ewhe nex ­ posed toA . mellea. Incontrast , Pinus radiata D.Do n isver y susceptible as ayoun gplan t andca nb ekille db yth e fungus ina littl e over amont h ininoculation s in aglasshous e (Raabe, 1967). Init snativ e areao f coastal California,thi splan ti srarel y infected andmor e rarely killed byth e fungus.Whe nteste d forsusceptibilit y ina nope nplot ,i twa s found tob ever y resistant (Raabe, 1979). Another reasonmigh tb e that knowledge ofth ebiolog y andgenetica l constitution ofpathogen s frequently iscorrelate dwit hprogram s forbreedin g or selecting forresistance .Suc h researchprogram s involve longter m researchproject swit h fewreward sre ­ garding researchpublication s andtherefor e researchers are reluctant tob e involved.On eothe r reason istha tth e difficulty ofproducin g basidiocarps ofthes e fungiunde rcontrolle d conditions is'adeterren tt oth e studyo f anygeneti cvariabilit y ofth eorganisms . Therema yb e other reasons forth e lacko fresearc h inthi s areabu t thepurpos e ofthi spape r ist obrin g together the information available regardingvariabilit y inthes e fungi.T od othi smos tefficiently , each funguswil lb ediscusse d separatelyunles s informationma y applyt obot h fungi. Information concerning generalvariabilit y willb e included though themai nemphasi swil lb evariabilit y inpathogenicit y andvirulence . Pathogenicity here,refer s toth e ability ofa norganis m toproduc ea disease.Virulence , asuse dhere ,refer st oth edisease-producin g ability of anorganis m asmeasure db yth e severity ofth edisease .

FOMES ANNOSUS

Although foundmostl y intemperat e zones throughout theworld , F. annosus generally isno tconsidere d tob e anexceedingl y variable fungus. Roll-Hansen (1940)note d the absence ofdistinc tstrain s ineaster nan d westernNorway .Dimitr i (1974)reporte dmino r differences inth evirulence s ofdifferen trace s ofth e fungus.Kuhlma n (1969c)use d 8isolate s ofth e fungus from 2host s from southernUnite d States to inoculate seedlings of Pinus taeda L. in aglasshous e and foundn ovariation s invirulence .Lan e& Witcher (1974)use d 19isolate s to inoculatepin e seedlings andthe yvarie d onlyslightl y invirulence . Possibly related topathogenicit y andvirulenc ewa s therepor to f Bassette t al. (1967), who found atoxi nproduce db y F. annosus. Ofth e9 isolates used, 7produce d thetoxin .Th e authors concluded thattoxi npro -

252 auctionwa sno trelate d topathogenicit y orvirulence .Gibb s (1972)sub ­ jected 10 isolates frompin e and 10 fromnon-pin e hosts tovolatil ecompo ­ nents ofpin e oleoresin andt ovariou s concentrations ofpinosylvi n and pinosylvinmonomethy lesther .N o detectable differenceswer e foundbetwee n the2 group s inregar d tothei rreaction s toth e substances tested. Incontras tt oreport s oflittl evariatio n inpathogenicit y and/or virulence aresom ereport s ofmuc hvariatio n inthes echaracteristics . Kuhlman (1970), in furtherexperiments ,use d 23 isolates ofth e fungus collected fromvariou spart s ofth eworl d and fromvariou shosts .H e found the isolatesvarie d considerably inthei rvirulenc e on seedlings of P. taeda and inaddition , found theyvarie d invirulenc e ondifferen thosts . James (1977)use d 5isolate s to inoculate large rootso f P. ponderosa Laws, and found considerable differences inth epercentage s of infection andth e amounts ofcolonization .H e alsouse d 10 isolates to inoculate seedlings of pine and found considerable variations inpercentage s of infection,number s of trees killed andcolonizatio n rates.I nanothe r experiment,usin gmas s conidial inoculum from 2isolates ,h e found thatth econidi a from themor e virulent isolateprove dt ob emor evirulen ttha tthos e fromth e lessviru ­ lentisolate .Negrutsk y (1978)als o reported differences in aggressiveness to affected plantsb y different formso fth efungus . Piatte t al. (1965)studie d thewood-rottin g ability of different isolates onexcise dbranche s androots ,an d found considerable differences existed between the isolates used.The y also found that singlebasidiospor e isolatesvarie d inthei r ability to rotwoo d and thatthe ywer e generally lessvigorou s thantissu e isolates.Simila r resultswer e reportedb y Kaufmann& Wellendor f (1978),wh o reported aprocedur e toby-pas s the years involved intestin gtree s forresistance .The y found thatth efungus , whengrow no nheartwoo d sawdust in1 % agar,varie d ingrowt h rates depend­ entno tonl y onth e sourceo fth ewoo dbu t alsoo nth estrai no fth e fungus used. Ifthi s couldb e used todetermin e accurately the resistance of trees, great strides couldb emad e insolvin g the F. annosus problem. Of interest areth eresearche s ofKorhone n (1978a)an dWorral l (1979). The former,i nFinland , divided the fungus into2 groups .One ,calle dS , from sprucewa s foundmostl y on Picea abies (L.)Karst .Thi s type alsowa s found to attack Pinus sylvestris L. saplings near infected sprucestumps . Itwa sno t isolated frommatur epine s andther ewer e few records ofth eS group onplant s othertha n spruceo rpin e inFinland .Anothe r group of isolates, frompine ,wa s called theP group .Thes ewer e common onpin eo f all ages and some alsower e found on Juniperus communis L., Betula sp., Alnus sp., Calluna sp.,an dothe rplants .I nfurthe r studies,th e Sgrou p was found tob epresen t inNorway ,Denmark ,Germany , Italy, India,an d Japan.Th eP grou pwa s found tooccu r inNorway ,Denmark , Scotland,France , Italy,Madeira ,Canada , andth eUnite d States. Inaddition ,culture swer e received fromAustralia ,Ne w Zealand, theFij i Islands andon e from Japan

253 thatdi dno tbelon g ineithe rgroup .Worral l (1979)foun d inCaliforni a thatisolate s frompin ewer emor evirulen t onpin e thano n fir andtha t thoughmos t isolates from firwer emor evirulen t on firtha no npine ,som e isolates from firwer emor evirulen t onpine .H e suggested thatth epin e isolatesmigh tb eexclude d from firwherea s the firisolate s could infect bothpin e andfir . An exciting contributionb yKorhone n (1978a)wa s the finding that mating in F. annosus cantak eplac e inculture .Th e fungus showed abipola r mating system andheterocaryosi s was indicatedb yth epresenc e ofclam p connections.Thi s informationwit htha to fSyche v &Negrutsk y (1978), in which they reported theproductio n of fruitingbodie s inpur eculture , should lead toman y studies regarding thegenetic s of F. annosus andit s variability inman y characteristics includingpathogenicit y andvirulence . The facttha t F. annosus produces aconidia l stagemus tals ob econ ­ sidered. Little iskno w ofth e importance ofth econidi a inth e disease cycle. Itha sbee nreporte d they areno t found frequently innatur e (Risbeth, 1951)thoug hRisbet h (1957)an dKalli o (1971)reporte d natural production ofconidi a on stumps covered bybranches .Morri s &Kno x (1962), Kallio (1967)an dNuortev a& Lain e (1968)also-hav ereporte d theproductio n ofconidi a on stumps. Ifthe y areimportant ,thi smigh t account,i npart , forth e lacko fvariability . The facttha tth enumber s ofnucle i inth e conidiama yvar y from 1t o4 o rmor e (Griffin& Wilson , 1967;Ahrberg , 1975)als omigh thav e aneffec tupo nth evariabilit y ofth e fungus. Iti sknow ntha tunde r laboratory conditions,th e conidia can infect seedlingso fpin e andspruc e (Hüppel, 1970). Kuhlman (1969b)als ouse d conidia for infecting roots ofpine s under natural conditions. Iti sals o knowntha tconidi a canb euse d toinfec tstump s (Kuhlman& Hendrix ,1964 ; Ross, 1968;Hun t et al., 1974,an d others). Inthei r experiments,Kuhlma n andHendri x foundbasidiospore s weremor e than60 0time s aseffectiv ea s conidia incolonizin gstumps . Of interesti sth e facttha tth econidi a aremor e resistantt ohea t thanbasidiospore s

254 inEurop e couldno tb e distinguished from those originating inNort h Americab y colony appearance,growt h rate,p Hoptimu m or cellulitic activ­ ity.H e divided them into 3growt hrat e groups andnote d thatthoug h some variability occurred, allcoul db e included inthes egroups .Courtoi s (1974),base d uponcultura l characteristics,reporte d 2ecotype s ofth e fungus.Roll-Hanse n (1940)an dCowlin g& Kelma n (1964)reporte d littleef ­ fecto ftemperatur e onth egrowt h of different isolates inculture .Th e variability ofthi s fungus incultur e thus issomewha t inquestion . Of interest areth ereport s ofDimitr i (page26 0o fthi sbook )an dvo n Weissenberg (inprep.) . Dimitri reported thatth e fungus isknow nt ovar y considerably inin-vitr o characteristics.However ,usin g atota l of 10iso ­ lates ofth e fungus ininoculatio n studies,h e foundtha twit hhi smetho d ofusin g only 3isolate s to inoculate asingl e tree,ther ewer en odiffer ­ ences inth egrowt h ofth edifferen t isolates inan ygive n tree.Vo n Weissenberg used singlebasidiospor e isolates,crosse s of singlebasidio - spore isolates andhypha l tip isolates from Finland.H e also found they varied considerably inin-vitr o characteristics butdi dno tvar ymuc hi n their growth rateswhe nuse dt o inoculate trees.Delatou r (page26 8o fthi s book)showe dvariou s isolates ofth e fungus grew differently in'culturean d he found thatther ewa s anegativ e correlationbetwee n the growth ofth e funguso nmal t agar andth e growtho fth e isolate intree s fromwher e they hadbee n isolated.

ARMILLARIA MELLEA

Although there is apresen ttendenc y tous e thebinomia l Armillariella mellea (Vahl)Karst. ,i nthi spape r the suggestion ofThier s & Sundberg (1976)wil lb e followed, thus retaining thenam e Armillaria mellea (Vahle x Fr.)Kummer . Incontras tt o F. annosus, A. mellea isa nexceptionall y variable fungus. Isolates from infectedplant s throughout California, single-spore isolates from asingl e sporophore (Raabe, 1966a), and single-spore isolates fromvariou s California crops (Maclean,1950 )showe d considerable vari­ ations inman y characteristics whengrow no npotat o dextrose agar.Thes e variations weremuc hmor epronounce d thanthos e reportedb yHintikk a (1973) where thecolonie sproduce dmainl y anaeria lmyceliu m withusuall y nobrow n crustose formation. The firstrepor to fvariatio n inpathogenicit y in A. mellea was thato f Childs &Zelle r (1929)wh o reported anoa k strainwhic hwa spathogeni c and a firstrai nwhic hwa snon-pathogenic .Va nVlote n (1936)foun dvariation s inth e ability of different isolates toattac kpotat o tubers.Th e facttha t the isolatemos tvirulen to npotat o also infected Ligustrum sp., Pinus sp., and Rosa sp.,le dhi m tostat etha t specializationb y the funguswa sno t apparent.Blis s (1946)als onote d differences inth epathogenicit y ofiso -

255 lateso fA . mellea. Raabe (1955,1967 )reporte d distinct differences inth e pathogenicities andvirulence s of field isolatesof-A . mellea when3 dif ­ ferenthos t species were inoculated underglasshous e conditions.Thes e differenceswer e also shownt ooccu rwhe n single-spore isolates froma single sporophorewer euse d to inoculate the same 3hos t species (Raabe, 1972). Intha texperiment ,th e single-spore isolateswer e comparedwit hth e parent isolate andnon ewer e asvirulen t asth eparen t and only 1wa sa s pathogenic asth eparent .Th e results ofth e inoculations using field iso­ lates and thoseusin g single-spore isolateswer e tabulated (Raabe, 1969) and ingeneral ,th e single-spore isolateswer e lesspathogeni c andmuc h lessvirulen ttha nisolate s from infectedplants .Thi s apparentdecreas e in vigor isals o foundwhe nth e isolates aregrow n inculture .Th e sameha s been shown for F. annosus (Kuhlman& Hendrix , 1964;Piat te t al., 1965). Thisma yb e due togeneti cvariabilit y alone,th e small sample of isolates selected orma yb e due toth e facttha thypha e ina haploi dmyceliu m are lessvigorou s thanwhe n inth ediploi d condition.Also , iti simportan tt o consider thati ncomparin g single-spore isolateswit h those from infected plants, the latterwer e selectedbecaus e ofthei rpathogenicity , andposs ­ iblybecaus e ofthei rvirulence . In1973 ,Hintikk a showed thatmatin g of single-spore isolates ofA . mellea could takeplac ewhe npairin g compatible isolates inculture .Wit h a compatible reaction,th eresultan tne w diploidproduce d adistinctiv e different growthpatter n incultur e and thereforewa s detected easily. Thisha s ledt oth edeterminatio ntha t inFinland , there are3 distinc t intersterile 'biologic species'i nA . mellea (Korhonen, 1978b)an d that inNort hAmerica , there are atleas t1 0 'biologic species' (Anderson & Ullrich, 1979). Therelationship sbetwee n the 10Nort hAmerica n 'biologic species' and 5 'biologic species' fromEurop e alsohav ebee n studied (Anderson et al., 1980). The discovery ofdifferen t 'biologic species'o fA . mellea inrathe r limited areas raises questions ast oho wman y other 'biologicspecies ' mightb e found throughout theworld . Itals o raises questions ast oth e pathogenicities andvirulence s ofthes eman y groups andth e individuals in thesegroups .Th e discovery ofa neas y laboratorymetho d for determining thedegree so fvirulence s andperhap sth edegree so fpathogenicitie s would bemos thelpful .A n attemptt od othi sha sno tbee n successful (Raabe, 1969)bu ti f found, suchmethod swoul db emos thelpfu l ina testin gprogra m for selecting resistantplants .Presently , inCalifornia , aprogra m isi n progress to find resistant species ofornamenta l plants and fruittree s (Raabe, 1966b, 1979). Insuc hexperiments ,plant s aregrow n inarea sknow n tob e infestedwit h the fungus. Inaddition , inoculum of2 pathogeni c and virulent isolates ofth e fungus arepu tnea rth e testplant swhe nthe y are planted.Plant s arerate d ast oth e length oftim ebefor e they arekilled . Those still alive after 10year s aredu g andth eroot s examined and rated

256 as to the amounts of infection. A most important aspect in the research regarding this fungus would be the discovery of a reliable method of producing basidiocarps easily. The closely related A. tabescens (Scop, ex Fr.) Emel . produces basidiocarps readily in culture and successful matings in culture have produced basidio­ carps (Tang & Raabe, 1973) but A. mellea fruits only occasionally and reluctantly, though there are reports of it fruiting in culture (Reitsma, 1932; Raabe, 1972). If a consistent and reliable method for this could be found, many studies on this important pathogen could be made including those regarding pathogenicity and virulence.

REFERENCES

Ahrberg,H.E. ,1975 .Untersuchunge nzu rCytologi eun dzu mWachstu mverschiedene rStämm e vonFome sannosu s (Fr.)Cooke .Europea nJourna lo fFores tPatholog y 5:287-303 . Anderson,J.B .& R .Ullrich ,1979 .Biologica lspecie so fArmillari amelle ai nNort h America.Mycologi a71 :402-414 . Anderson,J.B. ,K .Korhone n& R .Ullrich ,1980 .Relationship sbetwee nEuropea nan dNort h Americanbiologica lspecie so fArmillari amellea .Experimenta lMycolog y4 :87-95 . Azevedo,N.F .do sS .d e& M .d eF.M.A .Moniz ,1974 .Influenc eo ftemperature ,p Han d nutrientso nth egrowt ho fFome sannosu sisolates .Proceeding s4t hInternationa l Conferenceo nFome sannosus :163-168 . Basset,C , R.T.Sherwood ,A .Keple r&P.B .Hamilton ,1967 .Productio nan dbiologica l activityo ffomannosi na toxi csequeterpen emetabolit eo fFome sannosus . Phytopathology 57:1046-1052 . Bega,R.V. , 1963.Symposiu m onroo tdisease so ffores ttrees :Fome sannosus . Phytopathology 53:1120-1123 . Bega,R.V .& F.F .Hendrix ,1962 .Variatio no fmonobasidiospor eisolate so fFome sannosus . Phytopathology 52:3 (abstract) . Bliss,D.E. ,1946 .Th erelatio no fsoi ltemperatur e toth edevelopmen to fArmillari a rootrot .Phytopatholog y36 :302-318 . Childs,L .& S.M .Zeiler ,1929 .Observation so nArmillari a rootro to forchar dtrees . Phytopathology 19:869-873 . Courtois,H. ,1974 .Abou tth eoccurrenc eo fecotype so fth especie sFome sannosu s(Fr. ) Cooke.Proceeding s4t hInternationa lConferenc eo nFome sannosus :125-132 . Cowling,E.B .& A .Kelman ,1964 .Influenc eo ftemperatur eo ngrowt ho fFome sannosu s isolates.Phytopatholog y 54:373-378 . Dimitri,L. ,1974 .Resistenzforschun gbe ide rFicht egegenübe rde mFome sannosus . Proceedingso fth e4t hInternationa lConferenc e onFome sannosus .p .76-80 . Etheridge,D.E. ,1955 .Comparativ estudie so fNort hAmerica nan dEuropea nculture so fth e rootro tfungus ,Fome sannosu s (Fr.)Cooke .Canadia nJourna lo fBotan y33 :416-428 . Fedorov,W.I .& N.I .Staichenko ,1974 .[Rat eo fgrowt han dactivit yo foxidizin genzyme s ofdifferen tFomitopsi sannos astrains] .Proceeding s4t hInternationa lConferenc eo n Fomesannosus .p .106-113 . Gibbs,J.N. , 1972.Toleranc eo fFome sannosu st opin eoleoresin san dpinosylvins . EuropeanJourna lo fFores tPatholog y2 :147-151 . Griffin,B.R .& C.L .Wilson ,1967 .Asexua lnuclea rbehavio ran dformatio no fconidi ai n Fomesannosus .Phytopatholog y 57:1176-1177 . Hintikka,V. ,1973 .A not eo nth epolarit yo fArmillari amellea .Karsteni a13 :32-39 . Hunt,R.S. ,W.W .Wilco x& F.W .Cobb ,1974 .Resistanc eo fstum ptop st ocolonizatio n byFome sannosus .Canadia nJourna lo fFores tResearc h4 :140-142 . Hüppel,A. ,1970 .Inoculatio no fpin ean dspruc eseedling swit hconidi ao fFome sannosus . Proceedings3r dInternationa lConferenc eo nFome sannosus .p .54-56 . James,R.L. ,1977 .Th eeffect so fphotochemica lai rpollutio no nth eepidemiolog yo f Fomesannosus .Ph.D .Thesis ,Universit yo fCalifornia ,Berkeley . Kallio,T. ,1967 .Havaintoj amaannousemasientest akevaall a1967 .Metsätal .aikakausleht i 7-8:228-230 ,234 . Kallio,T. ,1971 .Incidenc eo fth econidiophore s ofFome sannosu s (Fr.)Cook eo nth e loggingwast eo fspruc e (Piceaabie s (L.)Karst.) .Act aForestali aFennic a124 .9 p .

257 Kaufmann,U .& H .Wellendorf ,1978 .Geneti cvariatio n inPice aabie s (L.)Kars tan dFome s annosus (Fr.)Cook eshow nb ygrowt ho fth efungu so nwoo ddust .Europea nJourna lo f ForestPatholog y8 :226-239 . Korhonen,K. ,1978a .Intersterilit ygroup so fHeterobasidio nannosum .Seloste : Juurikäävänristeytymissuhteet .Communicatione s InstitutiForestall sFennia e 94(6):1-25 . Korhonen,K. ,1978b .Interfertilit yan dclona lsiz ei nth eArmillari amelle acomplex . Karstenia18 :31-42 . Kuhlman,E.G. ,1969a .Surviva lo fFome sannosu sspore si nsoil .Phytopatholog y 59:198-201 . Kuhlman,E.G. ,1969b .Numbe ro fconidi anecessar yfo rstum proo tinfectio nb yFome s annosus.Phytopatholog y 59:1168-1169 . Kuhlman,E.G. ,1969c .Inoculatio no flobloll ypin eseedling swit hFome sannosu si nth e greenhouse.Canadia nJourna lo fBotan y47 :2079-2082 . Kuhlman,E.G. ,1970 .Seedlin ginoculation swit hFome sannosu ssho wvariatio ni nvirulenc e andi nhos tsusceptibility .Phytopatholog y60 :1743-1746 . Kuhlman,E.G .& F.F .Hendri xJr. ,1964 .Infection ,growt hrat ean dcompetitiv eabilit y ofFome sannosu si ninoculate dPinu sechinat astumps .Phytopatholog y 54:556-561 . Lane,C .& W .Witcher ,1974 .Studie so nFome sannosu san dth einfectio no fpin e seedlings.Proceeding s4t hInternationa lConferenc eo nFome sannosus :227-230 . Maclean,N.A. ,1950 .Variatio ni nmonospor eculture so fArmillari amellea .Phytopatholog y 40:96 8(abstract) . Morris,C.L .& K.A .Knox ,1962 .Fome sannosus :A repor to nth eproductio no fconidi ai n naturean dothe rstudie si nVirginia .Plan tDiseas eReporte r46 :340-341 . Negrutsky,S.F. , 1978.[Positio nan dperspective so ninvestigation sabou tFomitopsi s annosa (Fr.)Kars ti nUSS Ran di ncountrie so fEaster nEurope. ]Proceeding s5t h InternationalConferenc e onProblem so fRoo tan dBut tRo to fConifers :19-26 . Nuorteva,M .& L .Laine ,1968 .Übe rdi eMöglichkeite nde rInsekte nal sÜberträge rde s Würzelschwamms(Fome sannosu s (Fr.)Cooke) .Annale sEntomologic iFennic i34 :113-135 . Platt,W.D. ,E.B .Cowlin g& CS . Hodges,1965 .Comparativ e resistance ofconiferou sroo t woodan dste mwoo dt odeca yisolate so fFome sannosus .Phytopatholog y 55:1347-1353 . Raabe,R.D. ,1955 .Variatio ni npathogenicit y ofisolate so fArmillari amellea . Phytopathology45 :69 5 (abstract). Raabe,R.D. ,1966a .Variatio no fArmillari amelle ai nculture .Phytopatholog y 56:1241-1244 . Raabe,R.D. ,1966b .Testin gplant sfo rresistanc e tooa kroo tfungus .Californi a Agriculture20 :12 . Raabe,R.D. ,1967 .Variatio n inpathogenicit y andvirulenc e inArmillari amellea . Phytopathology 57:73-75 . Raabe,R.D. ,1969 .Cultura lvariation so fArmillari amelle ano trelate dt opathogenicit y andvirulence .Proceeding s1s tInternationa lCitru sSymposiu m3 :1263-1272 . Raabe,R.D. ,1972 .Variatio ni npathogenicit yan dvirulenc e insingle-spor eisolate so f Armillariamellea .Mycologi a64 :1154-1159 . Raabe,R.D. ,1979 .Resistanc eo rsusceptibilit yo fcertai nplant st oArmillari a rootrot . Universityo fCaliforni aDivisio no fAgricultura lScience sLeafle t2591 .1 1p . (review). Reitsma,J. , 1932.Studie nübe rArmillari amelle a (Vahl)Quel .Phytopathologisch e Zeitschrift4 :461-522 . Risbeth,J. , 1951.Observation so nth ebiolog yo fFome sannosu swit hparticula rreferenc e toEas tAnglia npin eplantations .II .Spor ereduction ,stum pinfectio nan d saprophyticactivit yi nstumps .Annal so fBotany ,Ne wSerie s15 :1-22 . Risbeth,J. , 1957.Som efurthe robservation so nFome sannosu sFr .Forestr y30 :69-89 . Roll-Hansen,F. ,1940 .Underskelsee rove rPolyporu sannosu sFr .saerli gme dhenblik kp a densforekoms ti sonnafjelsk eNorge .Meddelelse r fraDe tNorsk eSkogforsjzSksvese n 7:1-100 . Ross,E.W. ,1968 .Duratio no fstum psusceptibilit yo flobloll ypin et oinfectio nb y Fomesannosus .Fores tScienc e14 :206-211 . Ross,E.W. ,1969 .Therma linactivatio no fconidi aan dbasidiospore so fFome sannosus . Phytopathology 59:1798-1801 . Sychev,P.A .& S.F .Negrutsky ,1978 .[Th eformatio no ffrui tbodie sb ydifferen t strainso fFomitopsi sannos a (Fr.)Kars tan dthei rinteraction si nculture. ] Mikologiya iFitopatologiy a 12:148-151 .(Revie wo fPlan tPatholog y58:547. ) Tang,H .& R.D .Raabe ,1973 .Sporophor eproductio nan dheterothallis mi n Clitocybe tabescens.Phytopatholog y63 :121 8(abstract) . Thiers,H.D .& W.J .Sundberg ,1976 .Armillari a (Tricholomataceae,Agaricales )i nth e

258 westernUnite dState sincludin ga ne wspecie sfro mCalifornia .Madran o23 :448-453 . Vloten,H .van ,1936 .Onderzoekinge nove rArmillari amelle a (Vahl)Quel .Fungus , Wageningen8 :20-23 . Weissenberg,K .von .Between-isolat evariatio ni npathogenicit yo fHeterobasidio nannosu m toPice aabie s (inpreparation) . Worrall,J. , 1979.Ecolog yo fFome sannosu s (Fr.)Kars to nAbie sconcolo r (Gond.)Lindl . exHildebn .I .Developmen to fhos tspecializatio nt opin ean dfi ri nFome sannosu s isolatesfro mCalifornia .Repor tfo rMaster sDegree ,Universit yo fCalifornia , Berkeley.

259 Some host/parasite relationships between Norway spruce (Picea abies) and Fomes annosus

L. Dimitri

Hessische Forstliche Versuchsanstalt, 3510 Harm. Münden, BRD

ABSTRACT

Research onth eroo tro tcause db y Fomes annosus hasbee n underway formor etha n10 0years .However , experiments onhost - parasite interactions andth enor m ofreactio n (i.e.,th ere ­ sistance)agains t Fomes havebee n initiated only quiterecent ­ ly.Th epape rpresent s some results ofextensiv ewor k (Dimitri, 1980)o nth e interrelationships betweenhost/parasit e aswel l ashost/environment/parasite , inth ecas e ofNorwa y sprucein ­ oculatedwit h F. annosus. Inspit e ofconsiderabl e differences inmorphologica l andphysiologica l featureso fspecia l strains ofth eparasit e F. annosus invitro ,w ehav e so farbee n unable to find anysignifican tdifferenc e inth e spread ofthes e strains inth ehos tplant s invivo .W e should, therefore,no t imply thatstrain s showth e sameparasiti c activity inviv oa s invitro .O nth ebasi s ofpresen tresult sw eca n assume ahori ­ zontal resistance ofNorwa y spruce to F. annosus. Environment seemst o influencebot h height-growth ofth ehos tan d spread ofth emyceliu m inth etrees .Mos tprovenance s showth e same trend: thebette r theheight-growt h ofth ehost ,th ehighe r themycelium-heigh t inth e trees.Bu t ineac hprovenanc e a certainportio no findividual s is found,whic hcombin e out­ standingheight-growt hwit hhig hphenotypi c resistance against the spread ofth emycelium . Thus, invie wo fresistanc e to F. annosus, the selection ofNorwa y spruce individualswil lb e more efficienttha ntha to fprovenances .

INTRODUCTION

Stem rot isth emos t important andmos t serious fungal diseasewhic h afflictsNorwa y spruce (Picea abies L. Karst.), the silviculturally and economically most significanttre e species inth e Federal Republic of Germany.Th e causes ofthi sdamag e and theorganism swhic h induce itca nb e

260 verydifferent .Whe nth elowe rpar to fth e stem iswounded , the ensuing wound-rot iscause d mainlyb y 2 Stereum species.Heart-rot ,o nth eothe r hand,whic h spreadsu p from theroo tzone ,i susuall y causedb yth e root andbut tro t fungus, Fomes annosus (Fr.)Cooke .A substantial reduction ofwound-ro t definitely appears tob epossibl e ifsurfac ewound s receive anearl ytreatmen twit h suitablepreservative s (Dimitri& Schumann,1975 ; Schönhar,1979 ;Bonneman , 1979). Severalpreventiv emeasure s againstheart - rotar e alsopossibl e (Greig, 1980). Onepossibilit y forreducin g damage,whic hha s so farreceive d compara­ tively little attention and study, isth eutilizatio n ofthi s treespecies ' natural resistance to F. annosus. Sincethi sresistanc e isdependen tupo n several factors (virulence ofth ecausativ e organisms,environmenta lcondi ­ tions, etc.), itwa s investigated within the scope ofa nextensiv eprojec t (Dimitri, 1980). Only a fewresult s ofth eexperiments ,whic hhav ebee n described indetai l inth e above-mentioned paper, arespecifie dhere .

mycelium-height U0-icm

120

100- 1 m. il 80- i• 60- i

40 I i I 20 J J w i CM CM ** "O •CM •- «- C M M J.Q CM to in CM to in CM to tri cN s s Fa-strain„ . £ £ J Ü_ a a u. ^ xi u. ul £ U.OX ü_ox u_ o x u. ü a u. ir

Figure 1. Spread ofth emyceliu m ofdifferen t F. annosus strains,3 o f whichha dbee n inoculated simultaneously intoeac ho fte n 60-year-old Norway spruce trees 12month searlier .

261 RELATIONSHIP BETWEEN THE HOST PLANT AND THE PARASITE UNDER IDENTICAL SITE CONDITIONS

Iti sknow ntha t inlaborator y testsdifferen t F. anrtosus strainsvar y distinctly inthei rmorphologica l andphysiologica l properties (Courtois, 1980;Dimitri , 1980). More importanttha nth ebehaviou r invitr o isth e parasitic productive capacity ofth eparasit e strains invivo .Neithe r in simultaneously inoculated 8-year-old spruceclone sno r inolde r spruce trees,whic hha dbee n inoculated simultaneouslywit h2 F, annosus strains (obtained fromdifferen tpart s ofWes tGermany )a swel l as athir d check strain (F2-culture,obtaine d from abasidiospore )coul d any significant differences inth emyceliu m heightb e determined.A s ex'ampleso fth edif ­ feringvirulenc e of individual F. annosus strains invivo ,Figure s 1an d2 illustrate theresult s obtained in60-year-ol d and 67-year-old spruce stands. Inbot h figures the dominating effecto f thehos tplan tupo nth everti ­ cal spread ofth emyceliu m canclearl yb e seen:i fa tre eha s ahig hphe -

mycelium-height KO-icm

120

100- e^L 80- 60- J u ri 40

20 jLmmi . CM CO in CN| « N^^ N •- CM «3 W CM CD in CM •* CN -* "O CM .- _Q CM *- Fa.-strain u.o x u_ if u.o £ U.I f U. U.OX 11. O X u- o o U-Xc5 U.QUI u. x u_ TreeNr . 22 32 27 16 23 26 15 29 21

Figure 2. Spread ofth emyceliu m ofdifferen t F. annosus strains,3 o f whichha dbee n inoculated simultaneously into eacho fte n 67-year-old Norway sprucetree s 12month s earlier.

262 notypic resistance,th e spread ofal l F. annosus strains ischecke d toal ­ mostth e same extent (e.g.,tree sno .1 an d 24i nFig.1 , aswel l astree s no. 22an d 32 inFig .2) .Vic eversa ,al lparasit e strains candevelo pver y well ifth ehos ttre eha s alo wresistanc e (e.g.,tree sno .1 6an d 18i n Fig. 1,a swel l astree sno .2 1 and2 9i nFig .2) .I fon ecompare s thein ­ dividuals inthes e2 population s of spruce,eac h ofwhic hha dbee ninocu ­ latedwit h the same F. annosus strains,th epictur e ofTabl e 1develops . Onth ebasi s ofthes eresults ,whic h are stillt ob e substantiated by means of further experiments,i tca nb e assumed thaton ecanno treadil y draw conclusions frommorphologica l characteristics or from physiological behaviour intes tculture s aboutth eparasiti c activity of different F. annosus strains innature . From the facttha tolde r spruce treesreac t tosevera l F. annosus strains incompletel y the samewa y (thatis ,eithe rvulnerabl e orresis ­ tant), ahorizonta l (i.e.generalized )resistanc e canb e deduced.

Table 1. Resistance and reaction oftree st o Fomes annosus strains.

TreeNo . Strain Resistance Reaction

In Fig. 1 F 2 high K different b medium

24 F2 high Frbg different 25 Frbg low

16 F2 low 18 G 6 low identical 19 Hi 57 low

In Fig. 2 16 F2 medium Hi different 21 F.l low

22 F2 high different 23 G6 medium identical 26 Hi 57 medium

263 INFLUENCE OF PROVENANCE AND SITE ON THE RESISTANCE OF NORWAY SPRUCE TO FOMES ANNOSUS

For the investigation ofthes e interrelations,308 6 trees from 10 differentNorwa y spruceprovenance s were inoculated with theF _ F. annosus strain,hal fo fthes ewithin ,th eothe rhal foutsid e ofth evegetativ e period. These 10provenance s grewwit h approximately the samenumbe r of stems on6 differen t sites.A description ofth eprovenances ,th esites , themethod s of inoculation and investigation, aswel l asth e individual results canb e found inDimitr i (1980).Again ,2 diagram swil l servet o illustrate theseinterrelations . Fig.3 show sth emea nvalue s forth eheigh to fth eNorwa y spruce (HWL) andth eheigh to fth emyceliu m (MH)whic hwer e attained intree so f indi­ vidualprovenance s onrespectivel y the same sites after inoculation outside thevegetativ e period.A s expected, themea nHW Lo f allprovenance s (xh ) varied greatly from sitet o site,whic h isa nindicatio n ofth e differing sitequalit y ofth e experimental sites forth e spruceplantations .Amon g

7-1 m Winter 1974

tre«-heigh t 6-

S-

4-

3-

' 5 1 11 10 6 5 9 6 11 10 1 11 9 10 5 1 9 6 11 10 5 1 9 11 6 10 9 5 1 6 1 5 9 11 6 10 5 9 1 11 6 10 5 1 9 6 11 10 1 5 9 11 6 11) 10 provenances1 6 17 23 38 13 U0 niei 130 mycelium-height 120 110 100 90 80- 70 60 50 40- 30- 20 10 5 Ml ID 6 S I 9 6 11 10 1 11 9 10 5 5 9 11 6 10 5 9 1 It 6 10 5 1 9 6 II 10 10 16 M 17 23 38 provenances

Figure 3. Kv&r&q&heigh to fdifferen tNorwa y spruceprovenance s (xh )an d averagemyceliu m height (xm )i ntree so fthes eprovenance s inprovenance - order inoculated outside thevegetativ eperio d 12month searlier .

264 theprovenance s onth e same siteo feac h testplo t itwa spossibl y to ascertain substantial and statistically significant differences inth e meanHWL ,whic h clearlypoint s outth e differences inth ehereditar y pro­ ductionpotentia l ofth eprovenances . Both the individual values andth erespectiv emean s ofth emyceliu m height (MH)hav e amuc h greaterdeviatio n thanthos e ofth eHWL .Amon g different sites there canb e considerable variation inth emea nmyceliu m height (xm )o f allprovenances .Statisticall y significant differences couldb edetermine d forthi spropert y (xm )a swell . Thever y substantial differences amongth emea nvalue s forth emy ­ celium height ofindividua l provenances onth e same site (e.g.,betwee n provenances 10 and3 8i nNentershausen , siteno .11 )coul dno tb eprove d statistically significantb ymean s ofanalysi s ofvariance ,du et oth eex ­ tremely largedeviatio n ofth e singlevalues . Also ina nanalysi s ofcovarianc e (withheigh t ascovariant )ther ewer e no significantdifference sbetwee n theteste dvariables ;amon gth esites , however, significant differences inth emyceliu m heightwer e found. Siteso nwhic h almost allteste dNorwa y spruceprovenance s haveat - tained anabove-averag e HWLbu ta myceliu m height farbelo w average (e.g., Bieber,sit eno .6 )ar eespeciall y suited forth ecultivatio no fspruce . Ifon e compiles the data forth echaracteristic s 'height' and 'mycelium height',whic hwer e attained ondifferen t sites after inoculation ofth e respectiveprovenance s outside ofth evegetativ e period, the following observations canb emad e (Fig.4) . - There seems tob e apositiv e correlationbetwee nheigh t andmyceliu m height: allprovenance s with amea nHW L (xh )whic h reaches or surpasses theoveral l average (xH )als oconsistentl y have amyceliu m height (xm ) surpassing theoveral l average (x M). Ina calculatio no fcorrelatio n according toPearson ,th e interrelations ofHW L andM Hprove d tob estatis ­ tically significant. -Th ebest-growin g provenance (No.13 )ha d the lowestresistanc e toth e spread of F. annosus mycelium. -Th emea nHW Lo fprovenanc e No.7 rank s closebehin d thato fprovenanc e No. 13,however , the averagemyceliu m spreadwa s distinctly lower (by 20 %). - Individual provenances appear tob e suited fordifferen t sites:thus , for example,provenanc e 23ha d abelow-averag e HWL inKönigstei n (5)bu ta n above-average MH,wherea s thisrelationshi p was exactly the opposite inth e forestdistric tBiebe r (6).(Compar e alsoprovenanc e 3i nReinhardshage n (1)versu s inOber-Ramstad t (10).). Even ifth emea nvalue s ofth eHW L and theM L foreac hprovenanc e and repetition aretake n as arati o ofth erespectiv e sampleplo tmea n (=10 0% ) (relative tree andmyceliu m height), itca nb e seen thatal l factorswit h apositiv e influence onth eHW L (nutrient supply,moistur e

265 _ 7-im Winter197 4

t 6- xh

-xH

lo 17 7 23 13 10 3 7 „17 38 16 17 23 38 7 10 16 17 14 7 16 14 23 7 13 10 3 14 23 13 140- mm 16 14 36 6,—, 16 23 p, 13 6 6 14 3 13 3 23 38 13 10 17 38 6 16 17 38 7 130- 1,120- S •= no­ •x" m

•x M

Königstein(5 ) Reinhardshagen (1) Chausseehaus (9) Nentershausen(H) Bieber 16) Ober-Ramstadt(IO) Trial areas Figure 4. Average heighto fsevera lNorwa y spruceprovenance s (xh ) and averagemyceliu m height (xm )i nth etree so fthes eprovenance si nsite - order inoculated outsideth evegetativ e period1 2month s earlier. content,temperature ,etc. )als o appeart oinfluenc eth edevelopmen to f decayi nth esam edirection .Analyse so fregressio nbetwee n treeheigh t andmyceliu m heightfo reac h sitean dprovenanc e could establish onlya lowcorrelatio n coefficient (r)fo rthi stendency ,i nothe rwords ,n o close relationshipwa sfound . ' Itha sbee nmad e evidenttha ta selectio no findividual s appearst o promisemor e advantages,als ofo rF. annosus resistance,tha n selection ofprovenances .Th eproportio no findividual swit h above-average yield ingrowt h (HWL5 0% abov eth emea no fth erepetition )an dabove-averag e phenotypic resistancet oth esprea do fmyceliu m (MH5 0 %belo wth emea n ofth erepetition )i smuc hhighe r (6.4t o10. 3% )i nth ebest-growin g provenances (Cottbus,Chausseehaus ,Rothenkirchen ,Westerhof )tha ni nth e others (0.4t o4. 0 %). Theinfluenc eo fsit ei nthi s respecti sles sclear ­ lyrecognizable ;th eproportio no fpositiv e individualso nth ewhol ewa s nearlyth esam eo nal lteste d sites (approx.4. 5 %).

266 REFERENCES

Bonnemann,I. ,1979 .Untersuchunge nübe rdi eEntstehun gun dVerhütun gvo n"Wundfäulen " beide rFichte .Dissertatio nan dde rForstl .Fakultä tde rUniv .Göttingen .17 3p . Courtois,H. ,1980 .Pathoge nbiolog y (Fomesannosus) .In :L .Dimitri :Proceeding so fth e FifthInternationa lConferenc eo fRoo tan dBut tRo ti nConifers ,p .43-53 . Dimitri,L. ,1980 .Untersuchunge nübe rdi eWiderstandsfähigkei t derFicht e (Piceaabie s Karst)gegenübe rde mWurzelschwam m (Fomesannosu s (Fr.)Cooke) .Schriftenreih e Forstl.Fak .Univ .Göttinge nNr .66 .12 6p . Dimitri,L .& G .Schumann ,1975 .Di eWundfäul e -ei naktuelle sProble mde r Forstproduktion.Holz-Zentralblat t 101:803-806 . Greig,B.J.W. ,1980 .Chemical ,biologica lan dsilvicultura lcontro lo fFome sannosus . In:L .Dimitri :Proceeding so fth eFift hInternationa lConferenc eo nProblem so fRoo t andBut tRo ti nConifers ,p .75-84 . Schönhar,S. ,1979 .Erprobun gvo nWundschutzmittel nau fde rFichte .Forst -u .Holzwir t 34:12-14 .

267 Behaviour of Fomes annosus in the stem of Norway spruce and in the laboratory

C. Delatour

CNRF,Laboratoir ed ePathologi e forestière,Champenoux ,5428 0Seichamps ,Franc e

ABSTRACT

The growthrat eo f F: annosus inth este m ofNorwa y spruce isconnecte d with invitr o characters such asmycelia l growth rateo nmalt-aga r (negative correlation).Thi s demonstrates that fungusvariation s are atleas tpartiall y responsible for variations ofth ediseas e innature .Suc hvariation s havet o be takenint o account forbreedin g experiments.Observation s indicate that F. annosus may encounter limitations inheigh t extension instems ,thu s leading toth ePotentiall y Colonizable Column concept.Th e significance ofdat a achievedb y theste m inoculationmetho d arediscusse d andexperiment s comparing natural and artificial infections are advocated.

INTRODUCTION

Very little isknow n aboutth ebehaviou r of F. annosus (Fr.)Cook ein ­ side trees,e.g . inNorwa y spruce (Picea abies (L.)Karst.) . Iti s'know n thatNorwa y spruce sapwood canexhibi tstron g reaction againstdiametrica l growth of F. annosus inth e stem (Shain, 1971), butdoe s F. annosus encoun­ ter limitations in itsheigh tdevelopmen t intrees ?Thi s isprobabl y true because someworker s (Weissenberg, 1975;Dimitr i &Kliefoth , 1978)hav e found that F. annosus doesno tgro w equally inal l the inoculatedtrees , allowing thehypothesi s of aninterna l resistance.Bu ti sthi s resistance thesam ei nal lpart so fth este mwoo d (excluding resistant sapwood)an d duringth ewhol e life ofth etree ? Onth e otherhan d theparasit e itselfexhibit svariation s studiedb y someauthor s (e.g.Courtois ,1972 ;Kaufman n& Wellendorf , 1978)i nth e laboratory,bu tw e dono tkno w exactly their significance innature . Iper ­ formed inNanc y someobservation s and experiments inthi s field thatar e thesubjec to fthi spaper .

268 GROWTH RATE OF F. ANNOSUS IN THE STEM OF NORWAY SPRUCE

Growth in natural conditions My observations havebee nperforme d ina secon d generation stando f Norway spruce,6 0year s old,growin g oncalcareou s soil 60k mS.W.o fNancy . In197 3 and againi n late 1977 Idetermine d theheigh to f F. annosus in the stemso f4 6 treesb yth e incrementbore rmethod , andthu smeasure d the growth rate ofth e fungus inever y individualtree . Results:Variation s arever y large inheigh tan dals o ingrowt hrat e (Table 1). Thesevariation s areno t clearly explainedb y tree size (height and diameter). Theheigh treache db yth e fungus isprobabl y related, at leastpartially , toth e infection agewhic h isno tknown .Th egrowt hrat e mightb e connectedwit hth eheigh to f F. annosus inth e stem asw e can expect slowergrowt h atgrea theights .Thi s assumptionwa sno texactl y confirmed within our4 6 trees:indee d thecorrelatio nbetwee nthes e2 characters isno t significant (r= -0.11).

Growth following inoculation at different heights in the stem To achieve acleare rvie wo fthi sheigh teffec to nth egrowt hrat eo f the fungus Iperforme d trial inoculations of some trees.Th e inoculation method was similar toDimitri's ,i.e . aninoculate d darto fwoo d introduced ina bor ehole .Fou r trees,2 5year s old,wer e inoculated inApri l 1979 fromth e soil levelu p toth e living crown.On e strainwa s used, collected from aninfecte d Abies grandis Lindl. inDec .197 6 atth eArboretu m des Barres (Center of France), thenno t stored onartificia l mediumbu ti n living spruce.Thi s strainbelong s toth e Sintersterilit y group.Th e treeswer e felled 6month s later,disc swer e cutint o them and observed for F. annosus. Inspit e ofth ever y smallnumbe r oftree s studied some interesting phenomenawer e observed (Fig.1) .A nunquestionabl e result isth e identical growth rateo f F. annosus inever y tree at6 m high ,whic h is approximately the living crown level.Below ,th e growth rate ofth e fungus isver yer ­ ratic, althoughw e generally observe greater growth rates atlowe rlevels . Incidentally,w e can emphasize the annual growth of F. annosus insideth e inoculated stems is frequently greater thanth e 'naturalgrowth' ,whic hi s aver y common feature for inoculations ranging over aperio d of1 2month s or less.

BEHAVIOUR OF F. ANNOSUS IN THE LABORATORY

From everytre e inwhic h Imeasure d thegrowt h of F. annosus, was collected inpur e culture thecorrespondin g straino fth e fungus (atth e top ofth edeca y column). So itwa spossibl e toundertak e some studies on variations ofth eparasit e inconnectio nwit h itsnatura lperformance ,in -

269 eluding (1)th emycelia l growtho nmalt-agar , (2)th edeca ycapacity , (3)th e cellulolytic capacity, and (4)th e intersterility group ofeac h strain.Mycelia l growthtoo kplac e inpetr i dishes (diam.8. 5 cm)o nDifc o malt-agar (3 %) at2 3 °Ci ndarknes s during 8days .Deca y capacitywa s

Table 1. Heightgrowt h (cm)o f F. annosus inth e stems of4 6Norwa y spruces.

Tree Heighto f Height Annual Tree Heighto f Height Annual No. F. annosus increase growth No. F. annosus increase growth between rate between rate 1973 1977 1973-1977 1973 1977 1973-1977

1 380 605 225 56 24 260 460 200 50 2 270 407 137 34 25 285 447 162 41 3 312 437 125 31 26 335 410 75 19 4 85 272 187 47 27 210 335 125 31 5 210 260 50 13 28 -110 272 162 41 6 582 644 62 16 29 160 335 175 44 7 480 617 137 34 30 110 272 162 41 8 382 482 100 25 31 110 222 112 28 9 425 475 50 13 32 260 435 175 44 10 582 694 112 28 33 235 285 50 13 11 182 269 87 22 34 310 460 150 38 12 282 319 37 9 35 410 522 112 28 13 25 137 112 28 36 185 447 262 66 14 290 407 117 29 37 160 372 212 53 15 330 592 262 66 38 260 385 125 31 16 230 380 150 38 39 ' 185 347 162 '4 1 17 210 210 0 0 40 210 247 37 9 18 360 522 162 41 41 185 247 62 16 19 235 445 210 53 42 185 222 37 9 20 460 572 112 28 43 135 297 162 41 21 235 347 112 28 44 315 427 112 28 22 260 360 100 25 45 295 470 175 44 23 360 522 162 41 46 265 352 87 22

Generalvalues : Min. Mean Max. height in197 3 25 268 582 height in197 7 137 396 694 height increase 0 128 262 annual growth rate 0 32 66

270 heighto finoculatio n cm

60.

50.

40.

30. •0-"

1 2 3 4 5 6 7 m growtho fF .annosu swithi n6 month s Figure 1. Growtho f F. annosus in4 Norwa y spruces inoculated atdifferen t heights.

studied onwoo dblock s intes ttube s (Delatour& Sylvestre-Guinot, 1978). Cellulolytic capacity was studied following aclassica l method usedb y Eghbaltalab et al. (1976). Intersterility groupswer e defined following theKorhone nmetho d (1978). Results:Nearl y allth estrain sbelonge d toth eP intersterility group (No. 22 and2 9belonge d toth e Sgroup) , sotha tn ocompariso nbetwee n these 2differen t groups ispossible .Th e correlationbetwee n thegrowt h rateo fth e fungus inth etree s and the 3in-vitr o characterswa scalcu ­ lated.N o significant correlationwa s found eitherwit h decay capacity (r= -0.19 )o rwit h cellulolytic capacity (r= 0.01). Incontrast , asig ­ nificantnegativ e correlation existswit hmycelia l growth (r= -0.49) , whichmean s the strongest strains inth etree s generally growmor e slowly onmalt-aga r (Fig.2) .

DISCUSSION

Themos tconspicuou s result isth econnectio nbetwee n growth rateo f the fungus inth etree s ando nmalt-agar . Nevertheless some criticism exists.A ver y important question is:wha t reallywa smeasure d inth e stemsbetwee n 1973 and 1977? In facti ti sth e growth ofth edeca y columns afterhole sha dbee ndrille d inth e stemsi n

271 Mycelial growth of F. annosus on malt-agar (mm/day)

8.

0 25 50 75 Growthrat eo fF .annosu si nth etree s(cm/year )

Figure 2. Relationship betweengrowt hrat eo f F. annosus inth e stemo f Norway spruce andmycelia l growth onmalt-agar .Th e line isth eregression . Regression equation:y = -0.025 x +7.42 ;r = -0.49; levelo f significance < 0.01.

272 1973. Inthi swa y there arecertainl y introduced somedisorder s inth ein ­ ternalwoo d andthe n inth e fungus growth,bu ton edoesn' tkno w atal lth e extento fthem . Soi ti spossibl e thatth emeasure d growthrat eo fth e fungus isno texactl y the 'naturalgrowth ' inth e stems;bu ton e canexpec t anyerro r thatexist s isth e same fromon e tree to another.Furthermor e one may expecttha tove r a4 yea rperio d the trees overcame the disorders.I n spiteo fthes e restrictive remarks itma yb eemphasize d thatvariation s of the fungus itself are,a tleas tt o some extent,responsibl e forth evaria ­ tionso fdiseas e innature .Unfortunatel y invitr ovariatio no fth e fungus inthi s studywa s onlybase d onmycelia l growth onmalt-agar ,whic hi s quite aroug h character. Itcertainl y wouldb emor e interesting to observe correlationwit hmor eprecis ephysiologica l characters. Ina breedin gpro ­ jecti tappear snecessar yno tonl yt ocompar e thetree svs .on e individual straino f F. annosus, as iscurrentl y done,bu t also totak e into account theperformanc e ofth e fungusitself . As concerns liability ofth etree s toth e spread of F. annosus, some tentative hypotheses mayb eproposed .M y observations showtha t F. annosus probably doesno tgro wequall y fasti nal lpart s ofth eheartwoo d inNorwa y spruce.Generall y speaking, theslowes tgrowt h occurs atth e topo fth e stem;tha t isno t asurprisin g factan don ema y suppose theoretically there exists aheigh t limitt o F. annosus extension inth e stem atever ymomen t ofit s lifetime. Soon ema y conceive ofa Potentiall y Colonizable Column in theste mo fNorwa y spruce.Thi spotentia l column islikel yrelate d toth e existence of 'heartwood', and itsevolutio n depends onman y factors sucha s ecological,silvicultural , and genetic conditions.Moreove r thepotentia l column isno tnecessaril y homogeneous asregard s fungalgrowth : somepre ­ existing factors ini tma y adversely affectth e fungus such aswoo dstruc ­ ture, composition ofth e ligno-cellulosic compound, orpresenc e andchemi ­ calevolutio no fphenolics .Th enearb y living tissues (i.e.: sapwood, cambium)ma y alsopla y arol etha t isi nconnectio nwit h the current physiological status ofth etree . Ibeliev e thatthi sPotentiall y Colonizable Columnconcep tma yb euse ­ fult oconsider ,especiall ywhe n inoculatingyoun gtree s inwhic hth epo ­ tential column isprobabl y notye t largely developed. For instance,inocu ­ lations inside thepotentia l columno r above itma yno thav eth e samesig ­ nificance, asth eorigi no fth eresistanc e isprobabl y not the same.I tma y bepostulate d that inoculations forbreedin gpurpose s have tob eperforme d insideth ePotentiall y Colonizable Columnbecaus e iti sth enatura l areao f development ofth e fungus.Anothe r consequence ofthi sconcep t isth e fol­ lowing question: isi tutopi et o imagine thaton e canreduc e theheigh to f thepotentia l columnb y geneticmean s or silvicultural practices? Finally, another fieldo fdiscussio n isth e significance ofdat a col­ lected afterartificia l inoculations.Wha ti smeasure d inthi sway ?Whe n inoculating treesb y drillinghole s inth e stem iti swel l knowntha tdee p

273 perturbations occur within it. I believe that the growth of F. annosus measured by this method is really the growth in a traumatic area where resistance factors of the stem seem to have been weakened.-Indeed several unpublished observations show that the mycelial extent of F. annosus is at first large after inoculation and then decreases more or less constant­ ly; for example, I observed at 6 months the extent is greater than at 12 months; a large extension 3 years after inoculation then decreased till 5 years (Weissenberg, pers. commun. 1978). So the tree builds up its resis­ tance again. Possibly the phenomenon is different, depending on whether inoculation occurred in the Potential Column or outside it. Unfortunately the connection, if any exists, between'resistance measured by means of stem inoculations and that occurring in nature is not known at all. It would certainly be very important to study this con­ nection to determine the effectiveness and limits of breeding based on artificial inoculations. This knowledge will be achieved in a long term experiment only, by promoting natural infection of clones planted in an inoculum-managed area. Results may be compared then to those achieved in a stem inoculation ex­ periment.

REFERENCES

Courtois, H., 1972. Das Keimverhalten der Konidiosporen von Fomes annosus bei Einwirkung verschiedener Standortfaktoren. European Journal of Forest Pathology 2: 152-171. Delatour, C. & G. Sylvestre-Guinot, 1978. Microflore interne des tissus ligneux de l'épicéa commun sur pied. II - Etude in vitro de la microflore naturelle. Annales des Sciences Forestières 35: 285-298. Dimitri, L. & R. Kliefoth, 1978. Untersuchungen über die Widerstandsfähigkeit verschiedener Fichtenklone gegenüber dem F. annosus. Proceedings of the 5th International Conference on Problems of Root and Butt Rot in Conifers, Kassel, 1978. p. 85-93. Eghbaltalab, M., J.C. Debaud & G. Bruchet, 1976. Etude in vitro des activités pectinolytique et cellulolytique de Fomes annosus et de Rosellinia quercina. Bulletin de la Société Linéenne de Lyon 2: 85-96. Kaufmann, U. & H. Wellendorf, 1978. Genetic variation in Picea abies and Fomes annosus shown by growth of the fungus on wood dust. European Journal of Forest Pathology 8: 226-239. Korhonen, K., 1978. Intersterility groups of Heterobasidion annosum. Communicationes Instituti Forestalls Fenniae 94(5): 1-25. Shain, L., 1971. The response of sapwood of Norway spruce to infection by F. annosus. Phytopathology 61: 301-307. Weissenberg, K.v., 1975. Variation in relative resistance to spread of F. annosus in four clones of Picea abies. European Journal of Forest Pathology 5: 112-117.

274 Detection of disease resistance in Douglas-fir seedlings and variation in pathogenicity in Phellinus weirii by monitoring water stress after inoculation

Chas. H. Driver, D.R. Morse & R.L. Edmonds

College of Forest Resources, University of Washington, Seattle, Washington, USA

ABSTRACT

Natural resistance toeffect s ofdiseas e inDouglas-fi r seedlings inoculatedwit h Phellinus weirii was detectedb y water stress reactionmonitoring .A series of fourpattern s were discussed relative tomoistur e stress reactions exhibited by inoculated seedlings. Inaddition , itwa s demonstrated that a synthesized karyotype ofth epathoge nexhibite d virulence equal to thatexpresse db ynaturall y occurringwoo d decay tis­ sue isolate dikaryons.Homokaryon swer e found tob e oflowe r virulence thandikaryons .Thes e results indicate thati tshoul d bepossibl e todevelo p aprogra m forth edetectio n andbreedin g ofDouglas-fi r for improved natural resistance toth e serious rootro tpathoge n P. weirii.

PART I

Introduction Poria rootro to fDouglas-fi r (Pseudotsuga menziezii (Mirb.)Franco ) inducedb y Phellinus weirii (Murr.)Gilbertso n isa majo rproble m inth e intensivemanagemen t ofthi stre e species. Iti sestimate d thatth e disease results ina nannua l losso f 100 000 0m 3 ofDouglas-fi rwoo dvolum e inth e states ofWashingto n andOrego n inth eUS A (Childs& Shea , 1967). Such impacti sthough t toincreas e asth eyoun g growth forest standsbecom e cultured more intensively. Presentmethod s oflimitin gth e impacto fthi s diseasema yno tb e economically feasibleunde rmor e limitedmanagemen tpriorities .Th eus e of Douglas-fir seedlings,wit h improved natural disease resistance toth e pathogen, inartificiall y regenerating P. weirii infected siteswoul db ea sound silviculturalpractice . SinceDouglas-fi r isa nindigenou s treeo fth ePacifi cNorthwes t forest ofth eUnite d States and thepathoge n P. weirii isals o knownt ob enativ e toth e area,source s ofnatura l disease resistance shouldhav ebee ndeve -

275 lopedthroug hnatura l selection.T odate ,t oou rknowledge ,n ometho dha s beenreporte d forth e detection inDouglas-fi r seedlings ofnatura l disease resistance to infection anddiseas e effects inducedb y P. weirii. Therefore, aprogra m ofresearc hwa s initiated todetermin e ifseed ­ lings ofDouglas-fi r exhibited natural resistance to infectionb y P. weirii. A secondpar to fthi s effortwa s toinvestigat evariatio n inpatho ­ genicity ofhomokaryotic , dikaryotic andtissu e isolates ofthi shetero - thallicpathogen .A n initial step inth eprogra m was todevelo p ametho d of detection ofnatura l resistance inseedling s ofDouglas-fi r uponbein gin ­ fectedb y P. weirii. The following detailswil l describe the results of suchefforts .

Methods Rootvascula rpathoge n activities havebee ndemonstrate d to increase theresistanc e to flowo fwate r inth etranspirationa l stream inman y plants (Dimond, 1970;Helm s et al., 1971). Waring& Clar y (1967)demonstra ­ ted thatth e Scholanderpressur e chamber couldb euse d tomeasur e plant water stress inDouglas-fi r infectedb y P. weirii. Inadditio nRitchi e & Hinckley (1971)foun d thatneedle s instead oftwig s couldb euse dwit hthi s system formonitorin g seedlingwate r stress.Suc h techniqueswer euse dt o monitor seedlingwate r stress changes inou rresearch . The second step inth eprogra mwa s to accomplish astandar d pathogen exposure system to allow the expressiono fdiseas e resistance.A standard infectiontechniqu ewa s followed that incorporated themethod s ofWalli s & Reynolds (1962), Kuhlman (1969)an dNelso n (1972). Itconsiste d ofintrodu ­ cing a2 0t o2 5g infecte d red alderbloc k segment into seedling tissue woundedb y removing thebar k atth e rootcolla r area.Suc h aprocedure ,a s reportedb yNelso n (1972), resulted ina metho d alwaysyieldin gbette r than 60% initialinfection . Utilizing thesemethod s of seedling inoculation andmonitorin g ofwate r stress change asyste mwa s developed for following theresult s of infection byth epathoge n andhos tresistanc ereactions .

Results and discussion The following areth eresult swhe n some20 0randoml y selectednursery - run2-year-ol d Douglas-fir seedlingswer e tested forth eexpressio n of natural diseaseresistance . Seedlingswer e inoculatedwit hknow nkaryotyp e isolates of P. weirii. Using the Scholanderpressur e chamber,plan twate r stresswa smonitore d on secondyea rneedle s randomly selected from the leader ofeac htes tseed ­ ling.Pressur e chamber evaluationswer econducte d duringpredaw nmornin g hoursbeginnin g 1wee k after inoculation. Four generalpattern s ofmoistur e stresswer e detected. -N o abnormalwate r stresspatter nwa s exhibited different fromth econtro l

276 treatment seedlings.Thi swa s interpreted to indicate no infectionha d resulted from exposure toth epathogen . -Withi n 16day s after exposure toth epathoge n asignifican t increase in water stresswa s exhibited byman y seedlings (Fig.1) .B y2 2 daysman y seedlings exhibited over -30bar s (1ba r =0.98 5atm. , Slatyer, 1967)o f water stress andneve r recovered. Seedlings,whic h exhibited -30bar s reactions fora perio d of 10days ,wer e considered dead.Tissu e fromman y ofthes e seedlings yielded thepathoge nupo n isolation from sites distant from theplac e ofinoculation . - Some seedlings continued toexhibi t abnormally highwate r stressindica ­ tions for aperio d of 100t o20 0day s (Fig.2 )befor e exceeding the-3 0 barspoin to fn o recovery. Suchseedling s alsower e found toyiel d the pathogen on isolation.Thi styp e ofreactio n couldb e interpreted assom e indicationo frelativ e disease resistance.Furthe r work,however , isre ­ quired inthi srespect . -A smallnumbe r ofseedling s (7% )exhibite d amos t interesting water stress reaction (Fig.3) . A constantly higher-than-normalwate r stresswa s detected for aperio d of12 0t o24 0day sbefor e the seedlings returnt owha t appeared tob e

PLANT WATER STRESS [BAR] -40

-30 v' / / \ i -20 u -10- . . / A

—t— i ii i i—r—r 12 16 20 24 28 30 DAYS AFTER INOCULATION DAY OF INOCULATION Figure 1. Lethalplan twate r stresso frepresentativ e 2-year-old seedlings ofDouglas-fi r exhibiting typical disease reactions to infectionb y Phelli- nus weirii. The seedlingswer e inoculated witheithe r synthesized dikaryon isolatesD- lo rD-2 ,o r aDouglas-fi r decayedwoo d tissue isolate(DF) .

( )D 1-seedlingno .4 ; ( )D_-seedlin gno .3 ; ( )DF-seedlin gno .17 ; ( )averag e of2 0contro l seedlings.

277 PLANT WATER STRESS [BAR]

-40I

-30

-20

-10

" I ' ' '10' 20 30 ' '40 ' ' '50 ' 6080 120 160 200 240 DAY OF DAYS AFTER INOCULATION INOCULATION Figure 2. Latent, lethal plant water stress disease symptoms exhibited by 2-year-old Douglas-fir seedlings infected with Phellinus weirii. The seed­ lings were inoculated with either a synthesized dikaryon (D-l) isolate or a Douglas-fir decay tissue isolate (DE). ( ) D-.-seedling no. 13; ( ) DF-seedling no. 9; ( ) average of 20 control seedlings.

PLANT WATER STRESS [BAR] -401

-30

-20 rjjiÄ-...-..-.Ik . ;-i -10 •»axni-Mrti. 10 20 30 60 80 120 160 200 240 DAY OF DAYS AFTER INOCULATION INOCULATION

Figure 3. Water stress patterns typicalo f2-year-ol d seedlingso fDouglas - firexhibitin g natural resistancet o Phellinus weirii. Theseedling swer e inoculated with eithera synthesize d dikaryon isolate (D-2)o ra homokaryo n isolate (H-3). ( )D„-seedlin gno .11 ;( )H,-seedlin g no.4 ;( ) averageo f2 0contro lseedlings . comparablet oth econtro l seedlingswate rpotential .Thes e seedlingswer e exposedt oa secon d complete serieso finoculation s andwate r stressevalu ­ ations thatyielde d similar results.Mos to fthes e seedlings remained alive after1 yea ran dexhibite d vigorous growth. Such seedlingswer e thoughtt o exhibit some formo frelativ e disease resistance after exposuret o P. weirii. Inconclusio ni twa sdetermine dthat : -needl ewate r stressmonitorin g evaluationsca nb euse dt odetec tinfec ­ tiono fDouglas-fi r seedlings infectedb y P. weirii; -Douglas-fi r seedlings after inoculationwit h P. weirii thatexhibi t-3 0

278 barsneve r recover from theeffect s ofth edisease ; - and finally,Douglas-fi r seedlings afterbein g exposed toth epathoge n andexhibitin g thewate r stresspattern s ofFig .3 ar ethough tt odemon ­ strate some form ofnatura l disease resistance to P. weirii.

PART II

Introduction A second phase ofou rprogra m of investigationwa s to search forth e expression ofvariatio n inpathogenicit y by P. weirii. Utilizing the above described methods fordetectin g disease reactionsb y thehos ta serie so f investigations were conducted using knownkaryotyp e cultures ofth epatho ­ gen. Themajorit y ofth e infection centers inpresen tda yyoun g growth forest arethough tt ob e attributable toth evegetativ e persistence ofth e pathogen from thepreviou s stand's infected roots.T o daten o asexual spore stage isknow n and sexual spores haveno tbee ndemonstrate d tob e effective inth e spread ofth epathoge n (Childs, 1970). Awid e range ofmorphologica l variability hasbee nrecognize db yvari ­ ousworker s (Buckland et al., 1954;Clark , 1958;Childs ,1963 )studyin g vegetative cultures under laboratory conditions.Geneti c characteristics of thisprominen t forestpathogen ,however ,wer eno tdescribe d until recently (Gillette &Driver , 1974;Gillette ,1975 ;Drive r et al., 1979). These workers andHanse n (1979)hav e demonstrated P. weirii tob eheterothallic . Inaddition , dikaryotic cultureshav ebee ndemonstrate d to induce higher rateso fwhit e rottyp eo fwoo d decay thanhomokaryoti cculture s ofthi s pathogen (Charlermpongse, 1976). Inrespec tt o further culturalvariation ,Child s (1963)indicate d that isolates from decayed wood from trees from agive n infection centerwithi n a forest stand yielded asingl evegetativ e type,whic hwa s designated asa clone. Inaddition , itwa s found that2 differen t clones couldb e isolated, each from separate infection centers,withi n the same stand.Thes ewer e demonstrated tob e different clonesb y thedevelopmen t of adistinc tzon e reaction linebetwee n the different clones atth e contact areabetwee nth e mycelialmat swhe nculture d together ona plate . Since Childs' (1963) cultures were initially derived from infected tree tissue,i tcoul dprob ­ ablyb e assumed thatmos to fhi s cultureswer e dikaryotic,therefore , making genetic interpretation difficult. This limited review ofth eliteratur e establishes theexistenc e of cultural strains in P. weirii thatlogicall y lead topos e the questiono f theexistenc e ofvariabilit y inpathogenicity . Raabe (1967, andthi sbook ,pag e251 )showe d thatstrain s ofth eroo t rotinducin g fungus Armillaria mellea (Vahle x Fr.)Kumme r exhibited sig­ nificantvariatio n inpathogenicity . Inaddition ,Da y (1960)point sou t

279 thatheterokaryosi s often leadst ovariatio n inabilit y toattack-hos t species.Thes e indications for strainvariabilit y inpathogenicit y andth e in-depthwor k onbreedin g southernpin e species for-resistanc e to strains of Cronartium fusiforme Hedge.& Hun t (Snowe tal. , 1975,--Powerse t al., 1977; andthi sbook ,pag e 236 and427 )indicat e thenee d toconside r the potential for straindifference s in P. weirii forpathogenicity . Inthi s caseth eheterothalli c sexuality of P. weirii offered aninitia lopportun ­ ityt oconduc t suchstudies .

Methods Heterokaryotic strainswer e synthesized inth e laboratory andwer e demonstrated tob e compatible dikaryons (D-lan dD-2 )b y the abilityt o produce fertile fruitingbodie s inculture .Homokaryoti c strains (H-3an d H-5)wer e derived frommonospori c cultures,an dwer euse d toproduc eth e dikaryotic strain,D-l ,use d inthes estudies . Inadditio n tothes e knownkaryotyp e strains,tissu e isolateswer e obtained fromdecaye dwoo d from infectedDouglas-fi r (DF)an d fromwester n hemlock (WH) (Tsuga heterophylla (R.) Sarg.. )trees .Th e tissue isolates wereuse d to function asth ewild-type soccurrin g innature .Suc h isolates were usually found togiv e riset o fertile fruitingbodie s underlabora ­ tory cultural conditions andwer e therefore determined tob e compatible dikaryoticheterokaryons . Thethu s obtained cultureswer euse d toprepar e inoculum employed in the seedling infection and detection ofnatura l resistance techniques described inth epreviou spar to fthi spaper . To accomplish acomparativ epathogenicit y evaluation inducedb yth e described strains ofth epathoge n adiseas e rating systemwa s employed.Th e disease ratingswer ebase d onth e following seedling reactions afterinocu ­ lationwit hth eknow nkaryotyp e cultures of P. weirii. Disease ratingvalue swer e designated asfollows :

Pathogenicity Symptom characteristics rates

0 absence ofabnorma l growthconditions , 2 latentmortalit y (after 200 days), 3 averagemortalit y (approximately 120 days), 4 orrapi dmortalit y (before2 0 days).

Mortalitywa s indicatedwhe nseedling s exhibited above -30bar s of water stress fora tleas t1 0days .

Results and discussion These results (Table 1)indicate sn o statistically significantdiffer -

280 Table 1. Karyotype ofculture s of Phellinus weirii asrelate d to abilities toinduc e disease reactions inseedling s ofDouglas-fir . 0.0 =N o disease reactions,3. 6 =rapi dmortality .

Isolate Disease reaction ratings

Synthesized dikaryonD- l 3.6 Synthesized dikaryonD- 2 3.2 Douglas-fir tissue isolate (DF) 3.2 Westernhemloc k tissue isolate (WH)3. 1 HomokaryonH- 3 2.1 HomokaryonH- 5 0.0

ences inpathogenicit ybetwee nth esynthesize d dikaryons (D-lan dD-2 )an d tissue isolates (DFan d WH). Each ofth edikaryoti c isolates and the tissue isolates yielded significantly higher disease ratings thanth ehomokaryoti c isolates (H-3an d H-5). Inaddition ,i ti so finteres ttha tth e.homokaryo ­ tic isolateH- 3 was significantly morepathogeni c thanth eH- 5 isolate.Th e H-5 isolate failed toinduc e abnormalwate r stresspattern s in inoculated seedlings. Iti so fspecia l interestt onot e thatth esynthesize d dikaryon isolateD- l produced thehighes tdiseas e rating of all isolates tested, even including thenaturall y occurring tissue isolates.I nconclusion , it canb e stated that true differences inpathogenicit y canb e demonstrated between karyotypes of P. weirii. Inaddition , theresult s ofthes e initial trials indicate thati t shouldb epossibl e tomaintai n astandar d inoculum culture equal inpatho ­ genicity tonaturall y occurring strains;or ,a tleas tpossibl e tosynthe ­ size akaryotyp e to represent those combinations found innature . We feel thatthi s latter attribute offers arar e opportunity forth e plantbreede r of improved natural disease resistance in foresttree st o maintain astandar d inoculum ofknow npathogenicity . Inaddition ,i t appears that since P. weirii hasbee nreporte d tob e spread mainlyb y vegetative systems,littl eunexpecte d changes invirulenc e shouldoccur ; thereby offering assuranceo fmaintainin g established lines ofdependabl e disease resistance inartificiall y produced foresttre eprogeny .

REFERENCES

Buckland,D.C. ,A.C .Molna r& G.W .Wallis ,1954 .Yello wlaminate droo tro to fDouglas - fir.Can .J .Botan y32 :69-81 . Childs,T.W. , 1963.Pori aweiri iroo trot .Phytopatholog y 53:1123-1127 . Childs,T.W. , 1970.Laminate droo tro to fDouglasfi r inwester nOrego nan dWashington . PNWFores tRang eExper .Sta .USD AFor .Serv .Res .Pape rPNW-102 .

281 Childs,T.W .& K.R .Shea ,1967 .Annua llosse sfro mdiseas ei nPacifi cNorthwes tforest . USDAFor .Serv .Resource sBull .PNW-20 . Clark,J.W. , 1958.Th eoccurrenc ean dinvestigatio no fphysiologica lstrain so fPori a weiriiMurr .Ph.D .Thesis ,Yal eUniversity . Charlermpongse,A. ,1976 .A comparativ e studyo fth edecompositio ncapabilitie so fhomo - karyotic anddikaryoti cculture so fPhellinu sweiri io nDouglasfi rsapwood . M.S.Thesis .Univ .Wash .Seattle ,Wash . Day,P.R. , 1960.Variatio no fphytopathogeni c fungi.Ann .Rev .Microbiolog y14 :1-16 . Dimond,A.E. ,1970 .Biophysic san dbiochemistr y ofth evascula rwil tsyndrome .Ann . Rev.Phytopath .8 :301-322 . Driver,C.H. ,W.D .Gillett e& Yi-Me iKao ,1979 .Heterothallis mo fPhellinu sweiri iinflu ­ encedb ygene sa ton eo rmor eloci .Abst .Proceeding s52n dAnn .Meeting .Northwes t Sei.Assoc . Gillette,W.D. ,1975 .Biolog yo fPori aweirii :sexualit yan deffec to nheigh tgrowt ho f Douglas-firM.S .Thesis .Univ .o fWash .Seattle ,Wash . Gillette,W.D .& C.H .Driver ,1974 .Sporophor e formationb yPori aweiri ii nculture . Abst.Proceeding s66t hAnnua lMeetin go fth eAmer .Phytopath .Soc . Hansen,E.M. , 1979.Sexua lan dvegetativ e incompatibility reactions inPhellinu sweirii . Can.J .Botan y57 :1573-1578 . Helms,J.A. ,F.W .Cob bJr .& H.S .Whitney ,1971 .Effec to finfectio nb yVerticicladiell a wagneriio nth ephysiolog yo fPinu sponderosa .Phytopatholog y 61:920-925 . Kuhlman,E.G. ,1969 .Inoculatio no flobloll ypin eseedling swit hFome sannosu si nth e greenhouse.Can .J .Botan y47 :2079-2082 . Nelson,B.D. ,1972 .Effect so ffertilizatio nwit hure ao nroo tro tdisease so fDouglas - firan dwester nhemloc kcause db yPori aweiri ian dFome sannosus .M.S .Thesi sUniv . ofWash . Powers,H. ,F.R .Matthew s& L.D .Dwinell ,1977 .Evaluatio no fpathogeni cviabilit yo f Cronartium fusiformeo nlobloll ypin ei nsouther nUSA .Phytopatholog y 67:1403-1407 . Raabe,R.D. ,1967 .Variatio n inpathogenicit yan dvirulenc e inArmillari amellea .Phyto ­ pathology57 :73-75 . Ritchie,G.A .& T.M .Hinckley ,1971 .Evidenc e forerro ri npressure-bom bestimate so f stemxyle mpotentials .Ecolog y 52:534-536 . Slatyer,R.O. ,1967 .Plant-wate rrelationships .Academi cPress ,Ne wYork . Snow,G.A. ,A.J .Dinu s& A.G .Kais ,1975 .Variatio ni npathogenicit y ofdivers esource s ofCronartiu m fusiformeo nselecte dslas hpin e families.Phytopatholog y65 :170-175 . Wallis,G.W .& G .Reynolds ,1962 .Inoculatio no fDouglasfi r rootswit hPori aweirii . Can.J .Botan y40 :637-645 . Waring,R.H .& B.D .Cleary ,1967 .Plan tmoistur e stress:evaluatio nb ypressur ebomb . Science155 :1248-1254 .

282 Variability of host and pathogen in the pitch canker complex

L.D. Dwinell & J.B. Barrows-Broaddus

Principal Plant Pathologist and Plant Pathologist, U.S. Department of Agriculture, Forest Service, Southeastern Forest Experiment Station, Carlton Street, Athens, Georgia 30602, USA

ABSTRACT

Since 1974,ther e hasbee n considerable research onth e variability ofhos t andpathoge n inth epitc h cankercomplex . There istremendou svariatio n inth e susceptibility ofth e hosts andpathogenicit y ofth ecausa l fungus, F. moniliforme var. subglutinans. However, researchers haveonl ybegu nt o assessth eimpac ttha tenvironmen tha s onthi shost-'parasit e system.

INTRODUCTION

Pitch canker is animportan tdiseas e ofplante dpine s inth e Southern United States.Th ediseas ewa soriginall y attributed to Fusarium lateritium Nees emend. Snyd.& Hans . f. sp. pini Hepting (Snyder et al., 1949), but recentresearc h has demonstrated thatth ecausa l agenti s Fusarium monili­ forme Sheld.var . subglutinans Wr.& Reink . (Kuhlmane t al., 1978). This species of Fusarium occurs onman yhost s and inman y areas ofth eworl d (Booth, 1971). Inth epitc h canker complex,th eplasticit y ofth epathoge n andth e variability ofth ehost smak eth eproble m ofevaluatin g the host/parasite interactionextremel y difficult. Ina stud y of Fusarium moniliforme from corn, Leonian (1932)note d thateve nth emos tvigorou s strains exhibited theirpathogenicit y incycles ;a ton e timethe ywer e ablet o infectth e host, atanothe rtim e andunde ridentica l conditions they failed tod oso . Thepitc h canker fungus frequently demonstrates this same type ofvariabil ­ ity onpine .

VARIATION IN THE HOST

Several species ofpines ,includin g slash (Pinus elliottii Engelm.var . elliottii), southFlorid a slash (P. elliottii var. densa Little andDor - man), loblolly (P. taeda L.),Virgini a (P. virginiana Mill.), shortleaf (P.

283 echinata Mill.), longleaf (P. palustris Mill.)/ pitch (P. rigida Mill.), TableMountai n (P. pungens Lamb.), Monterey (P. radiata D.Don), scotch (P. sylvestris L.),easter nwhit e (P. strobus L.),pon d (P. serotina Michx.), and sand (P. clausa (Chapm.)Vasey) , havebee n reported ashost s of F. moniliforme var. subglutinans inth eUnite d States (Hepting, 1971;Heptin g & Roth, 1953). Pitchcanke rha s alsobee nreporte d onCuba npin e(P . occidentalis Sw.) i nHait i (Hepting& Roth , 1953). Ofthes epin especies , slashan d loblolly pines areth emos t important economically inth esouth ­ easternUnite d States. The incidence and symptomso fpitc hcanke r arehighl yvariabl e and depend onth e inherent susceptibility ofth epin e species andth eenviron ­ mental conditions inwhic h thetree s aregrowing . InFlorida , forexample , shootdiebac k isth eprincipa l symptom onplante d slashpine s (Dwinell & Phelps,1977 ;Phelp s& Chellman , 1976).However ,elsewher e inth eSouth , bole cankers areth epredominan t symptom ofth ediseas e on slashpines .I n slashpin e seed orchards,canker s are frequently associated with theus eo f tree shakers forcon eremoval .I nlobloll ypine ,pitc hcanke r israrel y observed inplantation s andnatura l stands.,bu tca ncaus e serious dieback insee d orchards,markedl y reducing seed crops'(Dwinelle t al., 1977). Bole andbranc h cankers areth e commonsymptom s onVirgini a pine,bot h insee d orchards andplantations .O neaster nwhit epine ,bol e cankers arenormall y found ontree splante d inurba nenvironment s outside the species'natura l range. To delineate the relative susceptibility ofsouther npines ,w e recently inoculated 7specie swit h4 isolates of F. moniliforme var. subglutinans. Based on shootmortalit y from girdling cankers,Virgini a pinewa s ranked as highly susceptible; slash, loblolly, shortleaf andpitc hpine swer e rated asmoderatel y susceptible; andpon d andeaster nwhit epine s ashighl y resistant.Th e isolatex pin e species interactionwa sno t statistically significant. Ina previou s study,utilizin g less quantitativeprocedures , shortleafpin ewa s rated ashighl y susceptible (Dwinell, 1978). Iti s apparent fromgreenhous e studies and fieldobservation s thatpin e species varymarkedl y inthei r susceptibility topitc hcanker . Withinpin e species,th e incidence ofpitc h is frequently related to thegeographi c source ofprovenanc e ofth ehost . In3 lobloll ypin e seed orchardso nth eCoasta lPlain , forexample , incidenceo fpitc hcanke ro n loblollypine swa shighe r onPiedmon ttha no nCoasta l Plain seed sources (Dwinell et al., 1977). InFlorida ,Blakesle e &Rockwoo d (1978)foun d that pine clones from central Floridawer emor e resistant thanclone s from other geographic areas. Individual clones alsovar y greatly inthei r susceptibility toinfec ­ tionb y F. moniliforme var. subglutinans. Thisphenomeno n hasbee nnote d for loblolly, slash, longleaf, shortleaf andVirgini apine s growni n southern seedorchard s (Dwinell et al., 1977;Phelp s& Chellman , 1976). In

284 aVirgini a pineprogen y test,Barnet t& Tho r (1978)als o found clonal variation inth e incidence ofpitc hcanker . Therema yb e sufficient geneticvariatio n inresistanc e toselec tan d breedpine s forcontro l ofth edisease .Open-pollinate d families ofslas h and loblolly pinevarie d considerably inthei r susceptibility to F. moni- liforme var. subglutinans (Dwinell& Barrows-Broaddus , 1979). Shootmortal ­ ity forth e 7slas hpin e families ranged from46-9 6% ,wit h amea no f8 0% . Recently, this studywa s expanded to include 43 open-pollinated familieso f slashpin e collected from trees innatura l stands inFlorida .O fth e4 3 families, 3wer e classed ashighl y susceptible,3 3 asmoderatel y suscep­ tible, and 7a shighl y resistant (unpublished data). These slashpin e families arecurrentl ybein g fieldteste d inFlorida .Fo r the 12 loblolly pine families,shoo tmortalit y ranged from 26-78% wit h amea no f 52% . However,ther ewa sn o correlationbetwee n therespons e ofth e families and the shootdiebac k ofth eparen tclone s (unpublished data). Inprogen y tests, stress,insects ,hos tnutrition , andphysiolog y willprobabl y have tob econsidered , alongwit h environmental factorswhic h influence the host:parasit e interaction.

VARIATION IN THE PATHOGEN

Although F. moniliforme var. subglutinans occurs on awid erang e of hosts and is found inman y areas ofth eworl d (Booth, 1971), notal l strains ofth e fungus arepathogeni c topines . Ina stud y onth epathogeni ­ city of F. moniliforme var. subglutinans fromnonpin e hosts,onl y isolates from Gladiolus corms grown inFlorid awer e capable ofinfectin g slashan d loblolly pines.Th eothe r 17isolate s from corn, Dracaena, sycamore,pecan , lily, Araucaria and Amaryllis were avirulent (Dwinell &Nelson , 1978). Instudie s on sources of inoculum,w ehav e isolated thevirulen tvar ­ iety of Fusarium from forestsoi l (Dwinell &Barrows , 1978), surfaces of pineneedles ,branche s andboles ,an d fromtree-shake r pads.Result s of pathogenicity tests on slash and loblollypine s indicate thatth e fungus occurs naturally inmixe dpopulation s of saprophytic andpathogeni c strains.Fo rexample ,5 5% ofth e isolates of F. moniliforme var. subglu­ tinans from seed orchard and forest soilswer epathogeni c on slashan d loblollypin e seedlings.Simila r results havebee nobtaine d with isolates frombar k andneedl ewashe s and fromtree-shake r pads (unpublished data). Isolates of F. moniliforme var. subglutinans areidentifie d asth epitc h canker strain ifthei rpathogenicit y is demonstrated. Withinpathogeni c populations ofth epin epitc hcanke r fungus,ther ei s considerable variation invirulence .However ,th evariatio n generally appears tob e among isolates rathertha n among geographic sources orpin e hostsource s of isolates (Barrows-Broaddus &Dwinell , 1979).

285 PATHOGENIC RACES

Most studies to date indicate that the relationship between the suscep­ tibility of the host and the pathogenicity of the causal ag'ent is additive (Barrows-Broaddus & Dwinell, 1979; Dwinell, 1978; Dwinell & Nelson, 1978; Dwinell & Barrows-Broaddus, 1979; Kuhlman et al., 1978). However, some exceptions have been noted. In a comparison of the pathogenicity of F. moniliforme var. svbglutinans on slash and loblolly pine, one isolate from loblolly pine in South Carolina was highly virulent on loblolly pine, but avirulent on slash pine (unpublished data). This condition may be caused by the racial difference that Hepting noted in 1971. Edwards (1935) reported pathogenic specialization in F. moniliforme var. subglutinans on corn in the New South Wales. There may also be pathogenic races of the pine pitch canker fungus.

REFERENCES

Barnett, P.E. &E . Thor, 1978. Effects of site and inheritance on Fusarium incidence in Virginia pine. Proceedings Symposium for management of pine of the interior South. (Knoxville, Tenn., Nov. 1978). USDA Forest Service Southeast Area State and Private Forestry Technical Publication SA-TP2. p. 159-161. Barrows-Broaddus, J.B. &L.D . Dwinell, 1979. Variation in virulence of diverse sources of Fusarium moniliforme var. subglutinans on Virginia and loblolly pine. Phytopathol­ ogy 69: 525 (abstract). Blakeslee, G.M. &D.L . Rockwood, 1978. Variation in resistance of slash pine to pitch canker caused by Fusarium moniliforme var. subglutinans. Phytopath. News 12: 207. (abstr.). Booth, C, 1971. The genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England. 237 p. Dwinell, L.D., 1978. Susceptibility of southern pines to infection by Fusarium monili­ forme var. subglutinans. Plant'Disease Reporter 62: 108-111. Dwinell, L.D. &J.B . Barrows, 1978. Recovery of the pine pitch canker fungus from pine plantation and seed orchard soil. Phytopathology News 12: 207. (abstract). Dwinell, L.D. &J.B . Barrows-Broaddus, 1979. Susceptibility of half-sib families of slash and loblolly pine to the pitch canker fungus, Fusarium moniliforme var. subglutinans. Phytopathology 69: 527. (abstract). Dwinell, L.D. &P.E . Nelson, 1978. Susceptibility of slash and loblolly pines to strains of Fusarium moniliforme and its variety subglutinans. Phytopath. News 12: 207. (kbstr.). Dwinell, L.D. &W.R . Phelps, 1977. Pitch canker of slash pine in Florida. Journal of Forestry 75: 488-489. Dwinell, L.D., P.L. Ryan &E.G . Kuhlman, 1977. Pitch canker of loblolly pine in seed orchards. Proceedings 14th Southern Forest Tree Improvement Conference (Gainesville, Florida, June 1977). p. 130-137. Edwards, E.T., 1935. Studies on Gibberella fujikuroi var. subglutinans the hitherto undescribed ascigerous state of Fusarium moniliforme var. subglutinans and on its pathogenicity on maize in New South Wales. Science Bulletin. N.S.W. 49. 68 p. Hepting, G.H., 1971. Disease of forest and shade trees of the United States. USDA Agricultural Handbook 386. 658 p. Hepting, G.H. &. E.R. Roth, 1953. Host relations and spread of the pine pitch canker disease. Phytopathology 43: 475. (abstract). Kuhlman, E.G., L.D. Dwinell, P.E. Nelson &C . Booth, 1978. Characterization of the Fusarium causing pitch canker of southern pines. Mycologia 70: 1131-1143. Leonian, L.H., 1932. The pathogenicity and the variability of Fusarium moniliforme from corn. West Virginia Agricultural Experiment Station Bulletin 248: 1-16. Phelps, W.R. &C.W . Chellman, 1976. Evaluation of "pitch canker" in Florida slash pine plantations and seed orchards, 1976. USDA Forest Service, State and Private Forestry, Southeastern Area. 22 p. Snyder, W.C., E.R. Toole &G.H . Hepting, 1949. Fusaria associated with mimosa wilt, sumac wilt, and pine pitch canker. Journal of Agricultural Research 78: 365-382.

286 Pest outbreaks as a function of variability in pests and plants

Nancy Lorimer

USDA-Forest Service, North Central Forest Experiment Station, 1992 Folwell Avenue, St. Paul, Minnesota 55108, USA

ABSTRACT

A model ispropose d thatdescribe spes toutbreak s asa functiono fvariabilit y inbot hplan t andpes tpopulations . Plantvariabilit y selects forpes tvariabilit y andpes tnumber s remain low.Environmenta l factorsma y reduce genetic andpheno - typicvariabilit y inplan tpopulation s sotha tth eplan tpopu - lationquickl y selects ahomogeneou s pestpopulation .Pes tnum ­ bers then increase rapidly. Crashes occurbecaus e lack ofvari ­ ability inth epes tpopulatio n increases itsvulnerabilit y to elements of itsenvironment , including itsow npredator s and parasites.Th emode l incorporates principles ofgenetics ,ecol ­ ogy, and evolutionary biology inth e attempt tounderstan d the economically andbiologicall y importantphenomeno n ofpes tout ­ breaks.

INTRODUCTION

The dynamical behavior ofpopulation s of some forest insectpest s (Malacosoma disstria Hübner, Choristoneura fumiferana (Clem.), Orygia pseudotsugata, Zeiraphera diniana Guén. and others)ca nb e characterized by quickchange s inpopulatio n size over time. Insectnumbers ,ver y lowfo r some time,suddenl y and rapidly increase,o routbreak .Th e outbreak stage continues for several yearsunti l thepopulatio n rapidly decreases,o r crashes.Th e outbreak stage is shorter thanth eperio d oftim e from crash tone woutbreak . Forest insects thatoutbrea k and crash also showpropertie s thatfa ­ cilitate rapid changes invariabilit y (Lorimer, 1979d). Therefore, Ipro ­ posed ina nearlie r paper thatchange s inpes tvariabilit y mayb e related tochange s inpes tpopulatio n size,wit hnatura l selectionplayin g anim ­ portant role (Lorimer, 1979d). Inthi spape r Iwis ht oexten d themode l of pestoutbreak s by adding athir dparameter ,variabilit y ofth eplan thos t populations. Some considerations for foresttre ebreedin g andmanagemen t emerge. 287 PEST AND HOST VARIABILITY

Populationvariabilit y isdefine d asth e following:th § space and time differences inpropertie s ortrait swithi n and among individuals inth e population. Spacerefer st odifference swithi npart s ofa nindividual ,an d differences from individual to individual over thephysica l areao fth e populationhabitation .Tim e refers todifference s within an individual's life cycle,an ddifference s from individual toprogeny . Factors thatdeter ­ mine differencesbetwee n individual x and individual y inpopulatio n Par e genotype, age,an denvironmen t duringdevelopment . The genetics of forest insects isbeginnin g tob e studied (e.g.Stehr , 1955, 1959;Harvey , 1957;Campbell ,1966 ;Morri s& Fulton , 1970;Morris , 1971; Maye t al., 1977;Lync h& Hoy ,1978 ;Stoc ke t al., 1979;Stoc k & Guenther, 1979;Lorimer , 1979a,b ,c) ,an d thepossibilit y ofgeneti c changes in forestinsec tpopulation s duringris ean ddeclin e isbein grec ­ ognized (Franz,1949 ;Baltensweiler , 1971;Lorimer , 1979d;Haukioja , 1980). When individuals from 2population s interact,lik eherbivore s and plants, orpest s and trees,variabilit y isdefine d interm s of individual differences inpropertie s ortrait stha taffect'th einteraction .Th eimpor ­ tantpropertie s ofplant s inthei r interactionwit hherbivore s ingenera l andwit h insectpest s inparticula r canb e divided into 2broa d classes:1 . properties thatsatisf ypes tneeds ,an d2 .defens eproperties .Propertie s inth e firstclas s include nutritional content (i.e.protein , aminoacids , vitamins, minerals), water content,shelter , and alarg e group ofothe r properties thatenabl e insects tosurviv e and reproduce.Defensiv eproper ­ ties areals oman y andvarie d andwoul d include secondary compoundproduc ­ tion andphysica l features likethornines s and leaftoughness . Plantpopulation s are alsovariabl e among individuals. Individualtree s x andy inpopulatio nP wil lno thav e identical amounts ofnutrient s (Shaw & Little,1977 )o r secondary compounds (Chew& Rodman , 1979)i nthei r foliage, forexample .No rwil l they have the same amounts ofthes ecom ­ pounds atdifferen ttime s (Seigler& Price , 1976).

VARIABILITY AND NATURAL SELECTION WITHIN THE INTERACTION

Consider aheterogeneou splan tpopulatio n inoppositio nt oa homogene ­ ousherbivor epopulatio n (Fig.1A) .Suppos e thateac hherbivor e can survive and reproduce successfully only onplant s that 'match'it ,meanin g plants thatmee tth eherbivore' s needs andwhos e defenses theherbivor e canover ­ come (Mattsone t al., page29 5 inthi s book). Thusy herbivore s can survive andreproduc e only onv plant s andno to nu andw plants .Th e offspring willb e y. Now consider the situationwher e theherbivor epopulatio n has increased invariabilit y (Fig. IB). Herbivore x survives andreproduce s onplan tu ,

288 T3 01 fi •H rt r-i a, x m

•P .s* c

X) fi m

4-1 a

o m fi o -r4 4-> 18 rH 3 ft O ft fi m ai

&) fi O -H -P u ai w -p fi D fi S» D ft 0) T>

>i (* Ü fi a)

m H 'O: Tfê' fi

Y w •H «ffV' > « 5~i •9 HSS/ Vs / \ x • •H ^^ /•< \ V / >. s \ (8 / N/ \ >

•-4 4-1 •^/^ N\ 3 4 S» » >s g, * © •c -S

289 butno to nplant sv andw . Similarly,y doe swel l onv ,bu tno to nu orw , and soon .Th eprogen y reflectth e successful reproductions.Hence ,hetero ­ geneity inth eplan tpopulatio n hasmaintaine d heterogeneity inth eherbi ­ vorepopulatio nb y aproces s of frequency dependent selection (Clarke, 1976). When theplan tpopulatio n ishomogeneou s (Fig. 1C),ther e israpi d selection forth eherbivor e genotypes thatgro w and reproduce successfully onthes eplants :i nou rexample ,herbivor ey . Thewhol e ofth enex tgenera ­ tionwil lb ey . Their offspringwil l show amarke d increase innumber s (Fig. ID). The increase inpes tnumber s has come aboutbecaus e ahomogene ­ ousplan tpopulatio n selected anadapted ,homogeneou s herbivore population. Theherbivore/plan t interactionwil lb egoverne db y ahig hdegre e ofuncer ­ tainty ifth eplan tpopulatio nmaintain s space andtim evariability . Vari­ ability keeps thenumbe r ofmatche s low.

DECLINING VARIABILITY IN THE PLANT POPULATION

The degree ofvariabilit y ina populatio n oftree s is firsta function ofth eorigina l geneticbase ,th evariabilit y ofth e founders ofth e stand. Apopulatio n established from ahomogeneou s seed source islikel y tob e lessvariabl e than apopulatio n from aheterogeneou s seed source.A spopu ­ lations age,variabilit yma yb ereduce d ifnatura l selectiono r thinning eliminates somemember s from thepopulation . Short-term geneticrecomposi ­ tion isimpossibl e forpopulation s of long-lived individuals. Phenotypic variability inplan tpopulation s ismor e changeable than geneticvariabilit y andma yb e a functiono f stress.Stresse sma y reduce thevariet y ofchemica l defensesproduce d inplant s (Rhoades,persona l communication).Environmenta l stresses like temperature changes and drought havebee ndocumente d asprecedin g insectoutbreaks .Bu trathe rtha naffect ­ ing the insects directly, these occurrencesma yb e affecting theinäect s indirectlyb y reducingvariabilit y inth etre epopulation . Stressesma y trigger simultaneousmetaboli c andphysiologica l responses among individual trees, thereby reducingvariability . For example,drough t stress decreased protein levels and increased levels of free amino acids in Eucalyptus prior topsylli d outbreaks (White, 1969). Although the data areno t available to ascertainbiochemica l vari­ ability among thetree sbefor e and after thedrough t stress stimulus,simi ­ larity ofrespons e is indicated. As asecon d example,occurrenc eo f flowering inbalsa m fir increases with the ageo fth e stand, and ispromote db y certainweathe rconditions , sotha tnearl y allo fth etree s flower atth e same time (Morris, 1971). Sprucebudwor m outbreaks havebee nknow nt ooccu r followingheav y flowering (Greenbank, 1963;Kimmons , 1971). Although flower feeding appeared to enhance larval development,nutritiona l value of flowerswa sno t alikel y

290 factor inth eoutbreak s (Greenbank, 1963). Inth e lighto fth e hypothesis proposed here,massiv e flowering represents synchrony ofrespons e to stress.Decreas e inth eplan tpopulation' s variability results inunifor m selection ofth epes tpopulation .Th epes tpopulatio n thenquickl yin ­ creases innumbe r (Fig. ID). Haukioja (1980)apparentl y observed adifferen tpatter n inoutbrea k areaso fth e autumnalmoth , Oporinia autumnata Bkh.o nbirch , Betula pubes- cens Ehrh., innorther n Finland,wher epoo r climatic and edaphic conditions prevail.Whil e thesepopulation smigh tb e subjectt omor e stresses than populations inmoderat e climates,interindividua l variance indefens ewa s noted asbein g 'high'.However , thedefensiv e characters wereno tspec ­ ified, andn o directcompariso n ofdefens evarianc ewit h other populations was supplied. The evidence forchange s invariabilit y ininsec t andplan t populations before outbreaks is sparse for several reasons. (1)Th e role ofth eplan t inth epopulatio n dynamics ofth eherbivor e has only recently beenrecog ­ nized anddocumented . With the realization thatth eplan t isno tmerel ya passivepartne r inth e interactionbu ta n active defender of itsow ntissu e hascom eth enecessit y tore-evaluat e former assumptions aboutth e factors controlling populationnumbers . (2)Entomologist s have tended toregar d variability asnoise .Lif e table studies have concentrated onmeans ,an d individuals inpopulation s were assumed tob enearl y identical. (3)Th e process ofnatura l selectionha sbee n investigated mostly on anevolution ­ ary,rathe r than anecological ,tim e scale.Th eproces s ofnatura l selec­ tion asa forc e inth erelationshi p between trophic levels hasno tbee n sufficiently recognized.

A CATASTROPHE APPROACH TO MODELLING PEST OUTBREAKS

Systemswit hvariable s thatcanno tb e averaged over time and space can bevisualize d withcatastroph emodel s (Thorn,1970) . Thus catastrophemodel s mayb eusefu l in solvingproblem s thatdea lwit hnon-linea r systems,pheno ­ mena involving sudden change,th ekin d ofproblem s that abound atth ein ­ terface ofgenetic s and ecology.Populatio n outbreaks exhibitth e 5qual ­ itieso fth e cusp catastrophe (Zeeman, 1976), soth emode l is applicable to this aspect ofpopulatio nbehavio r (Jones,1975) . The 5 features ofcus p catastrophes (Zeeman,1976 )share db youtbrea kphenomen a (Lorimer, 1979d) are (1)bimodalit yo fbehavior :population s atver y lowo rver yhig h levels, (2)sudde n jumpsbetwee nmodes :quic k increases innumbers ,o rout ­ breaks, and quick decreases innumbers ,o r crashes, (3)hysteresis :popu ­ lationcompositio nbefor e theoutbrea k dissimilar tocompositio n atth e end ofth ecrash , (4)inaccessibility : thepopulatio n seldom ata moderat e level, and (5)divergence : agraduall y changing force causing anabrup t change inbehavior .

291 vest variab'Uitu

Figure 2. Control surface (C)an dbehavio r surface (B)o fa cus pcatas ­ trophe ofpes toutbreak s andcrashes .Th e2 contro lvariable s arepes t variability andplan tvariability .Th ebehavio rvariabl e ispes tnumbers . Themode l isdescribe d inth etext .

Thecus p isdescribe db yth e functionx 4/4+ a x/ 2+ bx ,wher e aan db arecontro lparameters ,an dx i sth ebehavio rvariable . Ifplan t andpes t variability aremad e the 2contro l dimensions ina cus pmodel ,an dpes t numbers isth ebehavio r dimension, anoutbrea k andcras hcycl e canb e traced onbot hth ebehavio r surface (Fig.2B )an dth e control surface (Fig.2C) .A t (1),hig hvariabilit y inth eplan tpopulatio n and lowvari ­ ability inth epes tpopulatio n keep thepes tpopulatio n ata lo wlevel .A s variability inpes tpopulatio n increases,th e systempasse s intoan dou to f thebifurcatio n set,jumpin gu p to (2).Her evariabilit y islo wi nth e plantpopulation ,bu thig h inth einsec tpopulation , andpes tnumber s are high.Fro m (2)t o (3)insec tvariabilit y decreases,plan tvariabilit y remains low, andth epes tpopulation ,passin g through the other side ofth e bifurcation set,crashe s to aver y lowlevel . Why doesth epopulatio n crash?Th ehomogeneou s treepopulatio nha s selected forhomogeneit y inth epes tpopulation ,which , inturn ,make sth e pestsvulnerabl e tothei r enemies orth eweathe r orothe r factors inthei r environment.Th epopulatio n isn o longergeneticall ybuffere d against environmental influences.Secondly , inlarg enumber sth epest s are forced by density considerations tofee d onothe r species ofplant ,whic hwoul d

292 have the same effect as increasing the variability in the original plant population (Fig. 1A).

MANAGEMENT PRACTICES AND THE PEST OUTBREAK MODEL

Management practices will clearly affect a system represented by the model proposed here. In natural systems, diversity maintains dynamical equilibria (Browning, 1974), but agricultural pests encounter built-in homogeneity in food crops and the response is outbreak (Marshall, 1977). The same danger exists in forestry. Besides reducing genetic variability in stands, human-applied stresses could lead to reduction in phenotypic vari­ ability and then to pest outbreak. Presenting a homogeneous phenotype to pests promotes rapid selection and ultimate destruction of the tree crop.

ACKNOWLEDGEMENTS

I thank Drs. W.J. Mattson and R.A. Leary for helpful discussions, Drs. H.0. Batzer, F. Chew, E. Haukioja, M.F. Stock, J.C. Schultz, and conference participants for critical comments.

REFERENCES

Baltensweiler, W., 1971. The relevance of changes in the composition of larch bud moth populations for the dynamics of its numbers. In: P.J. den Boer &G.R . Gradwell (Eds): Dynamics of populations. Proceedings of the Advanced Study Institute on the Dynamics of Numbers in Populations. Oosterbeek, 1970. Pudoc, Wageningen, p. 208-219. Browning, J.A., 1974. Relevance of knowledge about natural ecosystems to development of pest management programs for agro-ecosystems. Proceedings of the American Phytopath- ological Society 1: 191-199. Campbell, I.M., 1966. Genetic variation related to survival in Lepidopteran species. In: H.D. Gerhold, E.J. Schreiner, R.E. McDermott &J. A Winieski (Eds): Breeding pest-resistant trees. Pergamon Press. Oxford, p. 129-135. Chew, F.S. &J.E . Rodman, 1979. Plant resources for chemical defense. In: G.A. Rosenthal & D.H. Janzen (Eds): Herbivores: Their interaction with secondary plant metabolites. Academic Press. N.Y. p. 271-307. Clark, B., 1976. The ecological genetics of host-parasite relationships. In: A.E.R. Taylor &R . Muller (Eds): Genetic aspects of host-parasite relationships. Symposia of the British Society of Parasitology 14: 87-103. Franz, J., 1949. Über die genetischen Grundlagen des Zusammenbruchs einer Massenver­ mehrung aus inneren Ursachen. Zeitschrift für Angewandte Entomologie 31: 228-260. Greenbank, D.O., 1963. Staminate flowers and the spruce budworm. In: R.F. Morris (Ed.): The dynamics of epidemic spruce budworm populations. Memoirs Entomological Society of Canada 31: 202-218. Harvey, G.T., 1957. The occurrence and nature of diapause-free development in the spruce budworm, Choristoneura fumiferana. Canadian Journal of Zoology 35: 549-572. Haukioja, E., 1980. On the role of plant defenses in the fluctuation of herbivore popu­ lations. Oikos 35: 202-213. Jones, D.D., 1975. The application of catastrophe theory to ecological systems. In: G.S. Innis (Ed.): New directions in the analysis of ecological systems. Simulation Councils Proceedings Series 5: 133-148. Kimmons, J.P., 1971. Variations in the foliar amino acid composition of flowering and non-flowering balsam fir and white spruce in relation to outbreaks of the spruce budworm. Canadian Journal of Zoology 49: 1005-1011. Lorimer, N., 1979a. Patterns of variation in some quantitative characters of Malacosoma

293 disstria (Lepidoptera:Lasiocampidae) .Annal so fth eEntomologica lSociet yo fAmeric a 72:275-280 . Lorimer,N. ,1979b .Genetic so fsuperoxid edisumtas ei nth efores tten tcaterpilla ran d otherorganisms .Journa lo fHeredit y70 :199-204 . Lorimer,N. ,1979c .Th egenetic so fmelanis m inMalacosom adisstri aHübne r (Lepidoptera: Lasiocampidae).Genetic s92 :555-561 . Lorimer,N. ,1979d .Geneti ccause so fpes tpopulatio noutbreak san dcrashes .In :M.A .Ho y & J.J.McKelvey ,Jr . (Eds): Geneticsi nrelatio nt oinsec tmanagement .Th eRocke ­ fellerFoundation ,p .50-54 . Lynch,C.B .& M.A .Hoy ,1978 .Diapaus ei nth egyp sYmoth :environmen tspecifi cmod eo f inheritance.Genetica lResearc h32 :129-133 . Marshall,D.R. , 1977.Th eadvantage san dhazard so fgeneti chomogeneity .In :P.R .Da y (Ed.):Th egeneti cbasi so fepidemic si nagriculture .Ne wYor kAcadem yo fSciences . p. 1-20. May,B. ,D.E .Leonar d& R.L .Vadas ,1977 .Electrophoreti cvariatio nan dsex-linkag ei n sprucebudworm .Journa lo fHeredit y68 :355-359 . Morris,R.F. ,1971 .Observe dan dsimulate dchange si ngeneti cqualit yi nnatura lpopula ­ tionso fHyphantri a cunea.Canadia nEntomologis t 103:893-906 . Morris,R.F .& W.C .Fulton ,1970 .Heritabilit y ofdiapaus eintensit yi nHyphantri a cuneaan dcorrelate dfitnes sresponses .Canadia nEntomologis t102 :927-938 . Seigler,D .& P.W .Price ,1976 .Secondar ycompound si nplants :primar yfunctions . AmericanNaturalis t110 :101-104 . Shaw,G.G .& C.H.A .Little ,1977 .Natura lvariatio ni nbalsa mfi rfolia rcomponent so f dietary importance tospruc ebudworm .Canadia nJourna lo fFores tResearc h7 :47-53 . Stehr,G. ,1955 .Brow nfemale ,a sex-linke dan dsex-limite dcharacter .Journa lo f Heredity46 :263-266 . Stehr,G. ,1959 .Hemolymp hpolymorphis m ina mot han dth enatur eo fsex-controlle d inheritance.Evolutio n13 :537-560 . Stock,M.W .& J.D .Guenther ,1979 .Isozym evariatio namon gmountai npin ebeetl e (Dendroctonusponderosae )population si nth ePacifi cNorthwest .Environmenta lEnto ­ mology8 :889-893 . Stock,M.W. ,G.B .Pitma n& J.D .Guenther ,1979 .Geneti cdifference sbetwee nDouglasfi r beetles (Dendroctonuspseudotsugae )fro mIdah oan dcoasta lOregon .Annal so fth e EntomologicalSociet yo fAmeric a72 :394-397 . Thorn,R. ,1970 .Topologica lmodel si nbiology .In :C.H .Waddingto n (Ed.):Toward sa theoreticalbiology ,Vol .3 .Aldine ,Chicago ,p .89-116 . White,T.C.R. ,1969 .A ninde xt omeasur eweather-induce dstres so ftree sassociate dwit h outbreakso fpsyllid si nAustralia .Ecolog y50 :905-909 . Zeeman,E.C. ,1976 .Catastroph etheory .Scientifi cAmerica n234 :65-83 .

294 Role of plant variability (trait vector dynamics and diversity) in plant/herbivore interactions1

William J. Mattson, Nancy Lorimer & Rolfe A. Leary

North Central Forest Experiment Station, 1992 Folwell Avenue, St. Paul, Minnesota 55108, USA

ABSTRACT

Plants and insects interact ina manne rprescribe d by their respective individual,population , and ecosystem levelproper ­ ties.A tth eindividua l level,a ninsect' spropertie s must match thoseo f itshos tplan tbecaus e insectperformanc ede ­ pends onth e goodness ofmatch .However ,th eplant' spropertie s areno tconstan tbu teve r changing,thereb y influencingth e quality ofmatc h andthu s insectperformance . Individual plants orthei rmodula r compartments eachpresen t ahighl y variable seto fpropertie s owing tothei r (a)rapi dphenologica l and ontogenetic change,an d (b)induce d change inrespons e to herbivory andvariation s inth eenvironment .W epropos e that allplants ,bu t especially long-lived ones (relative tothei r consumers)depen d uponrapi d changes orvariabilit y inthei r properties to thwartmatchin g withphytophagou s organisms.

INTRODUCTION

Each living organism mayb e characterized by thecollectio no fproper ­ ties or special traits itpossesses .However ,th e listo ftrait s assigned to anorganis m willvar y depending onth eorganis m whichperceive s it because eachobserver' s perception is afunctio n of itsow n seto ftrait s andn o twoorganism s have identical setso ftraits .Ultimatel y then,th e relationship between organisms or themanne r inwhic h they interacti s prescribedb y the seto ftrait s thateac hpossesses ,modified , ofcourse , byenvironment . Allplant/herbivor e coevolutionary argumentsbegi nwit h alarge ,im ­ portant assumption;namel y thatth eherbivor epopulatio n is somewhatpre - adapted toth e seto ftrait spossesse d by theplan tpopulation . Inothe r

1.Thi sresearc hwa ssupporte di npar tb yth eCanada-Unite dState s(CANUSA )Spruc eBud - wormsProgram ,USD AFores tService ,87 0Ree dRoad ,Broomall ,P A19008 .

295 words, theherbivor epopulatio nmus tconsis to f atleas ta fe w individuals which eachposses s ase to ftrait s that allow it to.interac twit h acorre ­ sponding seto ftrait spossesse d by somemember s ofth eplan tpopulation . Ifthi s isno tth e case,th eplan tpopulatio n isimmun e or incompatible withth eherbivor epopulation .Hence ,n ocoevolutionar y scenario isposs ­ ible.W e emphasize thatthi s isno ta trivia l assumptionbecaus e thematch ­ ing ofcorrespondin g plant andherbivor e traits is atth ehear to funder ­ standing thenatur e ofplant/herbivor e interactions.W epropos e thatth e variability inth e 'goodnesso f fit' ormatchin gbetwee n the trait setso f plant and animalpopulation s contributes significantly tothei r long-term coexistence, theirpopulatio n dynamics (Haukioja, 1980), andthei r evol­ ution. Traits arethos e individualpropertie s ofeac h individual organism that areimportan t init s interactions withmember s of itsow n and theothe r trophic level.Example s ofsom eplan ttrait s aregrowt h rate,phenolog y of development,composition s of amino-acids,carbohydrates , lipids,moisture , and allelochemics,physica l characteristics (toughness,hairiness ,etc.) , andassociation s withmember s ofothe rtrophi c levels such aspredator s and parasites ofherbivores .Example so f someherbivor e traits aregrowt h rates, timing ofemergenc e for feeding andmating , sensory apparata for detecting chemical and/or electromagnetic cues from thehos tplant , systems for sequestering or detoxifying harmful substances, and associationswit h members ofothe r trophic levels such asgu tmicro-organisms .

TRAIT VECTORS

Each trait (T.)ca nb emeasure d atsom e timet and assigned aparticu ­ larnumerica l value, (T-,t,). The reference totim e isexceedingl y import­ antbecaus e traitvalue s areno t invariant.Chang e occurs inrespons e to internal (i.e.spontaneous )an dexterna l (i.e.inducing )factors .Particu ­ lartrait sma yno tonl y change innumerica lvalue ,the yma yeve n submerge ordisappear ; justa sother sma y suddenly emerge or appear ina n individ­ ual's traitcollection . Suchchange s aredee p orqualitativ e changes in contrastt o ordinary orquantitativ e change (Bunge, 1977). Furthermore, eachtrai tma yhav e itsow ncharacteristi c rate ofchange ,an d sometimes the rates ofchang e ofsevera ltrait sma yb e correlatedbecaus e traits are notentirel y separate andindependen t (Gould& Lewontin , 1979). Traits are notnecessaril y independento fon eanothe rbot h inthei r origin and their effects onothe r organisms.Fo r example,severa l traitsma yb e linkedt o the same gene(s), and other traitsma yb e influenced bymor etha non egene . Moreover,w ewan tt o emphasize thatth e interactions between organisms depend onth ewhol e collection oftraits ,i.e . the seto ftrait spossesse d by eachorganism , notan y individual traits of andb ythemselves . Thus, thepropertie s ofeac hindividua lplan tan dherbivor e canb ede -

296 scribed by ase to ftrait s having specific traitvalues ;i nshort ,a trai t T T T T An y 2 vector [ T/t\< ?(t)' 3(t)'""'' n(tï^' individualswithi n apopula ­ tionca ndiffe rwit h respectt oth enumbe r oftrait s theyposse s aswel l as their specific traitvalues .A sw ehav e defined traits,som e are clearly pervasive,tha t is theywil l occur inth e traitvector s of allmember s of thepopulatio n (growthrates ,flushin gphenologies , etc.). Whereas,other s willno tnecessaril y occur inal l individual traitvectors .

FITNESS IN RELATION TO TRAIT MATCHING

Webeliev e thatth eoutcom e ofa particula r plant/herbivore interaction isa function ofth egoodnes s ofmatchin gbetwee n their respectivetraits . Anon-matc h occurs eitherwhe non eo rmor e ofth e instantaneous trait values (Act.)i nth eplant' s traitvecto r fall outside ofth e tolerance limits (Min.an dMax. ) forth ecorrespondin g traits ofth eherbivore ,o r when aplan tha s oneo rmor e traits forwhic h theherbivor e has nocorre ­ sponding traits.O n theothe rhand , amatc h occurswhe nth e instantaneous values for allth e individual traits of aplant' svecto r fallbetwee nth e tolerance limitvalue s forth ecorrespondin g individual traits ofth e herbivore.A matc h canvar y frompoo r togoo d depending onho wnea r the individual plant traits come toexceedin g thetoleranc e limits forth e corresponding herbivore traits.Fo r example,a splan ttrai tvalue s (Act.) approach the 'optimum'valu e (Opt.)o fth eherbivore' s corresponding trait tolerance range,th ematc hbecome s increasingly better.However , asth e instantaneous values forth eplan t traits (Act.)deviat e from the 'optimum' toth e limits (Min. andMax. )o fth eherbivore' s trait tolerance range,th e match becomes progressively worse.W e suggestth e followingmatchin g func­ tion:

n Opt• -Act . b. a *i 11 M. = I 1-k- Ifan donl y ifMin .S Act .S Max. , i=l Opt. otherwiseM ^„ j.= A0 3,m,•. t whereM - isth ematc hbetwee n herbivore,an dplan t atsom e time t;n ]/in t,t j * m ' isth etota l number oftrait s ofconcern , k. andb . aretrai t specific il ^ numerical constants and ai s anumerica l constant specific toth eparticu ­ larspecie s ofherbivor e andplant . Weconten d thatth ebette r thematc h (M. .)betwee n the herbivore v j,m,t' (H.)an dth eplan t (P ),th ehighe r the likelihood thatth eherbivor ewil l be able toexpres s its full reproductive potential (rpH.)an d therefore realized reproduction (rrH. )wil l bemaximal .O nth eothe rhand , apoo r matchbring s about theopposit e effect.Th e outcome ofth e interaction for theplan t isusuall y theopposit e oftha t forth eherbivore ;namel y plant realized reproduction (rrP •)approache s its reproductive potential (rpP ) 297 whenherbivor ematchin g isver ypoor .Th e likely relationbetwee nthes e variables is formalized inth e followingequations: .

t =deat hH . n 3 rrHj,m = rPHj (1"e'bƒ < Mj,m,t>

tQ =birt hH .

tn =deat hP m

rrPm,j = rPPm ^~e'C/ j dt

t0 =birt hP m

where E- t is anencounte r functionwhic h isa uni t step functiontha t hasvalu e onewhe nth eencounte r occurs and zerowher ether e isn oencoun ­ ter, andb and car enumerica lconstants . The equations explicitly consider the impacto fmatchin g "quality"ove r the entire life span (birtht odeath )o feac horganis m inth e determination ofrealize d reproduction. If,however , reprodu'ctionnormall y ceaseswel l before deathoccurs ,on ecoul d integrate thematchin g function (M. t)dt justu p totha tpoint ,unles s thepost-reproductiv e adultstil l contributes insom ewa y toth e survival of itsprogen y -henc e itsrealize d reproduc­ tion.Thes eequation s aremos teasil y interpreted whenbot hmember s ofth e interactionpai rhav e similar life spanso rgeneratio ntimes .However ,i f their life spans aredifferent ,the nth eevaluatio n and impacto fmatchin g becomemor e complicated because the longer-lived member outlives theinflu ­ ence ofth e shorter-lived member.T o adjust forthi s relational asymmetry, oneca nallo w the shorter-lived member toreproduc e (ifi tca n afterth e initial encounter)s oa st ocarr y the interactionthroug htim e toth een d ofth e life spano fth e longer-livedmember . Fitness (f)fo rbot h organisms ismerel y the realized reproduction (rr) ofeach ,ranke d relative totha to f allothe rmember s of itsow npopula ­ tion. Inothe rwords ,i ti ssimpl y relative realized reproduction (rrr). Thenotio n of fitness is importantbecaus e evolutionary theorypurport s thatthos eorganism swit hhighe r rrrwil l eventually supplantthos ewit h lower rrr.Give nenoug htime ,a populatio n should finally evolvet oconsis t of individualswhic hhav enearl y equal rrr. Inth ecas e of aplant/her ­ bivore coevolutionary scenario,th eultimat e survivors wouldb ethos e indi­ vidualswhos e traitvecto rmatchin g qualities andreproductiv e potentials resulted innearl y equal rrr.Fo r example,suc hplant smigh to nth eon e handb e individuals whichhav ehighl ymatchin g traitvector s andhig hrp , oro nth eothe rhan d individuals whichhav epoorl y matching traitvector s and lowrp .Thus , anorganism' s capacity to 'stayi nth egame' ,t ob e an effective competitorwit h itsconspecifics ,depend s onth e relationship

298 which existsbetwee n thematchin g quality of itstrai tvecto r and its reproductivepotential .Nex tw eexamin e changes inmatchin g quality of plant traitvectors .

TRAIT VECTOR DYNAMICS

Both the instantaneous values oftrait s andth emagnitud e andperiodic ­ ity ofthei rvariatio n are importantbecaus e the goodness ofmatchin gbe ­ tweenherbivore s andplant s has tob eevaluate d not justa t an instant,bu t over the entire life spano feac horganism . Thevariabilit y ofmatchin g is really aspecia l caseo f amor e general issue ofth evariabilit y orstabil ­ ityo f favorableness of ahabita t forparticula r occupants oftha thabita t (Southwood, 1977). Inthi s special case,w epropos e thatchang e inth e quality ofth eherbivor e habitat (i.e.plan ttrai tvectors )i sselecte d for as aconsequenc e ofherbivory .

Seasonal and ontogenetic change Perhaps themos t obvious (tohuman s anyway)change s inplan ttrai t vectors arethos e thatw e casually observe andrecogniz e asseasona l change:change s in leaf coloration, and leafmorphology , changes inwoodi - ness ofplan tparts ,frui tdevelopment , andth e like.Althoug h eachplan t has atrai tvecto r containing n individual traits,w ewil l focus onjus t2 ofthe m for amor e detailed examination ofseasona l change.Tw owhic h are extremely important toherbivore s are foliar organicnitroge n content (T,) andmoistur e content (T_). The trajectory forthes etrait s (Fig.1 )show s thatplant sbegi nwit hbot hhig hnitroge n andhig hmoistur e andthe n stead­ ilydecreas e over theduratio n ofth e growing season.Th epatter n isth e same forman y kinds ofplant s (Scriber& Slansky, 1981). The importance of such change forherbivore s becomes obviouswhe non eplot s theherbivore' s corresponding traitranges ,tha ti sth eminimu m (Min.)an dmaximu m (Max.) tolerable limits foreac hparticula rplan ttrai t (Fig.1) .Fo r example,w e know thatcertai n kinds ofherbivore s musthav ediet s containing at least 2.0 %nitroge n or survival iszero .O nth e otherhand , asplan tnitroge n levels approach theuppe rbounds ,aroun d 7.0 %,th e survival ofherbivore s likewise approaches zero,bu t fordifferen t reasons (Mattson, 1980). One canmak eth e same determinations formoistur e content.Th e resultwhe n plotted isth edomai no fplan tnitroge n andmoistur evalue s forwhic h herbivoryb y aparticula r herbivore ispossible . Itappear s asa rectangl e inou rtwo-dimensiona l plant state space. Ifth eplan ttrai t spacewer e instead n-dimensional,the nth eherbivore' s domainwoul db e ann-dimen - sional volume.W e call this rectangle orvolum e theherbivore' s 'window'i n theplant' s state space.Differen tkind s ofherbivore s have different windows andhence ,a splan ttrai tvector s changethroug h seasonaltime , plants canpas s inan d outo fth ewindow s ofdifferen tkind s ofherbivore s

299 T!—FOLIARN CONTEN T(% )

"WINDOW""O F r - n HERBIVOREZ- , V "WINDOW"O F HERBIVOREX Li V

..t.

30 405 06 07 08 09 0 30 405 06 07 08 09 0 To—FOLIAR MOISTURE CONTENT(% )

Figure 1. Seasonal change infolia rnitroge n (Trait1 )an d foliarmoistur e content (Trait2 )i nrelatio nt oth eminimu m andmaximu m tolerable limits ortrai trange s (TR.)fo rthes etrait sb y differentherbivores .Th e region delimitedb yth eherbivore' s traitrange s foral lplan ttrait s isth e herbivore's window onth eplant' strai tstat espace .Tim e (t^) isrepre ­ sentedparametrically .

(Fig. 1). Justa sther e istrai tvecto r changeove rseasona l time,ther e is similar changeove rth e lifespa no fth eorganisms ,i.e .ove r ontogenetic time.Particularl y importantar eth equalitativ e changes;i.e . theemerg ­ enceo fne wtrait s andth esubmergenc e ofol d ones.Thos ewhic h aremos t obvious (tohuma nbeings )ar eth eemergenc e ofreproductiv e traits during thecours eo fa norganism' s ontogeny such asthos e associatedwit hse x identification, andreproductiv e capacity.Othe r lessobviou schange s are those ina norganism' s immunity systems,growt hcapacities ,an d soon .

Induced change The facttha tplant s arehighl y responsive tothei r abiotic andbioti c environmentha s longbee nwel l known (Bradshaw, 1965). Forexample , iti s now commonknowledg e thatplant s react inman yway s towoundin gb ybioti c agents.Typica l changes are increased levels andkind s ofvariou s phenolic compounds,proteas e inhibitors,altere d levels ofphotosynthesi s andphyto -

300 synthateallocation , aswel l as altered phenology,morphology , electromag­ netic spectra,growt h rates,an d structural form orcomplexit y (Bradshaw, 1965; Knutson, 1979). Just assignifican t asth ebioti c inducing agents are the abiotic ones, e.g.moisture ,heat ,nutrient ,an d lightregimes .Change s inth e abiotic fluxca n elicit large,significan t changes ina plant' s traitvector .In ­ creasing levels ofnitrogen , forexample ,ofte nincreas egrowt hrate sa t theexpens e ofothe rprocesse s inth eplan ttha t compete forcarbo n and/or amino acidprecursors ,suc h asth eproductio no fpolyphenol s (Mattson, 1980). Inshort ,eac hplan tha s auniqu epotentia l toreac tt ovariou s kindso f inducing agentswit h theresul ttha t itha s auniqu e capacity to change its state or itstrai tvecto r (Bradshaw, 1965). Consequently, a plant's traitvecto rma y change in itsmatchin g relationship with anher ­ bivore. Ifmatchin g becomespoorer , thiswoul d be advantageousbecaus e it would increase theplant' s opportunity to fulfill itsreproductiv epoten ­ tial. On theothe rhand , some inducing factors could cause aplant' s trait vector tochang e inth e opposite direction,thereb ymakin g itbette rmatch ­ ing. Moreover,som e inducing factorsma y restrictth eplant' spotentia l to change, thereby 'locking' itont o arelativel y stable statewhic hma yb e more susceptible orhighl ymatchin g fora particula r population ofher ­ bivores. Similarly, various spontaneous ontogenetic changes inth eplan t may also induceplant s tomov e intomor e susceptible states orcaus e them to lose flexibility and thereby remain insusceptibl e states longer than usual. Ifon eexpresse s theduratio n ofplan t suitability (i.e.goo d matching) byD and theduratio n ofth eherbivor e lifecycl e asd , thenove rth e course ofevolutio nbetwee nplant s andherbivores ,on emigh texpec tD/ d to evolve toward values of 1. Inothe rwords ,on ewoul d expectth eplant st o evolve labiletrai tvector swhic hwoul dpermi tthe m toescap e from their 'adapted' consumers through timeb y quicklypassin g through theherbivore' s window, therebyminimizin g herbivore impacto nplan t fitness attainment.O n the otherhand ,becaus e herbivores themselves have acapacit y forchang e (cc), onemigh t expect thatplant/herbivor e coevolutionwoul d eventually result inbot h organisms evolving nearly equal capacities forchange : ccP/ccH =1 .The ynee dnot ,however ,emplo y the samebasi cmechanism s for generating change (e.g.,mutations ,recombination , traitvecto rdynamics , etc.).

TRAIT VECTOR DIVERSITY

Up tothi spoin tw ehav e emphasized thatth e susceptibility of anindi ­ vidual plantt o anherbivor epopulatio ndepend s onth epropertie s ortrai t vector oftha t individual with respectt oth e traitvector s ofth e individ­ ualherbivores ,al lwit hrespec tt o sometime ,t .Thus , ata give ntim et ,

301 aparticula rplan tma yb e apoo rmatc h fora ver y large fractiono fth e herbivorepopulation , andtherefor e isdefine d tobe.minimall ysusceptible . Astim epasses ,however ,no tonl y theplant' s traitvector, ,bu t alsoth e herbivorepopulation' s seto ftrai tvector s canchange .Th e 'direction' thatth eherbivor epopulatio n changes isparticularl y importantt oth e long term 'resistance'o fth eparticula r plant.Thi s direction is,o fcourse , duei npar tt obot hspontaneou s factors aswel l asinducin g factors inth e herbivore's environment.Especiall y important isth e selectionpressur e exerted onth eherbivore sb y theres to fth eplan tpopulation , i.e. the contemporaries ofth eparticula rplan t inquestion . Inth e shortter m an individual plant's susceptibility or resistance dependsprimaril y onit s ownparticula r properties,bu tove r the longer term (i.e.ove r successive generations ofth eherbivor epopulations ) its susceptibility depends also uponth epropertie s ofothe rplant s inth e samepopulatio nbecaus e they have amarke d influence onth edirectio no fevolutio n orth etrai tvecto r change inth eherbivor epopulation . Theeffec to f aplan tpopulatio n on an individual herbivore canb e viewed interm s ofth enumbe r ofdifferen tplan ttrai tvector s thata n individual herbivore encounters through attacks'i nit sgive n lifespan . When anherbivor e attacks aplant ,eac horganis m ischange d as aresul to f the interaction.Th e change ismor eo r lessuniqu e toth e specific trait vectors ofeac h interactingpair .Moreover ,th eorde r inwhic h different herbivores anddifferen tplant s interacti sals o importantbecaus e each different sequenceo fplant stha t aparticula r individual herbivore attacks hasuniqu e cumulative effects onth e survival and reproduction oftha t herbivore.Th eherbivore' s attack sequence,then ,reall y represents the cumulative selectionpressur e exertedb y theplan tpopulatio n ona nher ­ bivore. We canquantif y the totalpossibl e number ofdifferen t attack sequences or attackpathway s thatherbivore s could traceo r follow ina plan tpopula ­ tiongive n (1)th etota lnumbe r ofdifferen tplants ,tha ti sthos ehavin g differenttrai tvectors ,an d (2)th eusua l number ofplant s attacked per herbivore.Fo r example,i fth eplan tpopulatio n consists ofm different individuals, all ofwhic hhav e different traitvectors ,an d the herbivores normally attackn plant s each,the nth e totalpossibl enumbe r ofattac k sequences andhenc epossibl e different selectionpressure s facingher ­ bivores inth eparticula rpopulatio n ofplant s issimpl ym .I fth eentir e plantpopulatio n consistso fonl y3 differen ttrai tvector s sucha s (A,B ,C) ,an d the individual herbivore normally attacks only 2 individual 2 plants during its life span,the nther e areonl y 3 or 9potentia l attack sequences or selectionpathway s facing theherbivor epopulations : [AA,AB , AC, BA,BB ,BC ,CA ,CB ,CC] .O nth eothe rhand , asm increases thenumbe r ofpossibl e attack sequences increases exponentially. From theothe rdirec ­ tion, asth enumber s oftrai tvector s inth eplan tpopulatio n decrease to

302 approach one, that of the hypothetical resistant plant, the selection pressures facing herbivores become increasingly homogeneous and therefore convergent instead of divergent. In a nutshell, then, as the rest of the plant population becomes more and more like the 'minimally susceptible' plant, then more and more selection pressure will direct the herbivore population's evolution toward better and better matching with the here-to- fore minimally susceptible individual and hence with the plant population as a whole. This argument holds not only for within herbivore generations but also between generations. For example, if each individual herbivore attacks but one plant during its life span, it has the possibility of selecting any one of m different types in the plant population. Its progeny (let's say it produces a minimum of p of them) each also have the possibility of selec­ ting any one of the m different types of plants in the plant population. Therefore, over the course of n different generations there are rr differ­ ent attack sequences or pathways for a given herbivore's germ cell line. In conclusion, the type and intensity of interaction between plants and herbivores depends on the collective goodness of matching between the trait vectors of individuals comprising the plant and herbivore populations. Variations in the goodness of match depend on changes which can occur in the trait vectors of both plants and herbivores. Minimal goodness of match will occur when individual plants show high rates of trait vector change with respect to capacity for change in the herbivores, and high trait vector diversity among individuals. On the other hand, maximal goodness of match will occur when individual plants show low rates of trait vector change with respect to capacity for change in herbivores, and low trait vector diversity among individual plants.

REFERENCES

Bradshaw, A.D., 1965. Evolutionary significance of phenotypic plasticity in plants. Adv. in Genetics 13: 115-155. Bunge, M., 1977. Ontology I: The furniture of the world. Vol. 3 of Treatise on basic philosophy. D. Reidel Publishing Co., Boston. 352 p. Gould, S.J. & R.C. Lewontin, 1979. The spandrels of San Marco and the panglossian para­ digm: a critique of the adaptionist programme. Proc. R. Soc. Lond. B. 205: 147-164. Haukioja, E., 1980. On the role of plant defences in the fluctuation of herbivore populations. Oikos 35: 202-213. Knutson, D.M., 1979. How parasitic seed plants induce disease in other plants. In: J.G. Horsfall & E.B. Cowling (Eds): Plant disease. Vol. 4. Academic Press, New York/ London, p. 293-312. Mattson, W.J., 1980. Herbivory in relation to plant nitrogen content. Ann. Rev. Ecol. Syst. 11: 119-161. Scriber, J.M. & F. Slansky, Jr., 1981. The nutritional ecology of immature insects. Ann. Rev. Entomol. 26 (in press). Southwood, T.R.E., 1977. Habitat, the template for ecological strategies? J. Anim. Ecol. 46: 337-365.

303 Stability in a multi-component host-pathogen model

E.G. Bnttam

Botany Department, School of General Studies, Australian National University, P.O. Box 4, Canberra 2600, Australia

ABSTRACT

A4 componen tmode l of2 competin g organisms,eac hwit ha specific parasite,ha sbee nexercise d soa st o investigate its equilibria. Inadditio nt oth eequilibriu m towhic hth emode l was already known toretur n aftermoderat e disturbance, severe perturbation ledt oa ne w steady statewhic hha d the appearance of anequilibriu mbu twhic hwa snot'maintaine d inth e long term. Stability ofequilibri a andwha tconstitute s anequilib ­ rium are also discussed.

INTRODUCTION

In197 2Graha m Chilvers and I (Chilvers& Brittain , 1972)publishe d a model for asyste m of2 specie s incompetitio nwit heac hothe r andeac h subjectt o attackb y 1host-specifi c parasite.W epresente d thismode l in general termsbu t in facti ndevelopin g itw e thought interm s of2 com ­ peting andclosel y related trees,eac h subjected to aspecifi c leaf spot fungus disease,s otha t theequation suse d inth emode l are intended tob e appropriate fortha tparticula r system. Inth ecours e ofdiscussin g thismodel ,w e asserted thati twa s stable inth e sensetha t for agive n seto fparameters ,i twoul d returnth e amounts ofth e4 component s toth e same level from almost any seto f initialvalues . Since 1972ther ehav ebee n anumbe r ofpaper swhic hhav ebee n concerned with stabilityo fmulti-componen t systems.Mos to fthes ehav ebee nmathe ­ matical inpresentatio n andhav ebee nconcerne d withseekin g analytical methodswit hwhic h onemigh tpredic twhethe r agive nmode l shouldb e stable ornot .Discussin g theproble m from rather adifferen t standpoint.Ma y (1977)ha s considered thepossibilit y thatnatura lmulti-specie s assemblies ofplant s and animals and alsomodel s ofthem , are likely toposses s sever­ aldifferen tequilibriu mpoints .Th epossibilit y thata necosyste mma y possessmor e than one stable equilibrium iso fconsiderabl e interestbe -

304 cause itraise s the thought thatth e familiar stable state ofth e system may have some degree of instability, sufficient thati tmay , ifperturbe d sufficiently change to some alternative andperhap sver yunfamilia r equi­ librium. Forthes e reasons, Ihav erecentl y made aneffor tt oexercis e our 1972 modelmor evigorousl y thanbefor e inorde r totes tmor e stringently our contention thati tha d only oneequilibriu m point (Brittain, 1980). The equationswhic h constitute the4 componen tmode l arese tou tbelow . Id ono tpropos e todiscus s thedetail s ofthes enow .Th e assumptions made and theargument s used inderivin g themode l are setou ti n full inth e original paper.Fo r thepurpose s ofth epresen twork , themode l hasbee n implemented inACS Lo n aUniva c 1100/42 computer atth eAustralia n National University (ACSL -Advance d Continuous SimulationLanguage .Mitchel l and Gauthier Associates Inc.P.O . Box 685Concord , Mass. 01742). I shouldmentio n that inth emodel ,th e environment isconceive d tob e capable of supporting alimitin gquantit y ofbiot at owhic hw e ascribed the value ofunity , sotha tth e amounts ofeac ho fth ecomponent s asthe yvar y within the environment arecorrectl y described asproportions .However ,I propose loosely tocal l these quantities, sinceproportion s of'components ina multi-componen t system has anothermeaning .

MODEL EQUATIONS

HostX : dX/dt= R XX(1-(X+ Y) )- (Dxx +D nX- D xX-DnX) (i)

HostY : dY/dt= R yY(l-(X+ Y) )- (Dyy +D nY -D yyDnY) (ii)

Parasite x: dx/dt = R x(X-x) (l-x(A x+A Y)) - (D x+D x-D D x2) (iii) A y\y A n x n Parasite y: 2 dy/dt= R yy(Y-y)(l-y(A yy+AxX))- (Dyy+Dny-DyDny ) (iv)

Dead tissuep : 2 dp/dt= R xx(X-x)(l-x(A xx+AyY))- (Dxx+Dnx-DxDnx ) (v)

WhereX =amoun to fon ehos t Y = amounto f second host x =amoun to fX specific parasite y =amoun to fY specific parasite

RX,R ,R ,R =growt h constants Dx , Dy =deat hrat e constants forparasite s

305 D =deat h rate duet oothe r causes

Ax,Ay = absorption constants for collection of inoculum p =amoun to fdea dtissue .

EXERCISING THE MODEL

Themode lwa s firstru nusin g theparameter s and initial amountso fth e 4 components (Table 1)unti l the steady statewa s reached. Ishal l refert o this asth e firststead y state.Th evalue s ofth e statevariable s atthi s point are incolum n 2o fTabl e 2. Perturbation ofthi s firstequilibriu mb y reduction ofeithe rhos twit h its attendantparasit e from theequilibriu m amount toits 'initia l amount resultedpredictabl y ina temporar y upsurge ofth eothe r host-parasite pair duet oreductio no f competition, followedb y recovery ofth eperturbe d pair and, after some strongly damped oscillations,retur nt oth e firststead y state.However ,whe nth eperturbatio nwa smor e extreme,a swhe n Ireduce d onehos tt o1/1 0 of itsequilibriu m value,somethin g rather different occurred.Remova l of 1hos t and itsparasit e totha t low level allowed the otherpai r to surgeupward s asbefore .The y reached ane w steady level 120 timestep s afterth eperturbation .Thi sne wleve lwa smaintaine d fora further 880tim e steps.I shal l refer tothi s asth e second steadystate . Only after 1000tim e stepsha d elapsed sinceperturbatio n did theperturbe d component reappear ina namoun t sufficient tob e distinguished fromzero . Thenafte r aperio d ofmor e or lessviolen toscillation ,th e firststead y statewa s restored. Perturbation ofth e firstequilibriu m by reduction of 1parasit e to much smallerproportions , inal lcase s resulted ina temporar y upsurge in thecorrespondin g hostwhic hwa sthu srelieve d ofit sparasit e load.How ­ ever theparasit e always camebac k againbefor e anyequilibriu m couldb e attained sotha t after afe woscillations ,th e firststead y statewa sre ­ stored.Onl yb y reducing aparasit e exactly to zero could any other equi­ libriumb e demonstrated.

Table 1. Initial statevariabl e andparamete rvalues .

Statevariable s Growthconstant s Otherrat e factors

X 0.01 R 0.04 D 0 x X 014 Y 0.01 R,,R 0.03v 5 Dy° 014 x 0.0005 R 0.18 Dn 0 009 y 0.0005 R 0.19 AY 0 05 •* y Ayo 05

306 Table 2. Valueso fstat evariable s inth e firstan d second steadystates .

Statevariabl e Steady state

Xi = 0.01,Y i = 0.01 X 0.3353 Y 0.2243 X 0.2072 y 0.1028

Xi = 0.01,Y i = 0.4 x10" 8 X 0.5137 Y 0.25 x1 0 X 0.3853 -14 y 0.46 x1 0

Xi = 0.410" 8, Yi= 0.01 X 0.21 x1 0 Y 0.4516 •14 X 0.62 x1 0 y 0.3301

MODIFICATION OF THE MODEL

May (1977)ha spresente d curves forth e rate ofchang e ofvegetatio n biomassplotte d againstth e amounto ftha tbiomass .Th ecurv e appears tob e parabolic,whic h is anatura l consequenceo fplottin g the first derivative ofdat a for asymmetrica l sigmoid growth curve inthi sway .H eha s super­ imposed onthi scurve ,a se to fothe rcurve s forrat eo fremova l ofvege ­ tationbiomas sb yvariou s fixed levels ofgrazing .Thes e interceptth e parabolic curve ateithe r 1o r3 points .A teac h intersection, grazing removesvegetatio n biomass atth e same rate asi ti sproduce d sotha ta n equilibrium appears tob e indicated.Ma yuse s thisdiagra m asa mean s of emphasising hispoin ttha tmulti-componen t systemsma yposses s multiple equilibria. Itoccurre d tom e thati twoul db e fairlyeas yt omodif y ourmode l and arrange itsoutpu t insuc h awa y ast oresembl eMay' s diagrams and sot o checkwhethe rw e infac thav e thepossibilit y ofmultipl e equilibria inou r model. Inorde r tosimulat e this situation itwa s firstnecessar y toreduc e it to2 component s and thatwa s doneb y setting theothe rhost-parasit e pair tozero .The n inorde r toobtai n afixe d level ofparasitis m to compare

307 withMay' s fixed level ofherbivory , Ibrok e the feed-back linkbetwee n parasite andhost .Thi swa s readily doneb y changing,th e lefthan d sideo f equation (iii)fro mdx/d tt odp/dt .Th e initialvalu e ofx , amounto f X-specific parasite,whic h appears inequatio n (i)wa s then retained asa fixedvalue .W e thusha d a fixed amounto fparasit e reactingwit h agrowin g host.Th eproduc to fthi s interaction,whic h Icalle dp ,ma yb e thoughto f asdea d tissue,neithe r livinghos tno r infective parasite.Th erat eo f production ofp wa s thenequivalen t toth e rate ofremova l ofhost . A seto fvalue s fordifferen t fixed amounts ofparasit ewa sinvestigat ­ ed.These , Ihav e not superimposed onth e 1paraboli c curve forth e amount ofhos tbecaus e the amounto fparasit epresen t itselfha s aneffec to nth e hostcurve .Therefor e Ihav epresente d them separately. Apai r ofcurve s foreac ho f4 differen t levels ofparasit e is showni n Fig. 1.Th emode loperate s dynamically sotha t itactuall ybegin s atth e leftwit h asmal l amounto fhos tX andthe nprogresse s toth eright .Even -

RATEO F GROWTH

.002.

0 .2 .4 .6 0 .2 A AMOUNTO FHOS T Figure 1. Rate ofgrowt h ofhos t (0)an drat e ofproductio n ofdea d tissue (A)plotte d asa function ofth e amounto fhos tpresen t ina 2 componen t model. Fourdifferen t fixed amounts ofparasit ewer eused : a0.01 ,b 0.10, c 0.20, d 0.30.

308 tually thehos tcurv epasse s through anintersectio nwit h thecurv e forth e rateo f formationo fdea d tissue,p , andthe ni tcontinue s to decrease alongth e descending limb ofth eapproximatel y parabolic course.Remova l of hosttissu e todea d tissue alsocontinue spas tth e intersection and the2 curves crossbu tpresentl y come together again atwha t Ia mcallin ga confluence.A tthi spoin t therate s ofincreas e ofhos t and ofdea d tissue arevirtuall y zero andw ehav ewha t Ibeliev e tob e theonl y equilibrium for thesystem . Inn o casewa s there evermor e than 1crossove rpoint , contrary toth econtentio no fMa y thatther ecoul db e3 .Furthermor e the crossoverpoin twa sneve r anequilibrium . Ihav e checked that deliberately by settingth e quantities tothos eobtainin g atth ecrossove rbu tth emode l thenmove d away fromtha t toth econfluence . Atth ecrossove r points the2 firs tdérivâte s areequa l butnonzero . Put anotherway ,th e rate ofremova l ofhos tequal s therat eo fgrowt ho f hostbu tneithe r iszero .Ye tthes epoint s areno tequilibria .The yar e not,becaus e although therate s areequa l they ares oonl ybriefly .Bot h arechangin gmor eo r lessrapidl y sotha tthei rcourse s intersectan d continuebeyon d the intersection. Ius e the term confluence todescrib e thecircumstanc e where the2 curves approach eachothe r and come to anen dwhil e coinciding. Theyd o thisonl ywhe nbot h thene trat eo fgrowt h ofhos t andth ene trat eo f production ofdea d tissuebecom ezero .

EQUILIBRIA AND STABILITY

Ino wwan tt odiscus s asimpl ephysica l model.Conside r acubi cpris m atres to non eo f its faces ona stationar yhorizonta lplane .W ema ycer ­ tainly saytha tth ecub ei s inequilibrium , isi na stead y state andi s stable.Th e cube canb eperturbe d by tilting it.Becaus e iti shighl y stable itwil l returnt o itsorigina l state fromquit e largeperturbations . However ifth e limito f itsequilibriu m isexceede d itwil l assume asecon d steady state,differen t from the first.W ekno w thati tca n assume in all6 suchstead y states,restin g oneac ho f its 6 faces.Thu sth e cube isa system whichha smultipl e equilibria orstead y states ineac ho fwhic h it ishighl y stable.Th ecub e infac tpossesse s 20othe rpossibl e equilibrium positions which areunstabl e states,1 2o nth e edges and 8o nth eapices ; positions inwhic h itmigh tb ebalance d ifon eha d sufficientpatienc e and skill.Give n astead y environment, acub ebalance d inan yo fthos e2 0 unstableposition s mightb e saidt ob e in astead y state ori n equilibrium. But itwoul db ehighl y unstable,th eslightes tperturbatio n causing itt o revertt oon eo f itsmor e stablepositions . Thepoin t Iwan t tomak ewit h this simple illustration istha tstabili ­ ty isquantitativ e and thatequilibri a and steady states existwhic hhav e varyingdegree so fstability .

309 Inou r 1972paper ,Chilver s and Istate d that 'theequilibri aeventual ­ lyarrive d atar etypica l ofth erat e constants used inth emode lbu tar e quite independent ofth e startingproportion s ...o fth e components'.Thi s statement istru eonl y ifth emode lha s auniqu e equilibriumposition . Inth epresen twor k some quite extremeperturbation s wereuse d and it was found that someo fthes eproduce d ane w statewhic h appeared tob e steady fora significan t length oftime .Thi s second steady statewa s virtually indistinguishable from thatwhic h the system assumeswhe non eo f thehost s is reduced exactly tozero , fromwhic h ofcours eneithe r itno r itsparasit e caneve r recover.Howeve r slightchange swer e occurring,belo w the level ofresolutio n ofgraphi c outputbu tclearl yvisibl e inth enumer ­ ical results.Thes eultimatel y ledt o aretur n fromth esecon d statet oth e original equilibrium. Iti s interesting toconside r whether thatsecon d state,whic h canb e seen tohav ebee n transitory, should be called anequi ­ librium.Probabl y adecisio n onthat-woul d depend onone' s owntim escale . Ifthi swer e area l system andth e time stepswer eyears ,I believ ew e should allb ehapp y tocal l ita nequilibriu m ifw e lived atan ytim e between aboutyea r 200 andyea r 800.Ye t inth emode l situationwit h an arbitrary time scale,w e seetha ti ti sno t afina l equilibrium. Onemigh tsa ytha tther e isa ris ktha ta stead y stateo fsuc hlon g durationmigh tb emistake n fora nequilibrium . Alternatively itmigh tb e suggested that astead y statedeserve s tob e called anequilibriu m ifi t endures for longenough . Ineithe r case thecrucia lpoin twoul d involvea subjective decision aboutth e lengtho fth e apparent steady state.Th e dictionarymeanin g of 'equilibrium' is 'stateo fbalance 'wit hn oqualifi ­ cation ast oth e duration ofthi s state.Ye t there seemst ob e atendenc y inth emodellin g literature toreserv e theter m for situationswhic h have some elemento fpermanency . Certainly ifthi swer eno tdone ,i fth eter m couldb euse d for any fleeting condition, itwoul d notb e ofmuc hvalue .

CONCLUSIONS

Ihav e argued that forth einteractin gpair s ofrat ecurve so fFig .1 iti sno tsufficien t forth e ratest ob eequa l for anequilibriu m tob e defined. Iti s alsonecessar y thatth ene trate so fchang eb ezero .Equi ­ librium occurswhe n thequantit y ofa substrat e andth e quantity ofit s productbecom e stationary,whic h ist o saytha tthei r rateso fchang e becomezero . Whether orno t asyste m or amode l systempossesse smultipl e equilibria depends atleas tt o some extento nth e definition ofequilibrium . Ihav e concluded thata necessar y condition forequilibriu m istha tth e first derivatives ofth e amounts ofth e components shouldb ezero . Thismus tb eregarde d asa nidea l state fori nan yrea l system oreve n ina compute rmode l ifi ti soperate d withinrealisti c constraints oftim e

310 and cost, exactly zero rates of change seldom occur. Rather they may seem to approximate zero. Whether they will not continue to remain near zero or tend away from it again may only be resolved if a great deal of observation time is available. Meanwhile one may speak of the existence of an equilib­ rium although with the reservation that this may be transitory on some other time scale.

REFERENCES

Brittain, E.G., 1980. Equilibria in a model system of four species (in preparation). Chilvers, G.A. &E.G . Brittain, 1972. Plant competition mediated by host-specific para­ sites - a simple model. Australian Journal of Biological Science 25: 749-756. May, R.M., 1977. Thresholds and breakpoints in ecosystems with a multiplicity of stable states. Nature 269: 471-477.

311 Selection in host-parasite systems

D. Krusche

Bundesforschungsanstalt fürForst -un dHolzwirtschaf tHamburg ,Institu tfü rForstgeneti k undForstpflanzenzüchtung ,Siekerlandstr .2 ,D 207 0Grosshansdor f2 ,BR D

ABSTRACT

Amathematica lmode l for anatura lhost-parasit e system with genetic coupling is"developed.Th e geneticvariance s and theheritabilit y needed forpredictin g genetic gain inth e course of aselectio nprogramm e aredependen t onth eactua l genetic state ofth eparasit epopulation .Th e results suggest thatwithou t detailed knowledge of.th eevolutionar y stateo f the system, genetic improvementb y artificial selectionma yno t besuccessful .

INTRODUCTION

Inforestr y awidel y acceptedmetho d forbreedin g forresistanc e to fungal diseases is startedb y selecting someparen ttree swit h lowinfec ­ tionwithi n aheavil y infected area.Controlle d crosseswit h several tester trees aremad e andth eprogenie s aregrow n in field tests andobserve d for symptoms after artificial infection (Becker& Marsden , 1972;Bingha m et al., 1969). Themeasure d variation isattribute d to several sources,'suc h asadditiv e geneticvariance ,dominanc evariance ,etc. ,an dth eheritabili - ties andgeneti c gains arecalculated . An assumptionunderlyin g thisbreed ­ ingmetho d istha tth egeneti cbackgroun d ofth edisease-producin g parasite population doesno tchang e over time.Bu tthi s assumption isunrealistic , especially inreferenc e to foresttree swhos eparasit epopulation sma y reproduceu p to several hundred timeswithi n onehos tgeneration .Thu s therecoul db e aconsiderabl e change inth egeneti ccompositio n ofth e parasitepopulation .Breedin g fordiseas e resistance differs therefore ina fundamentalwa y frombreedin g forcharacter s such as frostresistanc e ora high growth rate. Inthi spape r Ipresen t amode l of ahost-parasit e system.Th e analysis showsho w artificial mass selection acts onth edynami cbehaviou r ofth e system. The dependence ofheritabilit y and genetic gaino fth e character 'infectiondensity ' onth etim e and stateo fth e system isdemonstrated .

312 Even though thismode lma yn o correspond indetai l to anyrea lhost - parasite system, itaim st oclarif y the inherentdynamic s andma y stimulate discussion inorde r to improve thetheoretica lbasi s ofresistanc e breed­ ing.

THE MODEL

Themode l is animprove d version ofa simila rmode l from Krusche (1975), and isbase d onth ewor k ofMod e (1958), Person (1967), Groth & Person (1977)an dth e 'genetic feedback'concep to fPimente l (1961).Con ­ sider aclose d host-parasite systemwit h the followingproperties : -Th ehos t andparasit e arediploi d andrando mmatin gwit h non-overlapping generations and alternate reproduction. - Thehos tpopulatio n carries alocu swit h 2alleles ,R standin g forresis ­ tance andr forsusceptibility , Rbein g dominantove rr . -Th eparasit epopulatio nha s2 allele so na correspondin g locus,A stand­ ing for avirulence and afo rvirulence ,A bein g dominant overa . -Th e interaction ofhos tan dparasit e is according toth e gene-for-gene concept (Flor, 1956).Wheneve ra parasit ewit h anA-gen e comes iritocontac t with ahos tpossessin g anR-gene ,th eparasit e cannotreproduce .Th ere ­ maining 3possibilitie s resulti nsuccessfu l infections ofth ehost . -Th e absolute fitness ofth ehos tgenotype s is adecreasin g functiono f thenumbe r of infectionspresen to n ahos tbefor e reproduction. Inth e following sectionR - denotes RR andR rgenotype s andA - denotes AA andA a genotypes, and relative gene frequencies are inbrackets . The fitness ofth eparasit e genotypes ondifferen thos tgenotype sis :

Parasite

A- aa

Host R- 0 P, Pl P2

Thetota l (net)fitnes s ofth eparasit epopulatio ni s

Wp = (A-) (rr)p1 + (aa)p2 (1)

Thenumbe r ofparasite s after reproduction isP , =W P„ , withP „ thenumbe r ofparasite s before reproduction.Le tH „b e thenumbe r ofhost s inth e presentgeneration , then a =P./H .i sth enumbe r ofparasite s coming into contactwit h asingl ehost , i.e. theparasit e density. Thenumber s of successful infectionsb y differentparasit e genotypes on different hostgenotype sare :

313 slope:Si,S 2

Ci C? infectiondensit y Figure 1. Fitness functions of the host genotypes.

Parasite

aa total

Host R- 0 a(aa ) a(aa ) rr a(A-) a(aa ) a

We assume the fitness functions: n. for a R- host and n„ for rr hosts as presented in Fig. 1. The fitness of the host population is

WH = (R-)n1 + (rr)n2 (2)

THE EQUILIBRIUM OF THE SYSTEM

The equilibrium condition for the system is met when the fitness of all genotypes is 1. From (1)w e have (r, a, etc., are the stationary values):

rr px.= 1, p2 = 1, px > 1

The stationary value of the susceptibility gene frequency is fully deter­ mined by the reproductive properties of the parasite. A requirement for the existence of an equilibrium is p, > 1, i.e. a fully differential fit­ ness pattern of the parasite. The difference of the fitness of the A- parasites on R- and rr hosts must exceed the difference of the fitness of the aa parasite on R- and rr hosts (Person et al., 1976). From (1)w e have the change in gene frequency for the virulence gene:

2 Aa = a (l-a)(p2-(rr)Pl)/Wp 2 = a (l-a)Pl((R-)-(R=))/Wp

Accordingly, the evolutionary speed of the virulence gene is proportional

314 top . andt oth eexces s ofth eresistanc e gene frequency over theequilib ­ riumvalue .Th e selective advantage ofth evirulenc e gene ispositiv ewhe n the related resistance gene exceeds itsequilibrium , whichha sbee n termed the 'genetic feedbackmechanism ' (Pimentel, 1961).

The equilibrium condition forth eR - gene is from (2):n.=n ?=l.W e

linearize n1 andn „ inth eneighbourhoo d ofth e equilibrium:

n. = (a(aa)- c.)s . +1

n2 = (a- c 2)s2 +1

Itfollow s that â =c ? and aa= c,/c~, c,

HERITABILITY AND GENETIC GAIN

The selection ofplant s apparently low ininfectio n asparent s forth e nextgeneratio n ina mas s selectionprogramm ewil l increase theR - gene frequency.A sw e dono tkno w theexac tstat e ofth e system, theconsequen ­ cesma yb equit edifferent . Inou rmode l thecharacte r tob e improvedb y selection inth enegativ e direction isth enumbe r of infectionspe r tree,i.e .th e infection density. Iti sa direc tmeasur e ofth e relevanteconomi c damaget o thehos tindivid ­ ual, andeas y to assess.Th e genotypic values ofth e infection density for thehos tgenotype sare :

host genotype RR Rr rr

infection density a(aa) a(aa) a

Thus theR - genotypes are less infected than ther rgenotypes ,a sw ewoul d expect.W e calculate theadditiv e genetic anddominanc evarianc eV . andV „ and assume thatther e isenvironmenta l varianceV „ forth e infectiondensi - 2 E ty.h denotes theheritability . 315 8 9tim e

= Figure 2. A typical sample runo fth e system (p=1.234,P 2 l< c,=6.4, c2=10). The time ismeasure d inhos tgenerations .

V =2r 3R a2(A-)2

2 2 2 2 VD =r R a (A-)

3 2 ? 2rJR a (A-T h2 = 2 2 2 VA+VD+VE r (R-)a (A-) +Vc

Thephenotypi c populationmea no fth e infection density is

M =a((R-)(aa )+ (rr))

The genetic gaindivide db yth e standardized selection differentiali andth epopulatio nmea nM formas s selection is

316 AG/i/M = h2 V '2 /M, whereV isth ephenotypi cvariance . The additive geneticvarianc e and theheritabilit y are large forinter ­ mediate gene frequencies ofth eR - gene,an dten d toward 0fo rvalue snea r 2 0 or 1. Very important is the dependence of V and h upon the evolutionary state of the parasite population. For a very high virulence gene frequency, V and V_ are very low. The reason is that the difference between the high infection class (rr hosts) and the low infection class (R- hosts) decreases with increasing virulence gene frequency. Furthermore all genetic variances increase with the parasite density. As a result there is a strong dependence of the genetic variances upon the actual state of the parasite population. In Fig. 2 it is shown that for the same phenotypic mean infection density M, different heritabilities and genetic gains can be realized. Without knowledge of the internal evolution­ ary state of the system, the normal heritability may be a poor predictor of the realized genetic gain from artificial selection.

CONCLUDING REMARKS

- The present model should be extended to several loci. - A general quantitative genetic theory for the description of interacting host-parasite systems should be developed. - The influence of the breeding systems of the interacting populations is very important for the dynamic behaviour of the system.

REFERENCES

Becker, W.A. & M.A. Marsden, 1972. Estimation of heritability and selection gain for blister rust resistance in western white pine. In: R.T. Bingham, R.J. Hoff & G.I. McDonald (Eds): Biology of rust resistance in forest trees. USDA Misc. Publ. No. 1221. p. 397-410. Bingham, R.T., R.J. Olson, W.A. Becker & M.A. Marsden, 1969. Breeding blister rust resis­ tant western white pine. V. Estimates of heritability, combining ability, and genetic advance based on tester matings. Silvae Genetica 18: 28-38. Flor, H.H., 1956. The complementary genie systems in flax and flax rust. Advances in Genetics 8: 29-59. Groth, J.V. & CO. Person, 1977. Genetic interdependence of host and parasite in epide­ mics. Annals of the New York Academy of Sciences 287: 97-106. Krusche, D., 1975. Some remarks about stability management in host-parasite systems. In: B.R. Stephan & CS. Millar (Eds): Lophodermium in pines; Proceedings of the 5th European Colloquium for Forest Pathologists. Mitt. d. Bundesforschungsanstalt f. " Forst- und Holzw. No. 108. p. 97-104. Mode, C.J., 1958. A mathematical model for the co-evolution of obligate parasites and their hosts. Evolution 12: 158-165. Person, CO., 1967. Genetic aspects of parasitism. Canadian Journal of Botany 45: 1193-1204. Person, CO., J.V. Groth & O.M. Mylyk, 1976. Genetic change in host-parasite populations. Annual Review of Phytopathology 14: 177-188. Pimentel, D., 1961. Animal population regulation by the genetic feedback mechanism. American Naturalist 95: 65-79.

317 Non-specific interaction based on polygenes

C. Person, R. Fleming & L. Cargeeg

University of British Columbia, Vancouver, V6T 2B1, Canada

ABSTRACT

Polygenically-based systems ofhost-parasit e interaction areexpecte db yman y researchers toexhibit :non-specificity ; stability; andconstan trankin g ofhos t andpathoge ngenotypes . Thispape rbriefl y describes atheoretica l analysistha tals o leads tothes e expectations. Itals odescribe s experimental studies thathav e demonstrated therealit y ofpolygenically - basedpathogenicit y fora tleas ton eparasiti c system. Forthi s particular system (Ustilago hordei and Hordeum vulgare) the experimental results suggesttha tpolygene s areonl ypar to fa total genetic system that includesmajo r geneswit hwhic hth e polygenes interact.

'Field resistance todiseas e isextremel y common and appears tob eth e normal type ofresistanc e to those diseaseswhic h normally infectou rcrop s butd o little damage. Itappear s tob e evolutionarily stable,no tlikel yt o beupse tb y changes inth epathogen , ands o should be thetyp e ofresis ­ tanceuse db yplan tbreeders.. .Fiel d resistance appears tob epolygenical - lycontrolled , sotha tt oovercom e resistance, several gene changesma yb e necessaryb y the fungus.'Thes e statements aretake n from apape rwritte n almost2 0year s agob y CM. Driver (Driver, 1962). Thepostulate s they containhav e since received agrea tdea lo f attention:the yhav ebee n examined from theepidemiologica l (Vanderplank, 1963)an d ecological (Robinson,1976 )point s ofview ,an dne wterminologie shav ebee n introduced (loc. cit.). Iti sno wwidel y accepted that field, orhorizontal ,resis ­ tance isstable ,an d thatit s stability arises from the facttha t iti s non-specific andpolygenicall y controlled. Theevidenc e onwhic hDriver' spostulate swer ebase d was,an dha s

1.Presen taddress :Schoo lo fCivi lan dEnvironmenta lEngineering ,Holliste rHall , CornellUniversity ,Ithaca ,N.Y. ,14853 , USA.

318 remained, largely empirical.Muc ho f itoriginate s from thehistor y ofth e late-blight disease (Phytophthora infestans (Montagne)d eBary )o fpotato . To quote again fromDriver' spaper : 'Afamilia r example isth epotat o blight fungus onnon-immun evarieties .Thes evarietie s canb erate d inth e sameorde r andwit h the samedegre e ofinfectio n aswhe n first introduced eventhoug h some areove r onehundre dyear s old.Thi s istru e throughout theworld , sotha tther e isn oevidenc e ofpotat obligh tvaryin g thebal ­ ancebetwee ni tan dth e fieldresistanc e ofcommercia lvarietie s overth e pastcentury' .Thi s quotation alsopresent s theconcep to f 'commonranking ' whichwa s subsequently amplifiedb yVanderplan k and, later,b yRobinson . Constantrankin g isno w regardedb yman yresearcher s as adistinguishin g characteristic ofpolygenically-controlle d resistance. Themechanism s thatmak e for stability and forconstan trankin g of polygenically controlled resistance areneithe r knownno r understood. Fleming &Perso n (unpublished)hav e examined thisproble m ina theoretica l wayb y assuming: (i)tha tresistanc e andpathogenicit y werebot h non­ specific andpolygenicall y controlled; and (ii)tha t foreac ho fth e2 interactingpopulation s thevariabilit y was continuous and ofth ekin d described by anorma l curve.Sinc enothin g is actually knowno fho wthes e assumedpolygene s would interact,2 differen t 'models'o finteractio nwer e constructed (Fig.la ,b) .Th enorma l curves inth euppe rpar to feac hfig ­ ure represent thepolygenically-base dpotentia l forenablin g thediseas e to develop.Eac h ofth euppe rcurve s isdivide d (by1 ,2 an d 3standar ddevia ­ tions from themean )int o 6classe s towhic h are assigned arbitrary values (ranging from 0t o5 ,inclusive )tha tnumericall y represent thepotentia l forenablin g thediseas e todevelop .Th enumber s inth ematri x represent the levels ofdiseas e thatar e assumed todevelo pwhe nhost s andparasite s interact.Fo r the additivemode l (Fig.la )thes e 'realized' levels of dis­ ease areobtaine d by adding, and forth emultiplicativ e model (Fig. lb)b y multiplying, thenumber s thatwer e arbitrarily assigned. Thebotto m curves describe the 'realized' levels ofdisease .Fo rth e additivemode l itwa s shownmathematically : (i)tha tth e 'realized' levels ofdiseas ewoul d also conform to anorma l frequency distribution, and (ii)tha t ashif ti nth e mean ofth edistributio n curve for 'potential'diseas e development,occur ­ ring ineithe r ofth e2 interactingpopulations ,woul d resulti na rela ­ tively smaller shifti nth emea no fth ecurv etha trepresent s 'realized' disease development.Thi s reduction ineffec tha sbee n labelled 'phenotypic damping', iti sthough t tob e asourc e for stability inpolygenically - determined systems.Th emultiplicativ e modelwa s less easyt oanalyze : themea n level ofdiseas e favoured hostresistanc e and the effects brought onb y assumingchange s ineithe r ofth e2 interactin gpopulation s weremor e complicated.However ,examinatio no fth e2 contrastin gmodel sdi d support the following general conclusion: (i)polygenically-base d systems should display constantranking , and (ii)polygenically-base d systems shouldb e

319 ©

10 8 6 4 2 O DISEASE REACTION (D)

Figure 1. For these figures (laan d lb), f(P)an d f(S)represen tth e fre­ quencieswit hwhic h individualswit hvarying-level s ofpathogenicity'(P )o r susceptibility (S)occu r inparasit e andhos tpopulations .Becaus e ofthei r assumedpolygeni c determination these arerepresente d asnorma l frequency distributions.Fo r the additivemode l (Fig.la) ,wher e iti sassume d that the amounto fdiseas e isD =A + S ,th ematri x records the severity of disease forth evariou s indicated classes ofhost-parasit e interaction.Th e resulting frequency distribution forD , f(D),i snormall y distributed. For themultiplicativ e model (Fig.lb) ,wher e iti sassume d thatD =A x S,th e shape ofth e resulting frequency distribution fordiseas e reaction,f(D) , depends onth eparameter s ofth edistribution s forpathogenicit y and sus­ ceptibility, andth e distribution ison e thatwoul d favorhos tresistance . (Fig.l awa sused ,wit h ourpermission ,b yRobinson , 1976.)

320 25 20 15 10 5 0 DISEASE REACTION (D)

relatively stable.Thi s second conclusiondepend s onth e assumption that pathogenicity andresistanc ebot h correlatepositivel y with reproductivity. Thus thedirection s takenb ynatura l selection inth e 2interactin gpopula ­ tionswil lb e diametrically opposed. (This conclusion is in disagreement withVanderplank' s (1975)suggestio n thatth e expected stability derives from stabilizing selection operating independently ineac ho fth e2 inter ­ acting populations). Although therehav ebee nnumerou s studies ofpolygenically-determine d hostresistance ,relativel y little isknow no f polygenically-determined pathogenicity.A tth eUniversit y ofBritis h Columbia,Emar a (1972)an d Emara& Sidhu (1974)hav e demonstrated that strains of Vstilago hordei (Pers.)Kell . & Sw.diffe r inthei rpathogenicit y toward susceptible barley cultivars and thatthi svariabilit y ispolygenicall y determined. Theseexperiment s havebee ncontinue d incooperatio nwit hDr .Cate na t Birmingham and,a tleas t in1 strain ,th egene s involved showadditive ,

321 dominance andepistati c effects (unpublished data). For theremainde r ofm y talk Iwis h todescrib e another seto fexperi ­ ments thatillustrat e the combined actiono fmajo r genes andpolygene s in determining levels ofdiseas e development.Thes eexperiment s also involve U. hordei andtak e advantage ofth e facttha t U. hordei forms anordere d tetrad fromwhic h4 'selfed' (andobligatel yparasitic )dikaryon s canb e established.A 'selfing'tha tresult s ina 3: 1 ratio identifies the 2:2 ratio of 'V' and 'v'gamete s inth etetra d (seeTabl e 1), and theidenti ­ fied 2:2 gametic ratio canb e readilyverifie d ina tes tcross .A large number ofgamete s canthu sb e extracted from asingl e dikaryon, and for eacho fthes e thegeneti c content (V andv )i nrelatio n toth emajo r gene canb e determined.Wher epathogenicit y isals o determined inpar tb ypoly ­ genes iti sals o tob e expected thatth e gametes formedb y asingl edi ­ karyonwil lvar y inthei r contento fpolygenes .Thi sexpectatio n leads toth epredictio n thatth e V- and v-containing gameteswil lvar y inthei r pathogenic behaviour, and thatthi svariabilit y willb e polygenically determined. Totes tthi shypothesi s thepredictio nwa smad e thatth e V- and v-containing gametes would showconstan trankin g (Table2) .

Table 1. The 3kind s oftetrad , andth eprogen y obtainedvi a selfing from singleteliospores ,o f aV vdikaryo n forwhic h theorigina lmatin gwa s V+x v- .Parenta l ditype (PD)an dnon-parenta l ditype (NPD)tetrad s dono t segregatewhe n 'selfed'.Tetratyp e (T) tetradsyiel d 3:1 (V-:vv)segrega ­ tionstha tlea d to identification ofV andv sporidia .

Type oftetra d

PD NPD

1 2 V v v Sporidia 3 v~ v~ V~ 4 v~ v~ v

1x 3 Vv Vv W Matingsan d 1x 4 Vv Vv Vv •selfed' 2x 3 Vv Vv Vv a progeny 2x 4 Vv Vv vv

Segregation (V- :v 4:0 4:0 3:1

a.Thi s culture identifies thetw ov-containin g and,b y extension,th etw o V-containing sporidia ofth etetrad .

322 Table 2. Expectation of 'constantranking ' amongdikaryons :whe nplu s and minus sporidia withvaryin gnumber s ofpolygene s (range0 t o 5)ar ear ­ ranged inascendin g series the resultingdikaryon s (having0 t o 10poly ­ genes) ar eexpecte d tosho wconstan trankin g inrespec tt othei rpatho ­ genicity.

Numbero fpolygene s in '+'sporidiu m

t/I (1) G <1l tu >i r-\ S O 0 -H a T) -H

Theresult s of alarg e experiment are shown inTabl e 3.Mathematica l analysis ofthes edat a confirm theexpectatio n ofconstan tranking .Withi n eacho fth e 3majo r genotypes (W, Vv andvv )ther e is aconsiderabl e range ofpathogenicit y attributable topolygenes .Th e analysis ofvarianc e shows significantvariatio nbetwee n thegamete s inthei rcontributio n toth e pathogenicity ofdikaryons .Howeve r the analysis ofvarianc e also showed significant interactions,detecte d inth e W group and inon eo fth e Vv groups,whic h indicate thatth epolygene s governingpathogenicit y dono t always acti na nadditiv emanne r asassume d by thegenera l theory ofpoly ­ genes. These can alsob e seenb y directvisua l inspection ofTabl e 3.Wit h use ofsporidiu m 21B3+ theperformanc e of W dikaryonswa s enhanced while thato f Vv dikaryonswa s reduced,wherea swit hus e of sporidium 22G3+th e opposite resultwa s obtained. The geneticbasi s forthes eparticula r inter­ actioneffect s isno wbein g studied further. So far asw e areaware ,th eexpectatio n ofconstan trankin g among gametes represents anapproac h toth e analysis ofpolygene s thati sentire ­ lynew .Th emetho d is,o fcourse ,restricte d toorganism s such as U. hordei forwhic h iti spossibl e toestablis h gametic cultures.Th emetho d has yielded resultswhic h add supportt oth econclusion s thatwer e reached earlierb y using the traditional approach. To summarize:polygenically-base d systems ofhos t :parasit e interac­ tion areexpecte d byman y researchers todispla y 2importan tcharacteris ­ tics,viz .constan trankin g ofhos t andpathoge n genotypes,an d stability. Although agoo ddea l isknow no fpolygenically-base d hostresistance , rel­ atively little isknow no fpolygenically-base d pathogenicity, and almost

323 CU rH a >rH ^ eo *ƒ CN in Ol OD CM KO vn CM * co co co CM CM CM CM H rH rH CN >1 SH CO M O KD CO CO CO CM •# •H •o CO CM vO t"^ CO 00 O CM Ol 00 CO <* o CO CO H CM CM •^ in CO CM CM CM rH CO ca •O r-i fi u a (0 o +J cu o en r-t r> r- •C! H S m ro in co CM CU o VH bi co H CO CM CO CM rH rH r-l Û ^ cü < •O CD rf 1 co KO a> r~ •* eO •* rH rH CM rH ^ rH «1 CM CM CM C•M* H 3 •* CÖ <+H O o "* 1 CO ^ CO Ol H rH ^ 00 O CO O •* CO eO in — > > CM CM •* CO H CM CM p ca fi eu (O rH i eo H m CO rH eß O C- CM CM CM •* 00 eO Ol CM •tf CM CO CO rH PS ^ •* <* o o CJ T3 -H O) £ efiu rH 1 CM m co rH Kû <* CN co in in oo in O rH P > Oi CM CM •* CO C^O CO CO rH CM u co CU U-I 4-1 O o Ö o CM r- en m t~ 00 CM co [-- Ct! in CM •* CM ^ CM rH CO CO CM rH rH UH i o [0 Cef p fi rH I CO •* ^ rH CJl in CO O- rH CO CO o O CM Ol fi o CU CM 1 <* n u co p oo in co ^ O sf eO ca •p en Oi eo •* in in "* in m <* <* >* co CM in •* c 10 o Q O -H <+H •P • U -H ca * .Q Cl) 01 o Oi (0 >i -.H ^ + + + + + + + + + + + r-t N rH rH CO CM CO T-^ CO CO T-\ CM CO CM r-< co M O •H W CO 00 10 M U CU Q m PÛ CU O fi eu CO -H •P a-H 00 rH rH o CO rH Ol CM O <3< •* CM > > •H P CU rH CM CM CM CM CM rH CM CM CM CM CM Ê > > O 3 .fi u ia co A sdfoousS A sdÄ^ousß S cu cu >-H rH -P CO rO ( + )Y 'sdÂ^ 5uTq.Biu 'ç-pp-fjods SUBS U Ei £1 •p

324 nothing is known of how polygenically-based systems of host and parasite would actually interact. Constant ranking has received very little study, and the mechanisms that would lead to stability have yet to be discovered. Our experimental studies have demonstrated the reality of polygenical­ ly-based pathogenicity for at least one parasitic system. These studies suggest that polygenes are only part of a total genetic system that also includes major genes with which the polygenes interact. Our theoretical analysis leads to the expectation that polygenically- based systems will show constant ranking, and that such systems will be relatively more stable than those that are based exclusively on major genes and specific gene-for-gene interaction. However, there is both practical and theoretical investigation to support the view that major genes could be used more efficiently than they have been in the past. Our overall conlusion is that the question of major vs polygenes is still an open one that requires extensive study. At this particular point in time it seems that both kinds of resistance (if indeed they exist sepa­ rately) are valuable, and that for both kinds of resistance the objective should be to breed for genetic heterogeneity rather than for a monoculture.

ACKNOWLEDGEMENT

The work described in this paper was financed by a grant from the Natural Sciences and Engineering Council of Canada.

REFERENCES

Driver, CM., 1962. Breeding for disease resistance. Scottish Plant Breeding Station, Research Report. 1-11. Emara, Y.A., 1972. Genetic control of aggressiveness in Ustilago hordei. I. Natural variability among physiological races. Canadian Journal of Genetics and Cytology 14: 919-924. Emara, Y.A. & G. Sidhu, 1974. Polygenic inheritance of aggressiveness in Ustilago hordei. Heredity 32: 219-224. Robinson, R.A., 1976. Plant pathosystems. Springer-Verlag, Berlin. 184 p. Vanderplank, J.E., 1963. Plant disease: Epidemics and control. Academic Press, New York. 349 p. Vanderplank, J.E., 1975. Principles of plant infection. Academic Press, New York. 216 p.

325 Monoculture versus mixture: interactions between susceptible and resistant trees in a mixed stand

HansM .Heybroe k

"Dorschkamp"Researc hInstitut efo rForestr yan dLandscap ePlanning ,Wageningen ,th e Netherlands

ABSTRACT

Theus e ofdivers e genotypes isa neffectiv ewa y ofspread ­ ingrisks .Fo r thispurpose ,a mosai c ofpur e stands,eac h consisting of asingl e genotype,ma yb e adequate under certain conditions.Mixe d standsma y offer abonu s ifan d ins o fara s aninteractio nbetwee n resistant and susceptible trees ina standwil l reducedamag eb ypest ,disease ,wind , etc.A survey ofsuc h interactions andth emechanism s involved isgive nwit h regard to diseases,includin g Fomes annosus, Nectria cinnabari- na and Lophodermium pinastri. Beneficial effects forth e sus­ ceptiblepartner s inth emixtur e aswel l asharmfu l effects for thepartiall y resistantpartner s arenoted .Th ene teffec t for the stand isdependen t onman y factors,includin g the damage threshold. Consequences fortestin g designs arementioned .

INTRODUCTION

Inth e decisions toeithe rplan to r avoid stands ofmixe dgenotypes , many considerations mustpla y arole .Pur e stands ofa singl e genotype oftenma y cost less toestablish ,ten d andharvest ,the yma ypromis e to producemor e timber ofa highe rvalue ,the y seem torequir e lesssilvicul - turalexpertise .Ther ema yb en o alternative toa pur e stand incase swher e only oneresistan to r locally adapted genotype isavailable . Monocultures are,however ,widel y believed toattrac tdisease s and pests andt ob emor evulnerabl e thanmixe d stands,especiall y inth e long term.Multiclona l varieties havebee nregarde d asa mean s ofdeployin gnot - too-resistant clones (Schreiner, 1965). Further,mixe d standsma y haveth e advantage ofofferin g amor evarie d scenery and,unde r certainconditions , ofgivin g ahighe rproductio n (Heybroek, 1978). Monocultures havehealt hhazard s thatcom e in3 forms: firs tthe yma y constitute alarge ,undivide d risk, and itma yb ebette r tosprea drisks ; second, the concentration of susceptible plantspe r semigh t increaseth e

326 disease rates ofth e individuals,whil e the interactionbetwee n neighbours ofdifferen t susceptibility mightreduc e thoserates ;third , monocultures mightstimulat e theevolutio n ofnew ,mor evirulen t or aggressive forms of theparasite .Thi spape r dealsbriefl y with the firstaspec t andconcen ­ trates onth e second. Mixing ofdifferen t species that serve asalternat e hosts toth e same rust (oraphids ,etc. )ha sprofoun d buteviden teffect s andwil l notb e discussedhere . The severity of some diseases isconsiderabl y affected by themicrocli ­ mate inth e stand.Mixin g ofspecie sma y influence disease severity in either direction by influencing themicroclimate .Thi s effectwil l notb e discussed either.

SPREADING THE RISKS

Thenotio n iswidel y accepted thatth eexclusiv eus eo fa singl egeno ­ type (e.g.clone )ove r alarg e area entails anenormou s risk: ifth e clone fails for any reason,th e failurecoul db etota l over the entire area,thu s causing almost insurmountable problems forth emanagemen t ofth e forestan d for the industries depending on it.I tseem smor e acceptable tohav e 10 clones, eacho non e tentho fth earea :eve n ifth e chance of failureo f anyo fthes e is 10time s asgrea t (supposing thateac hha s the same chance of failure asth eearlie rmonopolis t clone)th eprospectiv e damage isonl y onetent h and canb emuc hbette r absorbed. Iti sth ewisdo m ofno tputtin g all eggs inon ebasket . Iti sth ephilosoph y of insurance:man y small risks are less serious than asingl ebi g risktha t could radically destroycon ­ tinuity. Spreading the risk isa goo d common senseprecaution . From this limitedpoin to fview ,ther e is little advantage inindivid ­ ualmixtures :plantin g 10block s of 1000h awit h 1clon e eachwoul d spread theris k justa seffectivel y asplantin g 10 000h awit h themixtur e ofth e 10clones . The individual mixturema yhav e anadvantage ,however , if compensation occurs: compensation being theproces s inwhic h theneighbour s fill thega p causedb y afailin g tree,thu s increasing their ownproductio n andmor eo r less compensating forth e loss. Thismechanis m mayb eparticularl y effec­ tive ifth e failure occurs early inth e stands'developmen t and ifinitia l spacingwa s narrow.O n theothe rhand , if2- 5 clones fail, themixe d forest would become defective over theentir e area.Tha t loss is less easyt o handle than ifth e failing clones hadbee nplante d inpur e stands,whic h couldb e salvaged and replanted. These considerations might applyparticularl y topopla r planted at finalwid e spacing,whic h reduces theeffec to fcompensation , and growni n short rotations.Th e latter silvicultural trait isamon g thosementione db y Kleinschmit (1979)a sreducin g thenee d formixing .

327 A relatively safe situationma y existeve nwhe nmonoclona l stands are used,provide d thenumbe r ofclone s usedpe r region isno t small,provide d theclone s havevaryin gbackgrounds ,an dprovide d they arebacke d upb ya wide array ofexperimenta l cloneswhic h arebein g keptunde r test for different sites andwhic h form areserv e fromwhic h old clones canb e replaced when failing forsom e reason (Heybroek, 1981).

THE EFFECT OF INTERACTIONS

This section dealswit h thequestio n ofho w farth e damaget o asuscep ­ tibleplan t isdecrease d ifi ti ssurrounde d by resistant instead of sus­ ceptibleplants . Itseem s importantt o study themechanism s involved. Treeswit h different levels ofresistanc e are foundmixe din : - stands consisting ofdifferen t tree species,differin g inresistance ; - stands consisting of aseedlin gpopulatio n of 1specie swit h variation inresistance ; - stands consisting ofclona lmixtures ,varyin g inresistance ; - stands inwhic h treeso f 1specie s ofdifferen t ages aremixed ,whil e susceptibility toth e disease inquestio n is limited to anarro wage-class . Thismean s that experience gained with traditional 'mixedstands 'ca n beuse d tounderstan d andperhap s topredic twha twil l happen ina clona l mixture.However , inspit e ofth ecustomar y interestb y foresters inth e advantages anddisadvantage s ofmixe d stands,literatur e doesno taboun d withwell-documente d examples ofdisease s that aremuc hmor e serious in pure than inmixe d stands.A case oftencited ,havin g thechar m ofth e exotic,concern s Hevea brasiliensis (Willd.)Muell. : this species issai d tob e severely damaged by leafdiseas e ifgrow n inpur e stands inSout h America,bu tt ob emainl y healthy inth eneighbourin g virgin forestwher e itoccur s singlybetwee n other species (Boyce, 1954). The search for examples iscomplicate d by the facttha tin.th eolde r literature, examples were collected anduse dmainl y toprov e ordisprov e the ideatha tdisease s etc. areboun d tob emor e serious in 'unnatural'stand s than in 'natural' ones (Boyce,1954 ;Peace , 1962',p .18) . The diseaseproces s canb e divided in2 phases ,an d inbot h neighbours canpla y arole . Inth e firstphase ,th e individual host-treemus tb e reachedb yth eparasite ,an d inth e second, theparasit e mustmultipl y or spread ono r inth ehos tunti l thedamage-treshol d isreached .

Reaching the host

Inth e firstphase ,i nwhic h thehos t isreache d by theparasite ,3 groups ofcase s canb e distinguished.

1. Inon e group ofcases ,th ehos tneed s tob ereache d only once:onc eth e

328 parasite has reached the tree,i tca n survive and spread ini to ro nit . This applies forexampl e toperennia l cankers,woo d inhabiting fungi,etc . Thepresenc e ofresistan t neighbour treesca nhav e adelayin geffect , whichma y orma y notb e ofpractica l use,a s illustrated inth e following examples: -Th e chestnutbligh t spread quickly throughmos t ofth e areao f Castanea dentata (Marsh.)Borkh. , scarcely differentiatingbetwee npur e andmixe d stands. Some outlying stands or individuals escaped at first,bu teve n isolated trees,plante d inth eMidwes to fth eUnite d States,fa routsid e thenatura l areao fth e species,wer e finally reached and succumbed. -Th eperennia l cankero fbeech ,cause db y Nectria ditissima Tul.,wa sa rarity inth eorigina lmixe d coppice-with-standards innorth-easter n France. Itincrease d toepidemi c levels afterthes e forestswer e converted topur e even-agedbeec hwood s (Perrin& Vernier , 1979). Apparently, under theol d regime,th epresenc e ofman ynon-host s allowed even susceptible beeches to escapeinfection . - Several treedisease s spread through live rootgrafts ; for some,eve n mechanical rootcontac t issufficien t (Epstein, 1978). Among them areoa k wilt,Dutc h elm disease and Fomes rootrot . Itseem splausibl e tnatplan ­ ting tree species susceptible tothes edisease s inmixture swit h resistant individuals, ofth e same oro fothe r species,wil l greatly reduce this type of spread ofth edisease . Indeed itha sbee n showntha tbot h oakwil t (Epstein,1978 )an d Fomes rootro t (Rennerfelt, 1947)ar emuc h less severe inmixe d than inpur e stands.Fro m themodel s inFig .la- ci tappears ,tha t theus eo f 50% resistant trees isenoug h tovirtuall y stop the spread from one focus,provide d transmission occursbetwee nneighbour s only.Root smay , however,exten dmuc h furthertha nth ecrow no fth etree ,makin g rootcon ­ tactspossibl e among trees thatar eno tdirec tneighbour s (Stiell,1970 ; Eis, 1978). Ifroo tcontact s occurbetwee n diagonal neighbours in5 0% o f thecases ,reductio n ininfectio n isstil lconsiderabl e (Fig.1) :i nth e sametim e required to infect2 0tree s inth epur e stand, only 5tree swoul d be infected inth e 50% resistant stand. Inth emodel s ofFig . 1,systemat ­ icmixin gwa smor e effective thanrando mmixing .Th eprotectiv e effectwil l clearly diminish asth eroo t systems rangewider , andtend s todisappea r whenth e initial infection ishigher ,tha tis ,whe nth enumbe r of fociin ­ creases.Ther e isals o anage-effec t asth e relative range ofth e root sys­ tem changes with age (Eis,1978 )an d asmixture s ofdifferen t species tend tob emor e difficult tomaintai nwit h increasing age.Perhap s thepossibil ­ ity ofreducin g the incidence ofthes edisease s throughmixe d plantings shouldb e explored further. Itma yb e difficult to findnon-host s thatca n bemixe d inwithou t silvicultural problems,bu teve nth e admixture ofpar ­ tiallyresistan thosts ,tha t istree s throughwhic h spread is slower,migh t give aworthwhil e reduction ofdiseas e inth estand .

329 o o o o o o o a. Spread ina homogeneousl y susceptible stand.2 0transmissions .

• O b. Spread ina stan dwit h 50%,evenl y distributed, resistantplants .5 trans ­ missions. O

O

o

c. Spread ina stan dwit h 50%,randoml y distributed resistantplants . Intw o examples 6an d 9transmission s are realized.

Legend: # resistantplan t O susceptible plant »~sprea d ofdiseas e 1unsuccessfu l attempt atspreadin g © infected plant © focus

Figure 1. Spread ofdiseas e through rootcontacts ;assumin g thatal l direct neighbourships leadt otransmission ,bu tonl y 50%o fth ediagona lneigh ­ bourships;ove r atim eperio d sufficient fortw odiagona l transmissions.

330 2. Ina secon d group ofcases ,disease s thatrequir e anannua l reinfection from outside,th epopla r rusts Melampsora medusae Thuem. and M. larici- populina Kleb,ca nb e taken asexamples . Inthese ,th e fungus oftenha s tocom e from far.A mixtur e of susceptible poplarswit h some non-hosts might atbes t cause amino r delay inth e firstinfectio n and the onset ofth ebuild-u p ofth e epidemic.Thi s effectma yb e even non-existent ifth ewhol e stand is infected atth e same timeb y aclou d ofspores . (Theeffec to fmixin g onth ebuild-u p ofth e epidemic isdiscusse d in thenex t section.)

3. Between these 2groups , Nectria cinnabarina (Tode)Frie sprovide s an intermediate case.Pathogeni c strains ofthi s fungus form annual cankers thatcontinu e tosporulat e for 1o r 2years .Fo r asuccessfu l infection, woundsmus tb epresen t atth erigh t time ofth eyea r andunde r the right weather conditions.Geneti c resistance occurs. Ifgenotype ,conditio n and environment ofth ehos t are favourable for disease development, asingl e tree canreinfec t itselfyea r afteryear ;i fnot ,th e treewil l loseth e parasite for fewo rman yyear s until ane w colonization occurs.Then ,a larger closedpopulatio n ofhos ttree s isneede d tomaintai n aloca lpopu ­ lationo fth epathogeni c strain ofth e fungus.A single suchtree ,sur ­ rounded bynon-hosts ,migh tremai n freeo f infection forman yyear s asth e fungusdi d nothappe n to reach ita tth erigh tmoment . For this intermediate category, inwhic h atre ema y shed or loseth e parasite for sometim eunti l thenex t recolonization,parallel s canb e found inth e island-theory ofMacArthu r& Wilso n (1976). Theseauthors , comparing thenumber s of animal speciespresen t of islands ofdifferen t size and atdifferen t distances from themainland , conclude thatth e chance forextinctio n of aspecie s on an island increases asth e island is smaller and asth epossibilit y forrecolonizatio n decreaseswher e other islandso r themainlan d aremor e remote.Chance s forextinctio n alsodepen d onth e size ofth epopulation : allpopulation s fluctuate insiz e over theyears , a smallpopulatio n can easily disappear completely insuc h afluctuation . Now, thegroup s ofhost-tree s mayb e regarded asecologica l islands ina 'sea'o fnon-hosts ;loca l extinction ofth eparasit e onth e group ofhos t treesmigh t incritica l cases depend onth e size ofth egroup ,an do nth e distance from the 'mainland',th e sourceo f reinfection. Itfurthe r depends onth e intensity of attack onth ehos to rth e amounto fparasit epresen to n the single tree:th e 'populationsize' .

Returning to Nectria cinnabarina, itca nb eexpecte d that atlo w levels ofdisease , amixe d stand ofhos t andnon-hos t treesma y lead tobette r health ofth ehosts .Matter s arecomplicated , however,b y the facttha t pathogenic strains can survive for some time assaprophytes .Loca lextinc ­ tion of N. cinnabarina ispursue d artificially by thenurseryma nwh oprac -

331 tices sanitation rigorously.Onc eth enurser y is 'clean',man y susceptible momentswil l not lead toinfectio nbecaus e ofth e absence ofth eparasite . In forestrypractice ,thi smechanis m of local extinction ofan d delayed recolonizationb y theparasit e seems of limited value, asi tma y require thereductio no fth eproportio n ornumbe ro fhos ttree st o aleve l thati s not interesting economically.

The build-up of a parasite population

Once theparasit eha s found thehos ttree ,generall y abuild-u p ofit s population isneede dbefor e thediseas e canreac hth e dajnagetreshold .Her e again, resistantneighbour s mayhav e aneffec to nth eoutcom e ofth epro ­ cess. The classical and successful example is themultilin e inoats , as describedb yBrownin g &Fre y (1969). Itconsist s of amixtur e of several separatehost-parasit e combinations, inwhic hhos tA iscompatibl e with parasite race 1,an dB wit h 2,etc. ,bu tno tA wit h 2,no r Bwit h 1,etc . Thusth e sporeso f1 generate d onA and landing onneighbou rB ,wil lb e ineffective and lost forth eepidemi c onA , andvic eversa .Thu s thebuild ­ up ofth epopulatio n ofparasit e 1o nth ecomponen tA wil lb ebase d ona severely diluted sporecloud , sotha tth ebuild-u p isdelaye d considerably. The conceptcanno tb e simply copied for allhost-parasit ecombinations . Success depends onwhethe r areductio n inth edensit y ofth e spore cloud willb e sufficient to delay thebuild-u p ofth epopulation , andwhethe r this delaywil lb e sufficient toreduc e thedamag e toth ehost .Then ,siz e is important:th e individual cerealplan t is small,lon g andnarrow ,s o thatspore sproduce d on ithav e afai rchanc e oflandin g onneighbourin g plants. Incomparison , thecrow no f asingl etre eprovide s alarg evolum e of leaveswit h the same genotype. Inth ecrown ,a smal l epidemic could develop independently, unaffected by thepresenc eo r absence ofa resistan t neighbourtree . Themultilin e isdevelope d toemplo yvertica l resistance. Infores t trees, however,withi n 1species ,mainl y somedegre eo fpartial ,horizonta l resistance canb e found.A priori ,mixin g suchtree s doesno t lookpromis ­ ing.Mor e effectmigh tb e expected frommixin g highly divergentgenotypes : hosts andnon-hosts ,differen tspecies . Certainly,no t anymixin g oftree swit h different levels ofresistanc e willb ebeneficial .Althoug h itmigh tb ehope d thatth epresenc e ofth e more resistantwoul d somehowprotec t the less resistantplants ,th e reverse mayhappe n aswell : the less resistantplant sma y acta sdiseas espreaders , overcoming the resistance ofth emor e resistantpartners .A n example ofth e latterpossibilit y isgive nb yMaslo w (1970,pag e62) : "Whiteel m (V. laevis) suffered inth eprefectur e Rostov,Ukraine ,muc h less fromDutc hel m disease than fieldel m (U. carpinifolia), atleas ti n

332 the earlieryear s ofth e epidemic. Inplanting s ofth e firstan d second growth class,wher eu p to10 0% o fth etree s [offiel d elm, seepag e 61] gotinfected ,n omor e than7 % ofth ewhit e elmsdied . The condition ofwhit e elmwa s dependent onth e abundance of spore infection ando f stem damaging insects,th e latterbein g dependent onth e presence of field elm inth e immediate neighbourhood ofwhit e elm: in all stands where white elm occurred in mixture with the severely diseased field elm, dying of white elm was of much higher significance, in cases even nearly total." (Italicsb yH.M.H. )

LOPHODERMIUMPINASTR I Some idea ofwha tca nhappe nwhe nhost swit hvaryin g degrees ofpartia l resistance aremixe d canb e gained fromdat ao nneedl e castcause db y Lophodermium pinastri (Schrad.e xHook. )Chev. ,collecte d inth eDutc h programme ongeneti c improvemento f Scotspin e (Squillace et al.,1972 , 1975;Krie k& Bikker , 1973). Inon eexperiment , 14Dutc hprovenance s werecompare dwit h 4Germa n provenances under severe disease-conditions.Th e experiment contained3 replicateblocks :eac h singleplo t consisted of 5rows , each0/6 0plants ; spacing 1.50 mbetwee n rows and 1.20 m inrows . Ineac hblock ,provenance s were randomized. Four andeigh tyear s afterplanting ,mortalit y and growth were assessed.Th eprovenance s clearly separated into2 groups : after8 years, theGerma nprovenance s suffered anaverag emortalit y of45. 5% ; theDutc hprovenance svarie d slightly around anaverag e of20. 1% .Mortal ­ itycoul db eprimaril y attributed toneedl e cast,thoug h Armillaria mellea (Vahl.)Quel. ,presen t throughout the area, accelerated the dying of weakenedtrees . Plots ofth e 'resistant'provenances,whe nsituate d nextt o aplo to f the 'susceptible'provenance s (Fig.2 )showe d aslightl y significanthighe r mortality.Th e influence ofth e susceptible plotswa sno t evidentbeyon d thewidt h ofon eneighbou r plot,tha ti s 10.5m .Unfortunately , the limited number of susceptible provenances didno t allow assessment ofth e reverse effect, thatis ,a possibl e lowerdiseas e rating insusceptibl e plotsbor ­ deredb y resistantplots .

Fig.2 i sbase d on assessments byKrie k& Bikke r (1973), following the method that Squillace et al. (1972)ha d usedwit h earlier data.Fo reac h plot ofa resistan tDutc hprovenance ,th e deviation of itsdiseas e rating from the averageprovenanc e ratingwa splotte d against itsdistanc e froma susceptible Germanplot .Thi s showed thato n average, aplo to f aresistan t provenance had a6. 5 %highe r disease rating thanth eprovenanc e average if itwa s adjacentt o asusceptibl eplot .Th emor eremot eplots ,necessarily , had aslightl y lower disease rating thanth eprovenanc e average.

333 mortality

fciiiiiiaaverag e susceptible provenance

50 I Iaverag e resistant provenance

40

neighbour plots

30- .furtherremot eplot s

/ mean forth e resistant provenances / 20-

10-

5 5 5 0 10. 2131 . 42 53. Distance from susceptible plot (meter)

12 10 10 20 Figure 2. Disease ratings after attackb y Lophodermium pinastri inplot s of relatively resistantprovenance s ofScot spine ,i ndependenc e onthei r distance from theneares tplo to fa susceptibl e provenance (based ondat a inKrie k& Bikker , 1973).

Itwa spossibl e to studybot h effects,however , ina larg e scalehalf - sib testo f29 4Dutc hplu s trees of Scotspine .Needl e castwa s assessed7 years afterestablishmen t ofth etest ,usin g ascal e of1 throug h 7,base d onth epresenc e ofneedl e spots and on loss of foliage.Th ehighes tratin g represented dead trees.Th etes tconsiste d of six 7x7 latticesquares , eachcontainin g4 replicates .On eo fth e lattice squares,containin g4 9 families,wa s analyzed indetai l forneedl e castoccurrence . Interaction between resistant and susceptible familieswa s evidentwhe nth erating s of the4 corne rtree s ofth e4x 4 treeplot swer e comparedwit hthos eo fth e 4 inner trees.Th e inner treeswer e always surrounded by their own kind; the corner trees,however ,b y treeso fothe r families. Itwa s found that corner treeso fresistan tplot sha dhighe r average disease ratings thanth e inner trees,a sthe ywer emostl y surrounded by trees ofmor e susceptible other families;th erevers ewa stru e forth e susceptibleplots ,whil e there

334 Resistant Average Susceptible families families families cornertree smor e cornertree sles s heavilyinfecte d heavilyinfecte d thaninne rtree s 100% thaninne rtree s

plot diagram (16 trees)

c o o c olio Olio c o o c

C =corne r trees I= inner trees 0 =othe r trees

Averageratin go finne rtree s _1 I 0% 29 68 100% Figure 3. Averagediseas erating s (Lophodermium pinastri on Pinus silves- tris) of 'cornertrees 'ove r 'innertrees ' (seeplo tdiagram )o fth e4 plots ofeac ho fth e4 9half-si b families inExperimen t 2o fth eprogeny - trial. The field ratings:1 = healthy , 7= dead , areconverte d to 0-100%. RL:hypothetica l slope (=1 )i fth e differences between inner and corner treeswer e independento fresistanc e of families,o = media n score forth e (relatively) 'resistant families',• = media n score forth e 'susceptible families'. Inth e former group,th e inner trees areles sdiseased , inth e latter group,the y aremor edisease d thanth ecorne r trees.Afte r Squillace et al.,1972 .

was nodifferenc ebetwee n corner- and inner trees inth e average families (Fig.3) . Ifth e resistant families ofFig .3 a s agrou p arecontraste d withth e susceptible families as agroup ,th edat a ofFig .3 ca nconvenientl y be represented ina diagra m like Fig.4 . Inthi s 'mixogram',diseas e ratings (ordinate)ca nb e given for aresistan thos t inpur e stand (left-hand 335 disease disease

100% 100%

Or' 50- 50

Res. 50/50 Res. 100% mixtures 0% Susc. Susc. 0% 100% Figure 4. Disease rating (Lophodermium pinastri) for agrou p of resistant families (x)an d susceptible families (o)o fScot spin e grown inmixture s ofdifferen tproportion s ( extrapolations).

side), for asusceptibl e host inpur e stand (right-hand side)an d foreac h ofthe m grown inmixtur e inan yproportion .Th e line forth e resistant family group shows that the inner trees,whic h represent amor epur e stand situation, are less diseased than thecorne r treeswhic h represent a,mor e mixed stand situation.Th e actualpositio n ofth e line inth egraph ,an d thus its slope,i sdependen t onsom e assumptions aboutth e reach ofthi s neighbour effect:eve nth e inner treesma yb e affected to acertai n extent byplot s ofdifferen t susceptibility inth eperiphery .

Fig.4 i sderive d from Fig.3 a s follows. InFig .3 i nbot h therela ­ tively resistant and susceptible group,th emedia n scorewa s drawn.The n themedia n disease rating ofth ecorne rtre e ando f the inner tree ofei ­ ther group couldb e read onth eordinat e and abscissa respectively. These were converted intopercentag e (1=0%, 7= 100 %), giving thevalue s on theordinat e inFig .4 . Some assumptions had tob emad e inorde r to assignt oth ecorne r trees and inner trees certainvalue s onth e abscissa. Itwa s assumed thatth e influence of aneighbou r tree isproportiona l toth e square rooto fit s

336 distance from thetre e inquestion .Thi smean s that,i fth e surrounding trees areregarde d asstandin g inconcentri c rings around thetre e inques ­ tion, the influence ofring s farther outdecrease s quickly inspit e ofth e facttha tthe y containmor e trees.Th erati o ofth e influence ofth e first 3 rings (of4 ,4 and 12trees )t otha to fth e following 3ring s (of16 ,2 0 and2 4trees )i s9 t o4 .Thi sproportio n iseve n increased ifno tonl y dis­ tance,bu t also interception or afilterin g effect isconsidere d toreduc e the influence. Itcoul db e assumed, rather arbitrarily, that interception is0 % forth e firstrin g (directneighbour) , 20% forth e second ring, 40% forth e third, 60% forth e fourth,8 0% forth e fifth,9 0% forth e sixth.B ydoin g so,th erati o ofth e influence ofth e first3 ring s totha t ofth e following 3ring s is 7.4 to 1.1.Ring s further outpla y aminut e role.Unde r these assumptions,a tre e surrounded byn omor e than3 ring s of its ownkind ,woul d still score 7.4/(7.4+ 1.1 ) x10 0 %= 8 7% onth e abscissa. Thehalf-si b testcontaine d families ofal llevel s of susceptibility, not justtwo .A corner tree,e.g . ina resistan tplot ,ha s init s first ring of4 neighbour s 2o fit s ownkind , and 2 'others'o f2 differen t fami­ lies. These are samples ofth eentir epopulation : 'resistant',^average ' and 'susceptible' families. Ina simplifyin g approximation the 'others'ar e regarded asconsistin g of5 0% 'résistants' and 50% 'susceptibles'.Th e situation isthe nreduce d to amixtur ebetwee n 'résistants' and 'suscepti­ bles', or 'ownkind ' and 'thedifferen t kind'.Th e composition ofth evari ­ ous rings ofneighbour s can thusb e identified ast o 'own'an d 'foreign'; this ismultiplie d by the 'weight'o rth e relative influence ofth ering , andvalue so fth edifferen tring s added.Usin ghighe r interception-factors than inth e aboveexampl e (0% , 50% , 80% , 90 %), theresul t istha tcor ­ ner treeswoul db e influenced 72% b y theirow nkin d and 28% b y theoppo ­ site kind,whil e forth e innertree s therelatio n wouldb e 97% versu s 3% .

A size-factor is also apparenthere :whe nth e stand is fullygrown , eachplo to f 16tree swil lb e replacedb y 1larg e tree,whic hwil l thusb e situated atth e 50-50poin to nth e abscissa ofth emixogram .Althoug h this tree could stillb e influenced by itsneighbours ,th eentir e situation of disease, susceptibility, spore flight and stand climatewoul db edifferent , whichwoul dprobabl y reduce theeffect . Iti sthu sclea rtha ta 'neighbour effect'doe s exist.Fro m thesedata , itca n evenb epredicte d that inth emor eresistan t families afurthe r decrease indiseas e ratingca nb eobtaine d byplantin g thetre e in anindi ­ vidualmixtur ewit h anon-host , aswa s reportedb y Fischer (1957).

CONSEQUENCES FORTESTIN G DESIGNS A closer inspection ofFig .4 , and atentativ e extrapolation ofth e lines shows severalpoint s of interest.Apparently ,whe ngrow ni na nindi -

337 vidual 50:50mixture ,th e differences indiseas e ratingsbetwee n theresis ­ tant and susceptible familieswoul d evenb e smaller thannow .O nth e other hand,whe ngrow n inpur e stands,th e resistant familieswoul db e less dis­ eased, and the susceptible familiesmor e diseased than the'averag eobtaine d inthi stes t field. Ina tes t field such asth e oneunde r consideration, the resistance ofth e relatively resistant families tendst ob eunderesti ­ mated, andthi s effectwil lb egreate rwhe nth eplot s aresmaller .Thi s bias iswell-know n inagricultura l plantbreedin g (Parlevliet, 1975, 1979). Squillace et al. (1972)discus s themerit s ofdifferen t testdesigns . As they found the diseasema yhav e apartiall y unexplained 'patchy'pat ­ tern,the ywoul d like randomized individual treeplot swit hman yreplica ­ tions andwid e spacing.Alternatively , toreduc e interactions between fami­ lies, they suggestrathe r large squareplot s (4x4 trees and more), in which onlyth e interior trees aremeasured . Intermediate blockswoul db e undesirable. Isolationrow s of arelativel y resistant familybetwee nplot s could alsoreduc e interactionsbetwee nfamilies . A similarbu trevers e effect isrecognize d inth etestin g forgrowt h rate in foresttre ebreeding , asdifference s ingrowt h ratebetwee n clones or families tend tob e overestimated whenplot s aresmall .

The use of mixograms

InFig .4 itca nb e seentha tth enegativ e influence ofth emixtur e on the resistantpartne r issimila r toth ebeneficia l influence onth esuscep ­ tiblepartner .Ho wwoul d this change ifa mor e resistantpartne rwoul db e involved? Suchplant swoul d be less affected by the sporesproduce d byth e susceptible plants,thei r line inth egrap hwoul d drop andbecom emor ehor ­ izontal.A tth e sametime ,a sth eresistan t treeswoul d contribute lesst o the spore cloud, the lineo fth e susceptible groupwoul d tend todescen d more steeply. Theparallellis m inFig .4 woul db e lost,th ene w graphwoul d approach that ofa mixtur ebetwee n ahos tan d anon-host .O n theothe r hand, ifth e susceptibility ofth e susceptible partnerwa s increased, its line inth e graphwoul d rise andten d tobecom emor ehorizonta l asmixin g with resistantmateria l would tend tohav e lesseffect . Thus the 'mixogram'ma yb e ausefu l tool forvisualizin g theeffect so f mixing genotypes.Mixogram s weretentativel y sketched for some ofth eexam ­ ples ofhost-diseas e combinations given above (Fig.5) .Not e thati nth e caseo fth emultiline , thepathogen s forth e 2host s aredifferent . Amixogra m by itself,givin g onlybiologica l information,canno ttel l whether acertai nmixtur e is advantageous ornot .Thi swil l depend notonl y onth e economic orothe rvalu e ofth epartner s inth emixture ,bu t alsoo n the level ofth edamag e treshold (Fig.6) .I fth edamag e threshold ishigh , atA ,th e 50:50mixtur e isver y advantageous asth ediseas e rating ofth e susceptible host isreduce d tobelo w that level. Ifth edamag e threshold is

338 others Castanea oak beech 100% 100

-5 0 50-

50/50 50/50

Chestnut blight on Castanea den- Nectria ditissima inN EFranc e tata (o) and other tree species (afterPerrin) . Susceptiblebeec h (x) in the eastern American fo­ (o)i sheavil y affected inpur e rest. Lines nearly horizontal: stands, little ifmixe dwit hre ­ hardly any interaction. sistantoa k (x).

Ulmuslaevi s U.carpinifoli a 100%100 % 4disease disease

50-

Res. A100 % 50/50 100% 100% 50/50 Simplified multilinewit htw o Dutch elm disease inUkrain e (af­ 'lines' (withthei rpathogens) . terMaslov) . Ifsusceptibl e V. Thedotte d linewoul d giveth e carpinifolia (o)i sadmixe d in average disease rating ofth e stands of 'resistant' U. laevis mixed stand atdifferen tcomposi ­ (x), the latter isbadl yaffec ­ tions. ted. Figure 5.Sketche s of 'mixograms'o f fourdifferen t host-host-parasite combinations.

339 disease disease 100% 100%

damage threshold — A

50%- 50?

Res. Susc. 100% 50/50 100%

Figure 6. Theeffec to fdifferen t levelso fth edamag e threshold onth e economic acceptability ofmixture s ofhost swit hpartia l resistances.Se e text.

intermediate atB ,mixin g iso f littleus e asth eresistan tpartne r remains good,th e susceptiblepoor . Ifth edamag e threshold islo w atC ,th emix ­ ture isunadvisabl e asdamag e canonl yb e avoidedb y growing the resistant partner ina pur e stand.

CONCLUSIONS

1. Indiscussin g thehealt hhazard s ofpur e stands,argument s stressing the danger ofth eundivide d risk shouldb ekep t separate from those stressing thatdiseas e incidencemus tb ehighe r inpur estands . 2.Broa d generalizations onth e effecto fmixin g ofgenotype s onth ehealt h ofa stan d or itscomponent s aredangerous .Th eeffec tma yb e differentfo r eachdisease ,site ,hos to rcase .Mixin gma y evenb edetrimental . 3.Th emixin g of ahos twit h anon-host ,whic hofte nmean smixin g different species,ma yhav emor e effecttha nmixin ghost swit hpartia l orhorizonta l resistance,bu t itma y entail greater silviculturalproblems . 4.Mixin g shouldb e doneconsciously , after astud y ofdiseas e andhos tha s allowed thepredictio n thatmixin g is advantageous.Blin d mixingwil l only inrar ecase s leadt ousefu l reductiono fdiseas eincidence .

340 5. Mixing of genotypes is a poor substitute for breeding for resistance in forest trees.

REFERENCES

Boyce,J.S. ,1954 .Fores tplantatio nprotectio nagains tdisease san dinsec tpests .FA O ForestDevelopmen tPape r3 .4 1p . Browning,J.A .& K.J .Frey ,1969 .Multilin ecultivar sa sa mean so fdiseas econtrol . AnnualRevie wo fPhytopatholog y7 :355-382 . Eis,S. ,1978 .Natura lroo tform so fwester nconifers .Symposiu mo nRoo tFor mo fPlante d Trees,1978 .p .23-27 . Epstein,A.H. ,1978 .Roo tgraf ttransmissio no ftre epathogens .Annua lRevie wo fPhyto ­ pathology16 :181-192 . Fischer,W. ,1957 .Zu rFöhrenschütt eLophodermiu mpinastri .Schweizerisch eZeitschrif t fürForstwese n108 :260-270 . Heybroek,H.M. , 1978.Primar yconsiderations :multiplicatio nan dgeneti cdiversity . Unasylva3 0 (119/120):27-3 3an d49-5 0 (acorrecte dreprin ti savailabl e fromth e author). Heybroek,H.M. ,1981 .Th erigh ttre ei nth erigh tplace .Dnasylv a (inpress) . Kleinschmit,J. , 1979.Limitation sfo rrestrictio no fth egeneti cvariation .Silva e Genetica28 :61-67 . Kriek,W .& G .Bikker ,1973 .Germa nan dDutc hprovenance so fScot spin e inth eNether ­ lands.Ned .Bosb .Tijds .45 :154-161 .Also :Dorschkam pMededelin gNo .131 . MacArthur,R.H . &E.0 .Wilson ,1967 .Th etheor yo fislan dbiogeography .Princeton . Maslov,A.D. ,1970 .[Insect snoxiou sfo rel mspecie san dthei rcontrol] .Moscow .7 6p . Parlevliet,J. , 1979.Wederzijds ebeïnvloedin gva nproefveldje se nd egevolge nhierva n voord eevaluati eva npartiël e resistentie.Zaadbelange n33 :329-332 . Parlevliet,J.E .& A .va nOmmeren ,1975 .Partia lresistanc eo fbarle yt olea frust , Pucciniahordei .II . Relationshipbetwee nfiel dtrials ,microplo ttest san dlaten t period.Euphytic a24 :293-303 . Peace,T.R. , 1962.Patholog yo ftree san dshrubs ,wit hspecia lreferenc et oBritain . Oxford.72 3p . Perrin,R .& F .Vernier ,1979 .L achancr ed uhêtre .Influenc ede scondition s stationelles surl agravit éd el amaladie .Revu eForestièr eFrançais e 31:286-297 . Rennerfelt,E. ,1947 .0 mrotröta n(Polyporu sannosu sFr. )i Sverige .Des sutbretnin goc h satta tupträda .Meddel .fra nStaten sSkogsforskningsinstitut .Ban d3 5fö r194 6(8 ) 1-88. Schreiner,E.J. ,1965 .Maximu mgeneti cimprovemen to ffores ttree sthroug hsyntheti c multiclonalhybri dvarieties .N EFores tTre eImprovemen tConferenc eProceeding s1 3 p.7-13 . Stiell,W.M. ,1970 .Som ecompetitiv erelation si na re dpin eplantation .Canadia nFores t Service.Publ .1275 ,1 0p . Squillace,A.E. ,J.G.A .l aBastid e& C.L.H ,va nVredenburch ,1972 .Breedin go fsuperio r Scotspin ei nth eNetherlands .Dorschkamp ,Inter nRappor tnr .10 . Squillace,A.E. ,J.G.A .l aBastid e& C.L.H ,va nVredenburch ,1975 .Geneti cvariatio nan d breedingo fScot spin ei nth eNetherlands .Fores tScienc e41 :341-352 .Also :Dorsch ­ kamp,Mededelin gNo .155 .

341 What is a safe number of clones per plantation?

W.J. Libby

Departmentso fGenetics ,an do fForestr y& Resourc eManagement ,Universit yo fCalifornia , Berkeley,California ,94720 ,US A

ABSTRACT

Thispape r discusses the following concepts:tha t a forest plantation is atris k from agrea tvariet y ofpossibl e physical andbioti c events,man yo fwhic h cannotb e anticipated, and eachha s itsow ndifferen tprobabilit y distribution of damaging aparticula r population oftrees ;tha tmanagement ,throug h its decisions onplantin g density, thinning schedule,an d salvage operations,ca n influence the amount ofdamag e ormortalit y that aplantatio n cansustai n and stillb emanage d economical­ ly; and,tha t cross-adaptation ofa short-generatio n pestma y bemor e likely amongman y clones than among fewunrelate d clones. Some ofth e (perhapsunexpected )conclusion s from analyses employing theseconcept s are:tha tmonoclona lplanta ­ tions are frequently thebes t strategy; thatmixture s of 2o r3 clones are frequently thewors t strategy, and arerarel yo r never thebest ;tha tmixture s of alarg enumbe r ofclone s are assaf e as seedlingplantations ; and, ifplantin g density and subsequent silviculture allow damage ormortalit y atlevel s comfortably above reasonable expected risk,the nmixture s of modestnumber s (7-25)o fclone s appear toprovid e arobus tan d perhaps optimum strategy.

INTRODUCTION

The question is simple and clear.Th e answer,i tseems ,i snot .Thi s paperwil l address some aspects ofth eproble m of assessing riskwhe n clones areuse d inproductio nplantations . Many thoughtful peoplehav e considered thata forestplantatio n ofa single clone isles s 'safe'tha n aplantatio n ofseedling s (Goodman,1975 ; Marshall, 1977;Singer , 1979). Ifth eclon e issusceptibl e to somephysica l event, such asdrought ,col d orwind , then asingl e sucheven tma y destroy or seriously damage the entireplantation .Worse , fastbreedingdisease s or

342 . insects,ove r severalo fthei rgenerations ,ma y adaptmor e closely toa singlewidely-plante d clone thant oth egreate r diversity ofphenotype s in a seedlingplantatio n (Day, 1974). Janzen (1970)use dpar to fthi s argument toexplai n species-level diversity intropica l forests. Planting asingl eclon e over large areas hasbee ndone ,an dwit h some success. Itprovide s operational simplicity, andma y alsomaximiz e genetic gain (Appendix Comment 1).Perhap s themos tnotabl e forestry example of monoclonal plantings isprovide db ypopla r clone 1-214.Bu teve nthi s unusually successful cloneha s encountered serious difficulties,no t allo f whichwer eperceive d atth e time ofplantin g (Heybroek, 1978;Herpk a & Guzina, 1979). There hasbeen ,particularl y inpopla r culture,som e resistance to planting mixtures ofclone s (Kolster, 1978;bu t see Schreiner, 1971an d earlier).However , seedlingplantation s of forest trees areusuall ymix ­ tures ofman y different genotypes.Thus , the ideao f intimately mixed genotypes in aplantatio n ishardl y new oruntrie d in forestpractice .

THE PROBLEM

The relationship between risk andgeneti c variation ina plantatio no r foresti s frequently presented asi nFig .1 . Ifsuc h afigur e iscorrect , one conclusion isclear :th emor e clones in aplantation , the safer itwil l be. Clonal forestry offersman y genetic andmanagemen t advantages (Ehren- berg, 1977;Libby , 1977;Lindgren , 1977;Heybroek , 1978), andthu sman y tree-improvement programs are exploring theclona l option (Wilcox et al., 1976; Kleinschmit & Schmit, 1977;Lepistö , 1977;Roulund , 1977;Werner , 1977; Libby &McCutchan , 1978;Rauter , 1979). Inconsiderin g clonalforest -

Risk

Number of Clones (Genotypes) in the plantation

Figure 1. Schematic relationship between geneticvariatio n and risk (after Kleinschmit, 1979).

343 ry, managers are asking: 'Whati ssaf e enough?'an d 'Howman y clonespro ­ vides that level ofsafety?' . Atthi spoint ,th e centralproble m seemst ob e abalancin g ofth egain s obtainable withone ,o r few,o rman y clones againstth e risks associated with thatnumbe r ofclones . Iti s frequently assumed thatbot h gainan d risk aremaximu mwit h 1clone ,an dminimu m ina seedlin g forest.Thi spape r will explorebot h endso f Fig.1 , andwil l suggest thatbot h endsma yb eo f a different shape than asshown .

AW APPROACH TO THE PROBLEM

Disasters certainly happen,eve n inth e safesto fmulti-specie s seed­ ling forests (Bormann& Likens , 1979). Diseases such asblister-rus thav e done enormous damage to seedling forests ofwester n whitepine ,an dchest ­ nutbligh tha s driven theAmerica n chestnutt ovirtua l extinction. Iti s unlikely that anyclona l strategyw emigh tdevis ewoul dhav eprotecte d such forests or species against suchdisasters .Le tus ,therefore , consider suchgenera l disasters asoutsid e thetopi c ofthi spape r (Burdon, 1977). Some damage andmortalit y arebot hnorma l and acceptable inmos tfor ­ ests grown asrenewabl e resources. Iti su pt o forestmanager s todecid e what level ofdamag e andmortalit y isacceptable ,an d topla nplantatio n spacings andthinning s accordingly (Appendix Comment 2).Wha tmanager swis h toguar d againsti sexceedin g someunacceptabl e level of loss, rather than attempting tominimiz e the losspe rse .

AN EXAMPLE

Letu s firsttak e asimpl e case (Appendix Comment 3).A s athinkin g framework inthi s example,an d inth eremainde r ofthi spaper , letu s consider the composition ofplantation s of2 h ao rmor e insize .Conside r some singlephysica l eventthat ,i fi toccurs ,seriousl y damages orkill s someproportio n ofth etree s inth egenera lpopulatio n (say 10 %), butdoe s little orn o damage toth e remaining 90% ,whic h areno tsusceptibl e toit . Managementha spreviousl y decided thatcontinuatio n ofa plantatio nwit h losses greater than2 5% woul d notb eeconomic . Ifsuc h aneven t occurs ina plantatio n ofgenetically-divers e seed­ lings, drawn from the generalpopulation , about1 0% ofthe m arebadl y damaged orkilled .Th e other 90% are sufficiently well-formed andhealth y sotha tthe y are acceptablewithi n themanagemen tplan ,an dmanagemen t of theplantatio ncontinues . Giventha tther eha sbee nn o selection affecting resistance tothi s event, there is a1 0% chancetha tth egenotyp e ina single-clon eplanta ­ tioni ssusceptible . Ifi ti sno tsusceptible ,mos to r allo fth etree s sustain little orn o damage from this event. Ifi ti ssusceptible , then

344 mosto fth e trees arekille d or seriously damaged. For susceptibleplanta ­ tions, this is adisaster , andmonoclona l plantations are (inthi s case) more likely to sustainunacceptabl e losses than are seedlingplantations . Ina 2-clon emixture , ifth eclone s areno trelated , the probability thatbot h are susceptible tothi seven t is0.01 .However ,on e orth e other ofth e 2clone s issusceptibl e in1 8% ofsuc hmixtures ,an d ifso ,abou t 50% ofth eplantatio n willb ekille d orseriousl y damaged ifth eeven t occurs (Appendix Comment 4). (Sinceman y live trees are still onth esite , butundamage d trees areno t insufficien t density tob e effectivelyman ­ aged,managemen tma yvie w 50% damage andmortalit y as agreate r disaster than ifal ltree s inth eplantatio nha dbee nkilled. )A 3-clon emixtur e is evenworse ,wit h a0.2 7 probability ofmor etha n2 5% ofth e treesbein g damaged or killedb y this oneevent . (Furthermore, thesecon d and third clones inth emixtur e areprobabl y susceptible to someevent s thatlittl e affectth e firstclone ,thu s further increasing the chance ofunacceptabl e loss ina mixtur e of2 o r 3clones ,compare d toa monoclona l plantation of anyon eo fthem. ) Nowconside r aplantatio n thati s amixtur e ofman y smallclones . (For instance,eac hclon ema yhav e 10member s dispersed inth eplantation ,an d thusther e arel/10t hth enumbe r ofgenotype s in sucha plantatio n as ina seedlingplantatio n of thesam e size.)I fth e clones came from abroa d geneticbase ,an d their selectionwa sno tcorrelate d with susceptibility to the event,the n theoccurrenc e ofth edamagin g eventproduce s similar results tothos e inseedlin gplantations .Abou t 10% ofth etree s are killed orbadl y damaged, and about 90% suffer littleo rn o damage.Manage ­ mentca ncontinue .

Probability

.3-

.2

.1

0

1 2 3 .In n Number of Clones (Genotypes) in the plantation

Figure 2. Probability ofunacceptabl e loss, given asingl ephysica l event towhic h 10% ofth e tree genotypes aresusceptible , and amaximu m accept­ able loss level of2 5% .Plotte d only for 1-5,n/10 , and n clones.

345 Fig.2 plot s theprobabilitie s oflosse s exceeding 25% from sucha 10%-probabilit yphysica l event, forth e5 point sw ehav eexplored , and for mixtures of4 and 5clones .Unde r theconcep to f 'acceptable loss', Fig.2 departs from Fig. 1a tbot h ends.Unde r the conditions describedabove , there isn o increase inris k ofunacceptabl e loss, compared toa seedlin g plantation, associated withusin g alarg enumbe ro fclones .Furthermore , mixing aver y smallnumbe r ofclone s ismor e likely toresul t inunaccept ­ able losstha ni sa monoclona l plantation.Bot ho f these results,i fgener ­ al, have important implications forth epractic e ofclona l forestry.

GENERALIZATION FOR PHYSICAL EVENTS

Binomial algebrawa s used tocalculat e theprobabilitie s inth e above example. Itallow s only 2classes .Surel y therewil lb e some genotypes that sustain intermediate levels ofdamage ,o r thatar e seriously damaged in somecircumstance s butno t inothers .Fig .3 present s 4 sets ofcurve s for different susceptibility levels inth egenera l population, and for 6-8 levels ofunacceptabl e loss.Thes ecurve swher e chosent o illustratesever ­ algeneralization s derived fromman y such curves.The ywer e generated by 1 000t o1 0 000rando m numbers drawn from each ofvariou s sets ofWeibul l distributions (Johnson& Kotz, 1970). (Tore Skr0ppa,o fNISK , greatly aided inprogrammin g and statistical interpretation.) The following interpretations were used when analyzing theprobabilit y curves generatedb y samples from theWeibul l distributions:A sampled clone withvalu e 1.0 meanttha t allmember s oftha tclon ewer e killed orserious ­ lydamaged ; 0.0 meant thatal lmember swer eundamaged , orwer eonl y damaged tosom e acceptable degree;0. 3 meanttha t3 0% ofth emember s were killed or seriously damaged,bu tth eremainin g 70% wer e damaged tosom eaccept ­ able degree orwer eundamaged . (Asampl evalu e of 0.3 wasnot ,however , interpreted tomea n thatth eclon e sustained a3 0% loss invalu e ori n growth ...Appendi x Comment 5.) 'Seriously damaged'mean tthat ,becaus e of the damage,th etre ewoul db e removed atth enex tthinning , ortha tmanage ­ mentwoul d consider such atre en o longer likely tocontribut e to aneco ­ nomic harvest. 'Acceptable damage'mean t thatth etre e couldb eeconomical ­ lymanage d to rotation, and that ifsuc h atre ewer e removed ina pre-har - vest thinning, thatdecisio nwoul d bebase d on spacing andothe rconsider ­ ations in addition toconsiderin g thedamag e sustained. Inshort ,th e Weibull distributions and samples from them allowed us togenerat epercent ­ ageso ftree spe rclon e andpe rplantatio n thatwoul d stillb e considered economically manageable after somedamagin g event(s). The analyses ofth eWeibul l samples included distributions thatar e normal, skewed, and (bytruncatin g and summingbot h tails)bimodal . The meanvalu e ineac hdistributio n isdefine d asth eRis k toa Rando m Genotype intha tcircumstance ,hereafte r called RRG. Repeated samples of 1,2 ,3 o r

346 more cloneswer e drawn from eachdistributio n andteste d againstvariou s levels ofMaximu mAcceptabl e Loss,namel y thehighes t loss level that management hasplanne d foro r iswillin g to accept,hereafte r called MAL. The following generalizations seempossibl e andappropriate : 1. At least3 factors strongly influence theprobabilit y ofhavin ga manageable plantation after some damaging event(s): (a)th e shape ofth e distribution ofdamag e sustained by random genotypes (clones)i nth epopu ­ lation; (b)th e RRG; and (c)th erelationshi p of MALan d RRG, i.e., MAL - RRGo rperhap s MAL/RRG. 2. If MALi s lesstha n RRG, then theprobabilit y ofexceedin g MAL, if theevent(s )occur , is 1.0 inplantation s of seedlings oro fmixture s of many clones.Monoclona l plantings provide thebes t strategy inthi ssitua ­ tion.Not e thatth eprobabilit y ofeve nmonoclona l plantings exceeding the MALi sgenerall y higher thanth e RRG(i tequal s RRGi fth epopulatio n isa mixture of 100% resistant and 100% susceptible genotypes,wit hn o clones sustaining onlypartia l loss, asi nFig .2) .Se eth euppe rportion s ofth e graphs inFig .3 . 3. If MAL - RRGi s about 0.2 (ormore ) ...o r if MAL/RRGi s about 1.7 (ormore ) ...i.e. , ifth emaximu m acceptable loss level iscomfortabl y higher thanth e risk, thenmixture s of 7-15 clones (or fewer)wil lprovid e aprobabilit y of less than 5% ofexceedin g the MALlevel .Se eth e lower portions ofth egraph s inFig .3 . Inthes ecircumstances , suchmixture s are safer thanmonoclona l plantations, and almost as safe asmixture s ofman y clones or ofseedlings . 4. When RRGi sonl y alittl e less than MAL, largernumber s ofclone s inmixtur e arenecessar y toreduc e toa negligibl e amountth eprobabilit y ofth eplantatio n sustaining unacceptable loss. Seeth e centralportion s of the graphs inFig .3 .However ,thi s apparentmathematica l reductionwit h largenumber s ofgenotype sma yb emisleading . RRGnea r MALi sa margina l situation, and ifsuc hdamagin g event(s)occur ,th edecisio n to terminate orcontinu emanagemen t isver y close inplantation s of seedlings oro fman y clones inmixture .A mosai c of smallmonoclona l plantations mayb e the better option insuc hcases ,a s therisk s to individual clones tend tob e more dispersed,bot hbelo w and above the MALlevel .Whil e some orman yma y be destroyed,monoclona l plantings of theclone s thatprov e tob e resistant will remain available forcontinue d effectivemanagement . 5. Ifther e is little differencebetwee n clones inaverag e damage sustained (characterized by adistributio nwit h asingl emod e and asmal l variance), thenclona l strategies areno tver y effective.The y doappea r simple. If MALi sgreate r than RRGb y even amodes t amount,the nmixture s of 5-10 clones are about assaf e asmixture s ofa larg enumbe r ofclone s or of seedlingplantations . If RRGi ssimila r too rgreate r than MAL, then monoclonal plantings offer thebes t chance ofhavin g some economically manageable plantations ifth e event(s)occur .

347 Probability 1.0 1.0 .9 A^

.8 a.RR G= .42 3 .7

.6

.5 .5 0.0 0.5 1.0 .4

•05

40- II 80 160

1.0

b.RR G= .32 5

-.05 •0 1 10 II 20 40 80 160II I

Figure 3. Curves showingprobabilit y ofunacceptabl e loss, given asingl e physicaleven tt owhic h 0.423, 0.325, 0.152, and0.04 4o fth e genotypes in thegenera lpopulatio n aresusceptibl e {RRG). Thecurve s indicate the probability ofplantation s exceedingmaximu m acceptable loss (MAL) levels of5 % , 15% , 25% , 35% ,4 5% , 55% , and, forth ehighe r RRGs, 65% and

348 Probability 1.0 -T— 1.0

.9

.8

.7

.6

.5 • - ' .5 c. RRG=.152

.3

.2 L 0.5 1.0 0.0 .1 0.5 •0.5 0 ' • •0

1 4 5 10 20 40 80 160'

Probability 1.0 1.0

.9

.8

d. RRG = .044 .7

.6

.5

.4 0.L0 _0.5 1.0 — ' - <

.05 •» m 0 II 10 II 20 II 40 II 80 II 160 III III n rxumber of Clones (Genotypes) in the Plantation 75% .Thes e aretypica l ofth ecurve sproduce dwit hnumber s drawn atrando m fromvariou sWeibul l distributions ofclona l damage levels.Th eparticula r distributions producing eacho fthes e4 setso fcurve s are indicated onth e right sideo feac hgraph .

349 6. Whenmos tclone s areeithe rhighl y resistant orhighl y susceptible toa neven t (thedistributio n isbimodal) , and MALi sgreate r than RRG, thenmixture s of2 (and sometimes 3o rmore )clone s areofte nmor e likely tosustai n damage exceeding the MALtha n aremonoclona l plantations.(Re ­ call thata secon d event intensifies this effect.)Th e closer RRGi st o MAL, the greater isth enumbe r ofclone swhos emixtur e has ahighe rproba ­ bility ofexceedin g the MALtha n amonoclona l plantation drawn from the samegrou p ofclones .However , if MALi sgreate r then 0.5 (i.e., 50% sustainable loss)an d also exceeds RRG, thenmonoclona l plantations are always thewors toption ,an d anincreas e innumbe r ofclone s inmixtur e leads to afairl y continuous decrease inth eprobabilit y of exceeding the MAL. See lower curves inth e graphs inFig .3 . 7. Athighe r RRG(greate rtha n about0.35 ) ittake smixture s ofmor e clones to reduce theprobabilit y ofexceedin g the MALt o anegligibl e level than itdoe s atlowe r RRG(les s than about 0.25). Itseem s likely that selection ofwell-adapte d clones will lowerth e average risk to such genotypesbelo w RRG. Butthi s effectma yb e smaller thanon emigh thope ,du et oth euncertai n andcomple x nature ofth erisks . Iti spossibl e thatecologica l gradients inrisk 'wil l affect regional strategies.Takin g species diversity asa cu e (Goodman, 1975;Burdon , 1977), RRGappear s lower athig h latitudes and increases toward the tropics.Th emajo r contributors tothi s increasing RRGappea r tob e biotic (Janzen, 1970).

BIOTIC EVENTS

Biotic events canb emor e complicated thanphysica l events (Harper, 1977). For instance,Da y (1974)liste d 19variable s that affectth ecoloni ­ zationan d subsequent spread of apest ,an dwhic hma y result ina nepidem ­ ic.Underlyin g atleas t some ofth e development that follows isa concep ­ tualmode l ofgeneti c influences onhos t resistance tocolonizatio n and susceptibility todamage ,an do npes thost-range ,virulence , andaggres ­ siveness,tha t includes several alleles ateac h ofsevera l loci (notneces ­ sarily ofequa l importance)fo reac hcharacteristi c (Brown,1975 ;Grot h & Person, 1977;Marshall ,1977 ;Griffiths , 1978). Giventh ebackgroun d of all thesevariables , and theirmuc h larger number ofpossibl e interactions,Fig .4 attempts toschematicall y and simplypresen t 2importan tprocesses ,th ecolonizatio n and subsequent genetic adaption of apes tt o asingl e clone.Usin g the same probabilities forcolonizin g ability as forth e above simplephysica l example,ther e isa 90% chance ofth eclon ebein g resistant (colonization fails)an d a1 0% chance of itbein g susceptible.However ,unlik e thephysica l event,i fth e insect ordiseas e successfully colonizes aplantation , its subsequent generations may evolve,an d inmor e thanon ewa y (Barrett, 1978).

350 Phenotypic Array of Trees 1 I I Pest Colonizing Ability (Host range)

Figure 4. The topportio n ofth e figure represents thephenotype s ofclon e 1 ina monoclona l plantation, arrayedwit h respectt o susceptibility toa particular pest. Ifth e colonizing ability of apes t includes the ability tocoloniz e clone 1, itwil lbecom e established andma y adapt increasingly precisely totha t singlehos t (pesta ) (Wolfe, 1978). However, ifth e invadingpes tha s thehost-rang e of 'b', itwil lno testablis h ina mono ­ clonal plantation ofclon e 1. (Itshoul db enote d thatth ephenotypi c array ofth ehos tpopulatio n isprobabl y notth e same forpest s 'a'an d 'b', particularly ifthe ybelon g todifferen t species.). / 1 1 Phenotypic Array of Trees ,» * , ! | | | Pest Colonizing Ability a C b (Host range)

Figure 5. Iti spossibl e thatclon e 2wil lb e increasingly safe from attack bypes t a, aspes t aadapt smor eprecisel y toth e interplanted clone1 (Appendix Comment 6). Clones 1an d 2wil lbot h escape colonization bypes t b. Butpes t ccoul d colonize and damage clone 2i fi tinvade s theplanta ­ tion, thus raising theoveral lprobabilit y ofdamag e exceeding 25% inth e 2-clonemixture .

Ifth epes t colonizes alarg emonoclona l plantation, itma y adapt increasingly precisely totha tclone .Thi sma y create anepidemi cmor e severe thanwoul d have occurred (ortha tmigh tno thav e occurred atall )i n a genetically-diverse seedling population (Day, 1974). Thus, itappear s that alarg emonoclona l plantation ismor e likely to sustain serious dam­ age, compared toa seedlin gplantation ,durin gbioti c events thandurin g physicalevents . Using the sameprobabilitie s ina 2-clon e mixture,9 0% ofth etim eth e insecto rdiseas epreadapte d tocoloniz e oneclon ewil l notb epreadapte d tocoloniz e thesecond . Furthermore, asth epes tadapt s increasinglypre -

351 ciselyt oth eon eclone ,i tma yb e lessabl et ocolonize ,o rwil lb e less virulent (oraggressive )on ,th esecon d clone (Fig.5 ) (Edmunds& Alstad , 1978) (Appendix Comment 6).However , following the same logic aswit hphys ­ icalevents ,th e risk ofunacceptabl e loss (inthi s example,2 5% o rmore ) isgreate rwit h 2-o r3-clon emixture s thanwit h amonoclona lplantation . And,a sindicate d inFig .5 ,a secon d clonei sprobabl y susceptible to pests thatcanno t successfully attack the firstclone ,furthe r raising the overall risk of anunacceptabl e loss in a2-clon emixture . Ina seedlin gplantation , the adaptation ofa n insect ordiseas epopu ­ lation following successful colonization may follow adifferen t coursetha n thatdescribe d above for1 clon eo r ina mixtur e ofver y,fe wclones .Whil e there aren o 2genotype s identical inmos t seedlingplantation s of forest trees, there areman y that arever y similar.Thes e form ageneti c continuum within theplantatio npopulation . Ifth epes tha s successfully colonized 10% ofth etrees ,th epes tpopulatio n isunlikel y to adaptnarrowl y to only 1o fthos egenotypes ,bu twil l itselfprobabl ymaintai n substantial genetic diversity (Day, 1974). Iti spossibl e (evenlikely )tha tove r several of itsgeneration s itwil l continue to adaptt ophenotype s similar tothos e justsuccessfull y attacked. Thus, ineac hgeneration , somemember s ofsuc h apes tpopulatio n can successfully attack trees (genotypes)resis ­ tantt opreviou s generations ofth epes t (Marshall& Pryor , 1978). Before the epidemic runs its course,i tma y infest and significantly damageo r killman ymor e thanth e 10% ofth eseedling-origi n trees initially sus­ ceptible to it,an d thus exceed higher MALlevel s (Fig.6) . The resultwil lb e little different ina plantatio n consisting ofman y clones, each cloned only afe wtimes .Thi sproduce s the relationship onth e right side ofFig .7 . Thus, 2rathe r differentgeneti cpossibilitie s aresuggeste d witha short-generation pest.Wit h 1o ronl y a fewclone s ina larg eplantation , thepes tma y adapt increasingly closely toth eclon e itsuccessfull y colo-

Phenotypic Array of Trees 1 "~ H Initial Pest Colonizing Ability

4 ^ Incremental adaptation to similar ». phenotypes in subsequent generations of the pest Figure 6. Colonization of 10% o f apopulatio n ofseedlin g origin, followed by incremental adaptation andsprea d ofth epes tt o initially-resistant trees (Wolfe, 1978).

352 Probability

"J?

12 3 4 5 .In

Number of Clones (Genotypes) in the Plantation

Figure 7.Relativ eprobabilitie s ofunacceptabl e loss, given asustaine d bioticeven tt owhic h 10% ofth etre egenotype s areinitiall y susceptible, continued adaptationb y thepest ,an d amaximu m acceptable loss level of 25% .Becaus e ofth e2-ste pnatur e ofbioti c events,colonizatio n followed by adaptation,probabilitie s cannotb e accurately identified withou r simpleassumptions .

nized,wit h anincreas e invirulenc e ontha tclon e and areduce d likelihood that itca ncoloniz e initially resistant clones (Wood, 1980). Withver y many clones or aseedlin gpopulation ,th epes tma ymaintai n andeve nin ­ crease itsgeneti cvariability , suchtha ti tca n adaptt o successfully attackpreviousl y resistant trees,althoug hperhap swit h reduced virulence asth eepidemi c spreads (Groth,1976 ;Wolf e& Barrett ,1977 ;Edmund s & Alstad, 1978;Gould , 1979). This suggests an interesting possibility. Therema yb emixe dplanta ­ tionswit h enough clones toacceptabl y spread general risk,bu twit hsuffi ­ cientgeneti c differences amongth e clones sotha tcross-adaptatio n ofa narrowly-adapted pest isunlikel y tocontinu e toman yo fth eothe r clones inth eplantation . The lowernumbe r ofclone s insuc h amixtur e isinflu ­ encedb y the MALdecide d bymanagement . Ifprotectio n againstcross - adaptation is attainable,th euppe rnumbe rwoul db e influenced by the number ofclone s that canb e included in aplantatio n and stillmaintai n genetic dissimilarities among them sufficient toimped econtinuin gcross - adaptationb y anarrowly-adapte d colonizingpes t (Fig.8) . The theoretical models ofMarshal l &Pryo r (1979)giv e somehop e that this iso nth e righttrack .Thei r theory isdevelope d forhomozygou s lines inth ehos t (most forest-treeclone swil lb ehighl y heterozygous), andthe y particularly wish toguar d againstth e evolutiono fa 'super race' ofpest , ablet o attack all lines.Thei r severalmodel s usedifferen t assumptions on

353 gene action,an d achieve somegeneralit y -i.e. , itappear s better tohav e fewlines ,al ldifferin g inman y alleles,tha nt ohav eman y lines,eac h differingb y only a few alleles from someo fth eothe r lines inth emix ­ ture.Th e somewhatdifferen tmodel s ofGrot h& Perso n (1977)als o support thisview . a i m i in ii

Phenotypic Array of Trees

o o o o o Clone phenotype o o o o o 'VMIJ/ o ° ^^° o ° O o o o Pest Colonizing Ability

Phenotypic Array of Trees

Figure 8. (top)Pes t awoul db e able tocoloniz ebetwee n 0an d3 clone s (depending onwher e inth ehos tphenotypi c array iti s initially adapted) ifonl y 12clone s aremixed , asi nth euppe rplantation .Th e genetic dis­ tancesbetwee nmos to fthes e 12clone s appear great enough sotha tlittl e cross-adaptation ofthi snarrowly-adapte d pest islikel y tooccur , (middle) Ifth eplantatio nmixtur e includes 23 additional clones,the n colonization bypes t aseem s almostcertai n ifi tinvades ,an d substantialcross - adaptationseem spossible , (bottom)Th eclon ephenotypi c arrays andpes t colonizing abilitiesma yb emor erealisticall y shown inmor etha n 1dimen ­ sion.Th eabov e2-dimensiona l illustration allows greater distances between the3 5clone s inth emixture ,wit h less apparentprobabilit y ofpes tcross - adaptation,eve nthoug h its initial host-range includes about1 0% ofth e population.

354 ProbabiliK

I ! ! —

I b c d n

Number of Clones (Genotypes) in the Plantation

Figure 9. Probability ofunacceptabl e loss, given acombinatio n ofphysica l andbioti c events ofvariou s kinds,an d amaximu m acceptable loss level thati sno tto o low.Regio n 1 - b ischaracterize d by anincreasin gproba ­ bility thata nunacceptabl e proportion of theplantatio nwil l be killed or seriously damaged, asth enumbe r ofclone s inmixtur e is increased from 1 to b. The size and shape ofregio n b - c is sensitive tomanagement' s judgement concerning theproportio n ofth eplantatio ntha t isdesigne d to allowdamage ,mortality , or eventually be thinned.Regio n c- c ii swher e someprotectio n againstcross-adaptatio n by dynamicpes tpopulation s mayb e afforded bymixture s ofrelativel y fewdissimila r clones.Regio n d - n is essentially flat,offerin g aboutth e same safety andris k as apopulatio n of n seedlings.

INTEGRATION OF PHYSICAL AND BIOTIC EVENTS

Without specifically identifying numbers other than 1 and n, agrap hi n thegenera l shape ofFig . 9ma yb epredicte d for amixtur e ofunknow n damaging events. Ifth e gainmaximu m isno t at1 clon e (Appendix Comment 1), but ato rnea r thenumbe r associated with theminimu m risk of exceeding acceptable loss, thendecision-makin gbecome seasy .

SOME SUGGESTED GUIDELINES

The relationships developed inFigure s 3an d 9no w allow afe wguide ­ linest ob e considered. 1. Managementma y choose MALlevel s that arever y low,wher e most treesplante d areexpecte d tob eharvested .Whe n such MALlevel s areap ­ plied togener awit h knownbioti c orphysica lvulnerabilities , sucha s Populus (Schreiner, 1971), i.e., whenth ereasonabl e expectation of RRGi s

355 higher thanth e MAL, thenmonoclona lplanting s appear tob e thebes tstrat ­ egy. Itseem spruden t tous e several different clones ina mosai c ofmono ­ clonalplantings ,s otha t anentir e enterprise isno t incapacitated by one or afe wdamagin g events (Roister, 1978). 2. Thehighe r the acceptable loss level thatmanagemen t iswillin gt o assume, themor e flexibility itwil l have inchoosin g a 'safe'numbe r of clones. This argues forplantin g arelativel y highnumbe r oftree spe runi t area,wit h anee d to thin several timesdurin gdevelopmen to fth eplanta ­ tion. Inth e absence of significantmortalit y ordamage , itallow sth e forester toselec t and favor those treesgrowin gbest . Ifseriou s damageo r mortality occurs,thinning swil l remove damaged andmalforme d trees,an d adjustspacin gt o favorth ebes to fth esurvivors . 3. Several factors argue formixture s ofrelativel y fewclones .Thes e include:th epossibilit y ofmixin ghighly-selecte d well-known complementary clones to increase unit-areaproductivity ; easier andmor e efficientnurs ­ erymanagement ; andth epossibilit y ofreducin g cross-adaptation ofnarrow ­ ly-adapted pests following colonization.A mosai c ofdifferen tmixture s in a regionma yhav e additional beneficial effectswit h respectt oth e first and third ofthes e factors (Ehrenberg, 1977;Libby , 1977). 4. Muchplant-breedin gwor k attemptst ocreat e cropmixture sbase do n knownvirulenc e and resistance genes inth ehos t and inspecifi cpest s (Wolfe, 1978). This,particularl y ifth emultilin e isbre d for agronomic uniformity, has atendenc y toreduc ebackgroun d variability inth ecro p with respectt oothe runanticipate d events orepidemic s (Day, 1974;Wolf e & Barrett, 1977;Marshall , 1977). With the longgeneratio n and rotation times of foresttrees ,i tappear s wise toavoi d astrateg y thatconcentrate s on breeding forresistanc e to aspecifi c seto fknow nproblems . Itma yb e better todevelo p astrateg y that includesprotectio n ofth eplantatio n againstunknow nproblem s (Allard& Hansche , 1964). 5. Heybroek (1978)properl y noted thati tshoul d bepossibl e tomain ­ tain ahighe rproportio n ofunusua l recombinants and divergent genotypes clonally thanca nb e repeatedlyproduce d inpopulation s of sexually recom- bined seedlings.A s astrateg y fordealin gwit hunidentifie d possible disasters,stric tpedigre e control from abroa d geneticbas e appears tob e a good approach,wit h the added restriction that allclone sb ewell-adapte d toth e site (Kleinschmit, 1979). Onecoul d construct rules againstmor e thana certai nnumbe r orproportio no fclone s from the same familybein g in the same clonalmixture . 6. Ifther e isvalidit y inth e above lines ofreasoning , itwoul db e useful if laws andguideline s affecting clonal forestrywoul d avoidpre ­ scribing orencouragin g eitherto oman yo rto o fewclone spe r plantation (Marshall, 1977;Wolfe ,1978 ;Fowler , 1979/80). As anearl y guess,2 o r3 clones inmixtur e isto o few,an d inman y cases iswors e than amonoclona l plantation.Number s near 30o rlarge r substantially erodeth epossibl e

356 advantages inpoin t (3)above .Onl y 1clon epe r family shouldb e allowed in mixtures of fewer than about3 0clones . Finally, amixtur e ofclone s from 2o rmor e species should reducepes t cross-adaption notonl ybetwee n the species,bu twithi n eacho fth e spe­ cies, since fewer clones from each species canb eused .

ACKNOWLEDGEMENTS

Consultations onthi s topicwit h K.Aastveit ,R .Burdon , J.Dietrichson , L. Fins, A.Franclet ,V . Guzina,D .Harry , J.Harper , I.Herpka ,H .Heybroek ,M . Jost,M .Karlman , J.Kleinschmit ,R .Koster , A.Krstinic ,H .Muhs , S.Papadopol ,H .& F .Roll-Hansen ,T .Skr0ppa , P. Spieth,H .Weisgerber ,D . Zagory, andB .Zobe l areacknowledge d and appreciated. Themanuscrip twa sprepare d while atTh eDepartmen to f Forest Genetics andDendrology , Forestry Faculty, Zagreb,Yugoslavia ; and atth e Norwegian ForestResearc h Institute,As ,Norway .Acces s toth eNIS Kcom ­ puter and librarywa sparticularl yhelpful .

APPENDIX COMMENTS

1. There are at least3 alternative conceptsbearin g onth e question ofproductivit y per unit area (Harper, 1977). At issue isth enatur e of biotic interactions betweenneighborin g trees.On e alternative istha tsom e 'best' cloneproduce smor epe runi t area inpur emonoclona l culture than does anymixtur e of clones (Kolster, 1978). A second istha t genetically identical neighborsmak e similar demands onth e site atabou tth esam e times;bu ttha tneighborin g treeswit h complementary genotypes interfere which each other less, andus e thesit ebette r thanth ebes t ofthe m ina monoclonal planting (Allard& Hansche , 1964;Challinor , 1968;Wolfe ,1978 ; Kleinschmit, 1979). Included inthi s second alternative isa possibl e interactionbetwee n theplant s and thehost-specifi c insects and diseases thatdamag e them (Chilvers& Brittain , 1972;Burdo n& Chilvers ,1974 ; Marshall, 1977). Itshoul d benote d thatmuc ho fth eevidenc e now available onth e relativeperformanc e ofmixture s andthei r components inpur eplan ­ tings isdraw n from agronomic crops,selecte d togro w aspur eplanting s (Marshall, 1977), rather than frompopulation s ofplant s (such as trees) thatgro w andhav e evolved ingeneticall y diversepopulation s (Harper, 1977). The third alternative istha tproductivit y peruni t area iscon ­ stant;tha ton etree' s gain is aneighborin g tree's loss; and that,i fth e trees areadapte d toth e site,abou tal l atre ebreede r cand o ischang e the allocation ofexces sphotosynthat e within selected trees.Th e second alternative is appealing, and suggests thato nproductivit y groundsalone , amixtur e of afe wwell-understoo d excellent complementary cloneswil lb e

357 moreproductiv e than amonoclona l planting ofth ebes to fthem .A t square spacing,th eminimu m mixturewit hn o identical neighbors iso f5 clones ;a t hexagonal spacing, of7 . 2. Levels ofacceptabl e lossma yb e different atdifferen t stages ofa plantation's development. Forexample ,i na youn gplantation ,managemen t may accept ahighe rpercentag e ofdamag e ormortalit ybefor e thinning than following thinning.Late r inth erotation ,whe n someo fth e dead ordamage d trees canb e effectively salvaged, ahighe rpercentag e of lossma y againb e acceptable. 3. More than 1damagin g orpotentiall y damaging eventma yb e expected during the life of aplantation . Someo fth eexample s and calculations in thispape r takethi s intoaccount .Other s simplifyth eproble mb y summing2 ormor e independent events over thegenotype s they affect.However ,on e eventma y change theprobabilit y of asecon d eventoccurring , orchang eth e severity of itsdamage .Thi s seemsequall y true forplantation s ofseed ­ lings ando f clones,an dtherefore ,i slargel y ignored inth e development ofthi spaper . 4. Evenwit h aphysica l event,th epresenc e of aneighborin g genotype may change theprobabilitie s ofdamage .Fo r instance,a windfir m clonema y bedamage db y fallingmember so fa clon esusceptibl e towindthrow . Or,th e damage ina drought-susceptibl e clonema yb e intensified by neighboring drought-resistant trees ifthe y extract scarcewate r from the soilwit h greatereffectiveness . 5. When interpreting curves such asthos e inFig .3 ,on ema y consider loss invalu e orgrowth ,rathe r thanpercentag e ofmanageabl e trees.Suc h lossesma yb e general ina clon e orplantation .Fo r instance,a defoliatin g caterpillar or leafrus tma y reduce average growth 30% ,wit hindividual - treelosse s ingrowt hvaryin g from2 0% t o4 0% , andwit hn otree skille d by thatpes to r free of attack anddamage .A clonal strategy seems of littlehel p insuc hcases . Ifth e levels ofgrowt h loss thatd oo rd ono t allow continuedmanagemen t canb e identified foreac htre eb y foresterso n theground , thenthi s canb etreate d asi nth emai npaper . 6. Ingrains ,perhap sbecaus e ofth esize ,shap ean d shortlifespa no f individualplants ,ther e isevidenc e ofprotectio n ofsusceptibl e plantsb y interplanted resistant lines (Browning& Frey , 1969;Burdo n &Whithread , 1979), whichusuall y includes areductio n of susceptible-host density (Burdon& Chilvers, 1977), both effects leading toreduce d rates ofpatho ­ genspread .Suc hprotectio n effects areinfluence d byth enumbe r andper ­ centage ofresistan t lines andplant s (see,fo r instance,th emanipulativ e experiments ofLuthr a& Rao , 1979). However,Heybroe k (1978)argue d that thelarg esiz eo ftre ecrown s and longrotatio ntime swoul d greatly reduce such aneffect .Within-tre e variability must alsob e considered. Asuccess ­ ful attack on susceptible portions ofa tre e (say,shad e leaves)ma y allow thediseas e or insectt obuil dpopulatio n levels thatca n adaptt oo r

358 overwhelm more resistant parts of the tree. Adaptation across physiological states within a single tree, or among members of a clone occupying differ­ ent local environments, is very much like adaptation among similar geno­ types. Finally, if the population size of a pest population attacking a susceptible clone becomes large, it may generate mutants, unusual recom­ binants, or overwhelming numbers, that then successfully colonize previously-resistant neighbors (Browning & Frey, 1969; Dietrichson, 1969; Day, 1974; Groth & Person, 1977; Barrett, 1978; Edmunds & Alstad, 1978).

REFERENCES

Allard,R.W .& P.E .Hansche ,1964 .Som eparameter so fpopulatio nvariabilit y andthei r implications inplan tbreeding .Advance si nAgronom y16 :281-325 . Barrett,J.A. ,1978 .A mode lo fepidemi cdevelopmen ti nvariet ymixtures .In :P.R .Scot t &A .Bainbridg e (Eds):Plan tdiseas eepidemiology .Blackwel lScientifi cPublications , Oxford,p .129-137 . Bormann,F.H .& G.E .Likens ,1979 .Catastrophi c disturbancean dth estead ystat ei n northernhardwoo d forests.America nScientis t67 :660-669 . Brown,J.F. ,1975 .Factor saffectin gth erelativ eabilit yo fstrain so ffunga lpathogen s tosurviv e inpopulations .Journa lo fth eAustralia n Instituteo fAgronomica lScienc e 41:3-11 . , Browning,J.A .& K.J .Frey ,1969 .Multilin e cultivarsa sa mean so fdiseas econtrol . AnnualRevie wo fPhytopatholog y7 :355-382 . Burdon,J.J. , 1977.Mechanism so fdiseas econtro li nheterogenou splan tpopulation s- A n ecologistsview .In :P.R . Scott& A .Bainbridg e (Eds):Plan tdiseas eepidemiology . BlackwellScientifi cPublications ,Oxford ,p .193-200 . Burdon,J.J . &G.A . Chilvers,1974 .Funga lan dinsec tparasite scontributin g tonich e differentiationi nmixe dspecie sstand so feucalyp tsaplings .Australia nJourna lo f Botany22 :103-114 . Burdon,J.J .& G.A .Chilvers ,1977 .Controlle denvironmen texperiment so nepidemi crate s ofbarle ymilde w indifferen tmixture so fbarle yan dwheat .Oecologi a28 :141-146 . Burdon,J.J .& R .Whitbread ,1979 .Rate so fincreas eo fbarle ymilde w inmixe dstand s ofbarle yan dwheat .Journa lo fApplie dEcolog y 16:253-258 . Challinor,D. ,1968 .Alteratio no fsurfac esoi lcharacteristic sb yfou rtre especies . Ecology49 :286-290 . Chilvers,G.A .& E.G .Brittain ,1972 .Plan tcompetitio nmediate db yhost-specifi c parasites -A simpl emodel .Australia nJourna lo fBiologica lScienc e25 :749-756 . Day,P.R. , 1974.Genetic so fhost-parasit e interaction.W.H .Freema n& Co. ,Sa n Francisco.23 8p . Dietrichson,J. , 1969.Provenanc ean dresistanc e toScleroderri slagerbergi iGremmen . NorwegianFores tResearc hInstitute ,Vollebek k92 :398-410 . Edmunds,G.F. ,Jr .& D.N .Alstad ,1978 .Coevolutio ni ninsec therbivore san dconifers . Science188 :941-945 . Ehrenberg,C , 1977.Geneti cconseguence so fclona lforestry .Genetik a9 :159-171 . Eliasson,L.A. ,A .Dunberg ,G .Eriksso n& K.R .Samuelson ,1977 .Vegetativ epropagatio no f foresttree s- Physiolog yan dpractice .Institut eo fFores tImprovemen tan dDepart ­ mento fFores tGenetics ,Swedis hColleg eo fForestry ,Uppsala .15 9p . Fowler,C , 1979/1980.Plan tpatenting .Coevolutio nQuarterly .Winte r79/80 :34-38 . Goodman,D. ,1975 .Th etheor yo fdiversity-stabilit y relationships inecology .Quarterl y Reviewo fBiolog y50 :237-266 . Gould,F. ,1979 .Rapi dhost-rang eevolutio ni na populatio no fth ephytophagou smit e Tetranychusurticae .Evolutio n33 :791-802 . Griffiths,E. ,1978 .Plan tdiseas eepidemiolog y -Retrospec tan dprospect .In :P.R .Scot t &A .Bainbridg e (Eds):Plan tdiseas eepidemiology .Blackwel lScientifi cPublications , Oxford,p .3-9 . Groth,J.V. ,1976 .Multiline san d 'superraces' :A simpl emodel .Phytopatholog y 66:937-939 . Groth,J.V .& CO . Person,1977 .Geneti c interdependence ofhos tan dparasit e inepidem ­ ics. Annalso fth eNe wYor kAcadem yo fScience s287 :97-106 . Harper,J.L. ,1977 .Populatio nbiolog yo fplants .Academi cPress ,London .89 2p .

359 Herpka,I .& V .Guzina ,1979 .Th epresen tstat eo fblac kpopla rimprovemen ti nYugo ­ slavia. (Danasnje stanjeoplemenjivanj acrni htopol ako dNAS. )J .Graca n(Ed.) : Proceedingso fAnnua lMeetin go fYugoslavia nForestr yGenetics ,Jastrebarsk o (in press). Heybroek,H.M. , 1978.Primar yconsiderations :Multiplicatio nan dgeneti cdiversity . Unasylva30 :27-33 . Janzen,D.H. ,1970 .Herbivore san dth enumbe ro ftre especie si ntropica lforests . American'Naturalist104 :501-528 . Johnson,N.L .& S .Kotz ,1970 .Continuou sunivariat edistributions .Volum eI ,Chapte r20 . HoughtonMifflin ,Boston .30 0p . Kleinschmit,J. , 1979.Limitation s forrestrictio no fgeneti cvariation .Silva eGenetic a 28:61-67 . Kleinschmit,J .& J .Schmit ,1977 .Experience swit hPice aabie scuttin gpropagatio ni n Germanyan dproblem sconnecte dwit hlarg escal eapplication .In :Eliasso ne tal . (Eds):Vegetativ epropagatio no ffores ttree s-Physiolog y andpractice .Institut eo f ForestImprovemen tan dDepartmen to fFores tGenetics ,Swedis hColleg eo fForestry , Uppsala,p .65-86 . Kolster,H.W. ,1978 .Mengin gva nklone ni npopuliere nbeplantinge n I& II .(Mixtur eo f poplarclones. )Populie r 15(1):3-11 , (2):27-32 . Lepistö,M. ,1977 .Vegetativ epropagatio nb ycutting so fPice aabie si nFinland .In : Eliassone tal . (Eds):Vegetativ epropagatio no ffores ttree s- Physiolog y andprac ­ tice.Institut eo fFores tImprovemen tan dDepartmen to fFores tGenetics ,Swedis h Collegeo fForestry ,Uppsala ,p .87-95 . Libby,W.J. ,1977 .Roote dcutting si nproductio nforests .In :Proceeding so fth e14t h SouthernFores tTre eImprovemen tConference ,Gainsville .p .13-19 . Libby,W.J .& B.G .McCutchan ,1978 .'Taming 'th eredwood .America nForest s84(8) :18-23 , 37-39. Lindgren,D. ,1977 .Possibl eadvantage san drisk sconnecte dwit hvegetativ epropagatio n forreforestation .In :Eliasso ne tal . (Eds):Vegetativ epropagatio no ffores t trees- Physiolog y andpractice .Institut eo fFores tImprovemen tan dDepartmen to f ForestGenetics ,Swedis hColleg eo fForestry ,Uppsala ,p .9-16 . Luthra,J.K .& M.V .Rao ,1979 .Multilin ecultivar s- Ho wthei rresistanc einfluence s leaf-rustdisease s inwheat .Euphytic a28 :137-144 . Marshall,D.R. , 1977.Th eadvantage san dhazard so fgeneti chomogeneity .Annal so fth e NewYor kAcadem yo fScience s287 :1-20 . Marshall,D.R .& A.J .Pryor ,1978 .Multilin evarietie san ddiseas econtrol.I .Th e 'dirty crop'approac hwit heac hcomponen tcarryin ga uniqu esingl eresistanc egene .Theoret ­ icalan dApplie dGenetic s51 :177-184 . Marshall,D.R .& A.J .Pryor ,1979 .Multilin evarietie san ddiseas econtro lII .Th e 'dirty crop'approac hwit hcomponent scarryin gtw oo rmor egene sfo rresistance .Euphytic a 28:145-159 . Rauter,R.M. , 1979.Spruc ecuttin gpropagatio ni nCanada .In :J .Kleinschmi t (Ed.):IUFR 0 JointMeetin go fWorkin gPartie so nNorwa ySpruc eProvenance san dNorwa ySpruc e Breeding.Lowe rSaxon yResearc h Institute,Escherode .p .158-167 . Roulund,H. ,1977 .Vegetativ epropagatio no ffores ttree sa tth eArboretu m inH0rsholm , Denmark.In :Eliasso ne tal . (Eds):Vegetativ epropagatio no ffores ttree s- Physio ­ logyan dpractice .Institut eo fFores tImprovemen tan dDepartmen to fFores tGenetics , SwedishColleg eo fForestry ,Uppsala ,p .103-128 . Schreiner,E.J. ,1971 .Genetic so feaster nCottonwood .USD AFores tServic eResearc h PaperWO-11 .1 9p . Scott,P.R .& A .Bainbridge ,1978 .Plan tdiseas eepidemiology .Blackwel lScientifi c Publications,Oxford . Singer,M. ,1979 .Th ethir dforest .Ne wWes t1 7 (Dec): 43-48. Werner,M. ,1977 .Vegetativ epropagatio nb ycutting so fPice aabie si nSweden .In : Eliassone tal . (Eds):Vegetativ epropagatio no ffores ttree s- Physiolog yan dprac ­ tice.Institut eo fFores tImprovemen tan dDepartmen to fFores tGenetics ,Swedis h Collegeo fForestry ,Uppsala ,p .97-102 . Wilcox,M.D. ,I.J .Thuli n& T.G .Vincent ,1976 .Selectio no fPinu sradiat aclone si nNe w Zealandfo rplantin gfro mcuttings .Ne wZealan dJourna lo fForestr y21 :239-247 . Wolfe,M.S. ,1978 .Som epractica limplication so fth eus eo fcerea lvariet ymixtures . In:P.R . Scott& A .Bainbridg e (Eds):Plan tdiseas eepidemiology .Blackwel lScientifi c Publications,Oxford ,p .201-207 . Wolfe,M.S .& J.A .Barrett ,1977 .Populatio ngenetic so fpowder ymilde wepidemics . Annalso fth eNe wYor kAcadem yo fScience s287 :151-163 . Wood,T.K. , 1980.Divergenc e inth eEnchenop abinotat acomple xeffecte db yhos tplan t adaptation.Evolutio n34 :147-160 .

360 Balance in indigenous plant populations

1 .1 .2.3 1 A. Segal ,J .Manistersk i ,J.A .Brownin g ,G . Fischbeck & I.Wah l

ABSTRACT

Studies invariou spart s of theworl d have showntha ti n undisturbed natural ecosystems,disease s of indigenousplan t populations caused by indigenousparasite s are seldomdestruc ­ tive.Th eprolonge d host-parasite coevolution associated with reciprocal selectionpressur e culminates ina stat e ofbalance d polymorphism. The 'protection of indigenousness' isbase d on genetic diversity ofhos t andparasite s adapted toa wid e range ofbioti c and abioticparameter s inth e ecosystem. Israel islocate d inth ecente ro forigi n and genetic diversification of Hordeum spontaneum and Avena sterilis, the putative ancestors ofthei r cultivated counterparts. H. spon­ taneum is annually attacked by Erysiphe graminis hordei and Puccinia hordei. A. sterilis isaffecte d every yearb y P. coronata avenae and P. graminis avenae. Populations ofth e four fungi representbroa d spectra ofvirulenc e inwhic h strains combining numerousvirugene sprevail . Inth enatura l defense structures,th e following mechanisms participate: (i)conventiona l resistance accompanied byhyper ­ sensitivity, (ii)slo w rusting or slowmildewing , (iii)toler ­ ance, and/or (iv)escape .Thei rproportion s inth epopulatio n varywit h thehost ,parasite , andenvironment . Forexample ,th e firstmechanis m isno tuncommo n inprofile s ofdefens eoper ­ ating againstcrow nrus t andpowder ymildew ,bu t iti svirtual ­ ly absent on Ornithogalum plants strickenb y the gametophytic stage of P. hordei. The diverse geneticpatchwor k ofnatura lplan t populations minimizes the danger ofpreferentia l selectionpressur e onth e

1.Facult yo fLif eSciences ,Te lAvi vUniversity ,Te lAviv ,Israel . 2.Departmen to fPlan tPathology ,Iow aStat eUniversity ,Ames ,Iow a50011 ,USA . 3.Lehrstuh lfü rPflanzenba uun dPflanzenzüchtung ,Technisch eUniversitä tMünchen ,805 0 Freising-Weihenstephan,BRD .

361 parasitepopulation s andenable s theestablishmen t of awid e gamuto f fungus strains.Urediospor e production inth eplan t populations ismarkedl y reduced andth ediseas e isthu smiti ­ gated. The relevance ofknowledg e aboutnatura l ecosystems to genetic disease control inagroecosystem s is indicated.

INTRODUCTION

Natural,undisturbe d ecosystems arecharacterize d bybiologica l bal­ ance.Plan tpopulations ,beneficia l andharmfu lmicroorganisms , and abiotic environment areenmeshe d ina comple x systemmaintainin g dynamic equilib­ rium regulated byhomeostati cmechanisms . 'Plantdiseas e occurswhereve r plants grow' (Zadoks& Schein, 1979). Plant diseases inindigenou s plantcommunitie s are anatura lphenomenon , andd ono t indicate 'thatsom e aspecto fth ebiologica l balance isno ti n equilibrium' (Baker& Cook, 1974). They areconsidere d by some researchers assubsystem s inth e ecosystem (Zadoks& Schein, 1979).W e adopted this concept inthi s study.Th e disease seldom attains damagingproportions . This isparticularl y true ofcenter s ofhost-parasit e coevolution where both constituents ofth e subsystem reach astat e ofbalance d polymorphism asa resul to f along-lastin g reciprocal selectionpressure . Such anequi ­ librium is labeledb yHof f &McDonal d (1972) 'balancedsymbiosis' . Mechanisms regulating diseases innatura l ecosystems have attracted considerable attention.Accordin g toPerso n& Sidhu (1971), information for effective managing of 'man-guided' systems ofparasitis m 'cancom e only through studies ofnaturall y occurringsystems' .

STUDIES OUTSIDE ISRAEL

Zhukovsky (1959)analyze d the equilibriabetwee nwil d relatives ofcul ­ tivated crops andthei rparasite s inth e commonhomeland .A s arule ,th e hosts areno t immune. Instead, they areendowe d with 'fieldresistance 'o r tolerance.Th e fungusparasit e attacks only someplan tparts ,elicitin g limitednecrosi s and reduced sporulation.Thi sha s enabled host survival overmilleni a alongwit h theparasite .Th e coevolutionha sgive nris et o new andmor e effective resistance diversitymatche db yne w races ofth e parasite, referred to also as 'aggressive'races .Othe r examples ofhomeo ­ stasis innatura l ecosystems outside Israelwer e discussed by Segal et al. (1980). Balanced host-parasite interactionha sbee n ascertained innatura l forests.Accordin g toDinu s (1974), such forests, 'thoughno t immunet o catastrophe', arebuffere d by their 'ordered diversity against outbreaks ofpest s and other destructive agents'.Bingha m et al. (1971), dealingwit h

362 disease resistance in forests,emphasize d that 'inth ebalance d systems óf gene centers,rampagin g epidemics and immunity from disease are absentan d bothhos t andparasit emaintai n theirreproductiv e capacity'. Consequently, studies ofth e stabilized genepool s asmodel s arerecommended . A detailed analysis ofmechanism s mitigating diseases innatura l forestswa spresente d by Schmidt (1978).H e stressed thatepidemic s causedb y indigenouspatho ­ gens areno trar e inthes eecosystems . 'Afailur e todistinguis h between disease incidence and disease lossha s contributed toth emyt h thatepi ­ demics arerar e innatura l forests'.Nevertheless ,epidemic s causedb y indigenous pathogens areusuall y limited intim e and space.The y aremol ­ lifiedb ymechanism s collectively named 'ecosystem disease resistance',o r 'functional diversity' ofth ehost ,pathogen , climatic, edaphic,an dbioti c environmental parameters.

STUDIES IN ISRAEL

Our investigations weremainl y concernedwit hhomeostasi s insubsys ­ tems involving thewil dbarle y Hordeum spontaneum C.Koc h andwil dhexa - ploid oats Avena sterilis L. andthei r respective fungusparasites .Bot h species are indigenous to Israel,whic h is apar to fthei rcenter s of origin andgeneti c diversification. Both species aredistribute d country­ wide and consisto fnumerou s ecotypes. H. spontaneum isannuall y attacked by Ergsiphe graminis (DC.)Mera t hordei Em.Marcha i and Puccinia hordei Otth. P. coronata Cda. avenae Frazer &Ledingha m and P. graminis Pers. avenae Eriks.& E .Henn .ar e incidento n A. sterilis everyyear .Th e4 parasites have coevolved with theirhost s from remote antiquity. P. hordei and P. coronata avenae complete their life cycles onth erespectiv e indige­ noushosts , Ornithogalum spp.an d Rhamnus spp.Hence ,th e host-parasite subsystems implicating the 2fung i arecomple x and comprise the following biological components, (i) H. spontaneum - P. hordei - Ornithogalum spp. (ii) A. sterilis - P. coronata avenae - Rhamnus spp.Bot h subsystems are wellbalanced . Mosto fth eparasite s havebroa d spectra ofalternativ e hostsbelongin g toa numbe r ofspecie s oreve ngenera . Thebroa d gamuto fvariatio n invirulenc e is attributed to geneticre ­ combination thattake splac e inth ecommo n and functional cleistothecia of E. graminis hordei, ando nth e alternate hosts ofth ecrow nrus t andbarle y leaf rust fungi. P. graminis avenae hasn o alternate host in Israel.It s variation isascribe d to somatichybridizatio n onth ewid e rangeo frecep ­ tive grasses (Anikster &Wahl , 1979). Populations ofal l organisms are characterized by theprevalenc e ofstrain s combining awid e array ofviru - genes. The investigated plantswer e sampledwit h thetransec tmetho d at1- 2 m intervals, regardless ofthei r disease reaction innature .Thei r seedwa s

363 planted intes tnurserie s inth e order ofdispositio n ofth eparent s inth e original setting.Th enurserie swer eperiodicall y inoculated with cultures ofspecifi c parasites collected across thecountry . Four components regulating theprotectio n structureswer e identified, namely (i)conventiona l resistance associated with hypersensitivity anda lowtyp e of infection, (ii)slo wrustin g or slowmildewing , (iii)escap e- avoidance,and/o r (iv)tolerance .Th e first2 factorswil l be discussed in greater detail;ou r information onescap e and tolerance israthe r limited. Forth e sakeo fclarity , the4 disease s areclassifie d into 2group sbase d onth eprevalenc e ofhypersensitivit y indefens e structures.Hypersensi ­ tivity isrelativel y common inth epowder y mildew and leafrus to fbarle y aswel l asi noa tcrow nrust ,bu trar e inoa tste m rust and practically lacking inbarle y leaf rusto nth ealternat e Ornithogalum hosts. Thehost-parasit e subsystems ofth e firstgrou p arerepresente d inou r discussionb y oatcrow nrust .Othe r associationswer e analyzedb y Segal et al. (1980).

Subsystem: Avena sterilis - Puccinia coronata avenae - Rhamnus spp. Murphy et al. (1967)explaine d the evolutiono fhomeostasi s inthi s subsystem with this statement: 'Anatura lbalanc e ...appear s tohav ebee n established between A. sterilis, crownrust ,an d R. palaestina, inwhic hA . sterilis, although infected,produce s seed ofgoo d quality.Variou s levels ofresistanc e andtoleranc e tocrow nrus thav e apparently resulted from natural selectionunde r conditions ofregula r andheav y crown rustinfec ­ tion and arelativel y high levelo foutcrossin g inA . sterilis'. Notably, the fungus inhabits innatur e grasses of several genera (Dinoor, 1967). The defense structures innatura l communities ofA . sterilis have embraced the4 aforementionedmechanisms . Conventional resistance waspresen t inal l investigatedpopulations . Its frequency varied with theregio no forigin ,tim e ofth e season,an d ageo fth eplant . Itoperate d throughout thewhol e lifeo f someaccessions , while inother s alo wtyp eo f infectionwa s limited to some growthstages . Conceivably, restricted duration ofhypersensitiv e reactions offerssuffi ­ cientprotectio n toth ehos t and receptive substrate toth eparasite , facilitating theirbalance d coexistence. Slow crownrustin g ischaracterize d bymarkedl y reduced diseaseprog ­ ress, low infectability ofth ehost ,an ddiminishe d sporulation. Tolerance occurs inA . sterilis in Israel and theUS A (Segal et al., 1980). Inclimaticall y adverse regions,A . sterilis isonl y slightly affected orescape s the disease for alonge rperio db y early ripening. Inth epar ­ tially escaping hosts,th eduratio n ofdevelopmen t ofver y susceptible growthphase s isrelativel y short. Themor e commondefens e structures inth eA . sterilis communities are distinguished byprominenc e ofplant swit h infection type 3an d lowt omod -

364 erate infection severity,whil e inabou t3 0% ofth eplant s infectiontyp e 2 is associated with infection severity of 5-25 %.Al l investigatedpopula ­ tionsencompasse d fastcrow nrusters .Conceivably , someo fth e fastruster s wereprotecte d by tolerance.Ther ewa sn ovisibl e indication thatprotec ­ tionha s developed ata cos to f fitness inth ehost . The effecto fthe.defens e structures in A. sterilis populations onth e race composition of P. coronata avenae was compared with thato f Iowa Multiline 1-78,an d its recurrent A. sativa L.hos t represented bya mixture ofcultivar s C649 and Clintford. Intha tmultiline , resistance isimparte d by genestha tha dbee n extracted fromA . sterilis of Israeli origin.A tth e locationsHasoleli m andE nDor ,th emultilin e and itsrecur ­ renthos twer e growni nseparat e plots inproximit y to R. palaestina. Urediawer e collected from all 3oa tpopulation s andthei r racial identity determined inth e greenhousewit h the aid ofstandar d and supplemental dif­ ferential cultivars atth e seedling stage.Th e results (Table 1)demon ­ stratetha trac egrou p 276-264,whic hpredominate s in Israel,als opre ­ vailed onMultilin e 1-78,it srecurren thosts ,an dA . sterilis. However, theheterogeni c geneticpatchwor k of A. sterilis expedited theestablish - mento f almosttwic e asman y races as inth e other oatplots .Furthermore , theA . sterilis genes incorporated inth ewil d oatpopulation s and in1-7 8 stands seemed todilut e theconcentratio n ofth edangerou s racegrou p 276 & 264. This race group has constantly prevailed countrywide forman yyears . Itcombine s abroa d spectrum ofvirugene s and renders ineffective mosto f the knownhexaploi d sources ofresistanc e to P. coronata avenae. Hence,i t comes close toth e conceptual super-race.Th e lush stands ofA . sterilis

Table 1. Frequency ofoccurrenc e of Puccinia coronata avenae races infiel d nurseries atHaSoleli m andE nDo r onplant s of Avena sterilis, A. sativa mixed check: (C64 9 +Clintford )an d IowaMultilin e 1-78.Urediospor e iso­ lateswer e randomly sampled atHaSoleli m in197 8 and 1979,an d atE nDo r in1978 .

Host HaSolelim EnDo r

number number number of number number number of of of isolates, of of isolates, tested races racegrou p tested races race group isolates 276+ 26 4 isolates 276 +26 4

A. sterilis 235 37 62 79 19 22 Mixed check 224 21 153 53 11 37 MultilineI - •78 200 20 65 30 10 10

365 permanently exposed torac egrou p 276-264sugges ttha tth edefens emechan ­ ismsoperatin g inth enatura l ecosystem effectivelyprotec tA . sterilis againstparasit e strains thatconstitut e anea r super-race„as far ashexa - ploid oats areconcerned .Thi s situation appears todispe l the lingering fearso fth epotentia l hazards thatma y arisewit h theevolutio n ofsuper - races. The intensity ofcrow nrus t infectionwa s assessedb y determining the amounto furediospore s collected onrotoro d spore traps 9time sdurin gth e season inplot s sownt oth e following oataccessions : (i)th e 'standard' susceptible cultivarMarkton , (ii)Iow aMultilin e 1-78, (iii)Iow aMulti ­ lineM-73 , (iv)Iow aMultilin e E-77, (v)a mixtur e ofcultivar s C-649an d Clintford, recurrentparent s and agronomic checks tobot hMultiline s 1-78 andM-73 , (vi)cultiva r C-237-89-IV,whic h served asth e agronomic check forMultilin e E-77,an d (vii)5 A . sterilis populations sown separately. Eachpopulatio n consisted ofcomponent s whoseparent swer e sampledwit hth e transectmetho d aton e ofth e locations:Guara ,Be tNehemia ,Ginnaton , Hagor,Risho nLeZion .Annua l developmento fcrow nrus to nA . sterilis at RishonLeZio n ismor e intensive than inth e other4 locations,reflectin g amor e favourable environment and/or amor e susceptible sub-population of thehost .Th e results (Fig.1 )sho wtha turediospor e production inal l Iowamultiline s and inth eA . sterilis plots isdistinctl y lower thano n Markton and all recurrent hosts.Thus , resultswit hmultiline s inth eUS A (Browning, 1974)an d in Israel arecorroborate d by results from indigenous populations in Israel (Browninge tal. ,pag e37 1i nthi sbook) . TheA . sterilis populations constitute to some extent 'naturalmultilines' .Suc h studies enablequantitativ e estimation ofdefens e structures operating in natural ecosystems.

Subsi/stem: Avena sterilis - Puccinia graminis avenae The disease development season isshorte r forth e stem rusttha nfo r the crownrust ,an dver ybrie f inari dregion swher e thehos tvirtuall y escapes its impact.Conventiona l resistance,whic hprotect s theplan t throughoutth ewhol e lifeo fth eplant ,i sver y scarce. Itoccasionall y operates atsom egrowt h stages.Numerou splant sharbore d uredia ofsuscep ­ tible and resistant classes onth e same stems,sheaths ,o rblades .Thi s patterno freaction s ischaracteristi c of 'regional resistance' (Segale t al., 1980). Browning (1974)considere d 'regional resistance' as amean s of keeping stemrus t inbalance .A smentione d above,Zhukovsk y (1959)foun d thisphenomeno n tob e common incenter s ofhost-parasit e coevolution. Defenseo fth e slow-rusting type againstste mrus ti swidesprea d inA . sterilis. Itwa s discovered byDr .H.C .Murphy . This type ofprotectio n is manifested indiminishe d infectability ofth ehost ,retarde dprogres s of the disease, andreduce dmyceliu m growth inth ehost ,whil e uredia signify a susceptible reaction. Slow rusting hasprove n tob e stable and effective

366 NO. OF UREDIOSPORES/lOOL AIR

1 - Multiline E-77 2 - Hagor Avena sterilis populations 3 - Multiline M-73 4 - Bet Nehemya A.sterilis populations 5 - Multiline 1-78 ó - Guara A.sterilis populations 7 - Ginnaton A.sterilis populations 2500 8 - Rishon LeZion A. sterilis populations 9 -Clintford + C 649 -C237-IV- 89 - Markton

2000

1500

1000

500

21/4 23/4 25/4 27/4 29/4 1/5 5/5/1980

DATES OF UREDIOSPORES COLLECTION

Figure 1. Cumulative urediosporeproductio n ofcrow nrus to nteste dacces ­ sions. againstman y races. Defense structures against stem rust inpopulation s ofA . sterilis have beendescribe db y Segal et al. (1980). The relativelybrie f annual association ofA . sterilis with thepara ­ site during the season seems towar d offseriou s epidemics.Th ehost - parasite-environment interaction in Israel is lesswel l balanced than inth e case ofcrow nrust . Races of P. graminis avenae withbroa d spectra ofvirulenc e havebee n consistently prevalent overman yyears .Studie sb yGerechter-Amita i (1973)

367 demonstrated that suchrace sparasitiz e plants of 71 speciesbelongin gt o 36 genera ofGramineae .

Subsystem: Hordeum spontaneum - Puccinia hordei - Ornithogalum spp. Puccinia hordei iswidesprea d on H. spontaneum. The fungus completes its lifecycl e innatur e on indigenous Ornithogalum species.Th e alternate host is indispensable forth eperpetuatio n ofth eparasite .Th eprecocit y ofteliospor e formation restricts to some extentth epropagatio n of P. hordei duringth egrowin g season,bu tenhance s itsassociatio nwit h Orni­ thogalum plants,sinc e the spores germinate readily.Th ebalanc e ofth e funguswit hth emai n and alternatehost s and theexcellen t coordination in the development ofth e components inth e H. spontaneum - P. hordei - Orni­ thogalum sub-system isa resul to fcoevolutio nwel l adapted toth e semi- aridenvironment .Th e restricted range ofalternativ e hosts supportsth e hypothesis (Anikster &Wahl , 1979)tha tth e fungus iso f arelativel y recentorigi n and shortphylogeneti c history.Ther e is animportan tdif ­ ference inth emechanism s balancing the funguswit h themai n and alternate hosts.Anikste r (unpublished)ha s showntha t H. spontaneum isprotecte d against P. hordei by resistance accompanied byhypersensitiv e reactions and slow rusting. Incontrast ,hypersensitivit y isextremel y rare on Orni­ thogalum plantsharborin gpycni a and aecia ofth eparasite .Presumably , other types ofdefens e operate inth e alternatehost . Inartificia l inoculation greenhouse tests, P. hordei alternates with Dipcadi erythraeum Webb& Bert. ,an d Leopoldia eburnea Eig& Feinbr . (Anikster,unpublished) .However ,plant s ofneithe r species rusti nth e natural aridhabitats .The y seemt oescap e the diseasewhic h isunabl et o develop indr y regions.

CONCLUDING REMARKS

Innatura l ecosystems,disease s ofundisturbe d indigenousplan tpopula ­ tions causedb y indigenousparasite s areseldo m destructive.Bot hconstitu ­ entso fth ehost-parasit e couplethav eundergon e long-lasting coevolution thatculminate s ina stat e ofbalance d dynamicpolymorphism . They represent a subsystem thatpossesse s attributes of itsown ,bu ttha tmaintain s atth e sametim e interdependent relationswit h otherbioti c and abiotic components ofth ewhol e ecosystem (Zadoks& Schein, 1979). The equilibrium andcoexis ­ tence inth e subsystem areregulate db yhomeostati cmechanism s inherent in thenatur e ofth ehost ,parasite ,an dth e environment.Th e stabilizing fac­ torstha tdampe nexcessiv eperturbation s arecollectivel y manifested as 'ecosystemdiseas e resistance' (Schmidt,1978 )o r 'protection ofindige - nousness' (Browning, 1974). They operateb y engendering 'functionaldiver ­ sity' (Schmidt,1978 )o r 'patterned diversity' (Dinus, 1974). Some ofthes e components were listedb yBrownin g (1974)an d Schmidt (1978,Tabl e3) .

368 Israel is located in'th ecente r oforigi n andgeneti c diversification of Avena sterilis and Hordeum spontaneum, theputativ eprogenitor s ofthei r cultivated counterparts.The y are annually attackedb y indigenous obligate parasites,bu t theensuin g diseases neverbecom e very damaging.Mos to fth e causal agents inhabita wid e range of alternative hosts. Puccinia hordei and P. coronata avenae complete their lifecycle s on indigenous alternate hosts. Studies onnatura l defense structures wereconducte d by inoculating fungus cultures collected countrywide toplan tpopulation s reconstructed intes tplots .Th e reconstructionwa s attainedb y growing the entries according todispositio n ofthei rparent s innatura l ecosystems.Thes e entrieswer e sampledwit hth e transect technique.Thi smetho d synthesizes amultiplicit y ofprotectio npattern s and sortsou tth e components and processes acting innatura lhabitats . Fourcomponent s ofdefens ewer e identified: (i)conventiona lresis ­ tance, (ii)slo wrustin g or slowmildewing , (iii)tolerance , and (iv) escape.Th eprofile s ofthei r interaction ata give n site arestable , and depend onth egenetic s ofth ehos t andparasite ,thei r age,onto ­ genetic stageo fth erus t fungus,an denvironmen to fth eorigina l eco­ system aswel l aso fth e site ofth etest .Th e fundamentally different reaction of H. spontaneum to thesporophyti c stage of P. hordei ascom ­ pared with thato f Ornithogalum plants toth e gametophytic stage ofth e fungus reveals important aspects ofth ehost-parasit e balance.Alternativ e hosts enhance theparasiti c fitness ofth e fungib y augmenting theirsur ­ vivability andreproductivity .A tth esam etim ethe y increase thebufferin g capacity ofplan tpopulation s bymaximizin g their genetic heterogeneity. Studies ofnativ eplan tpopulation s with thetransec tmetho dprovid e a better understanding ofth e individual constituents ofth e defense struc­ ture andth epattern s ofthei r integration; quantitation of sporeyield s anddeterminatio n ofrac ecompositio n inth eparasit e illuminate thepro ­ tective actiono fth ebalance d ecosystem as acomple x entity.T oparaphras e the statemento fBake r& Coo k (1974):whil ema nma yneve runderstan d all the interactions inth enatura l ecosystem, theen d result -biologica l bal­ ance -can ,however ,b e appraised andexploited . Significantly, defenseaf ­ fordedb y thisbalanc e seems tominimiz e the damaging effecto fnea rsuper - races such asth erac e group 276& 264o f P. coronata avenae. Knowledge aboutnatura l ecosystems offersvaluabl e implications for management ofdiseas e inagroecosystem s (Browning, 1974)an d forunder ­ standing ofplan tepidemiolog y (Apple, 1977). 'Thedeterminatio n ofth e contrasting conditions underwhic h the samepathoge nma yb e eitherdestruc ­ tive orbenig nwil lb e amajo r task ofplan tpathologist s inth efuture ' (Yarwood, 1967).

369 ACKNOWLEDGEMENTS

Our studies were supported by research grants of the United States - Israel Binational Science Foundation (BSF), Jerusalem Israel; the Deutsche Forschungsgemeinschaft (DFG), Federal Republic of Germany; and the German - Israel Fund for International Research and Development (GIFRID).

REFERENCES

Anikster,Y .& I .Wahl ,1979 .Coevolutio no fth erus tfung io nGraminea ean dLiliacea e andthei rhosts .Annua lRevie wo fPhytopatholog y 17:367-403 . Apple,J.L. ,1977 .Th e theoryo fdiseas emanagement .In :J.G .Horsfal l& E.B .Cowlin g (Eds):Plan tdiseas e1 .Academi cPress ,Ne wYork/London ,p .79-101 . Baker,K.F .& R.J .Cook ,1974 .Biologica lcontro lo fplan tpathogens .W.H .Freema nan d Company,Sa nFrancisco .43 3p . Bingham,R.T. ,R.J .Hof f& G.I .McDonald ,1971 .Diseas eresistanc e infores ttrees . AnnualRevie wo fPhytopatholog y9 :433-452 . Browning,J.A. ,1974 .Relevanc eo fknowledg eabou tnatura lecosystem s todevelopmen to f pestmanagemen tprogram sfo ragro-ecosystems .Proceeding so fth eAmerica nPhytopatho - logicalSociety ,St .Paul ,Minnesot a1 :191-199 . Dinoor,A. ,1967 .Th erol eo fcultivate dan dwil dplant si nth elif ecycl eo fPuccini a coronataCda .var .avena eF .& L .an di nth ediseas ecycl eo foa tcrow nrus ti n Israel.Ph Dthesis .Hebre wUniversity ,Jerusalem .37 3p . (Hebrew,wit hEnglis hsum ­ mary). Dinus,R.J. ,1974 .Knowledg e aboutnatura lecosystem sa sa guid et odiseas econtro li n managedforests .Proceeding so fth eAmerica nPhytopathologica l Society,St .Paul , Minnesota 1:184-190 . Gerechter-Amitai,Z.K. , 1973.Ste mrust ,Puccini agramini sPers .o ncultivate dan dwil d grassesi nIsrael .Ph Dthesis .Hebre wUniversity ,Jerusalem .42 0p . (Hebrew,wit h Englishsummary) . Hoff,R.J .& G.I .McDonald ,1972 .Ste mrust so fconifer san dth ebalanc eo fnature .In : R.T.Bingham ,R.J .Hof f& G.I .McDonal d (Eds):Biolog yo frus tresistanc e infores t trees:Proceeding so fa NATO-IUFR OAdvance dStud yInstitute .Miscellaneou sPublica ­ tionsno .1221 .U.S .Departmen to fAgriculture ,Washington ,D.C. ,Fores tService , 1972.p .525-536 . Murphy,H.C. ,I .Wahl ,A .Dinoor ,J.D .Miller ,D.D .Morey ,H.H .Luke ,D .Sechle r& L.Reyes ,1967 .Resistanc e tocrow nrus tan dsoilborn emosai cviru si nAven a sterilis.Plan tDiseas eReporte r51 :120-124 . Person,C .& G .Sidhu ,1971 .Genetic so fhost-parasit e interrelationships.In :Mutatio n breedingfo rdiseas eresistance .Internationa lAtomi cEnerg yAgency ,Vienna ,1971 . p.31-38 . Schmidt,R.A. ,1978 .Disease si nfores tecosystems :Th eimportanc eo ffunctiona l diversity.In :J.G .Horsfal l& E.B .Cowlin g (Eds):Plan tdiseas e2 .Academi cPress , NewYork/London ,p .287-315 . Segal,A- ,J .Manisterski ,G .Fischbec k& I .Wahl ,1980 .Ho wplan tpopulation sdefen d themselvesi nnatura lecosystems .In :J.G .Horsfal l& E.B .Cowlin g (Eds):Plan t disease5 .Academi cPress ,Ne wYork/London ,p .75-102 . Yarwood,CE. ,1967 .Respons e toparasites .Annua lRevie wo fPlan tPhysiolog y 18:419-438 . Zadoks,J.C .& R.D .Schein ,1979 .Epidemiolog y andplan tdiseas emanagement .Oxfor d UniversityPress .Ne wYork ,Oxford .42 7p . Zhukovsky,P.M. , 1959.Vzaimootnoshenni amezd ukhozaino m igribny mparazito mn aik h rodinei vn eee .(Interrelation sbetwee nhos tan dfungu sparasit e inthei rorigi nan d beyond it).Vestni kSel ' skokhozïaïstvennoiNauki .Moskow .4 (6):25-3 4(Russian , withEnglis hsummary) .

370 Extrapolation of genetic and epidemiologic concepts from indigenous ecosystems to agroecosystems

J.A.Brownin g ,J .Manistersk y ,A . Segal2,G . Fischbeck3 & I.Wahl 2

ABSTRACT

Ingeographica l areaswher ebot hhos t andpathoge nar e indigenous, andwit h time and space forgeneti c maneuvering, diverse hostan dpathoge npopulation s have coevolved toa dynamically balanced polymorphism. Theen d resulto fthi s coevolution iscarrie d asgeneti c information ineac h organism. Learning and applyingthes e results to agroecosystems canhel p build superior cultivars,populatio n structures,an d cultural systems. Individualplant sma yb eprotecte d by thehost' sbasi c system ofresistanc e toth epathogen' s basic system ofpatho ­ genicity.Th ehos tpopulatio n (notindividua l plants)ma yb e protected by the incompatibility-compatibility system.Bot h systems functionepidemiologicall y ascomponent s ofecosystem - buffering by slowing therat e ofpathoge n developmentan d dispersal. Incompatibility geneshav ebee n found tob e highly effectivewhe ndeploye d indiverse ,indigenou spopulations . Suchgene shav e alsobee ndramaticall y effective inagroecosys ­ temswhe n similarlydeploye d againsthighl y epidemicpathogens .

INTRODUCTION

Manlearn sver y reluctantly, if atall ,fro mhistory . Inth e areao f cropprotection , for example, followingth eUnite d States' 1970 southern corn leafbligh tpandemic ,Ullstru p (1972)wrot e that 'the first andmos t important lessont ob e learned istha tneve r again should amajo rculti ­ vated speciesb emolde d into suchuniformit y thati ti ss ouniversall y vul­ nerable to attackb y apathogen ,a ninsect ,o renvironmenta l stress. Diver­ sity must be maintained in both the genetic and cytoplasmic constitution of

1.Departmen to fPlan tPathology ,Iow aStat eUniversity ,Ames ,Iow a50011 ,USA . 2.Facult yo fLif eSciences ,Te lAvi vUniversity ,Te lAviv ,Israel . 3.Lehrstuh lfü rPflanzenba uun dPflanzenzüchtung ,Technisch eUniversität ,München ,805 0 Freising-Weihenstephan,BRD .

371 all important crop species'. (Ullstrup'semphasis. )Ye t inth eUnite d States today,cor n and several other crops aremor ehomogeneou s (Day, 1977) thaneve n in197 0whe nhomogeneit y was recognized as 'thecru x ofgeneti c vulnerability' (NAS,1972 )t odisease .Neve rmin d thatth e 1970pandemi c couldhav ebee n avoided hadma n learned fromprevious ,equall ypoignant , lessons (Browning, 1972). Thebasi c lessontha tprobabl ywoul d haveprevente d allo fth emajo r plantdiseas epandemic s ison etha tcoul dhav ebee n learned fromwil d ecosystems.Tha t lesson istha tcoevolutio n ofhost s andpathogen s inth e regionwher ebot h are indigenous has resulted ina stat eo fdynami cba ­ lance, and thatbot h groups oforganism s obviously carry genetic infor­ mation that conditions thebehavio r thatresult s inth emaintenanc e ofth e dynamicbalanc e characteristic ofindigenou s ecosystems.Fo r example,th e wild oat, Avena sterilis L., in Israel 'knows't omaintai n acertai nvarie ­ tyo f different reaction types inth epopulatio n ina give nenvironment ; susceptible genotypes areno teliminate d from thepopulatio n and resistant genotypes dono tdominat e it.Similarly , the oatcrow nrus t fungus, Pucci- nia coronata Corda, 'knows't osporulat e only ata certai n rateo nwil dA . sterilis tomaintai n itself inth epopulatio n andneithe r dominate (and risk destroying itshost )no r riskbein g eliminated -a nevolutionar y tight wire for anobligat eparasite .Whe nth epathoge n finds itselfremove d toa susceptible,homogeneou s cultivated population,th e same rate ofsporula ­ tiontha tmaintain s the fungus inth ewil dpopulatio n could destroybot h thehos t and itsobligat eparasite ,wit h favorableweathe r conditions,i f thecultivate d hostwer eno tmaintaine d artificially byman .Man ,wh o manipulates pathogens via theirhost s (Johnson, 1961), must ferretou tho w host andpathoge n effectdynami cbalanc e inth ewil dpopulation .Wha t resistancemechanism s orpopulatio n structures areth eproduct s ofthi s coevolution and areresponsibl e forth edynami cbalanc e observed? Cansuc h knowledgehel p inbuildin g superior genotypes,populatio n structures',an d managementpractice s soa st ostabiliz e agro-an d forestry ecosystems and prevent future epidemics? Thetype s of studies reportedb y Segal et al. (page36 1 inthi sbook ) give importantclues .Th e Israelipopulation s ofwil d progenitors ofsmal l grains andthei rpathogen s areespeciall y instructive because they areth e samebiologi c species (Harlan& Zohary , 1966)an d areattacke db y the same array ofpathogen s asthei r respective counterparts in agriculture.Hence , anything learned from them canb e interpreted immediately interm s ofth e accumulated literature and appliedwithou tdela y tobuildin g superior disease-management systems for small grains andothe r crops (Browning, 1974). Inthi spaper ,w ewil l study thecoevolutio n ofhost s andpathogen s and extrapolate theprinciple s learned to agroecosystems, firsti ntheor y and theni npractice .

372 EXTRAPOLATIONS IN THEORY

Host range - A source of theory gone awry Mostcontemporar y pest-managementpractice s arebase d ontheor ydeve ­ loped from studies ofagricultura l systems.Th e concepts of 'formaespe ­ ciales' and 'race'reflec t specialization oncro p species andcultivars , respectively, of interestt oma nbecaus e ofhi s agriculture.Eve r since formae speciales and raceconcept swer e formulated, agricultural scien­ tistshav e come toexpec tgrea tspecificit y andnarro whos trange s inth e obligately parasitic rustan dman y other fungi.Fo rexample , P. graminis tritici commonly isconsidere d to attackprimaril y wheat, and P. graminis avenae primarily oats,whil e race 1migh tb evirulen t oncultiva r Abu tno t B, andrac e 2th eopposite .Flor' s (1956)gene-for-gen e hypothesis placed specificity ona soli d genetic footing and, expressed inpathogenicit y formulae,eventuall y enabled isolates ofth epathoge npopulatio n tob e describedmor ecompletel y (Browdere t al., 1980).Al l ofthi s concentrated onagricultura l systems andpai dminima l attention tohost-parasit e inter­ actions outside afarmer' s field. But agriculture is,firs t and foremost,biology .Biologica l theoryo f howhos t andpathoge n coevolved ando fho w topreven t epidemics (genetic imbalance in favor ofth epathogen )shoul d be obtainable from indigenous populations where all components have geneticmaneuverin g room, interact without disease usually beingver y severe,an dye t still result indynami c balance. Comparing 'hostrange 'i na nindigenou spopulatio nwit h that considered normal to anagroecosyste m is an instructiveexample . P. graminis avenae race 2is ,b y definition,virulen t ononl yon e standard oatste m rustdifferentia l cultivar,Jostrain ,whic hcarrie s gene Pg-3 (Stewart& Roberts , 1970). Ina wil d ecosystem in Israel,however , Gerechter-Amitai (1973)showe d that P. graminis avenae, i.e. strains of P. graminis thatca nparasitiz e oats,ha d aver ywid ehos t range,bein gviru ­ lento n 66 species in3 4gener a inth e field and8 2 species in4 1 genera in thegreenhouse .On e single-pustule isolate (ageneti c clone)o f P. graminis avenae race 2eve n sporulated on8 0specie s ofwil d grasses inth egreen ­ house, 69o fwhic hwer e rated susceptible!Gerechter-Amita i (1973)obtaine d similar data for P. graminis tritici and P. striiformis Westend., asdi d Eshed &Wah l (1970)fo r Erysiphe graminis DC.an dEshe d &Dinoo r (1980)fo r P. coronata andthei r respective hosts inIsrael . Theorymus tb e developed toexplai n such avas t discrepancy between an indigenous ecosystem andwha tma nha sexperienced , andcom et oexpect ,i n anagroecosystem . The discrepancy suggests thatth e differencebetwee na 'host' and a 'non-host',o ra 'pathogen'an d a 'non-pathogen',ma yb e one ofdefinition . Ifma nca n crossmember s of awil d specieswit h acultivate d host species thatinclude s some susceptible cultivars,bot h share acommo n genepoo l and aredefine d ashosts ;individua l plants are said tocarr y

373 genes for 'hostimmunity/resistance' . Ifma ncanno tcros s them, thenth e immune/resistant species is anon-hos t and any Rge.ne sfo rimmunity/resis ­ tance areno t available toma nwit hpresen t technology. Thus, genes that conditionmatin g compatibility mayb emor e important toou r agricentric concepto fhos t andnon-hos t thangene s for immunity/resistance orsuscep ­ tibility asth eterm s imply.

A proposed model of host-parasite coevolution Reasoning thathos tan dnon-hos t immunity/resistance couldb econdi ­ tioned by the same genes,Brownin g (1980)develope d amode l (Fig.1 )o f host-parasite coevolution and interaction. Starting fromnon-hos t immunity (Fig. 1,top )an dnon-pathoge n avirulence (bottom)tha th ereasone d charac­ terized progenitor species (justa simmunity/resistanc e and avirulence characterize thevas tmajorit y ofhighe rplant s andpathogens ,respective ­ ly, today; susceptibility andvirulenc e remainth e rare exceptions), the model leads toth ebasi c system ofhost-pathoge n incompatibility (left)an d

NON-HOST IMMUNITY

R1R1 R2R2 R3R3'--Rn

Genes for Incompatibility- Genes for Resistance-Susceptibility Compatibility (Specific toBasi c Pathogenicity (General Resistance-Susceptibility) Resistance-Susceptibility) |R1-R2---| |--R3-r<(rH rn| —-Hypersensltivlty—»-Hlgh Resistance—»-Moderate Resistance Dilatory Resistance Susceptibility—- ., "Stop or Go Interactions"-»-. "Extent and RateInteractions " - -, Freauently Oligogenic and Dominant •*—Probably Quantitatively Inherited » |A l—a2a2- | •••W| W °n| Genes for Incompatibility- Gene'sfo r Basic Pathogenicity Compatibility (Specific and Aggressiveness (General Avirulence-Virulence) Pathogenicity)

A1A1 A2A2 A3A3 ...An NON-PATHOGEN AVIRULENCE

Figure 1. Proposed scheme ofhost-pathoge n coevolution from non-host immunity andnon-pathoge n avirulence to the 2basi c systems ofhost - pathogen interaction; (left)th ebasi c host-pathogen incompatibility- compatibility system; and (right)th ebasi c pathogenpathogenicit y system and its counterpart, thebasi c system ofhos t resistance/susceptibility to basic pathogenicity. (FromBrowning , 1980.)

374 (right), thebasi c system ofpathoge npathogenicit y and of itscounterpart , hostresistanc e or susceptibility tobasi cpathogenicity . Thus, Fig.1 ha s a double dichotomy: 2organism s thatbecom e genetically interlocked, and2 basic systems thatgover n their interaction. InFig .1 , 'non-host immunity' and 'non-pathogen avirulence'describ e coexistingpopulation s ofprogenito r organismsbefor e the onseto fparasitis m andpathogenicity . Following the onseto fparasitis m andwit h thecontinuatio n ofcoevolution , the2 even ­ tually come together inth ehorizonta l center lines thatdescrib econtempo ­ rary agricultural systems.Doubtles s the left-right dichotomy isth e same onetha tVanderplan k (1968)sense dwhe nh e describedvertica l andhorizon ­ tal resistance incontemporar y agricultural systems. Essentially, thehost-pathoge n incompatibility-compatibility system determineswhethe r thepathoge n can 'unlock thedoor 'o fth ehost .B y signaling 'stopo rgo 'interaction s (Person& Mayo ,1977 )throug h aserie s of 'switchingpoints ' (Heath, 1974), the incompatibility-compatibility system either allows thepathoge nt o 'unlockth edoor 'an dprocee d through theincompatibilit y barrier toth enex t system or signals theen d ofth e line. Ifth e signal is 'stop',incompatibilit y may express itselfi nhyper ­ sensitivity. If 'go',th e secondbasi c system takes over;i tdetermine s the amount thatth e 'door'i sopene d and therat e andultimat e extenttha tth e 'room' isoccupied . Ini tth epathoge n expresses itsbasi c pathogenicity/ aggressiveness and thehos trespond swit h itsbasi c resistance/susceptibil­ ity tobasi c pathogenicity. The firstsyste m frequently iscontrolle d oligogenically; the second frequently ispolygeni c ininheritance .Th e firstsyste m frequently is under gene-for-genecontrol ;th esecon d systemma ybe .A s greaterresis ­ tance isepistati c to lesser resistance (Flor, 1956), soth e firstsystem , which usually conditions thegreate r resistance and controls theincompati ­ bility barrier toth e second system, doubtless canb e considered epistatic toth e second. The theoretical model (Fig.1 )suggest s thatsimila r R and Agenes , with similarmode s of action,ar e involved inhost-pathoge n andnon-host - non-pathogen interactions.An d they couldb e the same genes!Probabl y host Rgene s arecarry-ove r genes indistinguishable innatur e and agriculture from non-host Rgenes .Th emode lwa s suggested fromGerechter-Amitai' s (1973)hos trang e study;however , iti ssupporte db yultrastructur e studies (Heath, 1974,1977) , gene-actionmodel s (Day,1974 ;Callow , 1977), and recognition-non-recognition theory (Callow, 1977;Sequeira , 1978). An attractive feature ofth e incompatibility-compatibility system istha ti t seems similar toth euniversa l biological phenomenon ofrecognition-non - recognitionb ywhic h likerecognize s like and rejects unlike (Callow, 1977).

375 Epidemiologie role of the incompatibility genes Inagroecosystems , single incompatibility genes frequently havebee n used ina nattemp t toprotec t field crops from highly epidemicpathogens , sometimes overmillion s ofhectares .Us e ofincompatibilit y genes topro ­ tectth eplant ,o rove r large areas,i s foreign tonature . Innatura l populations, incompatibility genes dono t serve toprotec tth eplant ,bu t toprotec tth epopulatio nb y keeping thepathoge n frombecomin g tooaggres ­ sive (Parlevliet, 1980). Theyprotec t thepopulatio nb y reducing the dis­ persal efficiency of thepathoge n amongth e genetically diverse host plants.Epidemiologically , this ismeasure d thesam e asreduce d aggressive­ ness inth epathogen .Also ,the yprovid e afinel y tuned genetic feed-back mechanism tohel pmaintai nbalance . Iti sth erol e ofth e second basic system -th ebasi chos tresistance-susceptibilit y system -t oprotec tth e individual plant.Th e secondbasi c system accomplishes thisb y slowingth e rateo fdevelopmen t of thepathoge n"i nth eplan t and, therefore,contrib ­ utes alsot o slowing its development inth epopulation .W ewil l illustrate thesepoint s inth e Section called 'Frequencyo fincompatibilit y genes needed toprotec tcultivar si nagroecosystems' .

EXTRAPOLATIONS IN PRACTICE

Frequency of incompatibility genes in an indigenous population Transect studies in Israelhav e showntha tnatur e usesman y different types ofhos treactio n seemingly atrando m indynamicall y balancedpopula ­ tions.Thes ewer epresente d anddiscusse db y Segal et al. (1980).Her ew e will considerprimaril y the frequency and spatial arrangement ofplant s carrying incompatibility genes for iti s from the supposed 'failure'o f incompatibility genes due toman' smismanagemen t thatmos tresistanc eprob ­ lemshav e arisen,an d iti swis emanagemen to f incompatibility genes that canenabl ema n tomos tquickl y apply knowledge gained from studies'o f indigenous ecosystems. Incertai npopulation s ofwil dbarley , Hordeum spontaneum K.Koch , attackedb yth epowder ymilde w fungus, Erysiphe graminis, the secondbasi c system contributed very littleprotection ; theprotectio n thatthes epopu ­ lations enjoyed was due largely toth epresenc e ofgene s forincompatibili ­ ty.Whe nsee dwa s collected at1-mete rinterval s along atransec twithou t regard toth e disease reaction ofth emothe rplant ,a slittl e as1/ 3 ofth e plantsha d resistance characteristics ofincompatibilit y in subsequent testswit h thepathogen . See,e.g. , data forTransec t20 7 (Segal et al., 1980, Fig. 1)an dTransec t 217 (Wahl et al., 1978,Fig .2) .Simila r data were reviewed for P. coronata on A. sterilis by Browning (1974)an d Segal et al. (1980). Thus, itseem stha t in agive nenvironment , an indigenous population canbe ,an d is,adequatel yprotecte d and stabilized chieflyb y genes forincompatibilit y ifca .1/ 3 ofth eplant s carry effective genes

376 for incompatibility.

Frequency of incompatibility genes needed to protect cultivars in agroeco- sys terns Can knowledge ofth e frequency of incompatibility genes thatprotec ta n indigenouspopulatio nb e extrapolated inpractic e toa nagroecosystem ?Yes . Since 1957,th e IowaAgricultur e Experiment Stationha sdevelope d multiline cultivars ofoat s forprotectio n againstcrow nrus t (Freye t al., 1977). Thirteenmultilin e cultivars in2 maturit y classes,earl y andmidseason , havebee nreleased .Th eMultilin e E (forearly )Serie s isprotecte d by both resistance systems,an dth etw o systems augmenteac hothe rver y nicely inthi s cultivar.Bu tth eMultilin eM (formidseason )Serie s isespeciall y interesting, for iti sprotecte d from crown rustonl yb y incompatibility genes. Thesemultiline s havebee nteste d againstcrow nrus t inextensiv e tests in Iowa,whic hha s ashor tdiseas e season, ando nth eTexa s Coastal Plain,wher e thediseas e seasoni slonge r andmor e severe. Inbot hloca ­ tions, dramaticprotectio n from thepathoge n resulted when 1/3 to 1/2 of theplant s carried resistance toon ecomponen t or another ofth epathoge n population.Th e susceptiblepur e line recurrentparen twa skille üprema ­ turelyb y crown rusti nth e same experiments (Browning, 1974). The Iowamultiline s were tested in Israel also,i na nepidemiolog yex ­ perimenttha t included several transect-derived populations ofA . sterilis. For these experiments,a specia l Iowamultilin ewa s composited, MultilineI (for Israel)-78 .Multilin e 1-78i sver y similar toMultilin eM-7 3 except that itwa s composited equally of 10isoline s each ofwhic h carried adif ­ ferentgen e forincompatibilit y tocrow nrus t fromA . sterilis sotha ti t wouldb ecompletel y relevant toth e Israelipathoge npopulation .Th ere ­ sults, shown inFig .2 ,als ower e described by Segal et al. (1981). Markton isa pur e lineuniversa l suscept.C237-89IV , asiste r toth e recurrentpar ­ enti nth eearl ymultiline , carries nogen e for incompatibility but carries considerable protection from the secondbasi c resistance system character­ istic ofth eearl ymultilin e series.Th e 'Clintford+C649 mixed check'con ­ sisted ofC649 , recurrentparen tt oth eM-serie s multilines,an d Clintford, thever y similar recurrentparen t thatwa s used inbreedin g some ofth e isolines used inMultilin e 1-78.Thi smixe d checkwa sprotecte d by 2incom ­ patibility genes from donor cultivars Bond and Landhafer,bu tbot h ofthe m seemed tob e completely overcomeb y strains ofth epathoge n inth eexperi ­ ment.Th e Clintford +C64 9chec kha sn oknow ngene stha tconditio n thesec ­ ondbasi c resistance system forprotectio n from crownrust .Thi scharacter ­ izes thebackgroun d ofMultilin eM-7 3 andMultilin e 1-78a swel l astha t ofth emixe d check. Thus, thediseas eprogres s curves forMultilin eM-7 3 andMultilin e1-7 8 inFig .2 sho wth e results ofrus tbuildu p where thehos tpopulation s were protected solelyb y genes for incompatibility deployed indivers epopula -

377 NO. OF UREDIOSPORES/lOOL AIR

1 -Multiline E-77 2 - Hagor Avena sterilis populations 3 - Multiline M-73 4 - Bet Nehemya A.sterilis populations 5 -Multiline 1-78 6 - Guara A.sterilis populations 2500 7 - Ginnaton A.sterilis populations 8 - Rishon LeZion A. sterilis populations 9 -Clintford + C 649 10 -C237-IV- 89 11 - Markton

2000

1500

1000

500

21/4 23/4» 25/4 27'i 29/4 1/5 55/98 0 • DATESO FUREDIOSPORE SCOLLECTIO N

Figure 2. Diseaseprogres s curves for Puccinia coronata on Avena sativa pure line checks andmultilin e cultivars,an d on5 Avena sterilis transect- derivedpopulations .Be tDagan , Israel,1980 . (From Segal et al., page36- ï inthi sbook ) tions,muc ha si nnature . Susceptible-type urediawer e easyt o find inth e populations,bu tdamagin g increase ofth epathoge nwa snegligable , asi n Iowaan d SouthTexas .Curve s forth eothe rcultivar s orpopulation s show effects ofbot h systems ofgeneti cprotectio n ando fothe r components of ecosystemprotection . Curves (Fig.2 )fo rth e Iowamultiline s and forth e 5 A. sterilis popu­ lations show acontinuum , regardless ofwhethe r geneticprotectio nwa scon -

378 ditioned exclusively by the incompatibility-compatibility system (as in Multiline M-73 and Multiline 1-78), the basic host resistance-susceptibil­ ity system, or both, as in most other populations. This illustrates that 'nature uses primarily a single type of epidemiologic resistance - dilatory resistance - to protect populations, but it uses many different genetic systems and population structures to achieve it' (Browning, 1980). (See also Browning (1980) Fig. 2 for another illustration of this principle.) Others, especially Wolfe & Barrett (1980), also have used incompatibility genes effectively to buffer against highly epidemic pathogens.

REFERENCES

Browder, L.E., F.L. Lyon &M.G . Eversmeyer, 1980. Races, pathogenicity phenotypes, and type cultures of plant pathogens. Phytopathology 70: 581-583. Browning, J.A., 1972. Corn, wheat, rice, man: Endangered species. Journal of Environ­ mental Quality 1: 209-211. Browning, J.A., 1974. Relevance of knowledge about natural ecosystems to development of pest management programs for agro-ecosystems. Proceedings of the American Phytopatho- logical Society 1: 191-199. Browning, J.A., 1980. Genetic protective mechanisms of plant-pathogen populations: Their coevolution and use in breeding for resistance. In: M.K. Harris (Ed.): Biology and breeding for resistance to arthropods and pathogens in agricultural plan£s. Texas Agricultural Experiment Station, Miscellaneous Publication 1451. p. 52-75. Callow, J.A., 1977. Recognition, resistance and the role of plant lectins in host- parasite interactions. Advances in Botanical Research 4: 1-49. Day, P.R., 1974. Genetics of Host-Parasite Interaction. W.H. Freeman, San Francisco. 238 p. Day, P.R., 1977. The genetic basis of epidemics in agriculture. Annals of the New York Academy of Sciences 287: 1-400. Eshed, N. &A . Dinoor, 1980. Genetics of pathogenicity in Puccinia coronata: Pathogenic specialization at the host genus level. Phytopathology 70: 1042-1046. Eshed, N. &I . Wahl, 1970. Host ranges and interrelations of Erysiphe graminis hordei, E. graminis tritici, and E. graminis avenae. Phytopathology 60: 628-634. Flor, H.H., 1956. The complementary genie systems in flax and flax rust. Advances in Genetics 8: 29-54. Frey, K.J., J.A. Browning &M.D . Simons, 1977. Management systems for host genes to control disease loss. Annals of the New York Academy of Sciences 287: 255-274. Gerechter-Amitai, Z.K., 1973. Stem rust, Puccinia graminis Pers. on cultivated and wild grasses in Israel. PhD thesis. Hebrew University, Jerusalem. 420 p. (Hebrew, with English summary). Harlan, J.R. &D . Zohary, 1966. Distribution of wild wheats and barley. Science 153: 1074-1080. Heath, M.C., 1974. Light and electron microscope studies of the interactions of hosts and non-host plants with cowpea rust - Uromyces phaseoli var. vignae. Physiological Plant Pathology 4: 403-414. Heath, M.C., 1977. A comparative study of non-host interactions with rust fungi. Physiological Plant Pathology 10: 73-88. Johnson, T., 1961. Man-guided evolution in plant rusts. Science 133: 357-362. National Academy of Sciences, 1972. Genetic vulnerability of major crops. Washington, D.C. 307 p. Parlevliet, J.E., 1980. Disease resistance in plants and its consequences for plant breeding. In: K.J. Frey (Ed.): Plant breeding II. Iowa State University Press Ames. (in press). Person, C. &G.M.E . Mayo, 1977. Genetic limitations on models of specific interactions between a host and its parasite. Canadian Journal of Botany 52: 1339-1347. Segal, A., J. Manisterski, G. Fischbeck &I . Wahl, 1980. How plant populations defend themselves in natural ecosystems. In: J.G. Horsfall &E.B . Cowling (Eds): Plant disease V. How plants defend themselves. Academic Press, New York. p. 75-102. Sequeira, L., 1978. Lectins and their role in host-pathogen specificity. Annual Review

379 ofPhytopatholog y 16:453-481 . Stewart,D.M .& B.J .Roberts ,1970 .Identifyin grace so fPuccini agramini sf .sp .avenae . Amodifie dinternationa lsystem .U.S .Departmen to fAgricultur eTechnica lBulleti n 1416.2 3p . Ullstrup,A.J. ,1972 .Th eimpact so fth esouther ncor nlea fbligh tepidemic so f 1970-1971.Annua lRevie wo fPhytopatholog y 10:37-50 . Vanderplank-,J.E. ,1968 .Diseas e resistance inplants .Academi cPress ,Ne wYork .20 6p . Wahl,I. ,N .Eshed ,A .Sega l& Z .Sobel ,1978 .Significanc eo fwil drelative so fsmal l grainsan dothe rwil dgrasse si ncerea lpowder ymildews .In :D.M . Spencer (Ed.):Th e powderymildews .Academi cPress ,Ne wYork .p .83-100 . Wolfe,M.S .& J.A .Barrett ,1980 .Ca nw elea dth epathoge nastray ?Plan tDiseas e 64:148-155 .

380 New problems, trends and visions in agricultural resistance breeding

Vilhelm Umaerus

Swedish University of Agricultural Sciences, Department of Plant and Forest Protection, P.O. Box 7044, S-750 07 Uppsala, Sweden

ABSTRACT

Breeding forresistanc e will continue tob e thenumbe r one defense againstplan tdisease s andpests .Geneti c uniformity creates largeproblem s anddiversifie d defense againstpest s and diseases is advocated.Race-specifi c resistance shouldb e usedwit h care andmor e attention shouldb e givent ono nrace - specific resistance.Th eus e of induced resistance opensu pne w perspectives.

INTRODUCTION

Afterhavin g listened toth econtribution s atthi sworksho p concerned withth e difficulties encountered inresistanc ebreedin g Icoul dmak e it easy formysel fb y saying:bree d forresistanc ebu t avoid geneticunifor ­ mity andd ono ttrus tth ei?-genes .Bu trealit y isusuall ymuc hmor ecompli ­ cated. Itha sbee nsai dtha tbreedin g isth e arto fthrowin g away;i ti s alsoth e arto fcompromise s and so isagricultura l resistancebreeding .Bu t sometimes youca nno tcompromise .Ther e isn o future ina potat o youca n noteat . Letm e firststat e thatbreedin g forresistanc eha sbecom e apowerfu l tool in agriculture andwil l probably increase inimportance . Sinceth e turno fth e century,whe nplan tbreedin g started,w ehav e seens o faronl y the 'topo fth e iceberg' concerning the incorporation ofgene s forresis ­ tance inacceptabl ecultivars . Clearly disease resistantplant s areth enumbe r onedefens e against plantdisease s andpests ,an dth eus eo fresistan t cultivars appears tob e one ofou rmos t importantmeasure s inmoder nplan tcultivatio n to combat the attacko fplan tparasites .Th eus eo fplan tresistanc e presents little hazard toth e environment andpresent s toth e growers a 'built in' plant protection,whic hwithou trisk s andextr a costso reffort s isprovide d with the seed. Inman y casesresistanc e isth e onlymean s ofcontrol ,e.g . for wart disease ofpotato .Yo u are all aware thatchemical s areno t available

381 forth econtro l ofplan tdisease s causedb yviruses .I nothe rcase sresis ­ tance isth e only economically feasible controlmetho dbecaus e othermeth ­ ods areto oexpensive . Furthermore, resistancebreedin g isa goo d investment forth e society. A recentcalculatio n has shown ane tretur no f 1i n100 ,whic h canb e comparedwit h the farmer's netretur n inth eus e ofchemical swhic hi s about 1i n4 (Sundell, 1979). Thedrawbac k istha tplan tbreedin g is aslo w process and investments usually dono tpa ybac kearl yenough . What lessons fromth epas thav ebee n learned from thisproces s which canb e ofvalu e forsimila r efforts insilviculture ?Th emuc hdiscusse dus e ofclona lpropagatio n immediately leads toon edominatin g issue:geneti c uniformity and its influence ondiseas eresistance .

GENETIC UNIFORMITY

Uniformly high-yielding crops grown indens e stands ina n intensive agriculture areals ouniforml yvulnerabl e to anyparasit ewit h epidemic potentials.W ehav e learned thatth ehar dway , and Iwil l give 2classi c examples.A ne w oatvariety ,Victoria ,wa srelease d in Iowai n1942 . Victoria was resistant to allrace s ofcrow nrust ,an d also resistantt o stem rustan d tosmuts . Itbecam ever ypopula r andb y 1945 together with some derivativevarietie s covered 97 %o fth etota l oatacreage . In194 6 a seedlingbligh tcause d by Helminthosporium victoriae Mee.& Mur . appeared which in2 year s reached epidemicproportions .Victori awa s particularly susceptible tothi s fungal diseasewhic hpreviousl y had onlybee n knowna s amino rparasit e onwil d grasses.Unluckily , theunforesee n reasonwa sa pleiotropic effect ofth edominan tgen ePc- 2 forcrow nrus tresistance .Th e followingyear s the Iowa acreageplante dwit hVictori a and its derivatives was reduced; eventually the susceptiblevarietie s were replacedb y other resistantvarietie s andVictori abligh t ceased tob e aproblem . Thesouther n leafbligh to ncor nwhic h reached epidemicproportion s in 1970 is arepea to fth eVictori abligh thistory ,bu t this time aunifor m cornmonocultur e had developedwit h the introduction ofhybri dcor nbase d oncytoplasmi c male sterility. Themos t dependable source for cytoplasmic male sterilitywa s derived fromplant s found inTexa s andb y 1969 thisso - called Tmscytoplas m hadbee nintroduce d intou pt o 90% ofth e field corn grownthroughou t theUnite d States.Tha tyea r reports appeared thatTm s lineswer eunusuall y susceptible to leafbligh tan d in197 0th ehithert o passive leafbligh treache d epidemicproportions . Itappeare d firsti n Florida andthe nrapidl y spreadwes tan d thennorth ,unti lb y September it hadbee nreporte d throughoutth eeaster nhal fo fth eUnite d States.Th e epidemic was causedb y raceT o f Helminthosporium maudis Nis.& Miy . Later investigations proved thatrac eT existed earlier,bu tu p to196 9 only race 0 was generally known andthi srac e didno tdifferentiat e betweenTm s and

382 normal cytoplasm. In197 1 seedproducer swen tbac k tonorma l cytoplasm with resistance torace s0 andT , andhybrid swer eproduce d bymechanica ldetas - seling. Modernbreedin g techniquesma y increase geneticvulnerabilit y inothe r ways, too.Male-steril e barley andwhea t inhybri d seedproductio n fields areespeciall y susceptible toergo t (Claviceps purpurea (Fr.)Tul.) . The disease infects the flowers atth etim eo fpollination . If flowering is cleistogamousinfectio n israre .Fo rhybri dproductio n flowers remainope n a longtime ,greatl y increasing their chances ofbecomin g infected. Hybrid sugarbee t and sorghum also runa grea t riskbecaus e ofnarro w sources ofcytoplasmi c male sterility.Th e listca nb e longer.An y trait thatma n introduceswithou t assurance ofa varyin gbackgroun d ofothe r genes isrisky , and itcertainl y isno teas y topredic twha t special risk mayb e encountered. Breeding fordiseas e resistance should introduce toth ecro p important geneticvariatio ndesigne d topreven tepidemics .Th etraditiona l approach employs asuccessio no fmonogeni c resistantvarieties ,wher e eachon ei s replaced as it fails.Thi s relieves genetic uniformity by creating genetic discontinuity intime .A n alternative ist o creatediscontinuit y inspac e bydeliberatel y introducing variationb y growingmixe d populations.Anothe r approach isth eus e ofno nrace-specifi c resistance,whic h rarelyprovide s complete control but slows downth e infection rate ofa nepidemi cdisease .

RACE-SPECIFIC RESISTANCE

Theusefu l life of arace-specifi c resistance gene deployed againsta cropparasit e isdetermine d by the frequency ofth e allele forvirulenc e in theparasit epopulation . This, inturn ,i sdetermine d by themutatio n rate tovirulence , andb y the fitness ofth evirulen tmutan t incompetitio nwit h avirulentnon-mutant s andothe rorganisms . Race-specific resistance isusuall y controlled by one gene,o r afe w geneswhos e individual effects arereadil y detected.Thu s race-specific resistance iseas y towor kwith .Testin g isusuall y done on seedlings or smallplants ,whic h allows aconsiderabl e saving inspac e andtime .Selec ­ tion isusuall y performed under the severe conditions of agreenhous e or laboratory test,wher e specific resistance almost always is stronglyex ­ pressed as an 'either-or'reactio n favouring amass-selectio n technique.I t isunderstandabl e thatmos tbreeder s inth epas thav e favouredbreedin g for race-specific resistance,whic h also inth e farmer's fields showed upa s completeprotection . The limitation ofrace-specifi c resistance fordisease swit h epidemic potentials arewel l known forth e samereason s aspreviousl y discussed under genetic uniformity -th e system isver yvulnerable .Th evulnerabilit y isgreate r thanexpecte d because during theproces s ofbreedin g forrace -

383 specific resistance alos s ofgene s forno n race-specific resistanceusu ­ ally underpolygeni c control canoccur ,o r asDa y (11374)expresse sit : 'Plantbreeder s stripped oligogenic resistance ofassociate dprotectiv e polygenic effects and exposed thegene s one ata tim e inagricultur e ona tremendous scale,forseein g theparasit e to respond intime' . Thisma y give the impression thatrace-specifi c resistance iso f little long-termpractica l value,which , ofcourse ,i s ageneralizatio nwhic hdoe s nothold ;th epictur e isno ttha tblac k andwhite . Soil-borneparasites ,e.g . cystnematodes, orth e agento fwar t disease ofpotato ,hav e aslo w spread, only 1generatio npe rvegetatio nperio d and host-crop, and long-distance transport israre . Iti seas y forth eplan t breeder tob e one step aheadwit h introduction ofne w resistance genes providedprediction s canb emad e early enoughwhic hvirulenc e geneth e parasite will develop.Rac eo rpathotyp e surveys arestil l ofhig himpor ­ tance. Contrary toth eobligat eparasites ,non-obligat eplan tpathogen shav e oftenbee neffectivel y controlled by the introduction ofa singl e gene for resistance andphysiologi c specialization hasno tbee n aproblem . The obligateparasite s aredifferent .Th emor ewidel y astron g genei s used, the less itca nb e relied on forprotection ; thecro p selects the race thatma y ultimately destroyit . One examplewher e race-specific resistance hasno tbee n successfully used is latebligh to fpotato .Th ehos tplant s arepolyploi d and clonally propagated, and areconsequentl y difficult tobree d by themetho d ofcross ­ ing anestablishe d variety with asourc e ofresistanc e and of screening thousands of seedlings for agronomic characters thatmatc hth e susceptible parent.Thi smean s thatrapi dproductio n ofa successio n ofgoo d agronomic varieties carrying differenti?-gene si simpractical . Inth e case ofresistanc e torus to rpowder y mildew incereal s this strategyma yb e successful,provide d asufficien t source of resistance genesexists .However , thecombinatio n ofeve n afe wgene sbuy s time,sinc e virulentmutant s that arise independently mustb ecombined , orels e they must arise simultanouslyo r sequentially before infection canoccur . Themos t successful strategy forus e ofrace-specifi c resistance in epidemic diseases isth ecreatio n ofdiscontinuit y inspac eb y growing mixedpopulations . Themultilin e variety approachha sbee ncarrie d furthestb y theoa t breeders in Iowa (Browning& Frey , 1969). Theus e ofmixe d varieties has the samemotive ,an d is less sophisticated andmor e flexible tomee tth e needo fprotectio n againstmor e thanon edisease . Regional deployment ofresistanc e genes isanothe rwa y ofcreatin g discontinuity inspac e andha sbee nbes texemplifie d along the Puccinia path inUSA , also described byBrownin g et al. (1969).

384 NON RACE-SPECIFIC RESISTANCE

Thepro s andcon s ofno nrace-specifi c resistance havebee n discussed byPersso n et al. (page31 8 inthi s book). Successful useo fno nrace - specific resistance isresistanc e tolat ebligh t inpotat o (Umaerus, 1970), common rust (Puccinia sorghi Schweinitz)i ncor n (Hooker, 1969), yellow rust (Puccinia striiformis Westendorf)i nwhea t (Lupton& Johnson, 1970). Efforts areno wbein gmad e also to look forno nrace-specifi c resistance toothe r diseases incereals . Thevalu e ofno nrace-specifi c resistance hasbee nemphasize d strongly byWah l inhi s lecture onstudie s ofnatura l ecosystems (Segal et al.,pag e 361 inthi sbook) . Myvisio n istha t futurebreedin g forresistanc e against epidemic diseases should firstsecur e ahig h level ofno nrace-specifi cre ­ sistance and asa secon d objective introduce race-specific resistance,no t thereverse . Improvements must andca nb emad econcernin g selectionmethod s asha sbee ndemonstrate d incomponen tanalysi so fresistanc e to latebligh t inpotat o (Umaerus,1970 )facilitatin gbot h early selection onyoun g seed­ lings and screening for adiscret e character:plant swit h alon gminimu m accessionperiod .Measure s alsomus tb e takent oovercom e thecove r effect ofknow no runknow ni?-gene sb yusin gprope r races for inoculation orusin g testground swit h thehighes theterogeneit y forvirulenc e genespossible .

INDUCED RESISTANCE

If Ino whav e theprivileg e togiv e avision ,ma y Idra w attentiont o thepape r ofKu cpresente d atth e IX International Congress ofPlan tPro ­ tection 1979 inwhic hh e remarks that 'modern agriculture canno twai t for the 'evolutionary'developmen to fdisease-resistan tplant s thatma yb eef ­ fective forsurviva lbu tprov e totally ineffective economically inth e degree orpersistanc e ofresistance' .Evidenc e isno w rapidly accumulating thatresistanc e canb e induced inplant s treated withcultivar-non-patho - genic raceso fpathogens ,avirulen t forms ofpathogens ,non-pathogens , whichprotect s againstdiseas e causedb y subsequent infectionb ypathogens . The intriguing pointis ,an d this is sodifficul t tocomprehend , thatal l plants, eventh e susceptible ones, haveth e capacity ofresistance . Browning et al. (page37 1 inthi sbook )indicate d thatresistanc e isth e normal reaction, susceptibility isth e exception.Ca nw eus e the resistance which obviously prevents non-pathogens fromestablishin g arelationshi p with ahos tplant ?Ho w dow e release the signal thatcommit s cells ofeve n susceptible cultivars toresistance ?Wit h thatquestio n solved theperspec ­ tives ofdiseas e controlwil lb eenormous .

385 f

REFERENCES

Browning,J.A .& K.J .Frey ,1969 .Multilin ecultivar sa sa mean so fdiseas econtrol .Ann . Rev.Phytopathol .7 :355-382 . Browning,J.A. ,M.D .Simons ,K.J .Fre y& H.C .Murphy ,1969 .Regiona ldeploymen t forcon ­ servationo foa tcrown-rus tresistanc egenes .In :J.A .Brownin g (Ed.):Diseas econse ­ quenceso fintensiv ean dextensiv ecultur eo ffiel dcrops .Iow aAgr .Exp .Sta .Spec . Rep. 64.p .49-56 . Day,P.R. , 1974.Genetic so fhost-parasit e interaction.W.H .Freema n& Co .Sa nFrancisco . Hooker,A.L. ,1969 .Widel ybase dresistanc e torus ti ncorn .In :J.A .Brownin g (Ed.): Diseaseconsequence so fintensiv ean dextensiv ecultur eo ffiel dcrops .Iow aAgr . Exp. Sta.Spec .Rep .64 :28-34 . Kuc, J., 1979.Biochemical s andplan tprotection .I XInternationa lCongres so fPlan t Protection.Proceedings :Openin gSessio nan dPlenar ySessio nSymposium ,p .21-26 . Lupton,F.G.H .& R .Johnson ,1970 .Breedin gfo rmature-plan t resistance toyello wrus t inwheat .Ann .Appl .Biol .66 :137-143 . Sundell,B. ,1979 .Pests ,disease san dweed si nagriculture .Repor t2 :Economi cevalua ­ tiono fdifferen tcontro lmethods .Swedis hUniversit yo fAgricultura lSciences . Departmento feconomic san dstatistic sRepor t151 . Umaerus,V. ,1970 .Studie so ffiel dresistanc e toPhytophthor a infestans.5 .Mechanism s ofresistanc e andapplication s topotat obreeding .Z .Pflanzenzüchtun g63 :1-23 .

386 Breeding of pines for resistance to the wood nematode, Bursaphelenchus lignicolus

Kihachiro Ohba

Forestry and Forest Products Research Institute, P.O. Box 16, Tsukuba Norin Kenkyu, Danchi - nai, Ibaraki 305, Japan

ABSTRACT

Aprojec t ofbreedin gpine s (Pinus densiflora and Pinus thunbergii) forresistanc et oth ewoo dnematode , Bursaphe­ lenchus lignicolus, was started insouthwester n Japan in1978 . To develop the schemes forth eproject , apilo ttes t anda researchprojec twer eperforme d with thecooperatio n ofth e Forestry and ForestProduct s Research Institute,th eNationa l ForestTre eBreedin g Institutes,an dth eRegiona l ForestOf ­ fice. Inthes eundertakings ,practica l techniques for candidate pine selection,mas s cultivation ofth ewoo dnematode ,artifi ­ cial inoculation,propagatio n ofplan tmaterial s tob e tested, and testing thepathogenicit y ofnematode swer e developed. In thisproject ,abou t2 500 0candidat epine s areexpecte d tob e tested forthei r resistance toth ewoo dnematod e using their grafted clones ina 5-yea rplan .A second inoculation testwil l use the grafted cloneswhic h succeeded inth e firsttest ,an d draw comparisons with check seedlings of Pinus taeda.

INTRODUCTION

InJapan ,ther e are2 two-needle d pine specieshavin gwid enatura l distributions, Pinus densiflora Sieb.& Zucc. (Japanese redpine )an d P. thunbergii Pari. (Japaneseblac k pine). They areimportan t in forestry,an d about20 0 000h ao f forest land are inplantation s ofthes e species.Th e pinewoo d hasmultipl e uses, such asmateria l forhousing ,pulp ,an dpaper . Moreover, these species grow inles s fertile soils andhav e protective effects inreducin g soil erosion and inaidin gwate r conservation inthi s country. Extensivemortalit y ofth epine s hasprevaile d insouthwester n Japan since the 1940's.Th ecaus e ofthei r deathha sbee nattribute d for alon g timet oman ykind s ofpin ebar kbeetles . In1971 ,Kiyohar a &Tokushig e found thatth eepidemi c occurrence ofpin e death (pinewilt )resulte d

387 from aspecie s ofwoo d nematode, Bursaphelenchus sp.,an d thenematod e wasname d Bursaphelenchus lignicolus Mamiyae tKiyohar a (1972). With cooperative work ofman y scientists indifferen t fields, itwa s confirmed thatther ewa s avecto r insectspecies , Monochamus alternä'tus Hope,b y Morimoto& Iwasaki (1972).

A STRATEGY OF PINE BREEDING

Apresentatio n ofbreedin g aims ina certai nbreedin g district forth e pines (Table 1)indicate s theprimar y aims areexpecte d tob e solvedb y genetic improvement.Workin g toward general aimswoul db euseles s unless the specific one,resistanc e toth ewoo d nematode,wer e solved. So,i ti s very importantt ohav e insightint o factorswhic hhav edestructiv e effects on survival,bol e traits,an dgrowt h of individual trees -kinds ,distri ­ bution, and overlapping ofth e factors -an dt odetermin e economic tech-

Table 1. Breeding aims in acertai nbreedin g district.Tre e species: Pinus densiflora and Pinus thunbergii. District:Lowlan d ofKyushu ,lowe r than 500-600m above sealevel .

Specific aims General aims primary aims secondary aims primary aims secondary aims

Disease: Bursaphe­ Disease: Lophoder- Fastgrowt h Others lenchus lignicolus mium pinastri Persistent growth Mamiya etKiyohar a Schrad. Straightbol e Non-twisted bole Insects: Petrova Narrow crownwit h cristata Walsing- small branches ham Matsucoccus spp. Dendrolimus spec- tabilis Butler Thecodiplosis ja- ponensis Uchida et Inou e

Others a.Aim s forwhic hgeneti c improvement iso fprimar y importance. b. Aims forwhic hvariou sprocedure s sucha ssilvicultura l tending,use so f natural enemies,chemica l control and genetic improvement arefeasible .

388 niques toovercom e these factors.Priorit y ofsom e counter-measures for these factorswil lb e judged inth e followingorder . 1. Factors resulting inth edeat h ofth eindividual . 2. Factors resulting indamag e toth ebole . 3. Factors resulting ingrowt hreduction . The counter-measures seem tob e asfollows : 1. Use the forest land forpurpose s other thanproductiv e forestmanage ­ ment. 2. Use the forestlan d for forestrypurpose swit h some counter-measures alone or incombination : - silvicultural tending, - useo fnatura lenemies , -us eo fchemica lcontrol , - genetic improvement, - others (change toothe r tree species, etc.).

A PILOT TEST OF BREEDING PINES FOR RESISTANCE TO THE WOOD NEMATODE

After the discovery ofthi snematode ,a pilo ttes to fbreedin g pines forresistanc e toth ewoo dnematod ewa s started incooperativ e work among several institutions such asth eForestr y and ForestProduct s Research Institute,Kyush uBranc h Station,th eKyush u ForestTre eBreedin g Insti­ tute, andth eKumamot oRegiona l ForestOffice .Candidat epine s forresis ­ tancebreedin g were selected andpropagate d by grafting andb y using seeds totes tthei r nematode resistance.Meanwhile , inth eKyush u ForestTre e Breeding Institute, aresearc hprojec twa s started with the cooperation of 2 othernationa l foresttre ebreedin g institutes todevelo p apractica l breeding system forresistanc e toth ewoo dnematode .Thi s ledt oth eadop ­ tiono fth e followingtechniques :

(1)Graft s areuse d asth emai ntestin gmaterial s Atpresent ,w ehav en o information onth egeneti c constitutions ofth e pineswhic hmigh thav eresistanc e toth enematode .The ymigh thav e dominant gene(s)and/o rpolygenes .A s thepine s arewin dpollinatin g species,i ti s quiteprobabl e thata resistanc e testwit h graftedplant s raised from sci­ ons taken from the candidatepine swil lproduc emor e resistantplant s than a testwit h seedlings from openpollinate d seeds ofth ecandidates .Usin g Pinus taeda L.whic h isalmos t immune toth enematode ,a sreporte db y Kiyohara &Tokushig e (1971)an d Ibaraki et al. (1978a), experiments were madet otes tresistanc e toth enematod e afterexchangin g stocks and scions between Japaneseblac kpin e andtaeda-pine .Taeda-pine s grafted on stocks ofJapanes eblac kpine s showed sufficient resistance toth ewoo d nematode after artificial inoculation asreporte db y Toda et al. (1977).

389 (2)Th ebark-peel-inoculatio n forpractica l use After trials of several kinds oftechnique s for artificial inoculation ofth enematode ,th ebark-peel-inoculatio n methodwa s adopted forpractica l use (Nishimura et al., 1977). Techniques of artificial cultivation andin ­ oculationo fth enematod e had alreadybee nestablishe d by scientists atth e Pathology Laboratory inth e Forestry andFores tProduct s Research Insti­ tute,Kyush uBranc h Station,an ddescribe d ina manua l (1974). Thebark - peel-inoculation was developedb y simplifying conventional techniques.I n bark-peel-inoculation, thebar k ofth emai n shoot ispeele dwit h aknif e ata locatio n about2 0c m above thegrafte d point for about a5 c m length. Aftermakin g thepeele d surface roughwit h asmal l saw,1 m l of awate r suspension ofth enematod e containing ca.1 000 0nematode s isdroppe d on this surface with amicropipett e andn ocove r isused . Iti sver y simple andeas y and about 500graft s canb e inoculated by a2-ma ntea m in1 day .

(3)Mas sproductio n ofth enematod e Techniques forartificia l cultivation ofth enematod e were established atth ePatholog y Laboratory (1974).Th enematod e canb e cultivated ona fungus, Botrytis cinerea Pers., onpotat o agar.medium.Mas sproductio no f thenematod ewa smad epossibl eb y theus e ofpetridishe s forcultivatio n vessels. Iti sexpecte d that about 0.5 millionnematode spe rpetridis h can be raised in2 weeks .

(4)Contro l ofth epathogenicit y ofth enematod e Iti sver y important toclarif y thediversit y ofpathogenicit y among nematode strains,no tonl y fornematolog ybu t also forresistanc ebreeding . Thismake s itpossibl e tous e adequate strains ofth enematod e forth e test ofresistanc e ofpine sb y artificial inoculation.A ver ywid evariatio n in pathogenicity ofth enematod ewa s foundb yKiyohar a et al. (1977)an d Ibaraki etal . (1978b).Researc h onthi sproble m hasbee ncontinued'sinc e thebeginnin g ofthi sresearc hproject .Nematod e strainswhic hhav elow , medium, andhig hpathogenicit y arekep t inth e institutes forus e inresis ­ tance testing.

(5)Artificia l inoculation ofpin egraft s ina glass-hous e Artificial inoculations commonly havebee nmad e around the20t ho f July.Bu tthi stim e isusuall y atth een d ofth erain y season insouth ­ western Japan.Condition s ofhumi d andcoo lweathe r result ina highe r survival ofpine s after inoculation.Th e optimumperio d for artificial inoculation isexpecte d tob e from lateJul y toearl yAugus t inth e field nursery. Ifth e inoculationwer emad e ina glass-house ,w e could expectt o have amor e severe test forresistanc e underhig h temperature and limited water irrigation.Moreover ,w e could combine thetechnique s of selective use ofnematod e strains havingvarie d pathogenicity with environmental con-

390 trol during thetest .

(6)Saf e andeas y collection ofth e scions fromcandidat epine s The candidatepine s shouldb e 30-50year s of ageo rmore .Becaus e they willb e tall, therewa s anee d todevelo p safe techniques tocollec t scions from thesepines .Severa l techniques andtool s forclimbin g treeswer e tested and iti srecommende d thatcommo n andne w safetytool sb euse d in combination.

(7)Selectio n ofth e candidatepine s There arestil l several surviving trees lefti nman y spotswher ese ­ vere damage'> yth enematod e had occurred. Good growth and straight stem formwer e added ascriteri a for selection ofcandidat epine s forwoo d production.

Inth ecours e ofthi spilo ttest ,abou t1 0pine s showinghig hresis ­ tance toth enematod e were found,givin gu sbrigh tprospect s forth e coming breeding project (Ohbae t al., 1977;Ibarak i et al., 1978a, 1978c).

SURVEY OF THE NUMBER OF SURVIVING PINES IN AN AREA WHERE SEVERE DAMAGE BY THE NEMATODE HAD OCCURRED

A typicalprogressio n ofth edeat h inpin e stands suffering fromth e nematode is as follows:i nth e firstyea r asmal lnumbe r ofpine s died,bu t mortality increased exponentially until almost allpine s diedwithi n afe w years. The owners ofdamage d pine standsuse d to fell allpines ,includin g healthy ones. So ina larg e area,especiall y atlowe r elevations inKyush u and Shikoku,pine s largely disappeared except forprotecte d pine forests. The latterwer e sprayed with insecticides everyyea rbecaus e of theirpub ­ licimportance . Inarea swher emas s deatho fpine sha d occurred, asurve ywa smad et o obtain aroug h estimate of survivingpine s thatmigh tb e considered ascan ­ didates forresistanc ebreeding . Following directions givenb y the Forestry Agency, 14organization s made thesurve yunde r the followinglimitations : (a)pin e stands specifically surviving inth e severely damaged areashoul d be included, (b)a taltitude s lower than 100-200m , (c)withi n adistanc e of4 km from thesea-shore . About3 400 0pine swer ecounte d consisting ofca .6 00 0 from standso f under 1% survival,1 900 0wher e survivalwa s1.1-5. 0% , and 960 0pine s in standswit h5.1-1 0% survival .

391 OUTLINE OF THE PINE BREEDING PROJECT FOR RESISTANCE TO THE WOOD NEMATODE

After thepilo ttes t andth e survey ofsurvivin gpine *i nth epropose d pinebreedin g area,a practica lprojec twa splanne d and started in197 8b y theForestr y Agency,Ministr y ofAgriculture ,Forestr y andFisheries .

(1)Purpos e ofth eprojec t The aim ist obree dpine s (redpin e andblac kpine )resistan t toth e woodnematod e in southwestern Japan (Fig.1) .Concernin g the degree ofre ­ sistance,pine shavin g aresistanc e equal too r above thoseo f Pinus rigida Mill, and Pinus taeda L.ar eexpecte d tob e selected.

(2)Organization s responsible forthi sprojec t The ForestryAgenc ymad e the fin,alpla nt oconduc tthes ebreedin g activities.Thre enationa l foresttre ebreedin g institutes and 14prefec - tural organizations willparticipat e inthi sprojec twit h a5-yea rplan .

(3)Tre e species tob ebre d andth ebreedin g district Pinus densiflora and Pinus thunbergii areth eobjectiv e species.Tw o

Kyushu.? .——\'

Figure 1. Twobreedin g districts (hatched areas)wer e set forth ebreedin g projectwit h thepin ewoo d nematode,o ,FORI , Forestry andFores tProduct s Research Institute;• ,Nationa l ForestTre eBreedin g Institute.

392 Table 2. The 5-yearpla n ofpin ebreedin gwit h thewoo dnematode .

Fiscal year (April toMarch )

1978 1979 1980 1981 1982

Selection ofcandidat epine s OOO Scioncollectio n 0 0 0 Grafting 0 0 0 Raising grafts 0 0 0 The first inoculation test 0 0 0

breeding districts are set: I,nort hKyush u and Setouti;an d II,sout h Kyushu, Shikoku, andKink i (Fig.1) .

(4)A 5-yearpla no fth eprojec t Theprojec t isoperate d under a5-yea rplan ,wit h yearlyplan s sucha s selectiono fth ecandidat epines ,grafting , and resistance testswit h arti­ ficial inoculation (Table2) .

(5)Numbe r ofcandidat epine swhic hwil lb e tested ineac h organization A total of 25 010candidat epine s expected tob e selected (P. densi- flora: 1103 5 and P. thunbergii: 13 975)i sallote d to 17organizations .

(6)Breedin g scheme Abreedin g scheme ofmas s selectionwa s adopted asdevelope d by the research and surveymad e inprecedin gyears . - The candidatepine swil lb e selected from survivingpine s instand s having lesstha n 10 %surviva l (preferably 1% o r less). From thesecandi ­ datepines ,scion swil lb etake n and graftswil lb e raised. - Tengraft s from each candidatepin ewil lb e inoculated with the nematode by eachorganizatio n asth e firsttest .Th enematode swil lb e cultivated at the3 nationa l foresttre ebreedin g institutes,an dwil lb e distributed to eachorganization . -A s achec k ofth edegre e ofresistance ,2-year-ol d seedlings of Pinus taeda willb e treated the same asth egrafts . Success ofth e candidate pineswil l be judged by the survival rate (including graftswit hpartia l death)an dth erat e ofhealth y grafts incombination . -A n inoculation testwil lb emad e twice,th e firstb y 17 organizations under theguidanc e ofth enationa l foresttre ebreedin g institutes.Th e second inoculation testwil lb emad e at3 nationa l foresttre ebreedin g institutes,usin gthos e candidatepine swhic hwer e successful inth e first

393 test. Inth e second test,2 0graft s foreac hcandidat epin ewil lb e tested. After these inoculation tests,pine s resistant toth ewoo dnematod ewil lb e selected. -Th e artificial inoculationwil lb emad e onpotte d grafts ina glass ­ house. - Strains of thenematod ewhic h hadbee nteste d forthei rpathogenicit y in theprecedin g year at3 foresttre ebreedin g instituteswil lb euse d selec­ tively.Moderatel y pathogenic strains areplanne d forus e inth e firsttes t toavoi d over-kill ofth e grafts. Inth e second test,strain swit h ahig h pathogenicity will beused . -Afte r the selection ofresistan tpines ,a ne wprogramm e willb e sett o producebre d materials resistant toth ewoo d nematode.Som eo fth eresis ­ tantpine swil lb epropagate d by grafting and thegraft swil lb eplante d foremergenc y use insea-shor eplantations ,nationa lparks ,an dothe r places. Seed orchardswil lb eestablishe d with thegraft s concurrentwit h researcho nth eheredit y ofresistanc e toth ewoo dnematode .

(7)Seedlin g test Beside thetes to fgrafte d candidatepines' ,seedling swil lb e raised from open-pollinated seedstake n from some ofth ecandidat epines .A n inoculation testo nthes e seedlingswil lb e continued toprovid e informa­ tiono nth egeneti cbehavio r ofresistance .A sreporte d by Ibaraki et al. (1978c), seedling-progenies ofsom e candidatepine s inth epilo t test showedver y highresistance .

CONCLUSION

A constructive pathway forth eprojec to fbreedin gpine s for resistance toth ewoo d nematode hasbee nbriefl y described. The resulto fth e first inoculation testo ngraft s isexpecte d this'fal l (1980), and itwil l appear inth enex t2 years . Iti sexpecte d that asufficien tnumbe r ofresistan t pineswil lb e secured inthis,project . Meanwhile,w ehav e deep concern forth e followingpoint s related toth e project and futurewor k after theproject : -T o selecta sman ypine s resistant toth ewoo d nematode aspossible . -T o clarify thehereditar y nature ofth eresistance . - To clarify themechanis m ofresistance . -T o combine through artificial crossingth e different genes responsible forresistanc e in individuals forus e asseed-parents . -T o clarify theprovenanc e variation ofpathogenicit y ofth enematod e strains. -T omak eexperimenta l plantations with grafts and seedlings derived from open-pollinated seeds ofth eresistan tpines . - Toestablis h seed orchardswit hth eresistan tpines .

394 ACKNOWLEDGEMENTS

To the former directors, Mr. T. Niga and Mr. C. Ibaraki, and to all members of the Kyushu Forest Tree Breeding Institute; to the former head of the Silviculture Divison of the Forestry and Forest Products Research Insitute, Dr. R. Toda, and the present head of the Division, Mr. K. Hachiya; and to Dr. N. Ohyama in the Kyushu Branch Station of the Institute, I express my heartfelt gratitude for their guidance and cooper­ ation.

REFERENCES

Forestry Agency, Ministry of Agriculture, Forestry and Fisheries, Japan, 1978. [Directions for the breeding project of pines resistant to the wood nematode] (in Jap.). Ibaraki, C, K. Ohba, H. Yamate, T. Ritsusen, T. Toda, K. Nishimura & K. Matsunaga, 1978a. [Graft-tests of the candidate pines for pine wood nematode resistance] (in Jap.). Nihon Ringakukai, Kyushu-sibu Kenkyu-ronbun-syu 31: 59-60. Ibaraki, C, K. Ohba, T. Toda, H. Hashimoto & T. Kiyohara, 1978b. [Different patho­ genicity of 23 strains of the nematode, Bursaphelenchus lignicolus to seedlings of Pinus thunbergii] (in Jap.). Nihon Ringakukai, Kyushu-sibu Kenkyu-ronbun-,syu 31: 211-212. Ibaraki, C, K. Ohba, T. Ritsusen, K. Nishimura, T. Toda, N. Ohyama, T. Takagi & K. Kawanobe, 1978c. [Seedling tests of the candidate pines for pine wood nematode resistance] (in Jap.). Nihon Ringakukai, Kyushu-sibu Kenkyu ronbun-syu 31: 61-62. Kiyohara, T., H. Hashimoto, K. Ohba & K. Nishimura, 1977. [Pathogenicity of four isolates of Bursaphelenchus lignicolus to plus tree progenies of Pinus densiflora and Pinus thunbergii] (in Jap.). Transactions of the 88th Meeting of the Japanese Forestry Society, p. 329-330. Kiyohara, T. & Y. Tokushige, 1971. [Inoculation experiments of a nematode, Bursaphelenchus sp. onto pine trees] (in Jap.). Journal of the Japanese Forestry Society 53: 210-218. Mamiya, Y. & T. Kiyohara, 1972. Description of Bursaphelenchus lignicolus n. sp. (Nematoda Aphelenchoididae) from pine wood and histopathology of nematode-infected trees. Nematologia 18: 120-124. Morimoto, K. & A. Iwasaki, 1972. [Role of Monochamus alternatus (Coleoptera: Cerambycidae) as a vector of Bursaphelenchus lignicolus (Nematoda: Aphelenchoididae)] (in Jap.). Journal of the Japanese Forestry Society 54: 177-183. Nishimura, K., K. Ohba, T. Ritsusen, K. Matsunaga & M. Imamura, 1977. [Development of artificial inoculation techniques for pine breeding with the pine wood nematode] (in Jap.). Nihon Ringakukai, Kyushu-sibu Kenkyu-ronbun-syu 30: 61-62. Ohba, K., K. Nishimura, T. Toda, H. Yamate & N. Ohyama, 1977. [Artificial inoculation tests of the candidate-grafts of Pinus thunbergii for the pine wood nematode] (in Jap.). Nihon Ringakukai, Kyushu-sibu Kenkyu-ronbun-syu 30: 71-72. Pathology Laboratory, Forestry and Forest Products Research Institute, Kyushu Branch Station, 1974. [A manual of cultivation and artificial inoculation of the pine wood nematode, Bursaphelenchus lignicolus] (in Jap.), p. 1-5. Toda, T., T. Ritsusen & H. Yamamoto, 1977. [Resistance tests of pine-grafts and of waterpotted pine branches] (in Jap.). Nihon Ringakukai, Kyushu-sibu Kenkyu-ronbun-syu 30: 65-66.

395 The Swiss Endothia-resistance breeding program

G. Bazzigher

Swiss Federal Institute of Forestry Research, CH-8903 Birmensdorf, Switzerland

ABSTRACT

Inth e fightagains tth e chestnutbligh ti nSwitzerlan da selectionprogra m was started to find resistant individuals in native (Castanea sativa) and Asian (C. crenata) populations. Thegreates tnumbe rpossibl e ofblight-resistan t trees formth e basepopulatio n forreforestation .

INTRODUCTION

Sinceth ebeginnin g ofth e20t hcentur y chestnutbligh tcause d by Endothia parasitica (Murr.)And .& And. ,whic h comes from easternAsia ,ha s in lesstha n 50year s killed theAmerica n chestnut (Castanea dentata (Marsh.)Borkh. )i nit snatura l range ineaster nUnite d States tosuc ha n extent thatonl y small remnantstand s remain.Abou t 35-40year s agothi s fungal diseasewa s also discovered invariou s European stands;i n194 7i t was identified inth eCanto n ofTessi n insouther n Switzerland. Alarming descriptions ofth eprogres s ofth eepidemi c inth eUS Aprompte d forestry authorities totak emeasure s againstth ediseas e and its consequences.I n order tosav e thethreatene d chestnut, aselectio nprogra m was carried out byth e SwissFedera l Institute forForestr y Research,wit h thegoa lo f selecting (using suitable testingmethods )a sman y resistant individuals as possible fromth enativ e andAsia nchestnu tpopulations .Thes e selected treeswer e to formth ebasi cmateria l for futurereforestations . Inorde r tocarr you tthi s selection andnumerou s expensive experiments,nurserie s wereprovide d inth eCanto n ofTessi nwher ew ecoul d raise several thousand chestnutseedling s eachyea r for infection and selectionexperiments . Itrapidl ybecam e clear thatth eepidemi c inou rEuropea n chestnut (Castanea sativa Mill.)progresse d muchmor e slowly andwa sweake r thani n theAmerica n chestnut.N o doubtth eEuropea n chestnut ismor eblight - resistant thanit sAmerica n relative,an dthi shighe r resistance justifies amor e favorableprognosi s forth e future ofou rchestnu t species. Ifi t couldb eprove n thathighe r resistance isgeneticall y controlled, there

396 wouldb e thepossibility , through systematic selection ofresistan t indi­ viduals, to improve thechance s for survival ofth e species.I twoul db e evenmor eefficien t tosimultaneousl y crossth eEuropea nchestnu twit h suitable resistantAsia nchestnuts ,especiall y the Japanese chestnut (Castanea crenata S.& Z.).

TIMETABLE

Inretrospec t theprogres s ofth e selectionprojec t canb e divided into 3phases : 1951-1960:Propagatio n andpreparatio n ofplan tmateria l anddevelop ­ mento f inoculation andtes tmethods .Durin g thisperio d themai npar to f thepreliminar y phytopathological studieswer e alsocarrie d out.I naddi ­ tion,th emetho d forvegetativ epropagatio n ofchestnu twa s developed (Bazzigher, 1968). In196 0a n important stepwa s takenwit h furtherdevel ­ opmento f inoculation andtes tmethods . 1960-1972:Perio d ofintensifie d systematic selection.Th eeffec to f thene w testmetho dwa s studied (Bazzigher & Schmid, 1962), andw e learned how theselection s shouldb emad e inth e future. Several spontaneoushy ­ brids, especially hybridswit h the early-flowering C. crenata, were includ­ ed inth eprogram . Today there are about4 0 000tree s from this selection work.Fo rtechnica l reasons,onl ypar to fthes e trees couldb e propagated vegetatively. 1964-1982:Perio d ofcheckin g and additional selection ofblight - resistant trees.Th e total selected material ofblight-resistan tchestnut s nowha d tob e tested inplantations . Inth emid-1960 sw eha d decided to build up theseplantation s systematically asgen ebank s andt oobserv e and measure thisplan tmateria l further.Thes e data,whic hw ecollec t fromeac h tree 3,10 ,an dpossibl y also2 0year s afterplanting , giveu sdifferenti ­ atedpossibilitie s forevaluatin g the results ofselection .Thu sw e can make additional selections that satisfy the special requirements forrefor ­ estations.

PREPARATORY PHYTOPATHOLOGICAL STUDIES

Predisposition and resistance

We canonl y reach ourgoa l ofsavin g the threatened chestnutthroug h comprehensive knowledge aboutth ebiolog y ofbot hpathoge n andhost .Thi s knowledgemus tb e obtained from experimentalwor k (Bazzigher & Schmid, 1962). Results that allowed ust odra w conclusions aboutdiseas e resistance andpredispositio nwer e especially interesting.Resistance ,a sdefine db y Gäumann (1951), isconsidere d here tob e the genetically controlled ability torestric t disease development;whil epredispositio ni s atemporar y state

397 ofsusceptibilit y inth eplant ,cause d by environmental effects andwithi n the limits ofth e geneticvariation .

SEASONALVARIATIO N INPREDISPOSITIO N Ininoculatio n experiments we determined thatth e seasonalvariatio n in predisposition followed therhythmica l changes inth evegetativ e condition ofth ehost .Optimu m growth ofth epathoge n inhos ttissu e takesplac e duringth ewarmes t summermonth s (Fig.1) .Th ebes ttim e for infection in resistance tests,therefore ,i smid-Ma y (Bazzigher& Schmid, 1962). Forth e resistance tests iti simportant ,a sha sbee n shown inou rexperiments , thatonl y results arecompare d from experiments thathav ebee nperforme d at the same time andunde r as similarclimati c conditions aspossible .

EFFECTO FWOUN DAG EO N INFECTIONAN DDEVELOPMEN TO F SYMPTOMS Thepathoge n causing chestnutbligh t isparasiti c inwounds ,i.e. , for infectionth e fungus dependso nwound s inth ebar ko fth ehost .A woun d heals after acertai n time andth etissu e isregenerated . Ifa woun dpara ­ site like E. parasitica cannotente r thewounde d tissue early enough,th e infection fails,o r succeedsbu t isgreatl y delayed.Wit h olderwounds , i.e.,wit h increasing development ofcallu s tissue,th e initial conditions forth epathoge n arepoorer . Ina nexperimen t onwoun d age,e.g. , the growth rate ofth epathoge n in2-day-ol dwound swa s reduced toles s than halftha t found in freshwounds . Ifsuc h atre e recovers,i ti sno t duet o greater resistancebu t rather tounfavourabl e initial conditions forth e fungus. Suchsource s oferror s alsomus tb e excluded from tests onresis ­ tance ofchestnu t tobligh to rmus tb e accounted forwhe n theresult s are interpreted.

Lesion length, mm 40

; 20 -

j F M A M J J A S 0 N D

Month Figure 1. Averagemonthl y growth of E. parasitica inchestnu tbar k in nature. Inoculation experiments done in1960-1961 .

398 DIFFERENTIAL WOUNDHEALING :A CAUSEO FVARIATIO N INSUSCEPTIBILIT Y OF DIFFERENTAGE D SHOOTS ONYOUN G CHESTNUTS Invariou s experiments (e.g.,Bazzighe r & Schmid, 1962)considerabl e variation inresistanc ewa s foundwithi n the tree itself.W e thought,a s was shown ina nexperiment ,tha tthi swa s duet odifferen t rates ofhealin g forwound s indifferen tpart s ofth etree .A t2-da yinterval s theuppe r and lower ends of1- , 2-,an d3-year-ol d shoots on five 7-year-old chestnuts werewounded . Thewound s weremad e and coveredwit h adhesive tape inth e samemanne r asi nou r ordinary inoculationmethod . Inthi sexperimen tw e were thus able tostud y thewoun d healing in0- ,2- ,4 -t o12-day-ol d wounds.Th e amounto fcallu s tissue inmicroscopi c sections from eachwoun d wasuse d as ameasur e ofwoun d healing.Th e results indicated thatther ei s a rapid reduction inhealin g capacity from theyounges t shoots toth e older ones. The older the shootth emor e time itrequire d forth ewoun d toheal . This is,however , the caseonl y for 1t o3-year-ol d shoots; forolde r wounds these differences are reduced. From this experimentw e learned that for comparative studies the inoculationmus tb emad e oncomparabl e spots on shoots ofth e sameage .

ONTOGENETIC VARIATION INPREDISPOSITIO N The ontogenetic variation inpredispositio n iso fgrea t significance for interpretation of inoculation tests.Withi n the rangeo fvariatio n in genetic resistance,chestnu t trees aremaximall y susceptible up to age 6. Susceptibility isreduce dwit h increasing age,bu t increases again inolde r trees.Thes e changes inresistanc e duet o agear eclassifie d inFig .2 .U p toag e4 chestnu ttree s areextremel y susceptible.Thi s isexpresse d bya specialpreferenc e ofth epathoge n forth e cambium.Th e growth rate inthi s tissue isu p to3 time s faster thantha t inth eoute rbar k tissue.A tag e 4-6 the lesionclos e toth ecambiu m isabou t as large as inth e outerbark . Wehav etherefor ecarrie d outou r inoculationtest swit htree so fthi sag e

Wood Bark 'l.'iVli

Growtho f Endothia nfection

Chestnut< 4 yr . 4-6yr .

Figure 2. Ontogenetic changes inpredispositio n reflected ingrowt h of n. parasitica inbar k tissue ofchestnu ttree s ofdifferen t ages.

399 (error-freemeasurement s oflesio n size).Th etree sbecom e increasinglyre ­ sistantafte r6 years .Growt ho fth epathoge ni sincreasingl y concentrated inth eoute rbar k (atendenc y towards exclusiono fth epathoge n fromth e bark,a healin g process).

Inoculum and inoculation methods

PATHOGENICITYO FTH EFUNGU S Usually therear en odifficultie s infindin g isolateswit h sufficient pathogenicity. Since,however ,withi na pathoge npopulatio na fairl y large variationi st ob eexpected ,th esuitabilit yo fa nisolat e forinoculatio n testsmus tb eteste d inpathogenicit y tests (Fig.3) .I na ninoculatio n test3 3isolate s from chestnuts aroundBellinzon a (theCanto no fTessin ) were compared.Hos tmortalit y andlesio n lengthwer e takena smeasure so f pathogenicity.

STORING LIVE ISOLATESO FTH EPATHOGE N Thebes tmetho d forstorag eo fliv e isolatesha sprove nt ob efreez e

Mortality,%

uu- r—• •"• •• • • • « ••«.

• • • • 60- • ••

Ä •

• m - • • • o-l->—* l l***4 , r r*—I 1 50 100 140 Lesionlength ,m m

Figure 3. Pathogenicity testo f3 3 Endothia isolates (462inoculation so n 5-year-old C. sativa). Inoculation:Jun e15 ,1976 .Evaluation : lesion length Sept.16 ,1976 ,mortalit y Sept.7 ,1977 . •= virulen tisolates , A= isolate swit htransmissibl e hypovirulence.Transmissibl e hypovirulence (hypovirulence contagieuse,Grente ,1975 )i sa nepidemi c diseaseo n E. parasitica thatreduce sit spathogenicit ybu tno tit svitality .Th ehypo ­ virulencei scontrolle db ya geneti c factori nth ecytoplas m ofth efungus .

400 drying (withoutprefreezing )an d subsequent storage invacuum-seale d ampuls.N o losso fvitalit y andpathogenicit y hasbee n found.Method : smallpiece s ofnylo nmes h (pore size21 5 ^m) were cutint o smaller strips (3x 10mm) ,sterilized , and stored inabsolut e ethanol.Dr y stripswer e dipped ina suspensio n ofconidi a andplace d onmal t agar inPetr i dishes (1.2% Difc oMal tExtrac t+ 2. 0 %Difc oBact oAgar )an d incubated at2 4° C for abouta mont hunti lpycnidi a formed.Th enylo n strips thenwer ecare ­ fully removed from the agar andeac henclose d ina nampu l and dried in _3 10 Torr.Th e ampulswer e thenmelte d shuti na vacuum , labelled, and stored below 20 °C.

INOCULATION METHOD The inoculationmetho d and its statistical testshav e alreadybee n described indetai l (Bazzigher& Schmid, 1962). Productiono f inoculum: culture flasks (2.51 )eac hwit h 500m lKYG - nutrient solution (Knop's solution +2 % glucose +0. 5 %Difc oYeas tEx ­ tract)wer e inoculated with aspor e suspension of E. parasitica and then rotated horizontally at2 0 °C for 6days . Smallball s ofmyceliu m formed; their sizeca nb econtrolle d byvaryin g thenumber s ofspore suse d forin ­ oculating thenutrien t solution.Whe npigmen t starts to formo nth eballs , themyceliu m isseparate db y sterile filtration andwashe d insteril e water.Th e excess liquid isdecante d andthi s inoculum (shelflife :1 week , possibly longer)i s then stored inplasti cbottle s (100ml )i na refrigera ­ tor (+2 °C) . Inoculation: thetes t seedlings arewounde d inth e stemwit h aspecia l instrument.Th ebar k isremove d as asmal l roundplat e (diameter 0.5 cm)s o thatth ecambiu m isexposed . Inoculum ispu tint o thiswoun dwit h aspatu ­ la. The inoculation spoti sclose d with Scotch adhesive tape toprotec t it from contamination and dessication. Iti sbes t tomak e the inoculations in the second halfo fMay .Growt h rate ofth epathoge n isdetermine d byperi ­ odically measuring the length ofth elesion . Thisevaluation ,usin g growth rate ofth epathoge n inth ehos ttissue , hasbee n successful (Bazzigher& Schmid, 1962). Resistance toblight , i.e., the inborn ability to survive infection, ispartl y correlated withresis ­ tancet o spread ofth epathogen .Th eexten tt owhic h resistance occurs in clones andpopulation s canb e estimatedb ypercentag e mortality inrelatio n toduratio no f symptoms.Ste m diameter atth e inoculation sitemus t alsob e considered.

SELECTION OF BLIGHT-RESISTANT CHESTNUTS

Inou rprogra m we selectblight-resistan t individuals from different species of Castanea and theirhybrids .Th ehybrid s are,excep t forspecia l crossing experiments, formed spontaneously. Freepollinatio n occurring in

401 our conditions most likelygive s riset o considerable inbreeding. Everyyea r chestnut seed iscollecte d from trees selected forseed - collection (266provenances ) and theirprogeny .Th e seed is sownan d raised invariou s experimental nurseries inth eCanto no fTessin .W etes teac h4 - or5-year-ol dtre e in individual inoculation tests.Durin g our selection program wehav e testedmor etha n12 0 000plants .Durin g the last1 0year s wehav e included inthi sprogra m alarg enumbe ro fhybrids ,especiall y with theJapanes e chestnut (C. crenata), which isconsiderabl y more resistant thanth eEuropea n chestnut. C. crenata isespeciall ywell-suite d forcross ­ ingwit h our chestnut since itflower sprecociously , atrai t controlledb y dominance,makin gpossibl e aver y shortgeneratio ntime . Chestnuts thatsurviv e the inoculationtes tar epreserve d asclone s (vegetatively propagated through layering orgrafting )o r as individualsb y transplanting them togen ebanks . Everyban k has its ownnumber , andth etree s arenumbere d consecu­ tively.Usin g these 2number s the location ofeac htre e isunmistakabl y identified; eachchestnu t tree istagge dwit h itsnumbe r andmapped . In addition, thesenumber s and information onparen t (9)tree ,generatio n (F-., F_,etc. )an dvegetativ e propagation are filed onpunc h cards (ParentTre e Card forChestnu tSelection )fo rcomputers . Ina breedin g and selectionprogra m such asw ehav e carried out forth e last2 5years ,a lon g initiationperio d isneede dbefor e alarge rproduc -

Numbero fselecte dchestnu ttree s

4000 0

3000 0

2000 0

1000 0

Figure 4. In196 2 improved testingmethod s and intensification ofth e selectionprogra m resulted ina n increase inannua lproductio no fblight - resistantchestnu ttrees ,whic h couldb e checked ingen ebanks .Ordinate : number ofblight-resistan t chestnuttree s inth egen ebanks ;abscissa :yea r ofplanting .

402 tiono fresistan tplant s ispossibl e (Fig.4) .Toda y the ca.4 0 000select ­ ed and registered trees inth egen ebank s include treeswit hmediu m tohig h blightresistance . Thegen ebank s serveno tonl y forcheckin gth e selectedmateria lbu t also for additional selectionprocedures . Inthes ebank s the trees are controlled andmeasure d according tophytopathological ,phenological ,an d growth characteristics 3, 10, and 20year s afterplanting . These dataar e alsopunche d onth ecard s and arenecessar y forth esecond-generatio nse ­ lection.Wit h thesew ehop e tob e able tosho w thedifferen tbligh tresis ­ tance inth eparen t tree,progeny , and lines.Th e evaluation ispossibl e after completiono fth e secondmeasuremen t (10year s afterplanting , i.e., in1982 )i nal lgen ebanks . Because thetestin gmethod swer e too inexact forth e selectionwork ,i t wasnecessar y to improve themconsiderably . Inaddition , reasons forvaria ­ tion ininfectio n and symptom developmentbecam e apparent,promptin g addi­ tionalphytopathologica l studies.Onl ythe nwa s itpossibl et o interpret thetes tresults . Several thousand chestnutseedling swer e raised annually and inoculated with thepathoge n atag e4- 5 years.Survivin g oronl y slightly infected treeswer e transplanted asclone so rsingl e treesint ogen ebank s inorde r to facilitate checking and second-generation selection.Ove r 120 000tree s have gone through inoculation tests.Abou t4 0 000selecte d treeshav ebee n outplanted and registered individually. For additional selection,measure ­ ments andcontrol swil lb ecarrie d out3 , 10,an d 20year s afterplanting .

REFERENCES

Bazzigher,G. ,1968 .Di eSelektio nEndothia-resistente rKastanie nun dihr eVermehrung . Schweiz.Beitr .zu rDendrologi eNr .16-18 .p .29-38 . Bazzigher,G .& P .Schmid ,1962 .Methodi kzu rPrüfun gde rEndothia-Resisten zbe iKasta ­ nien.Phytopath .Zeits .45 :169-189 . Gäumann,E. ,1951 .Pflanzlich e Infektionslehre.Verla gBirkhäuser ,Basel .68 1p . Grente,J. , 1975.L alutt ebiologiqu econtr e lechancr ed echâtaignie rpa r "Hypovirulence contagieuse".Annale sd ePhytopathologi e 1:216-218 .

403 Engineering blister rust-resistant western white pine

G.I. McDonald &R.J . Hoff

Plantpathologis tan dplan tgeneticist ,respectively ,Intermountai nFores tan dRang e ExperimentStation ,USD AFores tService ;Forestr yScience sLaboratory ,Moscow ,Idaho , USA

ABSTRACT

Ametho d ofmeasuremen t ofwhit epin ebliste r rusthazar d isdescribed .Tw o illustrations ofapplicatio n are discussed. The firsti s aimed atreductio n of severity of selectionim ­ posed onth ewhit epin epopulatio n for improvement inrus t resistanceb ymatchin g hazard and resistance.Th e secondappli ­ cation isdirecte d toward increasing efficiency of selection forphenotypi c resistance through amor e exactknowledg e of rustintensity .Dat a areprovide d to show longter m field performance ofwester nwhit epin e developed from lightt o severe levels of selectionpressur e andplante d onlow ,medium , andhig hhazar dsites .

INTRODUCTION

Whitepin ebliste r rust,cause d by Cronartium ribicola J.C.Fischer , hasbee na majo r forestdiseas eproble m inNort hAmeric a since itsintro ­ duction into theNortheaster nUnite d States early inthi s century.Th e diseasebega nkillin g largenumber s oftree s inth eextensiv ewester nwhit e pine forests ofOregon ,Washington , and Idaho about1920 .B y 1950,develop ­ mento frus tresistan tpine swa s underway (Bingham et al., 1960)and ,b y 1972, the firstcommercia l plantations ofresistanc emateria l wereestab ­ lished innorther n Idaho.Man y kindso fresistanc emechanism s havebee n defined andpossibl e useo fsom e ofthe m inintegrate d rustmanagemen t programs determined (McDonald, 1979;Kinloch ,pag e 119 ofthi sbook) . The materials inthes e firstplantation swer e derived from alimite dnumbe r of parents (Hoff& McDonald , 1980). Inth emeantime ,th edesirabilit y ofa broadened geneticbas e hasbecom e evident.Th e increased basewoul d provide for future improvement invariou s cultural traits,serv e asa sourc e of variation inresistanc e toothe rpest s andenvironmenta l challenges,an d the additional geneticbreadt hwoul d facilitate development of integrated rustmanagemen t (Dinus,pag e45 2o fthi s book). Lastly,maximu mbreadt hi s

404 necessary towag e asuccessfu l campaign againstth e inevitable pathogenic variability ofth erus t (Powers,pag e23 6 ofthi s book). The quickest and leastexpensiv ewa y to increase geneticbreadt h ist o acceptreduce d levelso fresistanc e thatar e alsomechanis m non-specific. Inaddition ,wh y expend resources,bot hgeneti c andmonetary , inth epur ­ suito fnon-essentia l levels ofresistanc e and, atth e same time,ru nth e risk ofincreasin g the selectionpressur e onth e rust?Therefore ,us eo f rustresistanc e inth eenvironmentall y variable PacificNorthwest ,t oman ­ age ahighl yvariabl e rust,wil l requiremaximu m flexibility inkind s of resistance andmethod s ofdevelopment .Th eobjectiv e ofthi spape r ist o outline abreedin g and rustmanagemen tprogra m under developmentb y the USDA ForestService ,t oobtai n this flexibilityb y specification ofhazar d atparen ttre e selection sites andb ymatchin g resistance increate dpopu ­ lations (percento fpopulatio n andmechanism s incorporated)t oexpecte d hazard on asite-by-sit e basis.W ewan tsite-specifi c specification ofbot h selection andperformanc e levels (resistance engineering).

VARIATION OF RUST INTENSITY

Siteswer e knownt ovar y inrus t intensity early inth eNort hAmerica n history ofbliste r rust.Man yprogram s havebee ndevelope d tocapataliz e on thisvariation .Thes erang e fromth e long-standing ribes eradicationpro ­ gram (Ketchame t al., 1968)t oVa nArsdel' s (1972)climati c hazardzones . We know that factors such aspathoge n genetics,hos tgenetic s (pinean d ribes), weather, soils,an dcanyo nphysiograph y work together toproduc e theobserve d variation (McDonald et al., 1980). Table 1show s thatsuc h variation exists innorther n Idaho,ho w iti smanifest ,an d 2o f itsmajo r causes, ribes species and ribesplan tdistribution .Give n thatrus thazar d varies, how does oneobtai n ausefu l site specificmeasure .Sinc e stands vary inage ,stockin g density, growth rate,an dyear s ofexposur e and ori­ gin (natural orplanted) , aparamete r independent ofthes evariable swa s needed. Infection rateha smuc h appeal inthi s regard. Infection rate expressed asproportio n orpercentag e oftree s infected/unit time canb e determined from actualproportion s ofinfecte d trees or from infections/tree/unit time.Bot hmeasure swoul d produce the same infection rate ifspor edistri ­ butionwa suniform .W ekno w iti sno t (VanArsdel ,1972 )an dw ekno w that ribesbushe s areno tdistribute d uniformly (Fracker& Brischle , 1944). It hasbee n suggested thatth edifferenc e incurve s erectedb ybot hmeasure s from data obtained inth e same stand isa ninde x toth e amounto fribe s bush clumping intha tstan d (Fracker, 1936). Such aninde x couldb euseful , butou r concern now is the site-specificmeasuremen t ofrus thazard .Bot h ratesca nb ecalculate d from field datao rthe y canb epredicte d inth e absence ofribes ,pine ,o rrus tb y acompute r simulation that integrates

405 Table 1. Blister rust infection in3 plantation s of susceptible western whitepin e located onth ePries tRive rExperimenta l Forest (PR),Bonne r County, Idaho,U.S.A. , inth eMerr y Creek resistance outplanting near Clarkia, Idaho,an d the CanalGulc hnatura l standnea rPierce ,Idaho .

Site Seasons Number Percent Percent Actual Ribes population of of infec­ dead infec­ (bushes/acre) expo­ trees ted rust tion sure ratea R.v. R.l.

PRlo w elevation 8 1277 11.4 2.4 0.015 PRmi d elevation 8 916 97.7 88.8 0.471 1500 10 PRhig h elevation 8 1196 91 5 6.2 0.308 37 263 Merry Creek 5 204 92 0 66.2 0.505 400 0 Canal Gulch 10 825 55 0 20.1 0.080 NA NA

a.Actua l infection rate= [loge 1 -x . -z - ""2 b. R.v. = Ribes viscossissimum Pursh.,R.l . = Ribes lacustre (Pers.)Pour . various aspects of ribes,pine ,an d rustbiolog ywit hweathe r andtopogra ­ phic characteristics specific toa sit e (McDonald et al., 1980). Ourinter ­ esti nthi sdiscussio n is focused onth emeasuremen t andus eo f actualin ­ fectionrate s forth e 'simpleinterest 'typ eo fepidemic s ofwhic hwhit e pinebliste r rusto npin e is aclassi cexample . Therat e calculated from infectionspe rtre ewil lb ecalle d thetheo ­ retical infection rate (R)an dth eequatio n (Vanderplank, 1975)fo rit s calculationwa s derived as follows.Th etheoretica l proportion infected (Y) is:

1 m where: m= infectio n intensity expressed asaverag e infectionspe rtre e and isa producto f arate ,cankers/tree/yea r (r ),an dperio d ofexposur e inyears .

Thesimpl e interestinfectio nrat e (rg)derive db yZadok s& Schein (1979)i sgive nb y

406 rs = loge loge (1)

t2-tj_ 1- x 2 1- xx where: t,= tim e of firstobservation , t- =tim e ofsecon d observation, x. =proportio n infected attim e1 , x? =proportio n infected attim e 2. Forbliste r rustt. .= 0 an dx - =0 .Thus ,

loge x x t2 - 2

l Ifx .= 1- =y , then em

1 R = log S r t2 e" c* 2

lo% rct2 = rcfc 2= r c H fc2 andR = theoretica l proportion infected underunifor m sporedistributio n= rc. The equationuse d tocalculat e actual infection rate (r)wa s derived from equation [1]a s follows.Sinc e t. andx .= 0 i nou r discussion,the n

r = loge (2)

t2 1- x 2

Infection curves canb e calculated from eachrat e (Zadoks& Schein, 1979)

_p-i - Y = 1- e 2 (3) and

X =1 - e" rt2 (4)

407 where:

Y =proportio n infected att ? calculated from average cankerspe rtree ,

X =proportio n infected att ? calculated fromproportio n.o fstan d infected. Sinceth evariabl e distribution ofribe sbushes ,a swel l asth einter ­ actiono fothe r factors,cause suneve n distribution of spores,th e actual progresscurv ewil l fall shorto fth e theoreticalprogres s curve,bu ti t should notexcee d it.Regressio n ofr o nR for3 0 stands onth e St.Jo e National Forest was r= 0.1 8 R+ 0.02 6 (data from Region I,USD A Forest Service). For r, the regressionwa sbase d on64 4tree spe r stand and forR , on8 4tree spe r stand.Th e standsvarie d from 9t o4 4year s ofag e and,i n thiscase ,exposur e time also.Th ecorrelatio n coefficientwa sr = 0.72 (p< 0.01 , 28 d.f.). After 5year s exposure atth eMerr y CreekPlantation , OP controls (Table 1)showe d R= 0.48 andr = 0.505 .Th e difference is probably not significant. Inan yevent ,th e larger rcoul db e attributed to variations associated with ashor texposur e time and smalltrees . Actual infection rate canb e estimatedb y recording numbers ofhealth y andcankere d stems andnumbe r ofwhorl s foreac h stem. Inwhit epine ,th e number ofwhorl s is anexcellen t indicator ofexposur e timebecaus e one whorl isproduce d eachyear .S oth edat aca nb e sortedb y thenumbe r of whorls and rcalculate d foreac hclas s thatcontaine s 10o rmor etrees .Th e estimated rate for astan d isth e average ofth e rates ofwhor l classes that containsmor e than 10trees . Theparamete r ofmos tsignificanc e toth e forestmanage r ismortalit y rate.Bliste r rust-infected whitepin e iskille dwhe nth ecanke r girdles themai nstem .Relativel y small amounts ofdamag e result from eithernon - girdling cankers orbranc h cankers andtypicall y there isn ogrowt hreduc ­ tionunti l justprior-t o death. Iti sals o important tokee p inmin d that therei sa 5 -t o50-yea rperio dbetwee n infection andcompletio no fth e girdle, depending on ageo fth e tree attim e of infection and resistance mechanisms present.W ehav eno tye t accumulated sufficient datat ocor ­ rectly establish the relationship between infection rate andmortalit y rate fordifferen t classes ofresistanc emateria l undervariou s degreeso f hazard.W edo ,however ,hav esom epreliminar y findings and short-term comparisons (Table 1).Som epreliminar y simulations (describedb y McDonald et al., 1980)als o graphically illustrate thisrelationship . Inth emean ­ time, infection rateswil l servet o illustrate somepoints .

GENE MANAGEMENT

Knowledge about infection rates canb euse d to assist inbot hdevelop ­ ment andus eo fresistan tpopulations .A majo rproble m facing thoseselec ­ tingphenotypicall y resistant trees isth edegre e ofconfidenc eon eca n have ina give ntree' s status. Isi tresistan to r is ita nescape ? For example, astan dtha t averaged 7.5 cankers/tree after3 0year s exposure

408 wouldb e expected tob e 99.94% infected under idealcondition s ofspor e distribution (calculated fromEquatio n 3),o r about6 trees/1 0 000woul db e expected tob e canker freedu et ochance .Th etheoretica l rate (R)i sbase d onth ex numbe r ofcankers/tre e for thestan d (m)divide d by theyear s of exposure.Th e estimated actual infection rate according toou r regression isequa l to0.1 8 R +0,026 .S oth e7. 5 cankers/tree should yield our actual rate (r)o f0.071 . This intur n (Equation 4),give s 88.12% infection,o r about 1canker-fre e tree in10 .No tmuc h confidence here and alo to f resources couldb ewaste d inprogen y testing susceptibletrees . Ifsusceptibl e trees inselectio n areas averaged even 50 cankers/tree after3 0 year s thenth e expected actual infection ratewoul d be 0.33/year. Thismean s that,afte r 30years ,on e should expect99.99 5 %o fth e standt o be infected, or1 tre e in2 0 000shoul db e canker-free due tochance .A ta n infection levelo f 100cankers/tre e after 30years ,th e actual rate should be0.62 6an dth e expected ratio ofcanker-fre e toinfecte d treeswoul db e1 treei n 143million .Suc hspecificatio n ofselectio n intensitieswoul db e mosthelpful .Th e original Forest Serviceprogra m in Idaho found 1canker - freetre e in1 000 0 -afte r 30year s exposure thatproduce d about10 0 cankers/tree (R.T.Bingham ,persona l communication).Man y trees selected under these levels ofdiseas e transmitted high levels ofresistanc e asw e shall see.On ewoul d notb e too confident in selecting for canker-free whitepine s from stands growing innorther n Idaho and exhibiting much less than 50cankers/tre e after 30year s ofexposure . Determination of infection rates alsoha spotentia l forth e application ofresistanc e torus tmanagement .Variou s extensive and intensive options ofgen emanagemen t areavailable .Extensiv e options include selectiono f leavetree s fornatura l regeneration (Hoff& McDonald , 1977), creationo f natural selection seedproductio n orchards (Hoffe t al., 1976), andopen - pollinated (OP)see d fromunteste dphenotypicall y resistanttrees . Intensive options include OP seed from testedparents ,F .crosse s from testedparents ,F .see d from tested F,parents ,screene d seedlings fromO P testedparents ,an d ageneralize d immunepopulation . Theusefulnes s ofresistance-hazar d alignment (REHAL)i sshow nb yth e performance ofth e different classes ofmaterial s ina serie s offiel d tests (Steinhoff, 1971)an d anupdate d comparison of infection rates (Table 2)o nthes e samematerials . These infection rates are anaverag e of 19-,21- ,an d 23-year exposure forth ePries tRiver ,Deceptio n Creek, andEmeral d Creekplantation s and5 years exposure fromMerr y Creek. Several trends areevident .Performanc e of nearly allclasse s ofpin epopulatio n isconsisten t from site to site.Onl y OP controls atPries tRive r dono tconform . These rates alsoprovid e infor­ mation aboutth eeffectivenes so fprogen y testingwhit epine .Th epopula ­ tions composed ofcrosse sbetwee nnon-genera l combining ability (non-GCAx non-GCA)an dGC Ax non-GC A shouldb egive n littleweigh t inthi s analysis

409 -0 41 0) a •p p 0 •ri U 41 •rSi a 4> 0 o wa G •p g G • tO -H 3 •H ^41 H G 4) •P flS • la M D .4) •P O G m< tn 41 X. •p M D 10 l-i m 41 M 4-1 u >1 p CJ o •* CM CM Oi oo m o o m 41 p m o 0 CM CM H rH CM CM Ci o 1H P M 4) H 0 M 0) M en •^^ <4H •ri £ U CM to -p m •O (U M T3 v fö 4) M o H H 41 •P a (0 CM o in rH oi in o rH U tfl 0 l-l CO 01 r* •* co co CO (0 4) •P G 41 CM O o O o o O •P •ri Ol> . •a l H nï •P G £| M o 4) •H O 4) Oi +J O m 0 a -H G M m .q •^ a 0 "M ,4*) W W is 4) M >l 41 3 M !>i r-t 4) w s O 41 l-l a co W •p 4H 'H G CM G 0 +J 0 4) p !s •ri •P G .-~ m •P rH- G (0 M G a CM LT) Ol m r~ o> p « 0 41 H oo vO en ^CM ^CM rH •ri M 4) Is 0 H o o o O O O G •ri >i o 41 Ol* •H 10 Q H O 1 o o o o o O i i 4) \X ) o t) U G 4-1 4) tfl tn •P >i •p a> U tO r-\ 10 u M >i U rH 3 4) 0 (0 'M 10 co rH Ü 0 0 •H> CM •H a Pu G X 4(f)l S 0 4> P H * 41 •P to CM in U3 H CM rH m co U O •r•Pi T3 41 Ol in iß CO CM CM H 4-1 G M rH O o O O O O O 4) 0 O •rMi 01> . en ,a PH H O 1 o o o O o O i i G a Oa -t >i •~- o > p w m A! 3 •rrHi CD IH G 4) 10 •ri •P tfl 0 41 XI ta a) •H U S tfl M >i •p O o r0 (H Oi G co •P >i tn m r~ <£> CO «H G o CM G ^1 IH o CM o» *o •H tfl M >H m rH O O T3 •rGi •P O .H 41 • 4) •H O -P PH S m 1 o i 1 1 1 1 O O O 41 d) •i <*H LT) 0 G •a 0 •r^ M 1) 4) U< •P rH H -P Ü tO <0 tfl «H [0 1 G M 3 rt tn G rH •H 4) •p G os O < bj r-t G • u r-\ Il ^ Ü to •ri tn ,Q G o rH «3 < G G rH rH 1 bn U O Oi 4) o 0 T3 0 u< X G < O a 0 • -H •M 4) tn M o 0 U< U Il G CM -P 4-1 4) .H •P 1 G o U X G (0 tfl tfl CD 0 G G O< 41 < P 01 r-H r-H u O 0 u< Ü X X X H a cj ra ^ 3 0) 3 •Ö •P Ü G o 1 PH o Ü -ri •o a G a rH G G < < (0 t« 0 •ri 0 •|H 0 PH PH PU 0 U< O< U u • 4) h a PU £ « S u O o o 53 u O u u <0 XI n

410 because only 4 non-GCA parents were involved and most of these 4 were bor­ derline GCA. There seem to be significant increments of resistance between OP non-GCA and OP GCA, and between OP GCA and higher levels of selection. Also, significant gain appears to be associated with OP controls and OP non-GCA (about 25 parents) over wild seedlings. The OP controls consisted of open-pollinated seed from trees surviving 30 years of intense exposure to rust levels that produced 100 to 500 cankers/tree; so one would expect some increase in resistance. There is probably a significant reduction in infection rate with production of the F, population, as the Merry Creek rate shows and as would be expected from other analyses (Hoff et al., 1973). In conclusion, the findings clearly show that engineering of blister rust resistance of western white pine is feasible and should provide the basis for an effective integrated rust management program.

REFERENCES

Bingham, R.T., A.E. Squillace & J.W. Wright, 1960. Breeding blister rust resistant western white pine, II. First results of progeny tests including preliminary estimates of heritability and rate of improvement. Silvae Genetica 9: 33-41. Fracker, S.B., 1936. Progressive intensification of uncontrolled plant-disease outbreaks. Journal of Economic Entomology 29: 923-940. Fracker, S.B. & H.A. Brischle, 1944. Measuring the local distribution of ribes. Ecology 25: 283-303. Hoff, R.J. & G.I. McDonald, 1977. Selecting western white pine leave-trees. USDA Forest Service Research Note INT-218. Intermountain Forest and Range Experiment Station, Ogden, Utah. 4 p. Hoff, R.J. & G.I. McDonald, 1980. Improving rust-resistant strains of inland western white pine. USDA Forest Service Research Paper INT-245. Intermountain Forest and Range Experiment Station, Ogden, Utah. 13 p. Hoff, R.J., G.I. McDonald & R.T. Bingham, 1973. Resistance to Cronartium ribicola in Pinus monticola: structure and gain of resistance in the second generation. USDA Forest Service Research Note INT-178. Intermountain Forest and Range Experiment Station, Ogden, Utah. 8 p. Hoff, R.J., G.I. McDonald & R.T. Bingham, 1976. Mass selection for blister rust resis­ tance: a method for natural regeneration of western white pine. USDA Forest Service Research Note INT-202. Intermountain Forest and Range Experiment Station, Ogden, Utah. 11 p. Ketcham, D.E., CA. Wellner & S.S. Evans, Jr., 1968. Western white pine management pro­ grams realigned on Northern Rocky Mountain National Forests. Journal of Forestry 66: 329-332. McDonald, G.I., 1979. Resistance of western white pine to blister rust: A foundation for integrated control. USDA Forest Service Research Note INT-252. Intermountain Forest and Range Experiment Station, Ogden, Utah. McDonald, G.I., R.J. Hoff & W.R. Wykoff, 1980. Computer simulation of white pine blister rust epidemic: model formulation. USDA Forest Service Research Paper INT-258. Intermountain Forest and Range Experiment Station, Ogden, Utah. 136 p. Steinhoff, R.J., 1971. Field levels of infection of progenies of western white pines selected for blister rust resistance. USDA Forest Service Research Note INT-146. Intermountain Forest and Range Experiment Station, Ogden, Utah. 4 p. Van Arsdel, E.P., 1972. Environment in relation to white pine blister rust infection. In: R.T. Bingham, R.J. Hoff & G.I. McDonald (Eds): Biology of rust resistance in forest trees. USDA Forest Service Miscellaneous Publication 1221. Washington, D.C. p. 479-493. Vanderplank, J.E., 1975. Principles of plant infection. Academic Press, New York. 215 p. Zadoks, J.E. & R.D. Schein, 1979. Epidemiology and plant disease management. Oxford University Press. New York. 427 p.

411 The international IUFRO experiment on resistance of white pines to blister rust (Cronartium ribicola).Th e French trial

C.Delatour 1 &Y .Birot 2

Thepape r gives afirs trepor to f aFrenc htria ldevote d toth einter ­ national IUFRO experiment onresistanc e ofwhit epine s to Cronartium ribicola Fischer exRabenh . Inthi s test are 154see d lotsbelongin gt o 14species .Th etes tseed ­ lings ineac h species represented eitherbul kcollection s (provenances)o r families ofvariou s origins.Thes e families consisted of (a)F- .progenie s from openpollinatio n of single-tree selections, (b)F ,progenie s fromcon ­ trolled crossesbetwee n2 selecte dwil dparents ,an d (c)F „progenie s from controlled crossesbetwee n 2selecte dF- .parents . Inth enurser y (lining out stage), interplanted Ribes nigrum L. (Wellington clone)plant swer e used asa sourc e of rust inoculum after artificial inoculationb yaecidio - spores collected fromolde r infected easternwhit epines .Th edesig n inth e nurserywa s aful l randomized blockdesig nwit hplot s of 10tree spe rrow , 5 replications,an d spacingbetwee n treeso f 0.6 mx 0.2 m.On e Ribes plant was interspersed ineac h secondro wbetwee neac h 20seedling s intha trow . Only fast-growing populations or species (P. monticola Dougl., P. strobus L., P. strobiformis Engelmann, P. balfouriana Grev.& Balf. , P. flexilis James, P. lambertiana Dougl., P. armandi Franch., P. griffithii McClelland, P. koraiensis Sieb.& Zucc. )wer eplante d outa s (1-2)seedling snea r Orléans ina nincomplet ebloc k designwit h 20tree spe rplot ,3 repli ­ cations and aspacin g of3 m x 1.5 m. Ribes plantswer e also interspersed: onero w forever y 16row so fpines .Th e slowestgrowin g (mostly P. albi- caulis Engelm., P. parviflora Sieb.& Zucc, P. cembra h., P. aristata Engelm., P. sibirica Rupr.)population swer emaintaine d inth enursery . Threeyear s after natural inoculation ofpine s inth enurser ybar k symptoms only ofrus twer e assessed onth e 5-year-old seedlings. Whenwil dpopulation s wereconsidered , species fromEurop e andAsi a (thenativ e habitats ofth e rust)prove d tob e less susceptible to rust thanth eAmerica n oneswit h someexception s (Table 1). Comparisons ofrus t

1.CNR FLaboratoir ed ePathologi eForestièr eChampenoux ,5428 0Seichamps ,France . 2.CRF OStatio nd'Amélioratio n desArbre sForestier sArdon ,4516 0Olivet ,France .

412 Table 1. Frequency ofrust-fre e seedlings inbul k collections ofwhit e pines.

Geographic group Species Number of Rust-free seed lots (%)

European species P. cembra L. 12 100 P. peuce Grisebach 7 80--100

Asian species P. sibirica Rupr. 2 100 P. parviflora Sieb.& Zucc . 3 100 P. koraiensis Sieb.& Zucc . 7 91--100 P. griffithii McClelland 10 33--9 3 P. armandi Franch. 1 42

American species P. aristata Engelm. 1 98 P. albicaulis Engelm. 5 0--6 0 P. flexilis James 17 0--100 P. strobiformis Engelm. 9 0--6 0 P. lambertiana Dougl. 5 4--3 6 P. strobus L. 24 0--1 2 P. monticola Dougl. 5 0-- 6

infection inth ecollection s representing P. monticola, P. lambertiana, and P. strobus showed some discrepancies butals o some agreements inthei r sus­ ceptibility inFranc e andth eUnite d States (Table 2). According to acur ­ sory analysis ofthes e results 2differen t features appear: resistance ratings inUS A and inFranc e areeithe rquit e similar ofver ydifferent ; these erraticperformance s ofth eprogenie s arerelate d toth e selection areaswher eth e original,parenta l selections are found.Accordin g to Bingham's opinion (personal communication)i tappear s thatth epathogeni c raceso f C. ribicola foundbot h inFranc e and Idaho aresimilar ,bu tdif ­ ferent fromthos e found incoasta lWashingto n andOregon . More detailed informationo nth erust-pin e system ist ob e expected in the future,whe nresult s fromothe r cooperating countries (Finland,Wester n Germany, Japan,Sout hKorea )ar ebrough ttogether .

413 Table 2. Comparison ofrespons e tonurser y inoculation inFranc e andUnite d States ofprogenie s ofwhit epine s selected forrust .resistance a.

Seed lotN o Rust freepercentag e Rust freepercentag e inFranc e inUSA , Idahob

P. lambertiana 41C (FI) 0-1 - 4 10- 70* 41B (FI) 0-2 - 3 10 - 75* 41A (FI) 39 -4 8 - 55 50- 100 *

P. monticola 43B (FI) 0-4 - 7 60* 43E (FI) 2-7 -1 7 60- 7 0- 80* 43C (FI) 17- 31 60* 43A (FI) 68 70 43F (FI) 13 -2 7 - 51 20 -3 5- 60 43D (FI) 61- 69 50- 5 5- 65 47A (FI) 0-25 - 60 35 46H (F2) 37 65 46E (F2) 44 60 46B (F2) 45 55 46F (F2) 56 65 46G (F2) 60 70 46D (F2) 62 80 46A (F2) 63 70 46C (F2) 76 85 a.Som e seed lotscomprise d several separateprogenies ; inthi scas eth e rangeo fprogen ymean s andgran dmea nar egiven . b. *Discrepanc y between French andAmerica nperformance .

414 Relative blister rust resistance of native and introduced white pines in Romania

loanBlad a

StatiuneaExperimental aForestiera ,Padure aVerde ,Timisoara ,Romani a

InRomani a there are 7specie s ofwhit epine ,amon gwhic h Pinus cembra L.i sth eonl ynative ,al lth eother sbein g introduced. Among theintro ­ ducedpines , P. strobus L. (thougheconomicall y important)i s susceptible to Cronartium ribicola J.C. Fisch,e xRabenh .wherea s P. griffithii McClell. and P. peuce Griseb. areresistan t toth e sameparasit e (Patton, 1966; Bingham, 1972;S0egaard , 1972). C. ribicola was firstnotice d in Romania in193 4 (Georgescu et al., 1957), and severe attackshav e occurred since 1973 (Blada, 1978). Because ofth e importance of P. strobus ando fth epotentia l dangerth e pathogen represents,a geneti c resistance improvementprogra m was started in1977 .Th eprogra m includesbot h intra- and interspecific hybridization andha s fora final objective the establishment ofsee d orchards composed ofselection s withhig hcombinin g ability forresistance . Results ofth e firstste po fth eprogra m area sfollows : 1. Atpresen t C. ribicola is spread over allth ecountr ywit h theexcep ­ tion ofth emountai n regions,wit h ahighe r frequency on Ribes nigrum L. (92% attackedpopulations )tha no n P. strobus (51% attacked youngpopu ­ lations) . 2. The degree ofinfectio nvarie s from 'low't o 'veryhigh 'o nyoun gpop ­ ulations of P. strobus whereas thematur e populations are free fromC . ribicola even ifthe y are inth eproximit y of some centers ofinfection . But, whenintroduce d inth e seedorchards ,th e families of'selectionsan d the clones from suchpopulation s havebee n attacked. 3. Except for P. strobus and P. monticola Dougl., all the introduced pines (P. peuce, P. griffithii, P. flexilis James, P. koraiensis Sieb.& Zucc. ) are still free frombliste rrust . 4. Bothnatura lpopulation s of Ribes (e.g. R. alpinum h., R. petraeum Wulf.)an dth enativ e P. cembra thatgro w inth e samenatura l mountain habitatar e free from C. ribicola. This fact (andothers )d ono t support thehypothesi s ofth eexistenc e of agen e center for C. ribicola inth e RomanianCarpathians . 5. Because some attackedplantation s of R. nigrum are farther than10 0k m from anywhit epine ,i ti spresume d thatth epathoge ni seithe r transmitted

415 from Ribes to Ribes, or that it has the ability to form aecia on hosts other than the white pines. In Romania, Minoiu (1974) found aecia of a rust that he identified as C. ribicola on Senecio vernalis W. et- K. (a common plant with Eurasian habitat). Then, after artificial inoculation with these aeciospores he obtained uredia and telia on R. nigrum.

REFERENCES

Bingham, R.T., 1972. Taxonomy, crossability, and relative blister rust resistance of 5-needled white pines. In: R.T. Bingham, R.J. Hoff &G.I . McDonald (Eds): Biology of rust resistance in forest trees. USDA Forest Service. Miscellaneous publication No. 1221. p. 271-278. Blada, I., 1978. Ameliorarea pe cale genetica a rezistentei la boli si insecte a speciilor de rasinoase. Manuscris ICAS , 30 p. (unpublished). Georgescu, C. et al., 1957. Bolile si däunätorii pädurilor. EAS, Bucuresti. 638 p. Minoiu, N., 1974. Bolile si daunatorii coacazului, p. 201. In: Suta, V. et al. (Eds): Protectia pomilor si arbustilor fructiferi. Ceres, Bucuresti. 287 p. Patton, R.F., 1966. Interspecific hybridization in breeding for white pine blister rust resistance. In: H.D. Gerhold, E.J. Schreiner, R.E. McDermott &J.A . Winieski (Eds): Breeding pest-resistant trees. Pergamon Press, Oxford, p. 367-376. S0egaard, B., 1972. Relative blister rust resistance of native and introduced white pine species in Europe. In: R.T. Bingham, R.J. Hoff &G.I . McDonald (Eds): Biology of rust resistance in forest trees. USDA Forest Service. Miscellaneous publication No. 1221. p. 233-239.

416 Developing fusiform-resistant trees in the south-eastern United States

Bruce Zobel

ProfessorEmeritus ,Schoo lo fFores tResources ,Nort hCarolin aStat eUniversity ,Raleigh , NorthCarolina ,US A

ABSTRACT

Inth eN.C .Stat eCooperativ e Tree ImprovementProgram ,w e havebee nemphasizin g fusiform resistance forth epas t2 4 years.Result shav ebee ngood ! Several approaches appeart ob e successful.Th e following are suggested: (1)Mak e fullus e of species differences,usin g species resistant orquitt eresistan t to fusiform rust. (2)Withi n the limits ofadaptability , use better sourceswithi n thepreferre d species. (3)Selec tresis ­ tant individuals within suitable species:choos e resistantin ­ dividuals fromver yheavil y infected stands;choos e resistant individuals based onphenotypi c selection, followed byprogen y testing; after testing,establis h specialty disease resistance orchards;establis h 'two-clone'orchard s ofparent swit h spe­ cial good combining and specific combining ability fordiseas e resistance. (4)Produc ehybrids ,suc ha s P. taeda x p. echi- nata, when feasible. (5)Contro lth e alternate hostoak sa s much aspossible . Wehav e arul etha t ifrus t infection isn o greater than 15% ,w ewatc hi tclosel ybu ttak en o special action. Ifi ti s greater than2 5% ,w e thentak eon eo r allo fth e 5step slist ­ ed above.Despit e thecomplexit y ofth erus t and the differing kindso fresistanc e inth ehost ,gain s in fusiform resistance havebee nremarkabl y good.Resistan tmateria l iscurrentl ybe ­ ingplante d on atleas t 10000 0 acres eachyear . Iti sobviou s that fusiform rustresistanc ebreedin g isth emos t important resulto fth etre e improvement activities inth e south-eastern United States.

INTRODUCTION

Themos tdestructiv e forestdiseas e onpine s inth e south-eastern United States is fusiform rust (Cronartium fusiforme Hedge.& Hun te x

417 Cumm.). Losses from fusiform rust aredifficul t toasses sbu ton e thing iscertai n -th eeffect s ofthi sdiseas e arever y important. Inth esympo ­ sium on fusiform rusti n197 7 (Anon.)/ Phelps estimated thatther ewa s an annual loss of56 2millio nboar d feet (ca.1. 3 millionm 3)o fsawtimbe ran d 194millio ncubi c feet (ca.5. 5 millionm 3)o fgrowin g stock.Power se t al. (1974)estimate d the loss intimbe r tob e $2 8 000 000i n1972 ,wit h asub ­ sequentlos so f $15 000 000 0 infinishe dwoo dproducts .O fperhap s greater importance isth e facttha trus ti s spreading.Holle y& Vea l (1977)stat e 'Itsdamagin g effects are intensifying andth e timber resourcebein g lost torus t isbecomin gmor evaluable' .Thi s disease is aproble m thatwil l not go away andwhic hha s amajo r impacto n forestry operations and onth e total economy inth e southernUnite d States. Fusiform rust attacks several species ofpin ebu tprimar y damage ist o loblolly pine (Pinus taeda L.)an d slashpin e (P. elliottii Engelm.); species such as P. palustris Mill, (longleafpine )an d P. serotina Michx. (pondpine )ar esubjec tt odamag ebu to n amuc h lesser scale thanth e first twomentioned . Ofgrea t importance,a coupl e ofpin e speciesnativ e toth e south-easternUnite d States areresistan t toth edisease ; P. echinata Mill, (shortleafpine )an d P. virginiana Mill. (Virginiapine )ar eno tsuscep ­ tiblet o C. fusiforme althoughthe y are susceptible torelate dCronartiums . Thehistor y of fusiform resistancebreedin g inth e Southha sbee no f special interest since Ihav ebee ndirectl y involved fornearl y 30years . Although therewer e someexceptions , inth eearl yyear spathologist sas ­ suredm etha tther ewoul db e littlebenefi t frombreedin g for fusiform rust resistance.Thei r ideawa s thatth evariatio n amongtree s and standswa s chance or adirec t response to local environmental variations.Further ,I was led tobeliev e therewa sn ovariabilit y inth erus torganis m itself.A s a resultw ewer e strongly urged nott owast e time including fusiform rust resistance inou r appliedprograms . Based onearl y studies andobservations ,i tbecam e apparent thatther e was indeed alarg ecomponen to fresistanc e to fusiform rust inth e southern pines.Especiall y good data,includin g quantitative inheritancepatterns , was obtained fromth ever y largeHeritabilit y Studysponsore db yN.C .Stat e University, the International Paper Company, theNationa l ScienceFounda ­ tion, andth eNationa l Institutes ofHealt h (Stonecypher, 1966;Blair , 1970; Kinloch, 1968).

THE INCREASED INCIDENCE OF FUSIFORM RUST

Fusiform rustha s alwaysbee npresen t inth e southernpine sbu t isha s becomeprogressivel y worse inrecen tyears .Whe nnatura l regeneration is closely observed, rust isquit e evidentbu tth e sicktree s areusuall y overtopped andkille d through competitionwit hth ehealth y trees.Sinc e natural stands areestablishe d atsevera l thousandspe r acre,th e diseased

418 trees arelost .Th emovemen t towardplantation s does result ina greate r prevalence of fusiform rust for severalreasons : 1. Especially inth epast ,see dwer epurchase d from anyoneb y quantity; cone collectors took cones fromtree swit h theheavies t crops.Pine sheavi ­ ly infected with fusiform rustusuall y haveheav y cone crops.Sinc esuscep ­ tibility toth e disease has areasonabl y strong inheritance,th eproportio n oftree s inherentlymor e susceptible to fusiform rust isgenerall y greater fromnursery-grow n seedlings thanwithi n thepopulatio n ingeneral . 2. Seedlings areprotecte d by sprays inth enursery ; thisresult s inver y susceptible individuals being grownt oplantabl e size and thenbein g field- planted.Becaus e ofsit epreparatio n andplantin g atwid e spacing,th ein ­ fected trees aregive nth echanc e to survive andgro w rather thanb e killed by competition indens enatura l stands. 3. Sitepreparatio n and fertilization result ina nincreas e in infection (Miller,1977 ;Gilmor e &Livingston , 1958). Ia m convinced thati nsom e instanceswher e rustinfectio n isbad ,th e added loss from fusiform rust more thanoffset s the added growth andqualit y expected frombette r site preparation and fertilization. 4. Although fusiform infection inth enurser y iscontrollable/i tsome ­ times isno tdon e correctly anddisease d seedlings havebee n shipped and outplanted throughout theSoutheast . 5. Fire suppressionha s resulted ina stron gbuildu p ofoaks , thealter ­ natehos t forth e rust.Thi s isa n important reason for anincreas e inrus t (Squillace &Wilhite , 1977). 6. Thewidesprea d planting ofth ever y susceptible slash and loblolly pines is astron g reason for increased rust.Mixe d pine-hardwoods, longleaf pine andhardwoo d stands arebein g site-prepared andplante d toth esuscep ­ tiblespecies . 7. Someba d 'offsite'plantin g has resulted inheav y rust infection.Whe n loblollypin e isplante d inth eextrem e southern areas andwhe n slashpin e hasbee nplante d north of itsrange ,i ti sver y susceptible (Zobel &Zoerb , 1977). Thelatte rha sbee nparticularl y important inth epas tbecaus e slash pinewa s considered the 'wondertree 'an d itwa sextensivel y planted toth e north ofit s suitable sites,wit h disastrousresults . Fusiform rust,whic hwa s formerly not considered important orwa sa nuisance atbest ,ha sno wbecom e themos t serious forestpes ti nth eSouth ­ east.Withou t control ofsom e sort,whethe r silvicultural orthroug hresis ­ tancebreeding ,man ymillion s ofacre so fotherwis eprim e forest landswil l beeconomicall y marginal or submarginal forpin eproduction .Becaus e ofth e difficulty ofcontro l of fusiform rustthroug h forestmanagemen tmethods , thereall y onlyviabl e alternative isresistanc ebreeding . Luckily the resistance component isstron g (Blair,1970 ;Kinloch , 1968). Currently thever y rapid spread ofrus tha s slowedbecaus e ofgoo d attention toplantin g theprope r geographic source andt o good nursery

419 practices.Bu t inpreviou s years thereha dbee n agran dmixin g ofbot hpin e andrus tgenotypes . Inth epas t itwa s common togro w seedlings ina nurs ­ ery and thent o sendthe mwhereve r needed throughout the South.Sinc e at the same time therewa smuc hnurser y infection,differen t'race so f rustan d pineswer e scattered throughout the South.Ho wmuc h themixin gwil l affect futurebreedin gprogram s isno tknown ,bu t itprobabl y willcaus e added difficulty ina nalread y difficultbreedin gprogram .

THE SELECTION AND BREEDING APPROACH WITHIN SPECIES

Whenth e applied industrial programswer e started in195 1w e knew essentially nothing about fusiform resistance inheritance patterns.Lucki ­ ly, considerable fundamental research hasbee n done,s omuc h isno w known aboutth ediseas e (Jewell et al., 1962;Mille r et al., 1976;Dwinell ,1973 ; Powers, 1975). Inaddition , afe wperson s such as Siggers (1947), Czabator (1971), andWakele y (1954)wer econcerne dman yyear s ago aboutth epoten ­ tial of fusiform rust.Bu t little actionwa s taken relative to disease resistance inlarg eplantatio nprogram sunti l the 1950's. Ourearl y formulawa s simple - 'Dono tus e.an ytre e fora paren ttha t has rustwhatsoever' ,whethe r itb e alim b orbol e gall. Suchrigo rresult ­ edi nth e rejection ofman yotherwis ebeautifu l trees for seedorchar d use andw eencountere d considerable criticism forbein g socritical , alongwit h the comment 'After all,yo u don'teve nkno w ifresistanc e to fusiform rust is inherited'. Iti shar d to assess absolute gains from our earlypolic y of rejecting alldisease d trees.Base d oninheritanc e patterns (Kinloch, 1968), wehav e gained considerably. But an 'error'wa smade .W ecombine d trees selected from areaso f low incidence ofth e diseasewit h thoseparent s selected from heavilydisease d stands.Whe nprogeny-testin g was done inarea so frea ­ sonably heavy infection (resistance isno t assessed ifth e incidenceo f rusti s less than 15 %), anumbe r ofth eparent s inth e seed orchards from the lightly infected standswer equit e susceptible to fusiform rust.Th e method of intensive selectionwher en o infected treeswer euse d and all treeswer eprogeny-teste d resulted ingoo d gains indiseas e resistance. Over 1/3 ofth eplantin g of 50000 0acre s eachyea rb ymember s ofou r Cooperative isi n 'hot'fusifor m areas.Plantation s from seedorchar d trees,whic hno w supply allth e seedneede d forth e 50000 0 000tree s planted annually, confirm thebette r fusiform rustresistance . Ina serie s of tests,Goddar d et al. (1975)foun dprogen y ofdisease - freetree s fromheavil y infected stands tohav eonl yhal f asmuc h infection asthos e from lightly infected stands.Whe nteste d in4 differen tareas , the followingpercentage s oftree swit h rustwer e obtained:

420 Test location Fromheavil y From lightly infected stands infected stands

Test 1 61 85 Test 2 36 63 Test3 26 47 Test4 6 14

There still are foresterswh o question thewisdo m ofusin g only dis­ ease-freeparents ,bu t Istrongl y advocate doing this.Th e gaino fu pt o 35% (Blair,1970 )whe n added toth e2 0% ormor e gain from roguingbase d onprogen y testing (Kinloch& Kelman ,1965 )i swel l worthwhile .Base d on progeny test results,a numbe r ofspecialt y disease resistance orchards havebee nestablishe d by several ofth e forest industries inth eSoutheast . Seedlings from these areplante d in fusiform rustho t spots.Considerabl e quantities ofsee d areno w available,enablin g aprofitabl e forestenter ­ prise inarea swher e diseaseha dpreviousl y beens oba d that suitable tree cropscoul d notb egrown . When thecomponent s ofvarianc e from theHeritabilit y Studywer e used, gainprediction s weremad e ast oth e improvement expected fromrust-resis ­ tance orchards (Zobel et al., 1971). Allparent s usedwer e rust-free,an d gainsrepresen t improvement frommas s selection combined withprogen y testing.Usin g aselectio n intensity ofth ebes t1 4o f24 0 families tested, thepredicte d gainvarie d from 37% to5 5% ofth emea nvalues ,dependin g uponth eyea r ofplanting . Severalperson s areno wmakin g economic analyses ofth emonetar y gains tob e achieved,bot h from the standpoint ofgrowt h (Porterfield et al., 1975)an d from theeffect s onwoo dpropertie s (Veal et al., 1974). Light (30 %), medium (30% -6 0 %), andheav y (60% +)severit y class galls of loblollypin ewer epulpe d by thekraf tprocess .Compare d with non-infected stemwood,Vea l foundth e3 classe syielde d4 , 10an d2 3% lesspul p and required 4,8 an d 20% mor e alkali.Translate d to anacreag ebasis ,th e heavily infected standswoul d yield 3% lesspul ppe r acre ofplantation . The galled wood didproduc emor eextractives .Porterfiel d (1973)assesse d losses involum e forstand swit hvaryin g degrees of infection.Fo rth e moderately infected level,th egrowt h losswa s 13% ; for theheav y level it was 24% . Ifreductio n invalue s for lumber andplywoo d are added, losses from fusiform rust infection arelarg e indeed. IfI wer e to again start atre e improvementprogra m Istil lwoul d not use anydisease d trees directly inth eproductio n seed orchard; Iwould , however,modif y methods formerlyuse d asfollows : 1. Seeds from outstanding trees thathav e oneo r fewsmal l limb galls would be collected.Testin g canb e donequickl y and efficiently inth e United States ForestServic e Fusiform Test Center inAsheville ,Nort h

421 Carolina (Phelps,1977 ;Power s et al.,pag e42 7 inthi sbook) . Thene w methodsbein gworke d out (Walkinshaw et al., 1980)shoul db emuc hmor e efficient thanthos euse d inth epast ;goo d greenhouse-to-field resistance correlations arebein g obtained. 2. Iwoul db emor eparticula r inseparatin g disease-resistance andnorma l seedorchards .Th eeconomic so fa separatio nha sbee nwel l documentedb y Porterfield et al. (1975), who showed thata norganizatio nwit h landstha t arebot h rust-free andheavil y rust-infected would gainconsiderabl y by having separate orchards.Fo rth e lightly infected areas,rus twoul d notb e a criterion ofselectio n forth eorchard , allowing greater gain inothe r characteristics. Excellent studieshav ebee nestablishe d andmor e areunderwa y toclari ­ fyth e inheritance ofrus tresistanc e andgain s frombreeding . Forexample , theN.C .Stat eUniversit y - Industry Cooperative established aserie so f disease diallel testplantings .Th emos tresistan t treesthe nknow n (plusa fewsusceptibl eparents )wer econtrol-pollinated , using a22-tre ehalf - diallel.Th e crosseswer eplante d inarea s selected as fusiform rustho t spots.Th eplantation s areno w 5year s old;reading s ofrus t resistance havebee nmad e and resultswil lb epublishe d soon. Indications aretha tth e original choice ofresistan t cloneswa sver y effective.Sixtee n ofth e2 2 parents included inth ehalf-dialle l (selection ofparent swa sbase d on limited testing)hav eexhibite d outstanding resistance torus tunde rrig ­ oroustesting .Th epotentia l forgai n appears tob e extremelybrigh tan d promising. Perhaps themos tsoli d information onrus tresistanc ewa s obtained from theNort hCarolin a StateUniversit y -Internationa l PaperCompan yHeri - tability Study atBainbridge ,Georgia .Result s from allth e tests fromthi s area areto onumerou s to listbu tth emor e important after 20year swere : 1. Resistance to rustwa sver yvariabl e among theprogen y ofth e28 0 parenttree suse d inth e study.O nth ewors trus t sites,infectio npercen t by familyvarie d from 17% to 100% ,wit h gallspe rtre evaryin g from 0.5 to10.8 . 2. Therewer e greatdifference s ininfectio n rate inplanting s made in differentyear s and ondifferen t sites.Infectio n onth e different sites ranged from3 2% to8 0% ;however , family rankingswer e quite stable onth e 2majo r sitestested . 3. Heritabilities ofrus tresistanc ewer equit evariable ,dependin g upon years and families involved,varyin g fromh 2 =0.0 4 toh 2 = 0.85.Mos t valueswer e inth e areao fh 2 = 0.30. Iti sclea r there issufficien tin ­ heritance ofresistanc e tomak e good gains from resistancebreedin g (Zobel et al., 1971). Considerable ofth e genetic component ofrus tresistanc e is due to additivevariance ,a sshow nb y theheritabilities ,bu t insom ein ­ stances non-additivevarianc ewa simportant . 4. Different families respond differently torus tove r time.Som ewer e

422 essentially destroyed while others seemed tob e ablet o survive,gro wan d 'livewit h therust' ;a fe w families seemt ooutgro w andhea l over therus t infection. 5. Littlegenotype-x-environmen t interactionwa s found. 6. Open-pollinated and control-pollinated tests gave similar rankings for the families tested. Onemajo r conclusion from allth estudie s already assessed istha t althoughbreedin g for fusiform rustresistanc e isprofitable , iti sdiffi ­ cult, time-consuming andexpensive .Despit e themas s ofne w information available,wit hmor ebein g reported all thetime ,th emor e fusiformresis ­ tance isstudie d the clearer iti sho w littlew e really know aboutth e genetics ofth ehost ,o fth epathogen , and their interactions.Th eproble m iscomplicate d because of themagnitud e ofvariabilit y inbot hhos tan d pathogen.Variabilit y inth ehos ti sver ywel l documented; agoo d example ison e small group ofclone s ina compan y seed orchard inGeorgia . Infec­ tionb y family inth e seed orchardvarie d from 0% to9 2% andther ewa sa reasonably strong correlationbetwee nparenta l andprogen yperformance . However,th e same clones indifferen t orchards sometimesproduce ,progen y with differing infection rates (Powers& Zobel , 1978), probably indicating thedifferin g mix ofparent s within thedifferen torchards . Amajo r approachw ehav euse d successfully, andon e Irecommen d forth e future, ist obree d forresistanc ewithi n aspecie s despite the complexity ofhost-parasite-environmenta l relationships.

OTHER METHODS TO REDUCE FUSIFORM RUST

There areothe r usefulmethod s tohel p infusifor m rustcontro l that mustb euse d ifprope r results aret ob eobtained .Thes e are fromus eo f (1)geographi c variationwithi n species, (2)hybridization , (3)differen t species, (4)contro l ofth e alternatehos tthroug h silviculturalpractices .

Geographic variation One important difference inrus t susceptibility thatwa s earlyob ­ served isrelate d to source ofsee dwithi n aspecies . P. taeda from East Texas and fromth enorther nextreme s ofit srang e ismuc hmor e resistant than loblolly from thecente r ofth erang e (Wells,1966) . Trees fromth e LivingstonParis h area inLouisian a arenote d forthei rgoo d growthan d relatively high disease resistance (Wells& Switzer, 1975). Theus e of seed from suchknow nresistanc e areasha sbecom ewidesprea d andha sbecom e a standard and successful practice along theGul fCoas t area.Ther e are dangers inmovin g resistant sources too far,however , and some losseshav e occurred (Zobel& Zoerb, 1977). Useo fa proper ,prove n superior geographic source isth eeasies t andbes twa y toobtai n quick and large improvements in fusiform resistance inth e southernpines .

423 Hybridization Usually ahybri dha scharacteristic s intermediate between itsparent s butthi s isno talway s true.Fo rexample , although thehybrid sbetwee nth e susceptible slash and resistant shortleafpin e is intermediate insuscep ­ tibility, the loblolly * shortleafhybri d appears tob emor e resistant (Florence &Hicks ,1976 ;LaFarg e& Kraus , 1975). Themoderatel y infected longleaf x susceptible loblolly pine appears tover y susceptible.N omatte r which crosses aremade ,th e characteristics of theparticula rparent sbein g usedt omak e thecros s largely determine theperformanc e of anyon ehybrid . Thepossibilit y ofus e andvalu e ofhybrid s such asth e loblolly xshort - leafcros s foroperationa l planting isconsiderable . If-efficientmethod s tomak e thehybrid s canb edeveloped , theirus e in 'hotspots 'woul db eo f greatvalue .

Use of different species The southernpine svar y from thever y fusiform-resistant shortleafan d Virginiapine s toth ever y susceptible slash and loblolly.Ther e areothe r southernpin e species thatca nb e used onprope r sites thathav e only alo w susceptibility torus t (Powers, 1975). Many ofth eheavies t losses arei n slashpin e growno nth edee p sand sites that aremargina l forwoo dproduc ­ tion; sometimes the stands aretotall y destroyed by rust andmus tb eaban ­ doned.O n such sites,longlea fpin e canb euse d (Wakeley, 1968)o rsan d pine (P. clausa (Chapm.)Vase y L.); especially theChoctawhatche e variety which is immune to fusiform rust (Burns,1972 )grow sremarkabl y well. Sand pine israpidl y developing into amajo r species onth e deep sands.Short - leafpin e isresistan t to fusiform rust;i ti sclasse d as a slow-starting speciesbu tgeneticall y improved trees grow quitewel l and arenearl y com­ petitivewit h loblollypin e on some sites.Durin g thepas tyear s Iha dex ­ pected shortleafpin e tob emor ewidel y usedbu t thisha sno thappene d- thebia s against itb ymos t foresters isto ostrong .

Control of the alternate host- and good silvicultural practices Fusiform rustha s oak (Quercus sp.)a sth e alternate host.Althoug h oaks are foundwidesprea d throughoutth e South,the yhav ebecom emuc hmor e numerouswit h the strongpractic e of firecontrol .Recen t studies sucha s thoseb y Squillace &Wilhit e (1977)hav e showntha tther e isa relationshi p betweenth e incidence of fusiform rust and theabundanc e ofoak , especially water oak (Q. nigra L.).Th e authors consider thatth epresenc e of ahig h proportion ofoa k indicateshazardou s rustsites .Unfortunately , practical methods ofcontrollin g oak toth edegre e required areno t available.Contro l burning doesreduc e oak,bu tcanno tb e donebefor e themos t susceptiblepe ­ riod ina pin eplantatio nha spassed .

424 REFERENCES

Anon.,1977 .South .For .Dis .an dInsec tRes .Coun .Managemen to fFusifor mRus ti n SouthernPines .Symp .Proc . (Incooperatio nwit hU.S .Fores tService ,N.C .Stat e Univ.an dUniv .o fFla. )16 3p . Blair,R.L. ,1970 .Quantitativ e inheritance ofresistanc e tofusifor m rusti nlobloll y pine.Ph .D .Thesis ,N.C .Stat eUniv. ,Raleigh .8 7p . Burns,R.M. , 1972.Managemen to fsan dpin eplantations .Proc ,San dPin eSymp . p.124-133 . Czabator,F.J. , 1971.Fusifor m rusto fsouther npine s- a critica lreview .USD AFor . Ser.Res .Pap .SO-66 .South .For .Enpt .Sta .3 9p . Dwinell,L.D. ,1973 .Basidiospor ecas tfro mteli ao fCronartiu mfusiform eo nhalf-si b familieso fnorther nre dan dwate roaks .Phytopath .63 :441 . Florence,L.Z .& R.R .Hicks ,1976 .Fusifor m rustresistanc eo fprogen y fromnatura l loblolly-x-shortleaf pine hybrids. Proc, Twenty-Third NE For. Tree Impr. Conf. p.10-19 . Gilmore,A.R .& K.W .Livingston ,1958 .Cultivatio nan dfertilizin ga slas hpin e plantation:effect so nvolum ean dfusifor mrust .Jour .For .56 :481-483 . Goddard,R.E. ,R.A .Schmid t& F .Vand eLinde ,1975 .Immediat egain si nfusifor mrus t resistance inslas hpin efro mrogue dsee dproductio narea si nseverel yinfecte d plantations.Proc ,Thirteent hSouth .For .Tre e Impr.Conf. ,Raleigh ,N.C . p.197-203 . Holley,D.L . &M.A .Veal ,1977 .Economi c impacto ffusifor mrust .Symp. ,Managemen to f FusiformRus ti nSouther nPines .South .For .Dis .an dInsec tRes .Coun. ,Gainesville , Fia.p .39-50 . Jewell,F.F. ,R.P .Tru e& S.L .Mallett ,1962 .Histolog yo fCronartiu m fusiforme inslas h pineseedlings .Phytopath .52 :850-858 . Kinloch,B.B. ,1968 .Geneti cvariatio n insusceptibilit y tofusifor m rusti nlobloll y pine.Ph .D .Thesis ,N.C .Stat eUniv. ,Raleigh ,N.C .4 7p . Kinloch,B.B .& A .Kelman ,1965 .Relativ esusceptibilit y tofusifor m rusto fprogen y linesfro mrust-infecte dan dnon-infecte dlobloll ypines .Plan tDis .Reporte r 49:872-874 . LaFarge,T .& J.F .Kraus ,1975 .Resistanc eo fshortleaf-x-lobloll ypin ehybrid st o inoculationwit hfusifor m rust.Proc ,Thirteent hSouth .For .Tre eImpr .Conf . p.113-118 . Miller,T. ,1977 .Fusifor m rustmanagemen t strategy inconcept :sit epreparation .Symp. , Managemento fFusifor mRus ti nSouther nPines .South .For .Dis .an dInsec tRes . Coun.,Gainesville ,Fia .p .110-115 . Miller,T. ,E.B .Cowling ,H.R .Power s& T.E .Blalock ,1976 .Type so fresistanc ean d compatibilityi nslas hpin eseedling sinfecte db yCronartiu m fusiforme.Phytopath . 66:1229-1235 . Phelps,W.R. ,1977 .Incidenc ean ddistributio no ffusifor m rust.Symp. ,Managemen to f FusiformRus t inSouther nPines .South .For .Dis .an dInsec tRes .Coun. ,Gainesville , Fia.p .25-31 . Phelps,W.R. ,1977 .Screenin gcente rfo rfusifor mrust .For .Farme r36 :11-14 . Porterfield,R.L. ,1973 .Predicte dan dpotentia lgain sfro mtre eimprovemen tprogram s- Agoal-programmin ganalysi so fprogra mefficiency .Ph .D .Thesis ,Yal eUniv .24 0p . Porterfield,R.L. ,B.J .Zobe l& F.T .Ledig ,1975 .Evaluatin gth eefficienc yo ftre e improvementprograms .Sil .Gen .24 :33-44 . Powers,H.R. , 1975.Relativ e susceptibilityo ffiv esouther npine st oCronartiu m fusiforme.Plan tDis .Rep .59 :312-314 . Powers,H.R. ,J.P .McClure ,H.A .Knigh t& G.F .Dutrow ,1974 .Incidenc ean dfinancia l impacto ffusifor mrus ti nth eSouth .Jour .For .7 :398-401 . Powers,H.R . &B.J .Zobel ,1978 .Progen yo fspecifi c loblollypin eclone svar yi n fusiform rustresistanc e accordingt osee dorchar do forigin .For .Sei .24 :227-230 . Siggers,P.V. , 1947.Temperatur e reguirements forgerminatio no fspore so fCronartiu m fusiforme.Phytopath .37 :855-864 . Squillace,A.E .& L.P .Wilhite ,1977 .Symp. ,Managemen to fFusifor mRus ti nSouther n Pines.South .For .Dis .an dInsec tRes .Coun. ,Gainesville ,Fia .p .59-70 . Stonecypher,R.W. ,1966 .Estimate so fgeneti can denvironmenta lvariance san d covariances ina natura lpopulatio no flobloll ypin e (P.taed a L.). Tech.Bull . No.5 ,Southland sExpt .For. ,Internationa lPape rCo. ,Bainbridge ,Ga .12 8p . Veal,M.A. ,R.L .Blair ,J.B .Jet t& W.T .McKean ,1974 .Impac to ffusifor m rusto n pulpingpropertie so fyoun globloll yan dslas hpines .TAPP IBiol .Conf. ,Seattle ,

425 Wash.p .47-58 . Wakeley,P.C. ,1954 .Plantin gth esouther npines .USD AFor .Ser .Agri .Mono .18 .23 3p . Wakeley,P.C. ,1968 .Rus tsusceptibilit y oflonglea fpin eassociate dwit hbrow nspo t resistancean dearl ycommencemen to fheigh tgrowth .For .Sei .14 c323-324 . Walkinshaw,C.H. ,T.R .Del l& S.D .Hubbard ,1980 .Predictin gfiel dperformanc eo fslas h pinefamilie sfro minoculate dgreenhous e seedlings.USF SSouth .For .Expt .Sta .Res . Pap.SO-16 0(i npress) . Wells,0.0. r 1966.Variatio n inrus tresistanc eamon gpin e seedsource san dspecie si na Mississippiplanting .For .Sei .12 :461-463 . Wells,0.0 .& G.L .Switzer ,1975 .Selectin gpopulation so flobloll ypin efo rrus t resistancean dfas tgrowth .Proc ,Thirteent hSouth .For .Tre eImpr .Conf . p.37-44 . Zobel,B. ,R .Blai r& M .Zoerb ,1971 .Usin gresearc hdat a- diseas eresistance .Journ . For.69 :486-489 . Zobel,B.J .& M .Zoerb ,1977 .Reducin gfusifor mrus ti nplantation sthroug hcontro lo f thesee dsource .Symp. ,Managemen to fFusifor mRus ti nSouther nPines .South .For . Dis.an dInsec tRes .Coun. ,Gainesville ,Fia .p .98-109 .

426 Testing for resistance to fusiform rust of pine

12 3 H.R. Powers,Jr . ,Susa nD .Hubbar d &Rober tL .Anderso n

ABSTRACT

An automated system for artificially inoculating large numbers ofpin e seedlings under standardized conditions has beendevelope d and isno w inus e atth eResistanc e Screening Center atAsheville ,Nort h Carolina.Thi s isth eonl y center in theworl d atwhic h seedlings canb eteste d forresistanc et oa foresttre e disease ona routine ,commerciall y operationalba ­ sis.Th eCente r hasevaluate d therelativ e levels ofresistanc e to fusiform rusto fprogen y ofove r200 0clone s from seedor ­ chards ofman ypul p andpape r companies and states from across the southernUnite dStates .

A reliableprocedur e forevaluatin g the relative resistance ofhos t families andvarietie s is ake yelemen t inan ydiseas e control program utilizing genetic resistance.Fiel dprogen y tests areusuall y carried out forthi spurpose ,bu tther e aresom e inherentproblem s inusin g suchtest s for some foresttre e diseases. Inth ecas eo f fusiform rust,cause db y Cronartium quercuum (Berk.)Miyab ee xShira i f. sp. fusiforme, heavyinfec ­ tiondoe sno ttak eplac e everyyear .Thi smean sprogen y testsmus tb emain ­ tained for several years.Fiel dplanting s areals o expensive toinstal l and maintain.Artificia l inoculation tests carried outo n 6- to8-week-ol d seedlings canprovid e information to supplementprogen y test data.Artifi ­ cial inoculation isrelativel y inexpensive andprovide s data ina shorte r period oftim etha nprogen y testing. With fusiform rusto fsouther npines ,th enee d forartificia l inocula­ tiontechnique sbecam e apparent inth e late 1950's,whe nvariatio n in resistance to fusiform rustwa s detected among slashpin e (Pinus elliottii

1.Chie fPlan tPathologist ,Southeaster nFores tExperimen tStation ,USDA ,Forestr y SciencesLaboratory ,Athens ,Georgi a30602 ,US A 2an d3 .Resistanc eScreenin gCente rManage ran dSupervisor yPlan tPathologist ,Southeas ­ ternAre aStat ean dPrivat eForestry ,USDA ,Asheville ,Nort hCarolin a28803 , USA

427 Engelm.var . elliottii) selections (Barbere t al., 1957). The firstsyste m devised forthi spurpos e (Jewell,1960 )use d telia-bearingoa k leaves suspended overpin e seedlings inside adoubl e tentkep tmois twit h water sprays. Inman y tests thisprocedur e worked verywell ,bu t insom ein ­ stances therewer e difficulties, such as lightinfectio n inon e areao f theten t andver yheav y infection inanother .On eo fth ebigges tproblem s was that inoculations were limited to something lesstha n2 month s inth e spring ofth eyear ,becaus e oak leaveswit hteli acoul db e collected in nature only attha t time.Thi s isa tremendou s limitation for alarge-scal e screeningprogram . Inth e early 1970'sth eU.S .Fores tServic e forestdiseas e research project atAthens ,Georgia ,wa s asked todevelo p the technology forlarge - scale screeningwit h standardized numbers of sporesunde rcontrolle dcondi ­ tions.Th e goalwa s ates ttha twoul db e repeatable andwoul dprovid e simi­ lar results inan yyear .Fortunately , theAthen s groupwa s ablet o adapt many ofth eprocedure s utilized incerea l rustdiseas eprogram s toth e needs ofth e fusiform rustprogram .Man ypeopl e areno t awareo fth edeb t thatw eow eth ecerea l rustresearcher s inthe•developmen to fman y ofou r currentprocedures .Fo rexample ,th evacuum-dr y spore storageprocess ,no w widely used on aeciospores ofth e fusiform rustorganism ,wa s developed for long-term storage ofurediospore s ofcerea l rusts.Th e cyclone spore col­ lectorsw eus e for aeciospore collectionwer e alsodevelope d by thewhea t disease researchers.Ou rprogres swit h fusiform rustowe smuc ht oearlie r workers inth e field ofplan tpathology . Dr. ThomasMille rmad e ake y innovation inth edevelopmen t ofth ene w inoculation system for fusiform rust.H ewa s able toconcentrat e largenum ­ bers ofbasidiospore s ofth e fusiform rustorganis m byus e ofMillipor e filters (Miller, 1970). He foundtha tthes e spores couldb e stored forsom e time onth e filterpads .Als o importantwas 'th e adaptation ofth e Coulter electronic particle counter to standardize sporedensit y for inoculations (Dwinell, 1973). The firstatomize d sprayprocedur ewa s developed by Matthews &Rowa n (1972), utilizing the techniques for spore concentration andstandardization .Thi s isno w knowna sth econcentrate d basidiospore spray (CBS)inoculatio n system.Thi s system involves the followingsteps : 1. Aeciospores representative of specified areas arecollecte d (Fig.1) . 2. These spores areprocesse d by screening, drying, and sealing under vacuum. 3. The spores areuse d to inoculate seedlings ofnorther nre doa k (Quercus rubra L.)i norde r toproduc e abundanttelia l columns onth eunderside s of the largejuvenil eleaves . 4. Oak leaves are suspended over acidifiedwate r at2 0 °Ct opromot e the germination ofteliospore s andth edischarg e ofsporidi a onto thewate r surface (Fig. 2). 5. Sporidia arepoure d into aserie s ofMillipor e filters andcollecte d on

428 Figure 1. Aecidiosporecollectio n ina natura l foreststand .

Figure 2. Inducement ofteliospor e germination and collectiono fbasidio - spores.

429 Figure 3. Inoculation ofpin e seedlingswit hbasidiospor e suspension.

Figure 4. 8-monthol d slashpin e seedlingswit h gall development (inocu­ lated at6 weeks) .

430 the filterpads . Thepad swit h the spores arewashe d into asolution ,an d the solution isdilute d toth edesire d sporedensit yb yus e ofth eelec ­ tronicparticl ecounter . 6. Theresultin g sporesuspensio n isapplie db y spraynozzle sont o 6-t o 8-week-old seedlings (Fig.3) . 7. Response ofth ehos t isassesse d 6t o 9month s after inoculation, depending onth e tree species (Fig.4) . All ofthes eprocedures ,plu sman y other individual steps,wer ede ­ scribed ina procedura l manual (Matthews et al., 1971)an d turned overt o Forest Insect andDiseas eManagemen t inth eU.S .Fores t Service South­ easternAre a State andPrivat e Forestry.Tha torganization' s assignmentwa s tous e thisbasi ctechnolog y todevelo p alarge-scale ,operationa l screen­ ingcente rwhic h could handle several hundred thousand seedlings annually. The CBS system was found tob e effective forsuc h anoperatio n (Laird & Phelps, 1975), andth eResistanc e Screening Centerwa s established atBen t Creek,N.C . Itsassignmen twa s to screen seed lots for alltre e improvement programs concerned with selecting andbreedin g for fusiform rustresis ­ tance.Man y innovative improvements weremad e inth ebasi c system inorde r totes t largenumber s of seedlings under standardized conditions.Hand-hel d sprayerswer e replacedwit h spraynozzle smounte d over aconveyo rbelt ,s o the seedlingswer e exposed toth e spray inoculum for aprecis e numbero f seconds asthe ymove d alongth econveyor .Continua l refinement ofth e inoculation system has resulted inconsisten tdistributio n ofunifor m droplets of inoculum onth epin e seedlings.Thi sha sbee n achieved through experimentationwit hvariou snozzle s and airpressures ,an dus eo fperi ­ stalticpumps . TheResistanc e Screening Center routinely collects aeciospores from loblolly (P. taeda L.) and slashpin e in3 0two-thre e county areas from Texas toNort h Carolina. Samples of 30gall s in3 location s aremixe d from each area.Th e client selects the source areao fth e sporeswit hwhic hhi s seed lots aret ob e tested, usually tocoincid ewit h thebreedin g regiono f the families involved. Resistantpin e selections areeithe ropen-pollinate d or full-sib fami­ lies, andther e ismuc h geneticvariatio nwithi n afamily .Therefore ,re ­ sistance to fusiform rust is relative,no t absolute.Performanc e ofeac h seed loti spresente d toth euse r incompariso n to standard resistantan d susceptible checks (Table 1).Result s also include ananalysi s ofvarianc e and aDuncan' s multiple rangetes to nsee d lotmean s asaid st ointerpreta ­ tiono fth edata .Thes e analyses have shownth erepeatabilit y of family means over replications tob e quitehigh .A s in fieldprogen y tests,re ­ sults fora see d lotar emos treliabl y compared tothos e forothe r lots within the same test.Th e dataderive d from thesetests ,however , canb e used inconjunctio nwit h information coming from other sources,usuall y fieldprogen ytests .

431 Table 1. Informationprovide d users ofth eResistanc e Screening Center regarding the resultswit hthei r seedlots.

Infection SeedlotNo . Testmea n Duncan'smultipl e rank <%galled ) range onseedlo t testmean s

Check Resistant (Liv.Parish ) 40 1 Seedlot 1 52 2 Seedlot 2 54 3 Seedlot 3 57 3 Seedlot 4 57 3 Seedlot 5 57 6 Seedlot 6 60 7 Seedlot 7 61 7 Seedlot 8 61 9 Seedlot 9 63 10 Seedlot 10 64 10 Seedlot 11 64 10 Seedlot 12 64 Check Susceptible (11-23) 64 13 Seedlot 13 65 14 Seedlot 14 67 15 Seedlot 15 70

All seedlots 60

A key question about artificial inoculation isth e correlation between its results andth eperformanc e ofth e samematerial s inth e field.Obser ­ vations todat e suggesttha tth e resultsma y relatebette r to fieldperfor ­ mance forslas h than forlobloll ypines .Wit hbot h species,however ,resis ­ tantmaterial s canb e easily identified.Th ehighl y susceptible materials inth e greenhouse areusuall y highly susceptible in fieldplantings .Prob ­ lemsusuall y involve families rated asintermediat e inresistance .Thi s seemst ob eparticularl y true for loblollypine ;w ehav e found some loblolly familieswhic h appeared tob erelativel y susceptible ininocula ­ tiontests ,bu twer emoderatel y resistantunde r fieldconditions .However , wehav eneve r found the reverse tob etrue ;tha tis ,w ehav eneve r rateda family asresistan t ingreenhous e tests and found itt ob e susceptible in fieldplantings .Thus , the CBStestin gprocedur e isquit e rigorous,an d estimates ofoveral l resistance ofth e seed lots areconservative . Methods forobservin g and interpreting CBS results arebein g refined.

432 Preliminary data suggesttha tth epredictabilit y ofgreenhous e results can be improvedb y looking atsevera l symptom types rather thanonl y atth e presence or absence ofa gal l (Walkinshawe t al., 1980). These refinements appear to improve thecorrelatio nbetwee n greenhouse and fieldresults . No artificial inoculation system,whethe r on latebligh to fpotatoes , wheat stem rust,o r anyothe r disease, isanythin gbu t anartificia l sys­ tem.Perfec tcorrelation swit h fieldresult scanno tb e expected. Infact , field results arequit evariable ,an dprogen y tests in2 field locations seldom agree completely.Result s from theResistanc e Screening Center are meant togiv e additional information tohel p inth eevaluatio n of specific families ast othei r resistance torust . The CBS inoculation system isbein guse dver yeffectivel y atth e ItalianFores tDiseas e Center inFlorence , Italy, inwor k onpin ebliste r rustcause d by Cronartium flaccidum (Alb.& Schw.)Wint .Sinc emaritim e pine (Pinvs pinaster Ait.), and Italian stonepin e (P. pinea L.)diffe r considerably in susceptibility, scientists inFlorenc eus e different spore concentrations forinoculatin g these 2species .A muc h lower spore dosage isapplie d to Italian stonepine ,whic h isth emor e susceptible species (Raddi, 1976). During the firstsevera l years ofoperatio nth eResistanc e Screening Centerha sevaluate d progeny ofove r 2000 clones from state and industry seed orchards inth ehig hrus thazar d area.Thes e results havehelpe d tree improvementprogram s across theSout h inevaluatin g themateria l inthei r seed orchards.Man y currenttest s areo fsecon d generationselections , which should have considerably improved rustresistance .Preliminar ytest s have alreadybee ncarrie d outo nth e feasibility ofevaluatin g pines for resistance toneedl e spotdisease s andt owhit epin ebliste r rust.Wor k on these two areasma yb e initiated inth efuture . Inadditio n toevaluatin g resistance of seed lots from thevariou s tree improvement cooperatives,th e Screening Center inoculates and evaluates researchmaterial s forvariou suniversitie s and research centers acrossth e South. Iti srelativel y easy forth e Screening Center to setu p andcarr y outa ninoculatio n on alarg enumbe r ofseedlings .Severa l research studies have alreadybee ncarrie d outunde r special arrangements,an dman ymor e suchcooperativ eventure sprobabl y willb eundertake n inth efuture .

REFERENCES

Barber,J.C. ,K.W .Dorma n &E .Bauer ,1957 .Slas hpin eprogen y testsindicat egeneti c variation inresistanc e torust .U.S .Departmen to fAgricultur eFores tService , SoutheasternFores tExperimen tStatio nResearc hNote s104 .Asheville ,Nort hCarolina . 2p . Dwinell,L.D. ,1973 .Th e relationshipo fth eoa khos tt obasidiospor e casto fCronartiu m fusiforme.Phytopatholog y 63:44 1 (abstract). Jewell,F.F. ,1960 .Ne wpin ehost sfo rsouther nfusifor mrust .Plan tDiseas eReporte r 44:673 .

433 Laird,P.P .& W.R .Phelps ,1975 .A rapi dmetho dfo rmas sscreenin go flobloll ypin ean d slashpin eseedling sfo rresistanc e tofusifor m rust.Plan tDisease .Reporte r 59:238-242 . Matthews,F.R .& S.J .Rowan ,1972 .A nimprove dmetho dfo rlarge-scal einoculation so f pinean doa kwit hCronartiu mfusiforme .Plan tDiseas eReporte r56 :931-934 . Matthews,F.R. , S.J.Rowan ,T .Miller ,L.D .Dwinel l&H.J .Duncan ,1971 .Procedure suse d infusifor m rustresearch .Offic eReport ,U.S .Departmen to fAgricultur eFores t Service.Athens ,Georgia .1 6p . Miller,T. ,1970 .Inoculatio no fslas hpin eseedling swit hstore dbasidiospore so f Cronartium fusiforme.Phytopatholog y 60:1173-1174 . Raddi,P. ,1976 .Indagin esu itip id imacchi efogliar ie su llor osignificat oi n discendenzed iPinu spinaste rinfett ed aCronartiu m flaccidum.Rivist ad iPatologi a Végétale12 :91-98 . Walkinshaw,C.H. ,T.R .Del l& S.D .Hubbard ,1980 .Predictin gfiel dperformanc eo fslas h pine familiesfro minoculate dgreenhous e seedlings.U.S .Departmen to fAgricultur e ForestServic eResearc hPape rSO-160 .Souther nFores tExperiment-Station ,Ne w Orleans,Louisiana .6 p .

434 Italian studies on resistance to pine blister rust (Cronartium flaccidum)

P.Radd i& A .Ragazz i

Centrod istudi ope rl apatologi adell especi elegnos emontane ,CN RInstitut od i Patologiae Zoologi aforestal ee agraria ,Firenze ,Itali a

ABSTRACT

Thispresentatio nprovide s asummar y of studies ofth e biology of C. flaccidum andpoint s out someproblem swhic hw e meet inth ebreedin gprogra m forbliste r rustresistance .Th e biology studies dealwit h thepossibilit y ofstorag e ofaecio - spores,basidiospor e production incontrolle d environments,an d themod e ofpenetratio n ofpin eneedle sb y C. flaccidum. We describe thebreedin gprogra m forbliste r rustresistan tpine s and discuss theresult s obtained.

INTRODUCTION

Blister rusto f2-needl epine s iscause db y Cronartium flaccidum (Alb. & Schw.)Wint .an d iswidesprea d in several countries ofEurop ewithi nth e ranges ofScot spin e (Pinus sylvestris L.), maritimepin e (P. pinaster Ait.), Austrianpine ,Villett a Barrea andLarici opin e (P. nigra Arnold), Italian stonepin e (P. pinea L.), Swissmountai npin e (P. mugo Turra), Aleppopin e (P. halepensis Mill.), andBruti apin e (P. brutia Tenn.). In central and southern Italy inth epas t2 0year s epidemic outbreaks ofthi s disease have caused severedamag e inplantation s and innurserie s of2 - needlepin e species (Moriondo, 1975). Theprimar y objectives ofthi spresentatio n aret oprovid e asummar y of studies ofth ebiolog y of C. flaccidum and topoin tou t someproblem s which wemee ti nth ebreedin gprogra m forbliste r rustresistanc e inItaly . C. flaccidum isheteroeciou swit h onepar to fth e life cycle onth e leaves ofwhit e swallowwort (Vincetoxicum officinale Moench),whic h isth e most frequent in Italy, Gentiana spp., Grammatocarpus spp., Impatiens spp., Loasa spp., Nemesia spp., Paeonia spp., Pedicularis spp., Tropaeolum spp., Verbena spp.,an d the alternate generation onpin e stems andbranches .Th e most frequentpin e hosts are: Italian stonepine ,maritim epine ,Bruti a pine,Austria npine ,Villett a Barreapine ,Larici opine ,Swis smountai n pine, andAlepp opine .Othe r species ofexoti cpine swit hth eexceptio no f

435 ponderosapin e seem tohav e aver yhig h degree ofresistanc e tobliste r rust after artificial inoculation (Raddi& Fagnani , 1978a). Infection occurs throughcotyledons ,primar y and secondary needles and possibly tender stem tissue.Th e firstsign s ofinfectio n areyello w orre d needle spots.Thes e firstbecom evisibl e 8-10 weeks after inoculation,bu t arever y evident after 6-8 months.Infectio n occurs onth eneedle ,an d through thebranche s the fungusthe n invades the stem.Disease d seedlings usually diewithi n 2o r 3year s after inoculation,whe nth e cambium sur­ rounding thecanke r iskilled .Pine s aresusceptibl e from the cotyledonary stageunti l theybecom e old,bu t infections decrease astre e age increases andth eprogres s ofth e disease isslowed .Th e greatestmortalit y isi n pineplantation s up to4- 5 years ofage . Thewid e range ofhos t species suggests thatth e fungus evolvedsimul ­ taneouslywit h itshost . Incentra l Italy themai nhost s ofbliste r rust are P. pinea, P. pinaster, and P. nigra, eachwit hdifferen t climatic requirements.Sometime s inth eoverlappin g zones ofth ehabitat s of2 o f thesepin e species one is foundbadl y damagedb y rust andth eothe rper ­ fectlyhealthy . Itha sbee ndemonstrate d (Mittempergher& Raddi ,1977 )tha t incentra l Italybliste r rustexist s indifferen t environments aspopula ­ tions ofdivers epathogenicity , eacho fwhic h isbes t adapted to infectth e pine speciesprevalen t init sow nhabitat .Thi sha s animportan tbearin g on thewor k of selection forbliste r rustresistance .Ou r researchwor k deals with: (1)som e aspects of fungusbiolog y andmethodolog y connected withth e breedingprogram , and (2)th ebreedin g forbliste r rustresistan tpines .

SOME ASPECTS OF FUNGUS BIOLOGY ANDMETHODOLOGY

The goal of such studies ist ostandardiz e theproductio n ofbasidio - spores of C. flaccidum andt oprovid e abette r understanding ofth einfec ­ tionprocesse s andrelatio n andexpressio no fhos tresistanc e inindividua l pine selections.Th e use of inoculum collections from several areas insures a good representation of fungal sources. Incentra l Italy iti sdifficul t tocollec ttelia-bearin g leaves ofwhit e swallowwort from severallocali ­ ties atth e sametim ebecaus e ofit s different stages ofvegetativ e growth. Research is addressed toth e culture ofwhit e swallowwortplant s inth e greenhouse ori na controlled-environmen tgrowt hchambe r and toth eharves t andpreservatio n of aeciospores andbasidiospores .Aeciospore s are col­ lected frompin ecanker swit h acyclon e spore collector duringth ewarmes t hourso fclea r days.The y areprepare d immediately forlong-tim e storage andvacuu mpacke d in aluminium foil at5 °C.Wit hthi svacuu m storage techniqueth egerminatio n capacity suffered areductio n of2 5% after1 year of storage.Aeciospore s canb e stored for 90day swhe n lyophilized andkep ti na containe r full ofgaseou snitrogen . Inthi scas e astead y increase ingerminabilit y wasnote d during the firstmont h ofstorage ,bu t

436 itdecrease d rapidly after 90day s (Ragazzi, 1978). The storagepossibilit y of aeciospores haspermitte d inoculation ofth e white swallowwort anytime duringth eyear .Bu tther e are someproblem s concerning teliaproductio nwhe nwhit e swallowwort is artificially cultured incontrolle d environments.Th eunde r surface ofyoun g leaves ofwhit e swallowwort is inoculated with awate r suspension of aeciospores.Usuall y abundanturediospore s wereproduce d onleaves , andth eneighbourin g plants thenbecam e infected by theurediospores .Th epresenc e oftelia l columns wasneve rnoted . Infiel d experiments white swallowwortplants ,inoculate d eachmont h ofth eyear , always gaveurediospore s butonl y inoculationsmad e inlat e spring and summer developed urediospores andwell-distribute d crops oftelia .Fo rthes e reasons,til lno w artificial inoculations ofpin e necessarily havebee nconfine d toth eperio d from July to September,whe n temperatures areconduciv e togoo d telia formation onwhit e swallowwort. Another aspect ofth e studies onth ebiolog y dealswit h themod eo f penetration ofpin eneedle sb y C. flaccidum todetermin ewhethe rnon-spot ­ ted seedlings have escaped infection orwhethe r the absence ofneedl e spots may indicate anothermechanis m ofresistance . Infact ,th e open-pollinated families ofmaritim epin ewit h thehighes tpercentage s ofnon-spotte d seed­ lings also tended tohav eth ehighes tnumbe r of 'spotted-only'seedlings , i.e., seedlingswhic h areabl e tosto pth e fungusmyceliu m inth eneedl e tissue.Th evariabilit y ofthi scharacte r amongth e families seems to indi­ cate theexistenc e ofmechanism s which stop thepathoge n inth e first stages ofth e infectionproces s by influencing thewa y C. flaccidum mycelium behaves and grows inth eneedl e tissue ofresistan t maritime pines. A fluorescent labelling techniquewa suse d forobservatio n ofbasidio - spores of C. flaccidum onth eneedle s of5 specie s (maritimepine ,Austria n pine,Villett a Barreapine ,Larici opine ,an d Scots pine). Basidiospores on theneedle swer e labelled by soaking theneedle s for 10minute s ina solu ­ tiono fCalcofluo r (0.05m l on 100m lo fdistille d water). Observations of basidiospore behavior onth eneedl e surfaces ofth e above-mentioned pine speciespointe d outth eexistenc e of significant differences forth e fol­ lowing traits (generally, Scotspin e gave the lowestvalues) : -percentag e ofbasidiospor e germination after4 0hours :4 4t o93 , -beginnin g ofgermination : 5t o2 0hour s (Scots pine), - germ tube length after 40hours :1-1 0 urnt o200-40 0(jm , -percentag e ofbranchin g germtubes :1 3t o71 , -percentag e ofger m tubestha tpenetrate d between theguar dcells : 7 to64 . Themechanis m ofresistanc e thati sbein g expressed in 'spotted-only' seedlings isbein g investigated bymicroscopi c examination ofmycelia l growth inneedle s from susceptible and resistantmaritim e pines,bu t results areno tye tavailable .

437 Thenursery-be d inoculation gavegoo dresult s for identifyingresis ­ tancemechanism s and themos tresistan t families ofmaritim epine .Never ­ theless,variatio n ininfectio n intensity across alarg etes twa s associa­ tedwit hth e location ofth e seedlings inth enurser y beds. Itcreate d relatively highvariance s amongth ereplicates .T o reduce this variability theconcentrate d basidiospore spray system,whic hwa s developed byU.S . Forest Service scientists atth eForestr y Sciences Laboratory,Athens , Georgia,wa s tested on alarg e scale.Th e objectivewa s todetermin e the inoculum-density levels capable ofcausin g successful infection on Italian stonepin e andmaritim epin e seedlingswhic hha dbee ntransplante d into planter trays andthe nwer e inoculated atabou t10 0day s ofage . Onth ebasi s ofth e results (Raddi& Fagnani ,1978b )w e can suggesta concentrationo f 12 500basidiospores/m l or less forth e large scaleinocu ­ lationo f Italian stonepin e ando f7 5 000basidiospore/m l formaritim e pine.Thes e densities caused anaverag enumbe r ofneedl e spotspe rplan to f 18.4± 1.4 , aninfectio n leveltha tgav eu sth emos treliabl e results in producing 'spotted-only' seedlings.

BREEDING FOR BLISTER RUST RESISTANT PINES

Research started in197 3b y selectingparen ttree s free from rust infections inarea swher e the incidence ofdiseas ewa smor e than8 0% . Progenies from openpollinatio n of4 0 Italian stonepine s and3 3maritim e pineswer e transplanted intonurser ybeds . Fifteennaturall y infected leaves ofwhit e swallowwortbearin g teliao f C. flaccidum were attached to cellophane tape andplace d 20c m above thetes t seedlings ineac hro w across thenurser ybed .Th e seedlingswer e inoculated during their second growingseason . Italian stonepin e seedlings inoculatedi nth eprimar y needle stage were infectedwithou t anyevidenc e ofresistanc e (Table 1).Maritim epin e was less susceptible than Italianston epine :significan t differences were found among families forth etrai t 'seedlingswit h needle spotswithou t fungus fruitingbodie s onth e stem' (Raddi et al., 1979). Thebreedin gprojec t ofthi s Center isbase d onth e followingmethods : 1. Inoculation ofmaritim epin eprogenie swit h acontrolle d mechanical spray inoculation systemprovidin g aknow n andunifor m quantity of inoculum ina give ntim e (conveyor speed iscontrolle d at1 0m/mi n andvolum e pass­ ingthroug hbot h spraynozzle s is standardized at7 m l of spore suspension per tray). 2. Foreac h family, 2set s of 100seedling s each areinoculate d atabou t 100day s of age,i neac ho f2 run s 1mont hapart . 3. All seedlings are inspected forth epresenc e ofneedl e spots and spermagonia.Seedling swit h spermagonia onth e stem areremove d because none ofthe m recovered inpreviou s tests.

438 T3 tu 0) p U 3 3 lu ft o .Q h-, «H •H o S-l o U-I P 0) (Il 0) •H u •O D en P C fi •H rd rH Ü o> T3 -H ta <* to G 0> 4-1 Oi •H G a> •H O o O O a 0] fi Oi rH p P P P D s •ri T3 3 oi 0) s •o 0) CM co ro •H •'S p P o o •H o & G SH ui oi 0) rd 0) i rH .fi P d i-l (O p G (d '1-4 (d > O) o *p ro fi o Ü — ^ < Eu p S-l PI ft0) co m ro CO co ^lO r- fi P id CO (N id O ^ id m fio Un id U •H \ o o •H Oi fi fi fi o Ol •H >i o O •p rH rH P P oi T3 -H 0) Ë co co G fi oi 3 CN 10 id G (Il 4H fi o Ol •H •G •H >1 0) en P P id H Ol ü r-t d) ld ß u 3 > P «H P rd Ü •H fi o •H O O P 01 3 P fi U o fi •H O SH o CJ1 rH >i o oi •Ö ft 0) o 01 01 0fi rH •H 73 OJ »H oi rH -ri rH P 0> S O H iw m 3 P O SH tn P U) m rH o 0 01 U O i o U] U O) 4) 01 1 w Ol O! -H 'pM p id (-1 3 id ->1 -01 (Il P 3 T3 SH rH M 3 G fi 0) i 0) O 0> ft Ol P ft(Il •Û P 0) G G rd 3fi ^C P •fi tu 0) •.H rH 0) MH 01 0 P .fi 3 rH 0) •rH p fi T3 U T) .G •fiH (Iftl 3 O 0) o 01 P P •r>| fi •ri 0) 01 •rH rH - o Ul •rfii fi 3 rd

439 4. 'Spotted-only' seedlings are transplanted from the trays into nursery beds 18 months after the inoculation to obtain furter information on their behavior towards the blister rust. 5. Nonspotted seedlings, which we assume were able to stop the pathogen in the first steps of the infection process or are escapes, are transplanted into nursery beds and inoculated again by placing telia-bearing leaves of white swallowwort over them. 6. All 'spotted-only' seedlings and those exposed to 2 inoculations with­ out fungus fruiting bodies on the stem having formed and alive 4 years after the inoculation are transplanted into plantations located in areas of severe incidence of the blister rust. If it is necessary, artificial rust epidemics are created by planting white swallowwort plants adjacent to the plantation and inoculating these bushes with aeciospores of C. flaccidum from different areas. The most promising seedlings in these plantations are then subjected to another screening'test, which takes into consideration rust resistance and also seedling growth rate and form. 7. Undesirable trees, including all rust susceptible trees, are removed at regular intervals, thereby automatically converting the plantations into potential seedling seed orchards. Our current status of knowledge about the biology of C. flaccidum and the mechanisms of rust resistance is sufficient to obtain a generally good level of resistance to blister rust. Every effort of this breeding program is addressed toward providing a broad genetic base of resistance in the parental trees of maritime pine.

REFERENCES

Mittempergher, L. &P . Raddi, 1977. Variation of diverse sources of Cronartium flaccidum. European Journal of Forest Pathology 7: 93-98. Moriondo, F., 1975. Caratteristiche epidemiche della ruggine vescicolosa del pino, Cronartium flaccidum (Alb. et Schw.) Wint. in Italia. (Epidemic characteristics of pine blister rust by C. flaccidum in Italy.) Annali dell'Academia Italia Scienze forestall 24: 331-406. Raddi, P. & A. Fagnani, 1978a. Relative susceptibility to blister rust caused by Cronartium flaccidum of several' species of pine. European Journal of Forest Pathology 8: 58-61. Raddi, P. &A . Fagnani, 1978b. Miglioramento genetico del pino per la resistenza alia ruggine vescicolosa: controllo ed efficacia della densità di inoculo. (Blister rust resistance in maritime and Italian stone pine: effects of the inoculum density level in resistance testing.) Phytopathologia mediterranea 17: 8-13. Raddi, P., L. Mittempergher &F . Moriondo, 1979. Testing of Pinus pinea and P. pinaster progenies for resistance to Cronartium flaccidum. Phytopathology 69: 679-681. Ragazzi, A., 1978. Conservazione e germinazione di ecidioconidi di Cronartium flaccidum (Alb. et Schw.) Wint. (Storage and germination of aeciospores of C. flaccidum.) Rivista di Patologia Végétale 14: 17-28.

440 Lophodermella sulcigena in clones and progenies of scots pine in Finland

Risto Jalkanen

Finnish Forest Research Institute, Rovaniemi Research Station, Eteläranta 55, SF-96300 Rovanierai, Finland

ABSTRACT

The incidence ofgra y needle cast (Lophodermella sulcigena) was examined inprogen y trials and forest tree seed orchards of Scotspin e (Pinus sylvestris). Whenever needle castwa s present ina progen y test,al lprogenie s weredisease d atleas tslight ­ ly.Ther ewer ehighl y significant differences in susceptibility togra yneedl e castbetwee nth e 30progenie s studied. The further theprogen ywa s transferred toth e cultivationloca ­ tion, thehighe rwa s itsresistance .However , theprogenie s fromnort h ofth ecultivatio n locationwer e healthier thanth e ones south of it.Mor etha nhal fo fth eclone s inth e seed orchards were completely healthy, the differences among the clonesbein g considerably greater than inth eprogen ytrial . Bothhealth y andheavil y infected treeswer e foundwithi n diseasedprogenie s orclones .

INTRODUCTION

Epidemics ofgra yneedl e cast (Lophodermella sulcigena) (Rostr.)v . Höhn)hav e occurred every 10-15year s inFinland , (from thecollection s of prof.Kujala , theFinnis h ForestResearc h Institute).Th epresen t extensive epidemicbega n in 1976 and is still continuing. The situation ismos t serious ineven-age d 10t o20-year-ol d pineplantation s growing on fertile sites, where thediseas e has spread throughout entire stands. Innatura l stands almost 100% ofth etree s arehealthy . Gray needle cast infects growing needles ofScot spin e inJune-July .A completely infected tree loses allo fit s currentyea rneedles .Matur e hysterothecia develop within oneyea r inth e infected needles.Dissemina ­ ting sporeswil l again infectgrowin gneedles . The disease doesno t killa tree, itonl y causes severe growthlosses . The fungus is also common in field experiments,an dha sbee n found in seed orchards,too .

441 MATERIALS AND METHODS

Field trialno .23 2i scompose do f3 2plu spin eprogenie s withope n pollinated seed.Fro m these,1 1subtrial swer e establishedi ndifferen t partso fFinlan di n196 6(Fig .1) .Eac h subtrialconsist so f6blocks .A t the starto fth etrial ,ther ewer e2 5seedling so fth esam eprogen yi neac h plot.Th enumbe ro fprogenie s includedi nth edifferen t subtrials varies between 18-29.

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Figure 1. Subtrialsan dprogenie si nfiel d trialno . 232.

442 Grayneedl e castwa s found in4 o fth esubtrial s covering 30o fth e progeniesbein g tested. In2 o fthe m theepidemi cbega n in197 8 and inth e other2 i n1979 .Th estand swer einventorie d during theyea r the epidemic started. Two seed orchards about2 0year s oldwer e alsoexamined . Both the field trial and thesee dorchard swer e establishedb y theDepartmen t of ForestGenetic so fth eFinnis h ForestResearc h Institute. Theprogenie s and clones aresubdivide d into3 groups according to their geographical origin (Oskarsson, 1972): Erefer s toSout h Finland (to approximately N 62°), K toCentra l Finland (betweenN 62°-64°), andP t o North Finland (north fromN 64°) . The amounto fcurren tyea rneedle s destroyedb y L. svlcigena ineac h treewa s estimatedb y eyeusin g the following classification:

Class Needles destroyed (%) Class average

0 none 0.0 1 1-3 3 16.5 2 34- 66 49.0 3 67-100 83.5

Using theclas s averages,th edegre e of infection foreac hprogen y in each subtrial couldb e calculated. Whenth eprogenie s indifferen tsub - trialswer e compared with eachother ,th e degree of infection ofeac h progenywa s divided by the average forth e subtrial andthe nmultiplie d by 100. Thus the average ofbot h the subtrials ando fth ewhol emateria lwa s 100.Thi svalu e iscalle d the infection index.Whe n aprogen y ismor e infected than the average,it sinfectio n index ismor etha n100 .

RESULTS

Progenies All the infected subtrialswer e situated inCentra l Finland.Th esouth ­ ernmost andnorthernmos t subtrialswer e free from L, sulcigena. Although the degree of infectionvarie d considerably indifferen t subtrials,th e funguswa s found throughout allth e stands (Table 1).Th eprogenie sdif ­ fered (P< 0.01 ) from eachothe r in all subtrialsbu t subtrial 8.Al ­ though theprogenie s ineac h subtrial couldb eranke d according todegre e ofinfection ,th ewithin-progen yvarianc ewa shigh . The infection index ofth emos theavil y infectedprogen y (no.26 )wa s 169 and thato fth ehealthies t (no.7 )wa s40 .Infectio n indexbetwee n subtrials differed onlywithi n 3progenie s (P< 0.01) . The healthiest subtrial always deviated fromth eaverag e (nos.30 ,14 ,16 ) (Fig.2) . North-south transfer ofth eprogen y toa cultivatio n locationha dth e effecttha tth e furtherth edistanc ewhic hth eprogen ywa s transferred,

443 PROGENY NUMBER

12 4 5 6 7 8 9 10 11 12 13 14 15 16 180 160

LU 1«> Z » 2 100 JLM W M^^--^-|--^-^|--T3ir^!f--ifcf-Ä|-^Ä-Ä|- | O

PROGENY NUMBER

ieo x HO LD g 140. ~ 120 S loo P. BO

Figure 2. Infection indexo fprogenie s insubtrial s 2,3 ,4 ,an d

Table 1. Degree of infection insubtrials .

Subtrial Trees indifferen t infection classes Infection degree (%) No. healthy 12 3

232/2 16 1605 323 143. 27.3 232/3 3155 508 20 9 2.8 232/4 84 1048 744 976 46.9 232/8 786 1125 152 155 17.7

either southo rnorth ,th egreate r itsresistanc e to L. sulcigena (Table 2). However,th eprogenie s originating fromnort h ofth e cultivation locationwer e onth e average healthier thanth e ones from southo f it.Th e infection indiceswer e 79 forth e former and 115 forth e latter (t-value= 6.61***). By observing the zone division,progenie s from SouthFinlan d (E)wer e themos t severely infected.Ther ewer en o differences betweenK - andP-plu s treeprogenie s although theywer e clearly healthier thanth e E-trees

444 (Table 3). No differences were found insubtria lno .4 ,perhap s due toit s location onth e southernborde r ofth eP-area , over 200k mnort h ofth e othersubtrials .

Table 2. The effecto fnorth-sout h and south-north transfers onth e degree ofinfectio nb y L. sulcigena asindicate db y the infectionindex . F-value= 5.21*** .

Transfer toth eplantin g area expressed Infection index indegree s of latitude (L)

L <1 110 1 ëL < 2 112 2 èL < 3 90 3 £L < 4 85 4 èL < 5 84 L ä5 8 Mean 100 a.L mean s the distance of theprovenanc e from thecultivatio n location. One Li s about 110km .Thu s allprogenie s the origino fwhic h isles stha n 110k m from thecultivatio n locationbelon g toclas s1 .

Table 3. The effecto fprogen y zone onth e amounto f L. sulcigena indif ­ ferentsubtrial sa sindicate db y the infectionindex .

Zone,N .Lat . Subtrial number Average

232/2 232/3 232/4 232/8

E 60°- 62 ° 113 .37 101 117 117 K 62°- 64 ° 104 45 98 75 82 P 64° -69 ° 72 63 98 81 79

Mean 100 100 100 100 100

Infection degree (%) 27.3 2.8 46.9 17.7 F-value 22.94*** 10.72*** 0.31 4.66*** 20.98***

445 Clones Differencesbetwee n theclone s inth e tree orchardswer ever ymuc h greater thanth e differences between theprogenie s inth e field trial.Ove r 50 %o f allth eclone swer ecompletel y healthy and about3 6% of allth e graftswer euninfected . Completely healthy treeswer ever y rare inth e field trials.However ,th emos theavil y infected cloneha d lost6 8% ofit s currentyea rneedles .Thi s ismor e thanth evalue s observed onan y ofth e progenies inth e fieldtrials .Ther ewa s only 1clon e inwhic h allth e grafts were infected. Inth eothe r seed orchard only 18graft s hadbee n attackedb yneedl e cast.Al lo fthe mbelonge d toth e same clone.N ostatis ­ tical differences between clones couldb e tested because the layouto fth e seed orchardswa sno t fortesting .

DISCUSSION

Susceptibility to L. sulcigena was dependent onbot h theorigina l geographical location ando nth ecultivatio n location.Th e farther from its origin,th e less infectedwa s theprogeny .Resistanc e increased whentree s hadbee nmove d either southwards ornorthwards ,bu tmor e greatlywhe nmove d northwards.Th emor enortherl y itsorigin ,th e greater isth eresistanc e of Scotspin et o Phacidium infestans Karst. (Björkman, 1949). Itha s alsobee n demonstrated that L. sulcigena causes less damage tonorther nprogenie s (Lagerberg, 1910). He suspected that thereaso n forthi s lies inth e changes inth e growth rhythm oftree s atth e southern cultivation location. Thegrowt h rhythm oftransferre d progenies may deviate from the local progenies to such anexten ttha t infection cannot spread to itsmaximu m extentwhil e spores arei nth e air.Th e structure ofth e needle, andespe ­ cially the formation ofwax ,ma ypla y animportan t role inprotectin g against infection (Campbell,1972a , 1972b). The resistance of Scots'pin et o L. sulcigena cannothav e developed inth esam ewa y asit sresistanc e to P. infestans. As L. sulcigena occurs only sporadically and isno t fatal,th e selectionpressur e directed atth eprogenie s is small or absent.However ,a persistent epidemic indens e standshelp s less infected individuals to become dominant. P. infestans infects Scotspin e everywinte r inNort h Finland.Th e resistant individuals maintain theirvitalit y and infected parts dieoff . Ifresistan t or less infected trees aredesire d for foresttre ebreed ­ ing, itwoul db ewort hwhil e directing selection atth ehealth y individuals ofth ehealthies tprogenies .Clone s suitable for foresttre ebreedin gca n onlyb e selectedb y excluding themos tdisease d clones.Clone s infectedb y grayneedl e castcoul db e eliminated whenroguin g the seed orchards.How ­ ever, iti squit e adifferen tmatte rwhe nresistanc e toothe rpathogen s is examined. Sinceth e aim of foresttre ebreedin g ist o find treeswhic h grow as fasta spossible ,progenie s cannotb e transferred fromver y farnorth .

446 Therefore local progenies, as resistant as possible, should be found.

REFERENCES

Björkman,E. ,1949 .Studie rove rsnöskyttesvampen s(Phacidiu minfestan sKarst. )biolog i samtmetode rfö rsnöskyttet sbekämpande .Meddelande nfra nStaten sSkogsforsknings - institut37(2) :1-128 . Campbell,R. ,1972a .Electro nmicroscop yo fth eepidermi san dcuticl eo fth eneedle so f Pinusnigr avar .maritim ai nrelatio n toinfectio nb yLophodermell asulcigena .Annal s ofBotan y36 :307-314 . Campbell,R. ,1972b .Electro nmicroscop y ofth edevelopmen to fth eneedle so fPinu snigr a var.maritima .Annal so fBotan y36 :711-720 . Lagerberg,T. ,1910 .O mgrâbarrsjuka nho stallen ,des sorsa koc hverkningar .Meddelande n frlnStaten sSkogsförsökanstal t7 :127-174 . Oskarsson,O. ,1972 .Suomalaise tplusmänny tj apluskuuset .Finnis hplu stree so fScot s pinean dNorwa yspruce .Foli aForestali a150 :1-138 .

447 Resistance trials of scotch pine clones in the Latvian SSR

I.Baumanis ,D . Pirägs& Z .Spalvin s

LatvianScientifi cResearc hInstitut e forForestr yProblems ,229021 ,Rig aRegion ,11 1 Rigastr. ,Salaspils ,Latvia nSSR ,USS R

Inth e last2 t o3 decade scountrie spracticin g intensive forestma ­ nagementhav e seenwidesprea d improvements in forest seedmanagement , and particularly inth e establishment ofsee dorchards . Inth eLatvia n SSR forest seedorchard s ofth e first generation (1006.8ha )hav ebee nestablished , about8 0 %o fthe mbein g Scotchpin e (Pinus sylvestris L.). We areno wworkin g toward theestablishmen t of second generation orchards.A tpresen tw e areevaluatin g the existing clonalmateria l forperiodicit y and intensity of fruiting, increment,woo d quality,plan t survival andresistance . The analysis ofinsec tresistanc e traits inScotc hpin e inexperimenta l areas showed that selectiono ftree s fora certai n degree ofincrease dwoo d and resinproductivit y increased resistance to Aradus cinnamomeus Panz. Resinproductivit y andpin e shootmot h (Rhyacionia duplana Hb.)attac kwer e notcorrelated , butth eprogen y having thehighes tresi nproductivit y was resistantt obot hkind so finsects .Becaus e ofth egrea tvariabilit y of these traits objective evaluation ofth ecorrelatio nbetwee n resinproduc ­ tivity andresistanc e requires thata large r number ofclone sb e tested. Under thecondition s inLatvi apin eplantation s aremor e seriously damagedb yneedleeas t (pathogen: Lophodermium pinastri (Schrad.e xHook. ) Chev.).Unde r conditions favouringdiseas e incidence considerable mortality andgrowt hreductio nhav e occurred.Becaus e chemical control in forest plantations isno tver y efficient,selectio n forresistanc e isth eonl y effectivemetho d ofeliminatin g damage fromneedleeast . Family comparisons ofneedleeas tresistanc ewer emad eusin g deviations ofdistribution s ofdiseas e ratings from anorma l distribution.Th emod eo f class frequencies for familieswit h thehighes t amounto f infection ranged between0.5-0. 9 (1= completel y healthy). Arathe r good correlationwa s foundbetwee n amount ofinfectio n andth edistributio n ofphenotype s in families ofdifferen t seedyears .I nth eyounge r ageclasse so f2- 9 years the amount of infection in familieswa shighe r than inolde r ageclasses , butthei r ranking indistributio n ofphenotypi c resistancewa supheld .Th e distribution ofresistan tphenotype s changeswit h age,bu t ina populatio n of9-year-ol dprogenie s resistantphenotype s aredistribute d more orles s

448 according toth ecurv e for anorma l distribution.Th e amounto f infection was negatively correlatedwit h annual increment (r= -0.3 2 to -0.97), fast-growing p'henotypesbein g the leastinfected . Thus, we conclude that individuals inpin epopulation s differ inthei r degree of susceptibility to needle-cast. Completely resistant (immune)phenotype s haveno tbee ndisco ­ vered. Aparenta l analysis ofsib s shows thatth e introduction ofa resistan t partner into crosses inal lcombination s increases the resistance ofpro ­ genies anddecrease s the frequency ofsusceptibl e phenotypes. Consequently, the selection ofresistan tparen ttree sbot h forope n and controlledcross ­ esincrease s the resistance ofprogenies ,an despeciall y inoptimu mcross ­ ing combinations.Selectio n forresistan tphenotype s increased the juvenile growth ofprogen yb y 23-71% , depending onth ehistor y of infection and progenyage . Thepredicte d genetic effecto f selectionwithi n thepopulatio nfo r resistance toneedlecas twa s 18% .

449 Impact of western gall rust in natural stands of lodgepole pine

OweMartinsso n

Departmento fSilviculture ,Th eSwedis hUniversit yo fAgricultura lSciences ,S-90 18 3 Umeâ,Swede n

The influence ofwester n gall rust (Endocronartium harknessii Y.Hiratsuka )o ngrowt ho flodgepol epin e (Pinus contorta Dougl.)wa s investigated in3 natura l stands incentra l and southernBritis hColumbia . Treeheight ,diamete r atbreas theigh t and radial incrementwer emea ­ sured in3 stands ,Bald yHughes ,Puncha w andChilliwack . Inth e 2younge r anddense r stands theheigh t above ground ofth e oldest stem gallwa s measured.Th e following calculationswer edone : -Annua l radial growthwa smeasure d on increment cores and stemdiscs . Geometricmean s ofth ewidt ho fth e first5 an d last5 annua l ringswer e calculated separately fortree swit h andwithou ton eo rmor e stemgalls . -Th erelatio nbetwee nheigh tan ddiamete r oftree swit h andwithou tste m galls. -Th erelatio nbetwee n total treeheigh tan dheigh tabov e ground ofth e oldest stem gall (Fig.1) .

Total tree height, m

• •

r=0,60*** 3-

12 3 4 Height to oldest stem gall, m

Figure 1. Relationbetwee n tree agea tth etim e of attack (=th eheigh t abovegroun d ofth eoldes tste mgall )an d total treeheight .

450 Table 1. Means of4 variable s oftree swit h andwithou t stem gall in 3 stands of lodgepolepine .* = differenc ebetwee n the two groupssignifi ­ cant atp S 0.05, **= atp S 0.0 1 and ***= a tp S 0.001 ;n.s .= no tsig ­ nificant.

Stand Age Treeheigh t Stem dia- Average ra- Averagera - (years) (m)* meter dialgrowt h dial growth (cm)** first5 last5 years years (mm)*** (mm)n s.

with with­ with with­ with with­ with with­ out out out out

BaldyHughe s 36 9.66 10.48 7.8 8.2 0.65 0.86 1.20 1.10 Punchaw 22 5.29 5.78 3.2 3.6 1.10 1.19 0.42 0.48 Chilliwack 32 2.55 2.89 2.5 2.9 0.54 0.61 0.34 0.33

Themea nradia l growth forth e first5 year swa s loweramon g infected thannon-infecte d trees (Table1) . No significantdifferenc ewa s foundbetwee nth e2 group so ftree s in growth forth e last5-yea rperiod .Measurement s ofradia l growthwer emade , however,i nth este mbelo wgal l formation.Th emea nste mdiamete r andth e meantota l treeheigh twer e smaller for attacked trees than fornon - attacked trees.Th erelatio nbetwee nheigh t anddiamete r showstha ti nth e 2 younger andclose r stocked stands,Puncha w and Chilliwack, diseased trees ofa certai n diameter are shorter thanhealth y treeso fth e samediameter . Inth eBald yHughe s stand diseased trees of acertai n diameterwer e taller thanhealth y treeso fth e samediameter .Thi sma yindicat e that innatura l stands of lodgepolepine ,attack s ofwester n gall rusthav eth e greatest influence onheigh tgrowt h inyoun gstands ,wherea s inolde r andmor e open stands thenegativ e influence ofgal l rustinfectio n ismor e ondiamete r growth. Ifth etre e survives,gal l formation doesno t seem to decrease radial stemgrowt hbelo w the gall.Th e slowerheigh tgrowt h of attacked young treesresult s ina reduce d ability tocompet e for light andspace . Inthes e stands treeswhic hwer e attackedb ywester n gall rustha da slower growth ratebefor e the attack thanthos ewhic hwer eno t attacked.

451 Integrating resistance breeding and tree improvement

Ronald J.Dinu s

InternationalPape rCompany ,Wester nFores tResearc hCenter ,Lebanon ,Orego n97355 ,US A

ABSTRACT

Usable levelso fpes tresistanc e occur inman ytre e spe­ cies. Breeding for resistance can complicate the improvement process andeffort s shouldprocee d only after thoroughassess ­ mento fcost s andbenefits .To o little emphasis limits utility ofimprove dmaterial ;to omuc hredupe s overallgains .Wher e special resistance trials areneeded ,the y should fulfill severalpurposes ,includin g isolationo fmateria l for future breeding andcharacterizatio n ofth enatur e and extento f resistance.Frequen treport s ofadditiv e inheritance suggest thatresistanc e canb emanipulate d the same asothe r traits and thatlong-ter m effectiveness canb ehad . Individual resistance reactions, their inheritancepattern s and epidemiological consequences shouldb eunderstoo d beforewidesprea dusage . Resistance should notb euse d alone,bu t shouldb e deployed on anas-neede d basiswit h othermeasure s incomprehensiv e control programs.Thi s seemsbes t accomplished via organized teamso f specialists,balance d ast odiscipline , andwit h extension capabilities.

INTRODUCTION

Breeding foresttree sha s ashor thistor y compared totha to fagricul ­ tural crops.Eve nwher emanipulate d forcenturies ,tree s remainbu tfe w generations removed from theirwil d ancestors (Libby, 1973). Theirrelati ­ velyundisturbe d status and considerable geneticvariabilit y providea unique opportunity to improveproductivity .Despit e thenewnes s oftre e improvement, substantive programs abound,progres sha sbee nmade ,an dpes t resistance hasbee n animportan tconsideration . That integrationnee db e addressed seemsmor e a function ofhuma n institutions thano fhost s orpests .Scientifi c andtechnica l personnel tendnaturall y topursu ematter e ofindividua l interest andconcern .Lik e

452 others, forest scientists specialize,gathe r insocietie s ofcommo ninter ­ est, and areorganize d into discrete disciplines inth eworkplace .Suc h trends often inhibit,i fno tprevent ,meaningfu l collaboration. Forests and plantations,however , arecomplex , andpes tproblem s areunlikel y tob e resolved byunitar y action. Thispape r seeks toexplai nho w tree improvement and resistancebreed ­ ingca n andmus tb e integrated. Improvementpractice s aredescribe d and related tomethod s forincreasin g resistance.Proble m areas arenoted ,an d solutions aresuggested . Lastly, afe wthought s onprogra m organization and management areoffered .

THE IMPROVEMENT PROCESS

Genetic variation Treebreeder s use geneticvariatio n atsevera l levels.Variatio n among species isconsidere d inchoosin g the speciesbest-suite d to sitean d product requirements,an dma y alsob e exploitedvi a interspecifichybridi ­ zation.Geographi c variation oftenoccur swithi n species astree, sfro m differentphysiographi c regions evolve differently inrespons e tovaryin g selectionpressures .Geneti c differencesma y also arise amongpopulation s or standswithi n regions.Lastly , individual treesvary , oftengreatly .Th e relativeproportio n andpatter no fvariatio n ateac h level governth e scope andnatur e of improvementprograms .

Selection Selection isth e identification ofdesirabl e trees forus e asparents . Criteria necessarily varywit h species and objectives.Tree s are long-lived andmulti-purpos e crops,an dman y traitsmus tb e considered. Two important factors affecting selection efficiency are selection differential -th e differencebetwee n themea no fselecte d trees andtha to fth epopulation , andheritabilit y -th edegre e towhic h atrai ti s governedb yheredity . Genetic gain,th eproduc to fth etwo ,ca nb eenhance d by increasingeither . Differentials canb e raisedb y increasing numbers oftree s examined,an d selecting only a few.Tha t for anyon etrai twil l decline,however , asmor e traits areconsidere d ona se tnumbe r oftrees .Trait s affected greatlyb y the environmenthav e lowerheritabilitie s thanthos e influenced to lesser extents.And ,heritabilit y willb ehighes twhe nenvironmenta l variability is low.Thus ,heritabilit y canb eraise d andusefu l genotypes selectedmor e reliablyb y lessening environmental variability. Commonly used approaches areth e comparison tree andbase-lin emethod s (Ledig, 1974). Selecting forman y traits iscomplicate d by differences invariabilit y andheritability , correlations amongtraits ,an dvalue . Independent cul­ ling,on e solution,use s genetic andeconomi c data to setlevel s of sizeo r quality foreac h trait.Tree s having less thanth e desired rating foran y

453 trait arerejected , even ifotherwis e superior.Th e approach is somewhat subjective,bu twork s reasonably well. Selection indices havebee nuse d ineffort s tooptimiz « gain.Decision s areweighte d bymathematica l integrationo fheritabilities , geneticcorre ­ lations, and expected economic return (Bridgewater &Stonecypher , 1979). Selection indices haveman y advantages,bu twide rusag e awaitsmor e and better data (Faulkner, 1979). Tandem selection, selection foron etrai tpe r generation, isconsidere d impractical because of long generation intervals (Faulkner, 1979). The method couldb eusefu lwhe non e trait severely limits culture and/orim ­ provement,bu t largepopulation s would benecessar y to insure improvement overgenerations . Indirect selection, selection foron etrai tthroug h correlation with another,ha sno tbee nuse d frequently (Faulkner, 1979). Such correlations areno tofte nknow n andca nb eha donl yb y observing offspring ofknow n parentage atmeaningfu l ages.Th emetho d couldb euseful ,wher e onetrai t canb e assessed easily orearl y and anotherca nb e evaluated onlywit h difficulty ortime .

Production of improved material Improved seed is securedb y grouping selected trees insee d orchards (Faulkner, 1975). Mostorchard s areestablishe d with grafts orroote d cut­ tings arranged tomaximiz e crossing andminimiz e inbreeding. Locations are chosent o favorproductio n andminimiz e contamination from external pollen sources.Cultura l andprotectiv e measures areuse d tohaste n and increase production.Design s oftenprovid e for increasing gainb y removal ofmate ­ rials laterprove nundesirable . Seedlingorchard s generallyyiel d less improvement thanclona lor ­ chards,bu t areusefu l alternatives inspecie s thatproduc e seedearJL yo r aredifficul t tograft .A majo r drawback isth enee d toestablis h them on sites typical ofwher e improved seedwil lb eused . Such situations dono t always favor seedproductio n and areofte n subjectt opolle ncontamination . Vegetative propagation isa fas twa y tomultipl y improvedmaterial .A major advantage istha tal ldesirabl e genes canb e captured andreplicate d over space andtime .Propagation , however,become smor e difficult astree s age (Faulkner, 1979), andmultiplicatio n iseithe r anuncertai n proposition or individuals areselecte dbefor e desirability isknown .Ag e and crown position also affectpropagul e growth anddevelopment . Cuttings from atre e old enough tob e evaluated mayhav e the form oftha t individual,bu tgro w more slowly thangeneticall y similar seedlings.Th eman ybenefit swil lb e reaped only as suchproblem s are solved.

Testing and evaluation Improved material mustofte nb e releasedbefor e gains areknow no r

454 provisions aremad e forfutur e selection.Soone ro rlater ,however ,test s areestablishe d with clonal lines orope no rcontrol-pollinate d offspring. Onepurpos e ist omonito rprogres s andobtai n genetic information.Anothe r purpose, oftencompatibl e with the above,i st ovalidat eparenta l quality and eliminatepoo r individuals. Such tests aremos tusefu lwher eheritabil - ities are lowo r special needs occur (Libby,1973 ;Kelliso n& Dinus , 1977). A thirdpurpose ,perhap s themos timportant , ist o selectmateria l forus e insucceedin g generations. Suchtest s aremor e important toth e future than those designed toupgrad e seed orchards,bu tth e latter canpa y handsome returns.Test s compatiblewit hbot h ends can and shouldb eused . Testing, regardless ofpurpose ,i scostly , and separate trials foral l purposes can seldom be afforded.

' Hybridization Interspecific hybrids generally aremos tusefu l outsidenatura l ranges ofth eparent s orwher e desirable combinations oftrait s cannototherwis e be obtained. Longgeneratio n intervals,crossin gproblems ,an d lowsee d yieldspresentl y limitefficiency .Usefu l materials,however , could emerge fromhybridizatio n efforts,especiall y inadvance d generations andwhe n vegetative propagation is feasible.Greates tutilit yma yb e in increasing geneticvariation .

BREEDING FOR RESISTANCE

Decision and emphasis- Choosing to improve resistance isofte nno t adecisio npe r se aspro ­ ductivity would otherwiseb e lost.Mos ttimes ,however ,th e decisioncon ­ cernsemphasi s rathertha nbein g amatte r ofye s orno .Suc hdecision s are noteas y as aspectru m ofpossibilitie s exists.Situation s involvingintro ­ duced trees or innocuouspest s areon eextreme ,an d requiremino r though positive action -pest-fre eparents ,larg ebreedin gpopulations , andflex ­ ibleprograms .Breeder s should also lend expertise tothos e prescribing silvicultural practices,applyin g other controlmeasures ,an d monitoring pestimpact . Other situations aremor e complex. Including resistance impacts every phase of improvement.Onl yb yweighin gbenefit s againstlosse s canbreeder s determine howmuc h toemphasiz e resistance with respectt oothe rtraits . Sound decisions require answers tosuc hquestion s as:wha tpest s areimpor ­ tant;whic h ismos t important;wher e isth epes tmos tdamaging ;ho wmuc h loss occurs;wha t loss canb e tolerated; howmuc h can lossb e reducedb y othermeasures ;ho wmuc h losswil l resistanceprevent ;wha t combination of measures ismos t effective;an dd oreturn s justify resistance breeding? Such concerns are afe wo fth eman y thatmus tb e addressed before ongoing programs arealtere d orne wprogram s are started.

455 Too littleemphasi s cancaus ereduce d yield.To omuc h canhav e thesam e effectb y reducing gain inothe rtraits .Maximu m economic benefit canb e had onlyb ygivin g eachtrai t itsrightfu l emphasis.Breeder s should seek justenoug h resistance toreduc e losses toacceptabl e levels,othe r control measures considered. Seekingunnecessaril y high degrees ofresistanc ema y also jeopardize futureprogres sb y reducingvariatio n inothe rtraits , including resistance toothe rpests ,an db yplacin gundu e selectionpres ­ sure onth epes t inquestion .

Sources of resistance Usable levels ofheritabl e resistance to avariet y ofpest s havebee n found inmos t important genera.Som e species and seed sources aremor e resistant thanothers ,an d suchvariatio n canb e exploitedb y substituting resistantbu tadapte dmaterial s for-susceptibl e ones.Usefu l resistance can alsob e found atth epopulatio n orstan d level,especiall y inth ewak eo f severe infestations. Selecting forgrowt h and form amongpest-fre e resi­ duals canprovid e resistant seed andmaterial . Individual treevariatio ni n most species issuc h thatresistanc e canb e increasedb y judicious selec­ tion andbreeding , withoutlosin g adaptability orintroducin g undesirable traits.Whe notherwis eunavailable ,resistanc ema yb etransferre d from related species.Breeder s seekingt ointegrat e interspecific hybridization efforts into improvementprogram snevertheles s mustb eprepare d to face problemswit h susceptibility toothe rpests ,undesirabl e traits orpoo r adaptability, repeated andprotracte d testing, andmultiplicatio n ofim ­ provedmaterials . Little, ifan yo fth e foregoing isne w orstartling .Th epoin tt ob e made istha tinformatio n required tochoos e among andexploi tvariou s options isseldo m available whenneeded .A s aresult ,reactio n orcrisi s managementbecome snecessary .Delayin g aprogra m togathe r all information issenseless ,bu thavin g advance information onth e amounts andkind so f resistance aswel l aswher et o find itwoul db ebeneficial .

Testing and selection for resistance Selection forresistanc e requires thatresistan tmateria l be identified with ease and certainty. Individual populations ortree smus tb e given equal andhig hprobabilitie s ofbein g attacked. Suchcondition s dono t oftenoccu r inroutin e species,provenance , andprogen y tests,bu tdat aan d material from them shouldb euse dwhereve rpossible .Specia l resistance trials,however , areofte nneeded . Giventh e expense ofeve nroutin e tes­ ting, resistance trialsmus t fulfill severalpurpose s andprovid e all informationneede db ybreeders .Foremos t amongpurpose s shouldb e isolation ofmateria l for futurebreeding ,wit h attention givent odiscernin g nature aswel l asexten to fresistance . Field trials arebes ta s largepopulation s canb e tested, resistance

456 canb e examined where itwil lb eused , andyield s canb e contrasted. Effi­ ciencyma yb e increasedb ymodifyin g environments orprovokin g artificial epidemics.Test s in standard environments with knownpes t concentrations and sources can save time and enhanceprecision ,bu tma y cause selection foronl y afe wo r simply-inherited formso fresistance .Pes tnumber san d sources should thereforeb evarie d tomaximiz e information aboutth enatur e and strengtho fresistance .Utilit ymus tb e confirmed inarea swher eresis ­ tantmateria l willb eplanted .

Nature of resistance Previous remarks about thenatur e ofresistanc e require explanation, especially invie w ofdoubt s aboutdurabilit y ofresistance . Somewoul d argue thatpest swil l evolve faster thantree sca nb ebred .Pest s are variable,bu trisk s occasioned by suchvariabilit y andwidesprea d planting ofresistan t trees areno teasil y determined. Experience with self-pollinated annual cropsha sno tbee nreassurin g as breeders have oftenuse dvertica l resistance -a for m typically controlled byon eo r afe wdominan tgenes .Thoug hexception s occur,wid eusag e imposes selectivepressures ,an dpes tbiotype s ablet ocircumven t the resistance becomeprevalent .Ne w resistance geneso rsom emean s ofcombinin gan d deploying old genesmus t thenb e found.Breeder s ofothe r crops,suc ha s corn,hav e foundmor e lastingprotectio n inhorizonta l resistance -a for m thatdoe sno tprovok e great selectivepressure ,an d isconditione d byman y genes, eachhavin g small effects.Suc hresistanc e isadditivel y inherited, andno t sodifficul t tous e asi softe npresumed . Viewed from anotherperspective ,vertica l andhorizonta l resistance are extreme forms ateithe r end of acontinuum , asprofesso r J.C. Zadokspoin ­ tedou t inhi s address tothi sworkshop .Man y formsca nb e expected, each having aninheritanc e pattern andepidemiologica l consequences determined by itspositio n inth e continuum. Analyzing host/pest systemsma y reveal thepositio n ofeac h andhos tan dpes tcharacteristic s canthe nb euse dt o decide thebes t forms ofresistanc e touse .Th e foregoing suggests that treebreeder s shouldno trel y solelyo nvertica l resistance,an dtha t forms approaching horizontal resistance ora combinatio nwoul db ebest .Resis ­ tance insom etree s appears tob e additively inherited, sobreeder s are alreadyworkin g atth e appropriate end ofth e continuum and longlasting resistance iswithi n reach.Tre ebreeder s routinelywor kwit h additive traits, and should nothav edifficult y includingresistance . Formos t species,however , information on inheritance patterns and epidemiological consequences iswoefull y inadequate.Difficultie s also stem frommeasuremen tmethods ,a sth een d resulto fresistanc e ismonitore d and underlying reactions areofte noverlooked . Overall effectsma y seemquan ­ titative,bu t some components mayb e quantitative andother squalitative . Better informationwil l enablebreeder s to assemblemor edurabl eproducts ,

457 through directed combination ofresistanc e forms.Simila r considerations applyt o detection andus e oftoleranc e -tha tcapacit yo fà 'hos t toendur e severe attackwithou t serious yield loss. Evidence from annual cropssug ­ gests significantheritabilit y oftoleranc e and its independence from resistance.Toleranc e occurs in foresttree s (Griggs& Dinus , 1977), but littlemor e isknown . Much research isneeded , especially onmeasurin g resistance andtole ­ rance, quantifying inheritance patterns,an d evaluating epidemiological consequences.Clarifyin g these issueswil l enablebreeder s toprocee d more confidently andconvincingly . Helpingwit h such concerns needno t involve research onphysiologica l orhistologica l details ofresistanc emechanisms . Knowing theho w andwh y ofresistanc ewil l facilitate continuedprogress , but suchwor k canb e deferred untilmor e important questions are answered. Hastening release of afirs tusabl eproduct ,howeve rpreliminary , ismor e important. Attentionmus tals ob e drawnt oth edangerou snotio ntha tresistanc ei s anal l ornon eproposition .Horizonta l resistance canprovid e onlypartia l protection.Vertica l resistancema yhav eutility ,mainl y as asupplemen tt o other forms,an dprovide d itsrisk s areconsidered . Thus, resistance should beuse d incombinatio n with othermeasure s on anas-neede d basis andwithi n the framework ofa comprehensiv e pestcontro l program.

PROGRAM ORGANIZATION AND MANAGEMENT

Seriouspes tproblem s seembes tresolve d by aforma l team approach. Clear goalsmus tb e seta tth e start,an don e individual charged with choosing and coordinating the approach.Personne l mustb e chosen carefully andmad e to feel aspartner s rather than rivals.Nothin g inhibitscollabo ­ rationmor e than specialization, and individual objectivesmus tb e setsuc h thatworker s understand theprogra m andwha teac h ist ocontribute .Objec ­ tives should also foster inter-dependencies. Incentives shouldb epropor ­ tional totea m accomplishments aswel l as individual performance.Personne l arebes tconcentrate d aton ecentra l location toheighte n interaction and lessencosts .Fundin gmus tb ecommensurat ewit hproble m severity and stable across time.Th etea m should include essential disciplines,withou tbein g unwieldy insize .Quantitativ e sciences shouldb eemphasized . Talentneede d only onoccasio n canb eborrowed .Fo r lessurgen tproblems ,a les s formal approachma y suffice,bu tth e sameprinciple s shouldb e followed. Research,development , and application, likeresistance , arecontinu ­ ous.Th e continuum isa circle ,however , and slowing or ignoring oneaf ­ fects theothers .Scientist sma y notdesir e orb e ablet o translate theory into application,bu t areobliged , astea mmembers ,t ocommunicat e with co-workers inway s thatwil l facilitate theirwor k and engender feedback. Such linkages should be formedwithi n thetea m atth eoutset ,b y including

458 anextensio n specialist.Well-balance d teams are aptt omov e from startt o application with efficiency anddispatch .

REFERENCES

Bridgewater,F.E .& R.W .Stonecypher ,1979 .Inde xselectio no fvolum ean dstraightnes s ina lobloll ypin epopulation .In :Proceeding so fth e15t hSouther nFores tTre e ImprovementConference ,1979 .p .132-139 . Faulkner,R. ,1975 .See dorchards .Forestr yCommissio nBulleti nNo .54 .He rMajesty' s StationeryOffice ,London .14 9p . Faulkner,R. ,1979 .Th eenploitatio no fgeneti cvariatio nb yselectio nan dbreeding .In : E.D.Ford ,D.C .Malco m& J .Atterso n (Eds):Th eecolog yo feven-age d forestplanta ­ tions.Proceeding so fth eMeetin go fDivisio nI ,IUFRO ,Edinburgh ,.1978 .p .99-117 . Griggs,M.M .& R.J .Dinus ,1977 .Pattern so ffusifor m rustincreas ean dthei rimplica ­ tionsfo rselectio nan dbreeding .In :Proceeding so fth e14t hSouther nFores tTre e ImprovementConference ,Gainesville ,Florida ,1977 .p .43-53 . Kellison,R.C .& R.J .Dinus ,1977 .Recen tadvance s ingeneti c improvemento fPinu staed a andPinu selliotti ii nth esouther nUnite dStates .In :Proceeding so fth eThir dWorl d Consultationo nFores tTre eBreeding ,Canberra ,Australia ,Pape rFO-FTB-77-3/2 , Vol.2 .p .475-488 . Ledig,F.T. ,1974 .A nanalysi so fmethod sfo rth eselectio no ftree si nwil dstands . ForestScienc e20 :2-16 . Libby,W.J. ,1973 .Domesticatio nstrategie sfo rfores ttrees .Canadia nJourna lo fFores t Research3 :265-276 .

459 Progeny testing of Douglas-fir seedlings for resistance to western spruce budworm

G.I. McDonald

PlantPathologist ,Intermountai nFores tan dRang eExperimen tStation ,USD AFores t Service,Ogden ,Utah ,84401 ;locate da tIntermountai nStation' sForestr yScience s Laboratory,Moscow ,Idaho ,US A

ABSTRACT

Second instar larvae of Choristoneura occidentalis success­ fullymine dneedle s andbud s and fedo ncurren tan dyear-ol d needles of 1-year-old Pseudotsugae menziesii after inoculation into largecages .Th e introducedbudwor m population appeared to behave in anorma lmanne r includingmating , oviposition, and hatching.Percen to fstem sdebarke d andnumbe r ofeg gmasse s per treewer ebot h significantly correlated with treeheight . Larval feedingwa s tooheav yt oobtai n areliabl emeasur eo f either seed treeo r stand related indication of larval food preference.

INTRODUCTION

Resistance ofman y agricultural cropplant s tovariou s insects iswel l known (Kogan, 1975), and resistance tovariou s insects in foresttre e speciesha sbee n studied forman yyear s (Hanover, 1976). Foresters are especially attracted toresistanc ebecaus e itgenerall y doesno trequir e repeated attention and iseasil y incorporated into integratedpes tmanage ­ mentplans .Anothe r consideration for localitieshavin g extensivewil d forests isth epreservatio n ofcoevolve d balances asthes e forests come undermor e intensivemanagement . Apes to fgrea t importance toth econife r forests ofNort hAmeric a is sprucebudwor m -variou s species of Choristoneura. Inland Douglas-fir (Pseudotsuga menziesii var. glauca (Beissn.)Franco )o fth eNorther nRock y Mountains ofwester nNort hAmeric a isparticularl y susceptible to attackb y C. occidentalis Freeman (Lepidoptera:Tortricidae) , thewester n spruce budworm. In1975 ,w ebega n investigating theDouglas-fi rbudwor m system to elucidate thenatur e ofthi s interaction. A tour of several stands subjected toextremel y heavy defoliation from pastoutbreak s orcurrentl y inoutbreak s quickly demonstrated amplepheno - typicvariatio n onwhic ht obas e studies (McDonald, 1981). Two questions

460 that seemedmos t importantwere :i sther e ageneti c component inthi s variation; and, ifso ,ho w areth eresistanc emechanism s expressed and inherited?A preliminar yprogen y testo fDouglas-fi r indicated thepresenc e of ageneti c component (McDonald, 1979). Our firstpriorit y hasbee nth e development ofprogen y testprocedures .Th eobjectiv e ofthi spape r ist o describe theprocedur e developed andt opresen tth eresult s ofou r first full-scaleprogen ytest .

MATERIALS AND METHODS

Douglas-fir Thehos tmateria l consisted of4 2 openpollinate d seedlings fromeac h of7 mothe r trees from 7stand s togiv e 2058 seedlings.Bot h standsan d mother treeswer e selectedwithou tregar d tobudworm . Seedlingswer econ ­ tainer grown foron e full seasoni nplasti c tubes ina mixtur e of forest soil, sand, andpea tmoss . Theywer e overwintered inthes etube s and then placed ina 1 3 °Ccoole rbefor ebu dbrea k atth ebeginnin g ofthei r second growingseaso n inorde rt o synchronizebu dbrea kwit hlarva ldevelopment . Their average heightwa s 101mm .

Budworm Hibernating 2nd instar larvaewer ecollecte d on stem sections of Douglas-fir,wester n larch,an dponderos apin e growing onth eUniversit y of Montana's LubrechtFores t located 25mile s easto fMissoula ,Montana . Larvalpopulation s were sampledprio r toth emai ncollection .O nMa y 1, 1979, enough stem lengthwa s collected toprovid e anestimate d 5000 larvae and transported back toMoscow , Idaho.O nMa y 2,th e stem sectionswer e placed inlight-proo f containers andtrap s affixed tocatc h the larvae as theybrok ehibernatio n andmigrate d toth e lighted traps.O nMa y3 and4 , the 2058 seedlingswer e removed from the cooler andplace d inth e testbed .

Test procedure Two cageswer e constructed toprovid e 1044plantin g spots each.A 91.4c mx 304. 8c m sheeto fplywoo dwa s drilledwit h 10442.5 4 cm diameter holes ona 5.0 8 cm spacing.Th ehole swer e arranged into 58row swit h1 8 holes each.Thi s floorwa s then installed ina 91. 4c mx 304.5 cmx 30.5c m cagemad eo f 32mes h (32strands/2.5 4 cm)nylo n screen.Th e topwa s totally removable andwa shel d inplac eb y screendoorhooks .Thi s cagewa s then suspended over ameta l tray 91.4c mx 304. 8c mx 15.25 cm.Periodi c filling ofth etra ywatere d the seedlings frombelow .Eac hhol ewa s designatedb y row andcolumn .A computerwa suse d torandomiz e the4 2 seedlings from each tree-stand combination into 2completel y randomized single-tree-plotblock s (21seedlings/stan d mother tree combination). As the larvae emerged from theirhibernatio n sites.Ma y 4t oMa y11 ,

461 andmigrate d into the traps,the ywer e collected insmal lplasti cpetri - dishes, about 30t oth e dish.Thes e disheswer e then'placed ina regula r fashion among the seedlings togiv e about 1649 larvae inbloc k 1an d 1665 larvae inbloc k2 . The larvaewer e allowed tocraw l from thedishe s onto the foliage of thetes t seedlingswher e they developed through thevariou s larval instars toth e adultmoths .Damag e and oviposition datawer ecollecte dAugus t 1t o 14an dpupa l caseswer e counted.

Data Thepla ncalle d formeasuremen t oflengt h oftota l foliated anddefoli ­ atedbranc h and stem length of197 9growt h oneac h seedling toobtai na ratio ofdefoliated/tota l foliated length foreac h seedling.Thi spla n could notb e followedbecaus e 1600 larvae/block overwhelmed the seedlings and completely consumed the 1979 growth andmos t ofth e 1978needle s as well.W e recorded asubjectiv e estimate ofpercen tdefoliatio n ofth e 1978 needles foreac hseedling .Becaus e this foliagewa s consumedbefor e larval developmentwa s completed,man y larvae consumed strips ofbark .Consequent ­ ly,w e recorded thepresenc e or absence ofdebarke d stem andbranc h asa n alternative tomeasurin g defoliation. Inaddition ,w e removed all seedlings frombloc k 1t okee p for 1yea r tochec k survival after complete defolia­ tion and thenpu t afres hseedlin g ofth esam e family inever y otherhol e togiv e a5.0 8 cmx 10.16 cm spacing.Th e replacement operationwa s done afterpupatio n andbefor e adultemergence .Afte r egg layingwa scomplete , we recorded thenumbe r ofeg gmasses/seedling . Analysis ofvarianc e and regressionwer e completed according to Snedecor (1956).

RESULTS

Defoliation Thebudwor mpopulatio n overwhelmed thehos teve nthoug h only 595o f 1649 larvae formed pupae inbloc k 1an d 834o f 1665 inbloc k 2. Also,th e larvae tended to congregate onseedling s around the dishes;thus ,mos t needleminin g occurred onthes e seedlings.N one w foliage orol dbud s survived, and defoliation ofth epreviou s year's needles averaged 98% i n block 1an d 97% inbloc k 2.Th e regression of averagepercen t defoliation of 1-year-old needles on average familyheigh t after 1979 growthyielde d an r of0.3 0 (n.s., 47 d.f.). We completed an analysis ofvarianc e (Table1) . Both stand and family show some significance (Table2) .

Debarking Study ofth e summarized data indicated apossibl e correlationbetwee n percent ofstems/famil y debarked and the averageheigh to f afamil y after growth. Such aregressio nwa s runusin gmea n familyheigh t asdetermine db y

462 Table 1. Keyouto f factorial-hierarchal analysis ofvarianc e used onbud - worm defoliation data.Assum eblock s fixed; stands and familiesvariable .

Source d.f. Expected mean square

Blocks a-1 a2 + rCTAC,B+ rCaAB + rcbaA 2 2 2 Stands b-1 a +raa _ D + rcaa D Families instand s b(c-l) °Z + raa2C,B Blocks xstand s (a-1) (b-1) ff2 + rCTAC,B + rCCT2AB Blocks x families instand s b(a-l) (c-1) a2 + ra2 AC,B Within families abc(r-l)

Table 2. Analysis ofvarianc e ofpercen t ofbudwor m defoliation ofyear-ol d needles on1-year-ol d Douglas-firseedlings .

Source d.f. MS. P>F

Blocks 1 559.44 Stands 6 191.37 2.00 0.086 Families instand s 42 95.45 1.50 0.022 Blocks x stands 6 69.36 Blocks x families instand s 42 40.43 Within families 1960 63.76

the average height inmillimeter s ofth e seedlingsplace d intobloc k1 after larval feeding tosuppl y oviposition sites.Th e rwa s 0.66 (p< 0.05 ; 47d.f. ) (% debarked = 100 [0.29tre eheigh t- 14.4]).

Egg mass number The tallest family averaged-20 6m m inheigh t andha d 2.36 eggmasses / seedling or0.011 5masses/ 1m m ofheight .Th e shortest familywa s 89m m tall aftergrowt h andha d 0.69 masses/seedling or0.007 8masses/ 1m m of seedling height.Thes e data arebase d onabou t 10 seedlings/family. A regression ofeg gmasse s onseedlin gheigh t showed r= 0.69 (p< 0.05 ; 47 d.f.). Analysis ofvarianc e ofeg gmasses/ 1 mm of seedlingheigh tshowe d no significant sources ofvariatio n (eggmasses/seedlin g =0.015 3 seedling height -0.84).

Survival of defoliated trees There appeared tob e adefinit e relationship between stands andprob -

463 Table 3. Survival ofDouglas-fi r seedlingsaf ­ terbein gdefoliate d bybudwor m inthei r second growing season,i nrelatio n toheigh tbefor e regrowth inthei rthir dyear .

Stand Number Number Proportion Height dead living dead (mm)

1 5 142 0.03 108 2 5 142 0.03 134 3 10 137 0.07 88 4 13 134 0.09 86 5 18 129 0.12 69 6 11 136 0.07 121 7 5 142 0.03 88

d.f. =6 ;X 2 = 126.29;P o f largerX 2 =0.000 5

ability ofdeat h after defoliation,bu t again,muc h ofth e observedvaria ­ tionca nb e associated with seedlingheigh t (Table 3). Inan yevent ,onl y 7 % ofth ecompletel y defoliated seedlings died.

CONCLUSIONS AND DISCUSSION

Fiveobviou s conclusionsare : - seedlingheigh tmus tb eremove d from anymeasur e ofovipositio n behavior and is also animportan t component ofth edebarkin gdata ; -percen t defoliation ofindividua l seedlingsmigh tb e analyzable without removal of seedlingheight ; - larvae dispersed,mine d needles andbuds , and consumed foliage ina normalmanner ; - adults emerged,mated , and oviposited toprovid eworkabl e oviposition data; an d - the testprocedur e isworkabl e and should provide good data afterprope r insect levels anddistributio n areworke dout . Theresult s fromthi s firstprogen y testprovide d little specific information aboutth e genetics ofth ebudworm/Douglas-fi r interaction,bu t theydi d showtha t suchtestin g canb econducte d and reliable feeding and ovipositionpreferenc e data collected.Th emetho d shouldb e adaptable to progeny testing ofhibernacula r site selection,pheromon echemistry ,preda ­ tor andparasit e effectiveness,hos t andpes t phenologic-synchronization, andbu d andneedl eminin gbehavior .

464 REFERENCES

Hanover,J.W. ,1976 .Physiolog yo ftre eresistanc e toinsects .Annua lRevie wEntomolog y 20:75-95 . Kogan,M. ,1975 .Plan tresistanc e inpes tmanagement .In :R.L .Metcal f& W.H .Luckma n (Eds):Introductio nt oinsec tpes tmanagement ,Joh nWile yan dSons ,Ne wYork . p.103-146 . McDonald,G.I. ,1979 .Resistanc eo fDouglas-fi r towester nspruc ebudworm :result so fa preliminaryprogen ytest .USD AFores tServic eResearc hNot eINT-264 .Intermountai n Forestan dRang eExperimen tStation ,Ogden ,Utah .7 p . McDonald,G.I. ,1981 .Differentia ldefoliatio no fneighborin gDouglas-fi rtree sb y westernspruc ebudworm .USD AFores tServic eResearc hNot eINT-306 .Intermountai n Forestan dRang eExperimen tStation ,Ogden ,Utah .3 3p . Snedecor,G.W. ,1956 .Statistica lmethods .Iow aStat eUniversit yPress ,American .53 4p .

465 Testing larch clones for Adelges laricis resistance

I. Blada

StatiuneaExperimenta lForestiera ,Padure aVerde ,Timisoara ,Romani a

ABSTRACT

Onehundre d twenty-onephenotypicall y resistant treeswer e selected in2 0population s of Larix decidua and in1 o f L. leptolepis, extensively attackedb y Adelges laricis. Clones of thecandidate s were infested inth enurser y and thenplante d in 3 different locations inorde r totes tthei r resistance to . laricis under natural conditions of infestation.Afte r 9year s oftestin gw e drewth e conclusion that among theclone s of L. decidua, therewer ehighl y significant differences,an d that theresistanc e to A. laricis is acontinuousl y varyingcharac ­ ter controlled bypolygenes .Broad-sens e heritabilitycoeffi ­ cients for the3 location swer e as follows:0.41 , 0.68, 0.77 and theexpecte d genetic gains 7.9 %, 14.1% , 15.1% ,respec ­ tively.Th e clones of L. leptolepis weremor e resistant (p< 0.05 ) than thoseo f L. decidua. Finally, 34clone swer e selected forus e inth e improvementprogram .

INTRODUCTION

Larix decidua Mill., anativ e species inth eAlp s and theCarpathians , isimportan t for itshig hwoo dproduction , itswid e ecological amplitude and itshig h resistance to injurious factors.Nevertheless ,som einsects , Adelges laricis Vail, included, cause adecreas e invigo r ofattacke d trees and incrementlosses . The study ofresistanc e to insects aswel l asth eus e incultur e of genetically improved material as awa y ofreducin g losseswer e recommended about3 0year s ago (Painter, 1951). Two factors underline the importance of genetic resistance to injuriousparasites :th e inefficiency ofth eclassi ­ cal controlmethod s (S0egaard, 1964)an d thenecessit y for avoiding envi­ ronmental pollutionwit hpesticide s (Gerhold, 1970). Thevariabilit y inresistanc e oflarc ht o A. laricis hasbee n mentioned byvariou s authors.Thus , Larsen (1953)foun d numerous liceo n L. decidua,

466 andonl y afe wo n L. leptolepis (S.& Z. ) Gord. Ina tes to n2 5Europea n larch clones,Eidman n (1966)prove d thatther ewa s oneclon eo f relatively low susceptibility,whil e theother s hadvariabl eresistance .Considerin g trees exposed tonatura l infection, Sindelar &Hochmu t (1967)foun d highly significant differences inresistanc e among differentprovenance s ofEuro ­ pean larch.The y also confirmed thatresistanc e to A. laricis ofJapanes e larchwa shighe r thantha to fEuropea n larch. Investigations inRomani a on attacked populations proved thevariabilit y ofindividua l phenotypicresis ­ tance toth e sameparasit e (Blada, 1977). Inorde rt o avoid losses,i n196 9 aprogra m ofimprovemen to fgeneti c resistance to A. laricis was initiated inRomani amakin gus e of intra-an d interspecific hybridizationmethods .Th e final objective ofthi sprogra m was tocreat e seed orchards oftree shavin g general combining ability for resistance to A. laricis. Thispape r dealswit h theresult s ofa clona l testtha twa s carried out inorde r to find new sources ofresistance .

MATERIALS AND METHODS

Onehundre d twenty-one apparently resistant treeswer e selected outo f 20population s of L. decidua and 1populatio n of L. leptolepis, extensively attackedb y A. laricis. Selectionwa spossibl ebecaus e ofth evariabilit y inphenotypi c resistance inth eheterogeneou spopulations . Grafts onth e candidates ofth e2 specieso flarc hwer emad e onroot - stocks inpolyethylen e bags. Infestationwa s induced inth enurser y inth e period 1970-1972b yplacin g theclone sbetwee nhighl y attacked rowso f larch seedlings;unde r thesecircumstance s theparasit ewa s transmitted in anatura lway . Inorde r toexpos e them tocondition s favoring natural infestation,th e clones thenwer eplante d out (inspacin g 3m x 3 m )i nth e following locations:Sinaia ,Toplit a andHuedin .Graft s ofal l 121clone s (111 L. decidua and 10 I. leptolepis) wereplante d in eachlocatio n dis­ posed in3 block s (replications).Eac h replication contained aramet ,s o thatth eentir e experimental system (replications x locations)include d9 individuals ofeac hclone .Fro m 1972 to197 9 resistancewa s estimated on thebasi s ofpercentag e of attacked needles.Sample swer e collected at random from eachrame twhe nth e attackwa s atit speak ; thishappene d in early July,whe n the insectwa s inth e firstlarva l generation (estivale). Three replications with thehighes t attackwer e included inth e statistical analysis foreac h location andclon e (notconsiderin g the year). This procedurewa s considered tob e effective ineliminatin gvariatio n inth e amount ofinfestatio n inth eexperimenta l system inorde r toestimat e the true resistance or susceptibility ofeac hclone .Percentage s weretrans ­ formed into arcsin 4 %value s and analyzedb ymean s ofth e analysis of variance.Th e analysiswa sperforme d onth e speciesbot h separately and

467 together, inorde r toobtai n agenera l ranking according toresistance .Th e significance ofth e differences was established by themultipl e range test and finally thebroad-sens e heritability andth e expectedgeneti c gain (AG )wer e computed according toth e following formulas (Nanson,1967 ; 1970): h2 =s 2 -s 2/s2 andA G =ih 2 a ,wher e s2 =th evarianc e ofth e clones, s2 =th eerro rvariance ,i = th e selection differential, anda _= ri r phenotypic standard deviation.

RESULTS

Inth e 3location s considered, therewer e significant differences (p< 0.01 ) among the clones of L. decidua inthei r resistance to A. laricis (Table 1;3) . Among the clones therewere ,too , significant differences (p< 0.01 ) in their reactions toth e ecological conditions inth e 3location s (Table2 ; 4th column); thisemphasize s the strong interaction ofclon ex environment , onth e onehand , andparasit ex environment ,o nth e other.Nevertheless , onemigh tconclud e thatresistanc e ofth eclone s isunde r geneticcontrol , even ifthei r reactions toth eenvironmen twer edifferen t (Table 2;5t h column). Resistance ofEuropea n larcht o A. laricis, according toth e percentage ofattacke d needles (continuously variablebetwee n 16 and8 5% )appeare dt o be aquantitativ e character (Fig.1) .Th e frequency distribution ofth e clones according toth epercentag e of attacked needleswa sver y closet o thenorma l curve (Fig.1) . The amplitude ofth evariatio n inresistanc e ofth e clones of L. lepto- lepis was low (3t o 27.5 %), andth edifference s wereno t significant (Table2) . Themea nresistanc e ofth eclone s ofJapanes e larch (19% infection) wasmuc hhighe r (p< 0.05 ) thantha to fEuropea n larch (44.3% infection)

Table 1. Analysis ofvarianc e ofresistanc e ofEuropea n larchclone s for eacho f3 experimenta l locations.

Source ofvariatio n Degrees Variance (s2) of freedom Sinaia Toplita Huedin

Replications 2 Clones 110 486.4** 442.7** 461.8** Error 220 285.7 141.9 105.6

**= p < 0.01

468 Table 2. Analysis ofvarianc e ofresistanc e ofEuropea n and Japanese larch clones foral l 3experimenta l locations combined.

Source ofvariatio n Degrees Variance F-testi nrespec tt o

of (S2) freedom s£= erro r interaction variance variance

European larch replications 6 locations 2 clones 110 730.6 3.27** 2.21** clones x locations 220 330.3 1.48** error 660 223.2

Japanese larch replications 6 locations 2 clones 9 280.0 1.88 1.13 clones x locations 18 247.9 1.66 error 54 149.2

** = p < 0.01

Resistance Susceptibility 70

|601 O O50- Average a JO­ Selected s\ clones 'S 30 Ol Ol Averag ? •§20' Pm.

) i*iiooroaiaioi/i'-pNMjiiDin or-oiioc •^ o jo ir> LT> oi a *^™ h^!?Î!i<:

L.leptolepic L.decidua Clone number.

Figure 1. Ranking ofth e 121 Larix sp.clone s according toth emea nper ­ centage of attackedneedles .

469 Table 3. Analysis ofvarianc e ofresistanc e forbot hEuropea n and Japanese larchclone s andth e combination of alllocations .

Source ofvariatio n Degrees Variance F-testi nrespec tt o of (s2) freedom error s£ interaction variance variance replications 6 locations 2 clones 120 937.1 5.31** 2.87** clones x locations 240 326.0 1.85** error 720 176.3

**= p < 0.01

Table 4. Analysis ofvarianc e ofresistanc e incompariso n ofclone s of L. decidua and L. leptolepis.

Source ofvariatio n Degrees Variance F-testi nrespec t to of (s2) 2 2 freedom sE =erro r s =I interactio n variance variance replications 6 locations 2 species 1 1607.4 112.4** 53.2* speciesx locations 2 30.2 2.1 error 6 14.3

*= p <0.05 ; **= p < 0.01

Table 5. Expected genetic gain (AG )an dbroa d senseheritabilit y (h2)fo r L. decidua clones.

Location h2 AG

Sinaia 0.41 7.9 Toplita 0.68 15.1 Huedin 0.77 14.1

Themea n 0.62 12.4

470 (Table 4). L. decidua was characterized by an intraspecific variability much higher (see clones 11 to 121) than that of L. leptolepis (see clones 1 to 10), and it had some clones as resistant as the latter's (Fig. 1). The broad-sense heritability was 0.41 at Sinaia, 0.68 at Toplita and 0.77 at Huedin (Table 5). The expected genetic gain was 7.9 % at Sinaia, 14.1 % at Huedin and 15.1 % at Toplita (Table 5), with only the clones having up to 30 % at­ tacked needles being considered. Finally, 24 clones of L. decidua and 10 clones of L. leptolepis (Fig. 1) were selected as resistant and useful for the improvement program.

DISCUSSION

Because variability was continuous the hypothesis was proposed that resistance of European larch to A. laricis was polygenic. The varying values of heritability and, implicitly, of the genetic gain from location to location might be attributed to the environmental condi­ tions in the 3 locations that influenced the evolution of the parasite. The highest attack was at Toplita, whereas the lowest was at Sinaia. Because L. leptolepis had a ratio of 100 % resistant clones and L. decidua had a ratio of only 21.6 % resistant clones, this was proof that the latter did not lack resistance genes, but only that the frequency of thèse was lower than that in L. leptolepis. The existence of individual variability in both species of larch makes intraspecific selection possible; if hybridization follows it may lead to an accumulation of resistance genes and consequently to an extra genetic gain. In conclusion, resistance to A. laricis is present and variable both at the intra- and the interspecific level which presents an opportunity for using both sources of resistance in the improvement program.

REFERENCES

Blada, I., 1977. Ameliorarea pe cale genetica a rezistentei duglasului si laricelui la principalele boli si däunätori. Studii si cercetäri, I, XXXIV. p. 19-31. Eidmann, H.H., 1966. Zur unterschiedlichen Resistenz von Lärchen gegen Läusebefall. Anzeiger für Schädlingskunde 39: 8-10. Gerhold, H.D., 1970. Dix ans de progrès dans la production d'arbres forestiers résistants aux maladies. Unasylva 24(97-98): 37-44. Larsen, S., 1953. Studies of diseases in clones of forest trees. Hereditas 39: 152-179. Nanson, A., 1967. Tables de la différentielle de sélection dans la distribution normale (0,1). Biométrie-Praximétrie VIII: 40-51. Nanson, A., 1970. L'héritabilité et le gain d'origine génétigue dans quelques types d'expériences. Silvae Genetica 19: 112-120. Painter, R., 1951. Insect resistance in crop plants. Mac Millan, New York. 520 p. Sindelar, J. & R. Hochmut, 1967. Variability of larch (Larix sp.) invasion caused by woolly aphids Adelges laricis and Sacchiphantes viridis. XIV lUFRO-Kongress, München, III: 902-906. S0egaard, B., 1964. Amélioration génétique de la résistance aux attaques d'insectes chez les arbres forestiers. Unasylva 18 No. 73-74: 82-88.

471 Variation in susceptibility of native and introduced coniferous trees to some insects of eastern Canada

W.H. Fogal, E.K. Morgenstern, P. Viidik & C.W. Yeatman

Petawawa National Forestry Institute, Canadian Forestry Service, Chalk River, Ontario KOJ 1J0, Canada

ABSTRACT

Insect-tree interactions arediscusse d from theviewpoin t ofcoevolution .Th ehost-pes t interactions considered are: Pinus strobus, P. banksiana, and Picea abies with Pissodes strobi; introduced species of Larix with Pristiphora erich- sonii; Pinus strobus, P. banksiana, and P. resinosa with Neo- diprion and Diprion spp.Damage ,behavioral , and ecological characteristics foreac hinsec t areoutline d briefly anda review ismad e ofvariatio n insusceptibilit y ofhos ttrees .

INTRODUCTION

Susceptibility of foresttree s to insectdamag e is acomple x phenomenon intimately related to theevolutio n ofbot h thehos t andparasite .Tree s have evolved certain strategic defenses comprising acomple x ofbiochemi ­ cal,physiological , andmorphologica l properties thatenabl e some to resist and survive insect attack:ovipositio n or feedingma yb eprevented ; the insectma yb ekille d or its growth impaired; orth etre ema yb eable't o tolerateheav y attacks.Presumabl y the level of success achieved by the insectdepend s onth edegre e towhic h itha s adapted tothes e defensive strategies (Dethier,1976 ;Painter , 1951;Ster n& Roche , 1974).W e dono t yet fully understand the interactionbetwee n insects andhos t treeso rth e genetics ofdefens emechanism s underlying insect-hostpreference s and selection.However , knowledge ofnatura lvariatio n insusceptibilit y within species is aprerequisit e forexploitin g tree defense mechanisms in forest pestmanagement .

NATIVE PINES

White pine : White-pine weevil Easternwhit epine , Pinus strobus L., isth emos tvaluabl e lumber species ofeaster n Canada andoccur s inth eDeciduous ,Grea tLake s-

472 St. Lawrence,Acadian , andpart s ofth eBorea l Forestregion s (Rowe, 1972). Wheni ti splante d or regenerated onope n land, iti sheavil y and repeated­ ly attacked by thewhite-pin e weevil, Pissodes strobi Peck, an indigenous pesttha tcause s extreme deformation anddevelopmen t ofmultipl e stems.Th e result ofthi s damage is areductio n inmerchantabl e volume and lumber value (Brace, 1971). Selection ofweevil-resistan t genotypes hasbee non eo fth egoal s in breeding ofwhit epin e (Fowler &Heimburger , 1969;Heimburger , 1966). However,ther e aren o conclusive data fromprovenanc e studies thatdemon ­ strate differences insusceptibilit y amongpopulations .A study ofheavil y weeviled, 13-year-old trees ina provenanc e testo fwhit epin e including 27 sources from theMaritim e Provinces ofCanad athroug hOntari o andQuebe ct o Minnesota,USA , and southt oGeorgi a revealed no differences indamag e (Garrett, 1972). Inanothe r test,o f 12sources ,difference s inweevi l damage atag e1 3wer e detectable,bu t all sourceswer eheavil y weeviled (Garrett, 1973). Inye tanothe r test,o f2 1see d sources,ther ewa sn o evidence ofgeneti c resistance toweevilin g independent oftre esiz e (Pauley et al., 1955). Incag e experiments,difference s amongprovenance s were found on3-year-ol d trees,bu tthe ywer e dependent onweevi l density (Soles& Gerhold , 1968). The lasttw o experiments suggest thatdetectabili - tyo fvariatio n inweevi l susceptibility amongprovenance s depends uponth e size ofth eweevi lpopulatio n andma yb e confounded byvariatio n intre e size. An alternative method fordevelopin g weevil resistance is interspecific hybridization. Forexample ,hybrid s of P. strobus with thewester nwhit e pine, P. monticola Dougl., growwel l atsom e sites andma yb evaluabl e in areashavin g heavyweevi lpopulation s because ofresistanc e inherited from P. monticola (Wright,1970 ;Wrigh t& Gabriel , 1959).

Jack pine : White-pine weevil Jackpine , Pinvs banksiana Lamb.,i s apionee r species,i s intolerant ofshade ,an d commonly regenerates after fire to form even-aged stands of highcommercia l value. Significant seed sourcevariation s ingrowth ,woo d quality, form, and susceptibility to insects and diseases areknown ,an d genetic improvement ingrowt h and formb y selection andbreedin g within suitable seed sources isconsidere d highly feasible (Yeatman& Teich , 1969). The species issusceptibl e towhite-pin e weevil (Baker, 1972), and geneticvariatio n insusceptibilit y hasbee nreporte d atth eprovenanc e level intes tplantation s inth eLak e States (Arend et al., 1961;Batzer , 1961; King, 1971). Themor e susceptible provenances came from areas south andwes to fLak e Superior inWisconsi n andMinnesota , andth e least sus­ ceptible originated innorther nMinnesota .Ther e isn o clear explanation forth edifference s insusceptibilit y amongprovenances , and there appears tob ewid evariatio n among themor e susceptible southern seedsources .

473 Weevil injuryha s alsobee nrecorde d ata numbe r ofprovenanc e testloca ­ tions inCanada ,bu t innon eha sth e incidencebee n.hig henoug h toasso ­ ciate susceptibility with seed origin (Yeatman,pers . obs..„ data on file). Incontras tt owhit e pine,jac kpin e apparently has evolved aconsiderabl e tolerance towhite-pin e weevil and isno t alikel yhos t forth ebuild-u p of weevilpopulation s ofepidemi c level in forests oftemperat e andborea l NorthAmerica .

Pines : Pine sawflies Geneticvariatio n ingrowth ,woo d quality, form, and susceptibility to insects has alreadybee nnote d forwhit e and jackpine . Improvementi s possible.Re dpine , P. resinosa Ait.,ha s limited natural variation,s o genetic improvement isconsidere d more difficult (Fowler &Heimburger , 1969). Some diprionid sawflies are importantpest s incommercia l standso f thesepines :th ered-heade d pine sawfly, Neodiprion lecontei Fitch, andth e Europeanpin e sawfly, N. sertifer Geoff.,attac kyoun gplantations ;th e Swaine jack-pine sawfly, N. swainei Midd., is athrea tt o stands ofjac k pine,particularl y inQuebec ; and the introduced pine sawfly, Diprion similis Htg., attacks stands ofwhit epine ,bu t iti s also found on jack pine andre dpin e of all sizes (Davidson& Prentice , 1967). Pine sawflies are justa fe wo fman y species ofth e familyDiprionidae ,whic h arecharac ­ terizedb y adaptive associationwit h anarro w range ofhost s (Knerer & Atwood, 1973). Some species areapparentl y confined toa singl ehost ;fo r example, the Swaine jack-pine sawfly. Significant geographic variation insusceptibilit y ofjac kpin e toth e red-headed pine sawflyha sbee ndemonstrate d inprovenanc e studies (Arend et al., 1961). Henson etal . (1970)studie d susceptibility ofsevera l species andhybrid s ofhar dpine s toEuropea npin e sawfly and selected genotypes ofcomparativel y lowvulnerabilit yb y subjecting trees toa battery oftest stha tmeasure d oviposition, initiation of feeding,growth , and eggproduction .The y suggested thatrat e of adult strike,initia l establishment, and survival oflarva e couldb euse d ascriteri a to select stock ina breedin gprogram .

INTRODUCED CONIFERS

Norway spruce : White-pine weevil Norway spruce isgrow n ineaster n Canada asa nornamenta l tree andha s beenuse d inreforestatio n ina fe wlocalities . Iti sno twinter-hard y in boreal Canada and ishighl y susceptible to attackb y thewhite-pin e weevil intemperat e climates,bu tcarefull y selectedprovenance s planted ongoo d siteswil l outperform native spruces ingrowt h andyield .Behaviou r ofth e weevil onNorwa y spruce isessentiall y the same asi ti so neaster nwhit e

474 pine (Vandersar et al., 1977). Norway spruceprovenanc e and selectionwor k atPetawaw aNationa lFores ­ try Institute datesbac k to1924 ,whe nth e firstplantatio n ofth e species was established on anabandone d farm field. Itsoo nbecam e evidenttha t winterhardines s and theabilit y towithstan d weevil attackswer e the principal factors limiting its suitability forus e inth eOttaw aValle y and other areas ineaster n Canada.Observation s onweevilin g weremad e from 1963 to 1972 in aloca lplantatio n of2 2provenance s from the International Norway SpruceTria l of 1938 (Viidik,pers . obs., data on file). Fourprove ­ nances from northernEurop e confirmedHoist' s (1955)observatio n that narrow-crowned phenotypes are lesspron e thanother s toweevi l attack and/orhav e ahighe r recovery rate.Th ebes tprovenanc e was aloca lpopula ­ tion derived from the firstplantatio n established in192 4an d latersub ­ jected to intensive selection forwinte r hardiness andweevi l toleranceb y Dr. CarlHeimburger .Thi s suggests thatselectio n forweevi l resistance in this introduced species iseffective .

Larches : Larch sawfly Tamarack, Larix laricina (DuRoi )K .Koch ,th enativ e larch ofeaster n andborea lNort hAmerica ,wa s animportan t species atth etur no fth e century for lumber,railroa d ties, andmin e timbers.However , larch sawfly, Pristiphora erichsonii Htg.,whic h isthough tt ohav ebee n introduced from Europe,becam e epidemic towards theen d ofth e 19thcentur y and caused widespread losses as infestations advancedwestwar d (Davidson& Prentice , 1967; Drooz, 1960;MacGillivray , 1969). Exotic larches andthei r hybrids arebein g considered for reforestation because ofthei rpotentia l forrapi d early growth.However , fearo fsawfl y hasprevente d theirwidesprea d use. InEurop e the larch sawfly generally hasno tbee n sodestructiv e asi tha s inNort hAmeric a (Drooz,1960 ;Hunt , 1932); theexceptio n isBritain ,wher ebot h larch andth e sawflywer e introduced (Benson, 1950). Itma yb epossibl e to select species and develop hybrids ofEuropea n andAsiati c larches thatwil lb e relatively resistant to larch sawfly under Canadianconditions . Experiments were established atPetawaw a in 1960 and 1961t o compare the silvicultural potential ofexoti c larch species and to select superior phenotypes for furtherbreeding . In197 7 asever e outbreak oflarc h sawfly inthes eplantation s provided anopportunit y torat e the seed sources for defoliation (Fogal, pers. obs.,dat a on file). Therewer e significant differences indefoliation ,height ,an dvolum e among seed sources.Ther e was also atendenc y for fast-growing,high-yieldin g sources tosuffe r heaviestdefoliation .However ,th eweaknes s ofth e correlation between defoliation andheigh twa s illustrated by analpin e seed source growni n Denmark for several generations beforeplantin g atPetawawa ; itwa s among sources suffering lightestdefoliatio n eventhoug h itwa s oneo fth efas -

475 testgrowing .A seed source fromPolan d had arelativel y highyiel d and appeared less susceptible to sawfly than did other sources. Incontrast ,a hybrid, L. evrolepis (Sieb.& Zucc. )Gord. ,wa shig h inyiel dbu t appeared tob ever y susceptible tosawfly .Therefore ,combination s of lowsuscepti ­ bility andhig hyiel d mayb e rarebu tca nb e found.

Genys &Harma n (1976)foun d thatF ? hybrids ofJapanese , L. leptolepis Gord., andEuropean , L. decidua Mill., larchgre wmos trapidl y butwer e highly susceptible to sawfly.Amon gpur e species,Japanes e larchwa smos t susceptible;Dahurian , L. gmelini (Rupr.)Litvin. , and Siberian, L. sibiri- ca Ledeb.,larche swer e intermediate;wherea s western larch, L. occidentalis Nutt.,wa s leastsusceptible . Susceptibility washighl yvariabl e among geographic strains ofEuropea nlarch .

CONCLUSIONS

Available information suggests thatther ear evariation s insusceptibi ­ lity among closely related tree species,geographi c strains of aparticula r species, and interspecific hybrids,bu tmor e documentation isrequire d if such information ist ob eeffectivel y used in forestmanagemen tpractice . Useo f trees that areresistan t orwidel y adapted for survival inth e presence ofpotentiall y damaging insects is ahighl y desirablepest-manage ­ mentstrategy .Thi s isespeciall y truewher e other alternatives,suc ha s directchemica l control,biologica l control,o rstan dmanagement , failo r where riskst oth e environment and/or costs outweighbenefit s interm so f wood and fibre saved. Thepossibilit y foremployin g knowledge ofvariatio n in susceptibility inpes tmanagemen t strategies will depend onth ehost-pes t relationship. In the case of anindigenou spes t adapted to arang eo f indigenous tree spe­ cies,possibilitie s mayb e limited andothe r alternatives more appropriate. Forexample ,white-pin e weevil damage toeaster nwhit epin e canb econ ­ trolledb yplantin g inshad e (Berry& Stiell, 1976).Wher e ahost-pes t relationship ishighl y specific'i tma yb epossibl e to select trees outside therang e ofhos t acceptability.Wher e anindigenou s treeha sbee n shownt o beparticularl y susceptible toinjur y from anexoti c insect,exoti c trees andhybrid s that are less susceptible mayb e used toreplac e thenativ e tree. To take advantage ofdesirabl e growth andyiel d characteristics of exotic trees,selectio n forresistanc e tonativ epest s isdesirabl e and possible insom ecases .

REFERENCES

Arend,J.L. ,N.F .Smith ,S.H .Spur r& J.W .Wright ,1961 .Jac kpin egeographi cvariatio n - fiveyea rresult sfro mLowe rMichiga ntests .Michiga nAcadem yo fSciences ,Arts , andLetter s46 :219-238 . Baker,W.L. ,1972 .Easter nfores tinsects .Unite dState sDepartmen to fAgricultur eFores t

476 ServiceMiscellaneou sPublicatio n 1175.64 2p . Batzer,H.O. ,1961 .Jac kpin efro mLak eState ssee dsource sdiffe ri nsusceptibilit yt o attackb ywhite-pin eweevil .Unite dState sDepartmen to fAgricultur eFores tService , LakeState sFores tExperimen tStatio nTechnica lNot e595 .2 p . Benson,R.B. ,1950 .A nintroductio n toth enatura lhistor yo fBritis hsawflies .Transac ­ tionso fth eSociet yfo rBritis hEntomolog y10 :45-142 . Berry,A.B .& W.M .Stiell ,1976 .Contro lo fwhit epin eweevi ldamag ethroug hmanipulatio n ofstan dclimate :Preliminar y results.Canadia nForestr yServic e InformationRepor t PS-X-61.8 p . Brace,L.G. ,1971 .Effect so fwhite-pin eweevi ldamag eo ntre eheight ,volume ,lumbe r recovery,an dlumbe rvalu ei neaster nwhit epine .Canadia nForestr yServic ePublica ­ tion1303 .3 3p . Davidson,A.G .& R.M .Prentic e (Eds),1967 .Importan t forestinsect san ddisease so f mutualconcer nt oCanada ,th eUnite dState san dMexico .Departmen to fForestr yan d RuralDevelopment ,Ottawa ,Canada .24 8p . Dethier,V.G. ,1976 .Man' splague ?Insect san dagriculture .Th eDarwi nPres sInc. , Princeton,Ne wYersey .23 7p . Drooz,A.T. ,1960 .Th e larchsawfly .It sbiolog yan dcontrol .Unite dState sDepartmen t ofAgricultur eFores tServic eTechnica lBulleti n1212 .5 2p . Fowler,D.P .& C.C .Heimburger ,1969 .Geneti c improvemento fre dpin ean deaster nwhit e pine.Th eForestr yChronicl e45 :414-420 . Garrett,P.W. ,1972 .Resistanc e ofeaster nwhit epin e (Pinusstrobu sL. )provenance st o thewhite-pin eweevi l (Pissodesstrob iPeck) .Silva eGenetic a21 :119-121 . Garrett,P.W. , 1973.Geographi cvariatio ni nresistanc et owhite-pin eweevi l (Pissodes strobi)(Coleoptera :Curculionidae )b yeaster nwhit epin e (Pinusstrobus) .Canadia n Entomologist 105:347-350 . Genys,J.B .& D.M .Harman ,1976 .Variatio ni nlarc hsawfl yattac ko fdifferen tspecie s andgeographi c strainso flarch ,exhibitin gdivers egrowt hrates .Journa lo fEconomi c Entomology 69:573-578 . Heimburger,C , 1966.Geneti c improvement fordiseas ean dinsec tresistanc e offores t treesi nCanada .In :H.G .Gerhold ,E.J .Schreiner ,R.E .McDermot t& J.A .Winiesk i (Eds):Breedin gpest-resistan t trees.Proceeding so fa nadvance dstud yinstitut eo n genetic improvementfo rdiseas ean dinsec tresistanc eo ffores ttrees ,Universit y Park,Pennsylvania ,1964 .Pergamo nPress ,Oxford ,p .45-51 . Henson,W.R. ,L.C .0'Nei l& F .Mergen ,1970 .Natura lvariatio ni nsusceptibilit yo fPinu s toNeodiprio n sawfliesa sa basi sfo rth edevelopmen to fa breedin gschem efo rresis ­ tanttrees .Yal eUniversit ySchoo lo fForestr yBulleti n78 .7 1p . Holst,M.J. ,1955 .Breedin gfo rweevi lresistanc ei nNorwa yspruce .Zeitschrif tfü r Forstgenetikun dForstpflanzenzüchtun g4 :33-37 . Hunt,S.S. ,1932 .Europea nlarc hi nth enortheaster nUnite dStates .Harvar dFores tBulle ­ tin16 .4 5p . King,J.P. , 1971.Pes tsusceptibilit yvariatio ni nLak eState sjac kpin esee dsources . UnitedState sDepartmen to fAgricultur e ForestServic eResearc hPape rNC-53 .1 0p . Knerer,G .& C.E .Atwood ,1973 .Diprioni dsawflies :Polymorphis m andspeciation .Scienc e 179:1090-1099 . MacGillivray,H.G. ,1969 .Larche sfo rreforestatio nan dtre eimprovemen t ineaster n Canada.Th eForestr yChronicl e45 :440-444 . Painter,R.H. ,1951 .Insec tresistanc e incro pplants .Macmilla nCo. ,Ne wYork .52 0p . Pauley,S.S. ,S.H .Spur r& F.W .Whitmore ,1955 .See dsourc etrial so feaster nwhit epine . ForestScienc e1 :244-256 . Rowe,J.S. ,1972 .Fores tregion so fCanada .Canadia nForestr yServic ePublicatio n 1300.17 2p . Soles,R.L .& H.D .Gerhold ,1968 .Cage dwhit epin eseedling sattacke db ywhite-pin e weevil,Pissode s strobi,a tfiv epopulatio ndensities .Annal so fth eEntomologica l Societyo fAmeric a61 :1468-1473 . Stern,K .& L .Roche ,1974 .Genetic so ffores tecosystems .Springer-Verlag ,N.Y .33 0p . Vandersar,T.J.D. ,J.H .Borde n& J.A .McLean ,1977 .Hos tpreferenc eo fPissode sstrob i Peck. (Coleoptera:Curculionidae )reare dfro mthre enativ ehosts .Journa lo fChemica l Ecology3 :377-389 . Wright,J.W. ,1970 .Genetic so feaster nwhit epin e (Pinusstrobu s L.). UnitedState sDe ­ partmento fAgricultur eFores tServic eResearc hPape rWO-9 .1 6p . Wright,J.W .& W.J .Gabriel ,1959 .Possibilitie so fbreedin gweevil-resistan twhit epin e strains.Unite dState sDepartmen to fAgricultur e ForestService ,Northeas tFores t ExperimentStatio nPape r 115.3 5p . Yeatman,C.W .& A.H .Teich ,1969 .Genetic san dbreedin go fjac kpin ean dlodgepol epine s inCanada .Th eForestr yChronicl e45 :428-433 .

477 International bacterial canker testing programme on poplar

M. Ridé

Stationd ePathologi eVégétal e etd ePhytobactériologie ,INRA ,rout ed eSain tClement , 49000Angers ,Franc e

ABSTRACT

Onbehal f ofth e international group ofcooperators ,th e authorpresent s results of inoculationwit h Xanthomonas populi onPopulu s clones.Th e same series ofclone swa s distributed to eacho f4 participatin g stations inBelgium , France,Grea t Britain andth eNetherlands . Ineac h station aloca l straino f thebacteriu m was used. Inoculations weremad e following the samemethod s and atcomparabl e inoculation dates in September 1978, andMay ,June ,Jul y 1979,bu t adjusted to local stage of development ofclones . Inoculation inSeptembe r 1978prove d more stringent than inoculation inMa y andJun e 1979.Thes e spring andearl y summer inoculations allowbette rpredictio n of resistance under field conditions. Strains candiffe r greatly inaggressiveness .

INTRODUCTION

Thebacteria l canker ofpopla r due to Xanthomonas populi Ridéremain sa risk forpopla rplantin g inEurope .Variou s programmes fortestin g of resistance aredevelopin g mainly inBelgium , France andth eNetherlands , but also inGrea tBritain , andWes tan dEas tGermany . Inoculationb ydrop ­ ping abacteria l suspension into fresh leaf scarsproduce d by removing the leaves inSeptembe r waspropose d byRid é and Steenackers and adoptedb yth e FAO International working group onpopla r diseases (1966)t otes tvariou s clones and selected trees inEuropea n countries (Ridé, 1963;Steenackers , 1966; Zycha et al., 1967;Burdekin , 1972;Gremme n& Koster , 1972). This method, though useful for a first screening againstbacteria l canker,i s very severe and doesno toffe r anyindication s ofth edegre e of field resistance that some clonesdemonstrate . Moreover acuriou s lacko fparallelis m was observed between resistance testresult s obtained by the samemetho d inth e recentpas t inBelgium , France,Grea tBritai n and theNetherlands .Thi s ledu s to take into account

478 the levelo faggressivenes s ofbacteria l strains used inth edifferen t countries andth eprevailin g ecologicalconditions . Therefore aninternationa l cooperative programmewa spropose db yKoste r inWageninge n4 year sag oi norde rt oscree npreselected , promising poplar clonesi nth e4 countrie s andt ocompar e andstandardiz e inoculationme ­ thods. Resultsobtaine db yRid éabou tth erespons eo fth etre et o X. populi interm so finoculu m dose,physiologica l stageso fth epopla r andaggres ­ siveness ofth ebacteria l strainconstitute d abas e fortes tplannin g (Figures1 an d 2). Theai mwa st ostud y differences insusceptibilit yi n poplarplant s simultaneously growni neac ho fth e4 countrie s from cuttings originating fromth esam ese to fstool si nth eNetherlands .

MATERIALS AND METHODS

Test design Thetes twa sse tu pwit h2 4clone s (seeTabl e3 ,plu sN L2066 , 'Rochester';N L1310 , deltoïdes x nigra, andN L2752 , 'hybrida275 ' Poland).A tHee s3 Belgia n clones ('Hunnegem',Raspalje 'an d 'Boelare') were added. These cloneswer e tested simultaneously in4 countries :a tHee s(th e Netherlands), Grimmingen (Belgium), LaRétuzier eAnger s (France)an dGrang e Estate (England). Spacingwa s3 m x 3 m ,th enumbe ro fplant spe rclon ewa s23 .Thi s number corresponds to4 date so finoculatio no n5 plant s eachtime ;a tth e last3 date s1 plan twa sadde d andno tinoculate d inorde rt oserv ea s control.

Times of inoculation Inoculations weremad e inSeptembe r 1978an dMay ,Jun e andJul y1979 . Inorde r toaccoun t forclimatologica l differencesbetwee nth elocation si n 4 countries,th einoculatio n dateswer erelate d toflushin g stages.E.g. , theMa yinoculatio nwa st ob emad ewhe n 5-6 leaveso fa clon eha dflushed . Forpractica l reasonsth eclone swer e divided into3 groups :th efirs t group consisted ofclone s flushing (with5- 6 leaves)a tth esam etim ea s 'Oxford' (348); thesecon d group atth esam e time as 'Robusta' (2098);th e third group atth esam etim ea sclon e1266 . The inoculations inJun e andJul ywer emad ei nprincipl e 30an d6 0day s afterth eMa yinoculation .

Reasons for choosing these times of inoculation The choiceo fth emetho d ofan dtim e forinoculatin g resulted fromth e observations made inFranc e duringth epas t1 0years .A nexampl e isgive n inTabl e 1.Fro m theresult s inth ecolum n Sept.7 4i ti sno tpossibl et o obtaina nimpressio no fdifference s insusceptibilit y under fieldcondi -

479 DxT "S.6-2'

26.3 7.4 17 22 2730 7.5 16 20 28 10.6- 19 A A ABCDEF5

Robusta

4 1n Inoculation '" date 26.3 7.4 17 22 27 30 7.5 16 20 28 10.6 19 A A BCD E EF 6 8 9 12

Nigra'V.524' Cankers%

4 Inoculation date 26.3 7.4 17 22 27 30 7.5 16 20 10.6 19 A A A A A-B D 3 8 8

480 * pih ;'^*%-

S 620-96 S 5-3 S 239-12

Figure 2. Expression ofbacteria l canker onvariou spopla r clones in1980 , subjected to inoculation in1965 . (PoplarResearc h Center, Geraardsbergen (Grammont)B) .Clones :S 620-96 : P. deltoïdes (Michigan); S5-3 : P. del­ toïdes V5 (Iowa)x p. trichocarpa V21 (Montana); S239-12 : P. deltoïdes (Missouri)x P. grandidentata. Correlationbetwee n results of juvenile test and adultexpressio n isno w established.

Figure 1. Response of differentpopla r clones to X. populi interm s of inoculum dose andphenologica l stages. (Spring and summer inoculations.) P. deltoides x trichocarpa S.6-2; P. x euramericana cv. 'Robusta'; P. nigra V. 524. (afterM . Ridé& S.Ridé , 1978.) A =dorman tbu d B =swellin gbu d C = firstlea fti p D =hal f appearing leaves E =petiol e appearingbu t leaves still rolledu p F =leave s completely spread out 5-12 =numbe r ofpreforme d leaves

481 Table 1. Susceptibility ofpopla r clones tobacteria lcan ­ keraccordin g toinoculatio nperiod .Angers ,score d February 1978.Explanatio n of scores:1 = n oreaction ; 6 =attac k spreading to largerpart s ofth e stem; stem oftendies . 052-2t oGR M 51: P. deltoïdes clones obtained fromNationa l Center forFores tResearch ,Orlean s(F) . 70-039/110 to 3: P. trichocarpa hastata (Idaho)x p. trichocarpa (Oregon)obtaine d fromPopla rResearc hCenter , Geraardsbergen(B) .

Clone Inocu lation Sept. May June July 74 75 75 75 052-2 5.6 3.1 - 4.2 3.8 061-3 5.3 4.1 3.3 3 234-1 5.8 4 3.1 2.3 053-2 6 3 3.2 2.3 257-1 6 3 3.1 1.8 044-3 5.5 2.7 2.7 3 257-2 5.8 2.3 3 1.3 244-2 5.3 1.8 1.8 1.3 044-2 5.2 1.6 1.8 1.1 241-1 5.2 1.5 1 1.1 063-1 5.7 1.2 . 253.3 6 1 1.3 1.3 Alpaga 6 2.8 2.8 2.1 Albiruch 6 2.6 3 2.7 Bergerac 5.5 1.8 2 2.3 WGN/19 5 1.5 1.2 2.1 S.335-12x 5.3 3.8 4 4.1 S.332-1/11 V8x vi ] B/5 6 3 3.3 3.2 B/6 6 3.3 2.8 2.7 B/14 6 3.1 2.2 2.5 GRM5 1 3.2 1.1 1 1.2 70-039/110 5.7 2.7 3.6 4 /108 5.7 2.5 2.3 3.7 / 40 4.7 ,2.7 2.3 2.1 / 97 5 2 2 2.3 / 64 6 1.8 1.1 1.7 / 98 5.2 1.3 1.5 1.8 / 99 4.2 1.1 1 1 / 85 2.8 1.5 2 1.6 / 39 2.7 1.7 2.2 1.7 / 7 3 1.3 1.5 1 / 31 3 1 1.1 1 / 14 2.1 1.5 2 1.5 / 20 1.6 1.8 1.2 1 / 90 1 1.5 1.3 1.2 / 3 1.1 1 1.1 1

482 tions.Autum n inoculation hasver y severe consequences andshow s all clones (exceptGRM51 )t ob e susceptible.O nth eothe rhand , ifw e compare the results ofth e inoculations inMa y andJun ew e are ablet oestablis ha ranking ofclone s according tothei r degree ofsusceptibilit y under field conditions.

Inoculation methods On theshoo to fth epreviou s year theFrenc h incisionmetho d wasused : awedge-shape d cutwit h aknif e into thecambiu m (asfa r asth e sapwood); on thecurren tyear' s shoot,fres h leaf scarswer e inoculated. A drop of bacterial suspensionwa splace d onth e fresh leaf scar oro nth e fresh wound. For furtherdetail s seeTabl e2 .

Inoculum Local strains ofth ebacteriu m wereuse d ineac hcountry . Strainswer e notmixed ; their aggressiveness isbein g compared inAnger s on1 4referenc e clones. Strainswer e incubated at2 3 °Cdurin g two days.Bacteria l suspension o concentrationwa s 10 cells/ml. Isolates usedwer ein : - Belgium:M l (isolated from P. euramericana cv.Regenerata ,Apri l 1978). -France :S P (isolated from P. euramericana cv.Blan c dePoitou ,Apri l 1978). - GreatBritain :Ra p (isolated from 'Rap',Apri l 1976). - TheNetherlands : 115 (isolated fromP . 'Serotina',Apri l 1978).

Scores The score system used is identical toth e FAOworkin g group system (scale 0t o 5) (1966)bu trenumbere d byGremme n& Koste r (1972)t o 1t o6 forcompute r analysis.

Table 2. Inoculation scheme forth e international test.

Date Number Place of inoculations

Sept. 1978 2 at3 0% and 60% ofth elengt h ofth eplan t May 1979 2 at3 0% and 60% of the length ofth e shooto f '78 1 onth ene w (very short)shoo to f '79 June 1979 2 at3 0% and 60% ofth e length ofth e shooto f '78 1 at3 0% o fth e length ofth e shooto f '79 July 1979 2 at3 0% and 60% ofth elengt ho fth e shooto f '78 2 at3 0% and 60% ofth e length ofth e shooto f '79

483 RESULTS AND DISCUSSION

The trialwil lprobabl y havet ob e continued for4 to 5year s depending onth e development ofth ediseas e ineac hcountry .Nevertheless ,th e first resultswer e compared in1979 .Thes e only took into accountth e scores of the September inoculations inBelgium , France and theNetherlands .Rankin g was foundt ob e similar through allo fthes e firstobservations ,probabl y asa resul to fth ever y comparable level ofstrai n aggressiveness.Thi swa s contrary topreviou s experiments inwhic h the 'Rap'strai nprove dver y highly aggressive while strain8 8N Lwa s only slightly aggressive (strain 88N Lwa sno tuse d inth etrial s atHees) . In1980 ,result s (Table 3)showe d agoo d correlation forDutc h and Frenchexperiments ,bu tno t forth eBelgia nexperiment s inthes emonths . After discussion inth e field inGrimminge n (B),i twa s thoughttha ttw o factorsprobabl y canexplai n this difference:th e lownutrien t levelo f trees inth eBelgia n experimentalplo t (Steenackers,persona l communica­ tion)and , above all,th e facttha t inmos to fth e spring and summerin ­ oculations thebacteria l suspensionha dbee n dropped onth e outer-bark layero fth etree s (cortexparenchyma )instea d ofint oth ecambiu m as mentioned inth e experimental procedure.Oute rbar k contains inhibiting substanceswhic h could explain inoculation failure (Ridé& Ridé ,1978 ; Ridé, 1980) (Table3) . Inth e future,i twil lb e interesting tocompar e theresult s obtained by thismetho d tothos eobserve d after spraying clones insprin gwit h bacterial suspensions (Gremmen& d eKam , 1980). Inth eexperimenta l plot atHees , thenorma l disease expressionwa s sometimes disturbed by attacks ofcambiu mborer s (Dendromyza carbonaria Hendl.). The larvae of this insectca ndistribut e X. populi and other bacteria along the trunk,twig s andbranche sbu t they can alsob y their presence induce anappearanc e ofth e stem orbranc h similar totha t caused bybacteria lcanker .

ACKNOWLEDGEMENTS

Thispape r summarizes afiel d demonstrationpresente d byDr .an dMrs . Ridéi nth enurser y ofth eDorschkam p institute atHee s on 19 IX198 0o n behalf ofth e international working group.Othe rmember s ofthi s informal working group areR .Koste r andM . deKa m (theNetherlands) ,M . Jilbert (United Kingdom), V. Steenackers (Belgium). Iwan tt oexpres sm y gratitude toMrs .Rid é (F)primaril y forth e design anddrawin g ofposters ,t oMr .Va n Lokhorst (NL),Mr .Larsi n and Remy (B)fo rthei r kind and constant assistance inthi s cooperative pro­ gramme.

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J JJ J J J J ij J J JJJJ ij J J J J J J s a s a a a 55 a a a aa aa a a aa a aa co 485 REFERENCES

Burdekin,D.A. , 1972.Bacteria lcanke ro fpoplar .Annal sApplie dBiolog y72 :295-299 . Gremmen,J . &R .Koster ,1972 .Researc ho npopla rcanke r (Aplanobacterpopuli )i nth e Netherlands.Europea nJourna lo fFores tPatholog y2 :116-124 . Gremmen,J .& M .d eKam ,1980 .Researc ho npopla rcanke r (Xanthomonaspopul isubsp . populi)i nth eNetherlands .Europea nJourna lo fFores tPatholog y10 :48-53 . Ridé,M. ,1963 .Ou rpresen tknowledg eo fbacteria lcanke rcause db yAplanobacteriu m populi.FAO/CIP/MA L23 ,Casal eMonferrat o (translatedb yBatko) . Ridé,M . & S. Ridé,1978 .Factor saffectin ginoculatio nsucces si nwood yplants .Proc . 4thInt .Conf .Plan tPath .Bact .Angers ,Vol .2 .p .957-968 . Ridé,M. ,1980 .Interaction shôte-pathogèn e dansl arésistanc e despeuplier sau xinfec ­ tionsbactériennes .Proc .FAO/IUFR OWorkin ggrou po nPopla rdisease s- Poznan . Steenackers,V. ,1966 .L asélectio ne tl acréatio nde speuplier s résistantsau xdiverse s maladiese tplu sparticulièremen tà Aplanobacte rpopul iRidé .FAO/CI PWorkin ggrou p onPopla rdiseases .Versailles . Zycha,H. ,M .Rid é& H .Fröhlich ,1967 .Prüfun gde rKrebsresisten zverschiedene rPappel n inDeutschlan d (unpublisheddata) .

486 A revolutionary view of resistance breeding and research1

HenryD .Gerhol d

Schoolo fFores tResources ,Pennsylvani aStat eUniversity ,Universit yPark ,Pennsylvani a 16802,US A

ABSTRACT

Evolving relationshipsbetwee nresearc h andproductio n of disease-resistant trees arereviewed , using ananalog y with symbiosis.Researc hdevelopment s arecite dwhic h arelikel yt o expand the demand forresistan t trees,increas e theprecisio n ofmanipulatin g genetic resistance, and improve the/designo f breeding systems.Resistanc e breeding has received increased attention incooperativ e tree improvementprograms ,whic hhav e themselvesproliferate d andhav e also improved technology transfer. Constraints caused byeconomi cpressures , changing pestcontro l methods, andenvironmenta l concerns aresee na s increasing thenee d formor e resistantvarieties .Th ecoevolu - tiono fresistanc ebreedin g and research inth e futurehold s many opportunities,an d suggestions areoffere d formakin g the mosto fthem .

INTRODUCTION

The intento fthi s concluding paper inth e 'Workshopo nGenetic s of Host-Parasite Interactions inForestry ' ist oprovid e someperspectiv e on trends inresistanc ebreedin g andresearch .Tw orelate dmeetings , similarly comprehensive insubjec tmatte r and international inscope ,precede d this one. 'Biology ofRus tResistanc e inFores tTrees 'wa s discussed in196 9 at Moscow, Idaho (Bingham et al., 1972). In196 4a world-wid e summary of 'Genetic Improvement forDiseas e and InsectResistanc e ofFores t Trees'wa s attempted forth e firsttim e (Gerhold et al., 1966). Othermor e narrowly focusedmeeting s havebee nhel d too,man y ofthe m organizedb yWorkin g Groups ofth e International Uniono fFores tResearc hOrganizations .

1.Base dpartl yo nresearc hsupporte db yTh ePennsylvani aAgricultura lExperimen tSta ­ tion,USD ARegiona lResearc hProjec tNE-27 ,an dth eU SFores tService .Journa lSerie s PaperNo .6143 .

487 My instructions from theOrganizin g Committeewer e torevie wdevelop ­ ments since the 1964meetin g andt obrin g insom e constructive suggestions andvision s intoth e future.Tha t isquit e achallenge !The-impressiv e array ofpaper spresente d atthi sWorksho p gaveu s justa glimps e ofsom e ofth eman y accomplishments during thepas t 16years . In196 9 asurve y turned up over 50resistanc ebreedin gprogram s in1 9countrie s (Gerhold, 1969), andundoubtedl y thewor kha s expanded since then.Rathe r thanre ­ viewing technical aspects ofhost-parasit e interactions, Iwis h to comment onanothe r type ofinteraction ,namel y the interdependency that Ise e between research andproductio n aspects ofimprovemen t inpes tresistance . Progress inproducin g more resistanttree sha sbee ndependent ,o fcourse , onresearc h indiscipline s such asgenetics ,pathology ,entomology , and physiology. Thereha s alsobee n feedback from tree improvementwhic hha s enhanced research inthes e fields.B y.considerin gevolvin g relationships between research andproductio n ofresistan t trees,I hop ew e canperceiv e more clearlywha tha scontribute d toprogress ,especiall y in applications ofresearch . Prior to 1964th ewor k onpest ,resistanc e oftree swa smainl y inth e realm ofresearch .Lik e organisms invading abarre n landscape,investiga ­ torswer e exploring geneticvariatio n inresistanc e andho w itcoul db e utilized to improve adaptation tohostil eenvironments . Todayw e knowtha tresistanc ebreedin g of foresttree sca nb esuccess ­ ful andeconomical ,unde r appropriate circumstances andwit h theprope r techniques.Researc h results havebee n incorporated into tree improvement programs toproduc e realized genetic gains inresistance , i.e. gains that earlier had onlybee npredicted . Research andbreedin g programshav eadap ­ ted tone w knowledge and external forces,jus ta scolonizin g populations pursue adaptive strategies toexploi tNature' svacuum s and accommodate its pressures.Furthermore ,thes e complementary activities have adapted toeac h other.

A COEVOLÜTIONARY ANALOGY

Itma yb eusefu l to think ofth e interrelated development ofresearc h andproductio n asanalogou s toth econcep t ofcoevolution , inwhic h evol­ ving species exertadaptiv epressure s oneac hother .Th ebasi s ofth e analogy isth e similaritybetwee n information systems thatoperat e within organisms and thosewithi n organizations thatconduc t forestry researcho r production activities (Table 1).Informatio n stored ingene s isequate d with ideas,i.e . related facts orpiece s ofknowledge .Bot h kinds ofinfor ­ mation giveris e to aserie s ofevent s orspecifi c activities.Chromosomes , cells, organs,an dorganism s areincreasingl y higher levels ofbiologica l complexity that are analogous toconcepts ,plans ,projects ,an dprogram s of research orproductio n organizations.Meiosi sma yb ethough to fa s are -

488 Table 1. Analogy ofinformatio nsystem s inorganism s and organizations.

Organisms Organizations

genes ideas chromosomes linked ideas,concept s meiosis rearranging ideas mitosis propagating ideas cells plans organs projects individuals programs fitness utility ofidea s genetic gain technological progress

arrangement of ideas,e.g. , duringmeeting s such asthi sone ; anfi mitosis represents thepropagatio n of ideas such asi ntrainin gworkers .Throug h populationgenetic s the analogyma yb eextended , andon ema ythin k about the fitness of ideas orprogram s asbein g similar toth e fitness ofgene s orgenotypes .Th e implied value of ideas accordingly would depend onthei r survival and reproduction, andperhap s also their impacto n relatedorga ­ nizations.Technologica l progress,lik e genetic gain,coul db e defined asa functiono fintensit y ofselectio nan dth edegre e towhic hth ebes t ideas areutilized . Thatbring s ust o thecoevolutionar y parto fth e analogy. Reciprocal evolutionary changes inspecie s thatexer t adaptivepressure s oneac hothe r may involveprédation , competition, orsymbiosis .M y analogy draws upon varioustype s ofsymbiosis ,whic h areclassifie d asparasitism , commensa- lism,o rmutualism . Parasitism works toth edetrimen t ofth ehost , asyo u well know.Commensalis m benefits onepartner , and doesno thar m theother . Mutualism enhances the ability ofbot h species to survive andreproduce . Parasitism couldb e likened to atre eproductio nprogram , thehost , that sustains aforestr y researchprogram , thepes t (Table 2). Now,a researchermigh tresen tbein g called apest .Bu t anindustria l executive would bemor e aptt o agreewit hth e analogy, especially ifh e ispreoccu ­ piedwit h the company ledgers.Afte r all,researc h cancaus e quite adrai n onth ebudget ,an d this type of investmentwil lpleas emanagemen t and stockholders only ifresult s findpractica l applications thatimprov ene t earnings.Application s depend upontechnolog y transfer, represented byth e aegricorpus, ater m coinedb y Loegering (1966).Thi spseudo-organis m ac­ tually isth e interactiono fhos t andparasite ,leadin g to infection ofa susceptible host.Rejectio no fresearc h applications,reflecte d byresis -

489 Table 2. Analogy ofparasitis m with forestresearc h andproduction .

Parasitism Research& productio n host treeproductio n program pest forestry researchprogra m aegricorpus technology transfer resistance rejectiono fresearc h results susceptibility acceptanceo fresearc h results

Table 3. Analogy ofcommensalis mwit h forestry research andproduction .

Commensalism Research &productio n root production fungus research mycorrhizae research applications

tance inth ehost ,i sperhap s anatura l tendency ofproductio norganiza ­ tions toresis t innovation and change.Susceptibilit y thenwoul d take ona favorable connotation, as itimplie s anacceptanc e ofresearc hresults . Treebreeder smigh tregar d this asbackwards ,bu tperhap s notpathologist s orentomologists .Fo r theyhav ebee nhear d toexclai m more thanonce , 'There's abeautifu l epidemic!' Commensalism is apreferabl e formo f symbiosisbetwee n research and production (Table 3). Amycorrhiza l relationship seems apropos,fo ri t benefits the fungus in all situations,bu tbenefit s to thehos t aremos t apparento nharsh ,nutrient-deficien t sites.Similarly , research goeso n continually, but seems tob e appreciated mostdurin g animpendin g calamity orwhe nther e ispressur e toboos tproduction .Commensalis m mayb e themos t appropriate analogy formos tresearc h -productio n relationships,especial ­ lyi nthei r initial stages ofdevelopment . An example ofmutualis m servesm ypurpose s better,howeve r (Table4) . Itca n illustrate an idealrelationshi p between atre e improvementprogram , represented by a figtree ,an d the supportive researchproject s represented bywasps .The ybuz z about,disseminatin g research results (pollen),whil e helping toensur e the future of figtree s andthei r own kind.Th ewaspis h researchers arerewarde dwit hth e sweetnecta r of fame and fortune,thoug h iti susuall y doled out insmal l doses.I nthi s intricate relationship

490 Table 4. Analogy ofmutualis mwit hbiologica l research andtre e improvement.

Mutualism Tree improvement& research figtre e treebreedin g program wasps research (genetics,pathology , etc.) pollen research results bats,bird s liaisonwit h industries seeds knowledge and genotypes

(oversimplified here), ifth ewasp s failed totransfe r theresearc hre ­ sults, thetre e improvementprogra mwoul d notb ewort h a fig.Furthermore , let'sno toverloo k thebat s andbird swhic hprovid e liaisonwit h industrial tree growers.Afte r gorging themselves with the fruito f improvement pro­ grams, they disseminate information and selected genotypes.A sthe y deposit these seeds,the y alsohel p them togro wwit h ahealth y doseo fdung . This analogy couldb eelaborate d ingreate r detail,bu t thatwoul d detract fromm y assignment torevie wpas t developments and topee r intoth e future.Fo r convenience, Ishal lgrou p developments under research, tree improvement, andth eexterna l environment thatinfluence s these activities andthei r interactions.

DEVELOPMENTS IN RESEARCH

Most individual impacts of thegrea tmajorit y ofresearc h findings have notbee n dramatic.Bu t aspaper s inthi sWorksho p have indicated, the steady growth inknowledg e aboutpes tresistanc e of forest treesha sbee n impressive. Itha s expanded beyond themor e important diseases and insects to lesser ones, and also toviruses ,nematodes ,an dmycoplasmas .A cumula ­ tive effecto fthi s research and its applications hasbee n to firmlyesta ­ blish resistancebreedin g as anaccepte dmean s ofcontrollin g certain forest diseases causedb y fungi.Th e acceptance ofthi s controlmetho dwil l certainlyb e extended-to additional diseases asresearc hprogresses ,an d perhaps alsot o some insects andothe rpathogens .Bu t itwon' t happen automatically, inmos tcases .Whe na piec e ofresearc h iscompleted , fur­ thereffort s willb erequire d toenginee r the applications (Shigo, 1977). Thisnee d for involvement ofresearcher s withpractitioner s is soobviou s ithardl y seemswort hmentioning ,ye t administrators oftenneglec tt obrin g the twopartie s together. There are3 type s ofresearc hwhos e effects appear tohav e greatpoten ­ tial for influencing resistancebreedin g in forestry.Thei r effects are

491 likely to (1)expan d thenee d formor e resistanttrees , (2)increas e the precisiono fmanipulatin g geneticresistance , and (3)improv e the strategy and designo fbreedin g systemsb ymodellin g theoutcome só' falternativ e improvementstrategies . An expansion of thedeman d formor e resistant treesha sbee nunderwa y for some time,an d is likely toaccelerate .Researc h onvegetativ epropaga ­ tion,provenanc e experiments, andexoti c species trialsha scontribute d to thistrend ,mainl yb ypioneerin g silvicultural alternatives whichma yb e implemented whenwarrante d by industrial conditions.Clona lplantation s wereconsidere d anovelt y until recently, except forpoplars .No w there are extensive areasplante d toclone s ofMontere ypine ,eucalyptus ,Norwa y spruce, and other species.Ove rhal fo fth e landscape trees grown inth e United States arevegetativel y propagated, and allo fthes especie s are represented by justa fewclone s ofeach .Researc hwit h exotic species and provenances isanothe rpar to fa genera l intensification of silvicultural practices. Increasedproductivit y isth e goal,bu tne w disease and insect problemswil l surelyb e encountered, so at leasti nsom e cases genetic improvement inresistanc e willb e an integralpar to fth e intensified managementpractices . Several research developments some dayma y greatly increase thepreci ­ sion inmanipulatin g genetic resistance,compare d toconventiona l methods (Abelson,1980 ;Gwynne ,1980 ;Kleinhof s &Behki , 1977). New tissue culture techniques and isoenzyme analyses canb euse d toobtai nmor e definitive information aboutgen eactio nan d interaction,an d thust oelucidat e the genetic control ofresistanc emechanisms .Genotype sma yb e altered through cell fusiono rtransformation .Recombinan tDN A technologyma yenabl eth e splicing of individual resistance genes into chromosomes.Suc h techniques formor eprecis e handling ofgene sca nb eespeciall y significant inshort - cutting treebreeding .Man y yearsma yb erequire d toadap t suchtechnolo ­ gies to foresttrees ,bu t theresearc h isunde rway . The applicability ofth e gene-for-genemode l to forestdisease s isa findingtha t Ibeliev ewil l hâvemor e far-reaching consequences foru stha n anyrelate d research inrecen ttimes . Iti s apowerfu l concepttha tca nb e used notonl y for identifying specific resistance genes,bu tmor eimpor ­ tantly forpredictin g theoutcome so fdifferen tstrategie s inbreedin g and deploying improvedvarieties .T o attainthi s capability anextensio n ofth e modelwil lb erequire d to include fitness ofresistanc e andvirulenc e genes and associated genomes invariou s environments,an ddiseas e levels induced byvariou sgen ecombination s over aperio d ofgenerations .Non-specifi c types ofresistanc e would alsohav et ob e accommodated and insom e cases interactionswit hvectors .A synthesis ofsuc hmodellin g studieswit h other analyses couldb euse d todetermin e the optimum designo f aresistanc e breeding system. Severalpaper sher ehav emad evaluabl e contributions to such anapproach .

492 Ifanalogou s gene-for-genemodel s andmor epowerfu l selectionmethod s couldb edevelope d for insectresistance ,i tmigh tprovid e agrea t stimulus toprogres s inthi s field.Resistanc e researchwit h insects seemst ob e generallymor e difficulttha nwit hdiseases ,presumabl y duet o theinsects ' greatermobility , responses todivers e stimuli,an d complex relationships withpredators .Tha tma yhel p toexplai nwh y indirectselectio n forbioche ­ mical traits hasbee nconsidere d inman y cases,thoug h its effectiveness remains elusive aftermuc h research. Insectresistanc e isno tno w aprinci ­ pal goal inbreedin gprograms ,bu ti tma yb e inth efuture .

DEVELOPMENTS IN TREE IMPROVEMENT

Thegrea tproliferatio n ofcooperativ e tree improvementprogram s in NorthAmeric a (Kang,1980 )an d cooperative research inEurop e onel m and poplar diseases arephenomen a worthnothing .No tonl yhav ene wprogram s started,bu tolder ,establishe d programs have increased theirwor ko n resistancebreeding . There isa greate r awareness ofth evalu e ofpes t resistanceb y treebreeder s and an acceptance oftre e improvementb yfores ­ ters. Furthermore, theorganizationa l nature ofthes e cooperative programs helps toassur eeffectiv etechnolog y transfer,b yprovidin g closeworkin g relationshipsbetwee n researchers andpractitioners . Resistance testing centersma yb eviewe d as arelate d organizational development, facilitating interactionsbetwee npathologist s andtre ebree ­ ders.Thei r intellectual interplay andmeldin g ofexpertis ema yb e justa s important asth e specialized facilities for imposinghigher ,mor e uniform selectionpressures .Artificia l inoculation iscommonl y employed formas s screening and resistance studies,bu tther ei ssom econcer n aboutho w closely results arecorrelate d with resistance innature . Another significant development isth e survival andeve nexpansio n of programs thathav ebee nchallenge d bymor evirulen t strains of fungi.I n eachcas eth e initial impactmus thav ebee n shocking fortre ebreeder s and sponsors alike.Th e facttha tthes eprogram s survivedma y implyno tonl y thatth egeneti c approach todiseas e control isa vali d one,bu ttha t confidence ini tha sbee n strengthened.

DEVELOPMENTS IN THE ENVIRONMENT

Anessa y onevolutio nwoul db e incompletewithou t comments onth erol e ofth e environment, inthi scas emeanin g external influences onth ecoevo - lutiono fresistanc ebreedin g andresearch .Th epas t 16year s hasbee na timeo f fluctuations inth eeconomi c climate,upheava l indiseas ean d insectcontro lmethods ,an ddrif ti npubli c concerns.Thes e events are suggestive ofmajo r geologic changeswhic hhav e altered the course of biological evolution.

493 Energy shortages andeconomi c pressures havebee n feltb yresearchers , breeders, andpracticin g foresters alike.W e feel constrained invariou s ways, sotha t iti smor e difficult toge tou rwor k done.A mor e serious consequencema yb e the trend toward concentration and intensification of tree growing.Thi s is likely tolea d togreate r risks ofdiseas e and insect damage, and thus increase thenee d formor e resistantvarieties . A revolutionha sbee nunderway , ofcourse ,i nmethod s for controlling insect anddiseas e damage.Hazard s ofchemical s are stillbein g discovered, andpesticid e regulations arebecomin g evermor e stringent.Exaggerate d reactions ofenvironmentalists ,th enew smedia ,th epublic ,an d government agenciesma yb e annoying, and restrictive measuresma yno t always seem acceptable,bu tgenerall y theyprevai l and dobecom e accepted.Despit e some misgivings,w e canregar d this course ofactio n aspruden tbecaus esafe ­ guards againstchemica l hazards areneeded , and they do stimulate thedeve ­ lopmento fbiologica l controls andresistan tvarieties .However ,thi s favorable influencewil lb e counteredb y another seto f fears,probabl y heldb yman yo fth e samepeopl ewh o objectt opesticides . Apprehensions andmisapprehension s concerning genetic engineering, monoculture, endangered genotypes,an d the like*surelywil l hamper forestry practices, research, andparticularl y opportunities toutiliz e resistant varieties.Outcrie s have alreadybecom emor e strident againstrea lo r imagined hazards ofgeneti c involvements with human intelligence,geneti c diseases, abortion, sterilization, andvirulen tmicrobes .Thi s isprobabl y justth ebeginning , forther e arecertai n tob emor e scientificbreak ­ throughs inhuma ngenetic s anddramati cproposal s for altering our social structures.Critica l attitudes surelywil l spill over into agriculture and forestry,whic hhav e alreadybee ncriticize d forhar d tomatoes,monocul ­ ture, andclearcuttin g of forests (Popovich, 1980).

THE FUTURE OF RESISTANCE BREEDING AND RESEARCH

Whatdoe s the futurehol d forth ecoevolutio n ofresistanc e breeding andresearch ? Ia moptimisti c thatth eoperationa l environmentwil l be full ofopportunities , andwil lb e favorable ifw e don'tneglec t our educational responsibilities.Th emai ndeterminant s ofth e futureprobabl ywil lb e those evolutionary 'ideas'generate d by research and improvementprogram s thatprov e tohav ehig h 'fitness'.I nothe rwords ,futur e directions will be setmainl yb yne w knowledge ortechnique s that findwidesprea d applica­ tion inforestr ypractices ,o rwhos e significance isrecognize d and uti­ lizedb yman y researchers.Not e thati nthi sview , intrinsicvalu eha s little influence on 'fitness ofideas 'unti l theyhav ebee nwidel ydissemi ­ nated and accepted. Ifi ti stru etha tth epractica l value of innovations in forestryproductio n and researchwil l have astron g effecto nrat e and direction ofcoevolution , then Ica n suggest severalimplications :

494 - We can favorably influence our professional destiny by cooperating with our coevolutionary colleagues. - We should build stable organizations that provide effective interactions among those engaged in research, tree improvement, and forestry production. - Resistance breeding goals should be identified early, especially in new improvement programs. Appropriate weights should be given to all important selection criteria, including measures of yield, quality, and pest resis­ tance . - For any threatening disease or insect a research lead should be main­ tained to explore genetic variation in resistance and virulence. This will make it possible to develop effective strategies for selection, breeding, and geographic deployment of improved varieties. - Improved plants or techniques should be moved into production aggressive­ ly, despite some risks, to demonstrate their practical value. - We should assume responsibility for assuring that genetic gains in resis­ tance will be demonstrated and that technology transfer will occur to a sufficient degree. In applying these suggestions, the differing needs of new programs versus firmly established programs should be recognized. A principal con­ cern in new programs is to demonstrate the effectiveness of research or improvement methods. As a program matures, there may be a shift toward overcoming complacency about routine accomplishments, and justifying more complex procedures so that progress may continue. This workshop audience is well aware of the complex, long-term scientific work required to obtain durable types of resistance, and integrating these with environmental adaptation, improved yield, and higher quality. But are we sufficiently aware and active in educating colleagues, foresters, policy makers, and the public? If this symbiotic requirement is overlooked, we may be relegated to the evolutionary fate of the dinosaurs.

REFERENCES

Abelson, J., 1980. A revolution in biology. Science 209: 1319-1321. Bingham, R.T., R.J. Hoff & G.I. McDonald, 1972. Biology of rust resistance in forest trees: Proceedings of a NATO-IUFRO Advanced Study Institute. USDA Forest Service Misc. Publ. No. 1221. 681 p. Gerhold, H.D., 1969. A decade of progress in breeding disease-resistant forest trees. Unasylva 24: 37-44. Gerhold, H.D., E.J. Schreiner, R.E. McDermott & J.A. Winieski, 1966. Breeding pest- resistant trees. Proc. NATO & NSF Advanced Study Institute. Pergamon Press. 505 p. Gwynne, P., 1980. The cloning of Russett Burbank. Mosaic 11: 33-38. Kang, H., 1980. Research needs in tree breeding. North American Quantitative Forest Genetics Group Workshop Proceedings, USFS Inst. Forest Genetics, Rhinelander, Wise. Kleinhofs, A. & R. Behki, 1977. Prospects for genome modification by non-conventional methods. Ann. Rev. Genetics 11: 79-101. Loegering, W.Q., 1966. The relationship between host and pathogen in stem rust of wheat. Proc. 2nd Int. Wheat Genetics Symp., Hereditas Supp. 2: 167-177. Popovich, L., 1980. Monoculture: a bugaboo revisited. Journal of Forestry 78: 487-489. Shigo, A.L., 1977. Communication of knowledge and needs between forest researchers and practicing foresters. Northern Logger and Timber Processor 25: 7, 38.

495 Recommendations

Participants inth eWorksho p onGenetic s ofHost-Parasit e Interactions inForestr y -1 4t o 21September , 1980,Wageningen ,th eNetherland s- recommendthat : 1. Scientific and technical termsb euse dwit hprecisio n andconsis ­ tency. 2. International andnationa l agencies forresearc h anddevelopmen tb e encouraged toacquir e andtrai nscientifi c andtechnica l personnel to combatpest s (includes allbioti c agents thatdamag e trees)attackin g or threatening forestplantation s intropica l and sub-tropicalcountries . 3. Systems for integrated forestpes tmanagemen tb e designed such that they arebase d ongeneti c resistance andprovid e formaintenanc e ofecosys ­ tem stability. 4. Genetical,physiological , andepidemiologica l aspects ofhost - parasite interactions allb e investigated sotha tmechanism s underlying resistance canb e understood. 5. Legislative andregulator y agenciesb e alerted to thedange r that well-intentioned actions toensur e excellence ofclona lmateria l may have theundesire d side effecto frestrictin g thenumbe r andvariet y ofclone s legally available foruse ,an dtha tsai d agenciesb eencourage d tomodif y suchactions ,whereve rwarranted , soa st o fosterus eo fadequat e numbers ofclone s inmixe d ormosai cplantations . 6. Actionst oconserv egeneti c resources contained in forestecosys ­ temsb etake n soon,tha tconservatio nb eundertake n aton eo rmor e levels (alleles,co-adapte d gene complexes,populations ,o r ecosystems), and that ecosystems ofspecia l interestb e secured intacts otha t interactingpopu ­ lations cancontinu e toco-evolv e and remain available forstudy . 7. Procedures for international exchange andtestin g of agronomic host-plant germplasm be examined forapplicabilit y to forestrysituations , andb emodified , asnecessary , to increase effectiveness inmonitorin g and breeding forpes t resistance in foresttrees . 8. Those formulating theories,models ,an d strategies for forestpopu ­ lationsb e encouraged todra w ongeneti c concepts from thebroade r fields ofpopulatio n andcommunit ybiology , aswel l as fromth enarrowe rbu t

496 richer experience had inclosely-bre d agronomicsystems . 9. Population genetics ofmajo r tree species and theirpest sb einves ­ tigated soa st ocharacteriz e qualitative andquantitativ e components of environmental and geneticvariation . 10. Efforts todevelo p trees resistantt odeca y agentsb e increased, invie w ofrecent ,promisin g advances indeca yresearch . 11. Parallel non-protected experiments beconducte d todetermin e if resistance islos twher ematerial s inbreedin gprogram s aregive nartifi ­ cialprotectio n againstdamagin gagents . 12. Modelhost-parasit e systems,analogou s to Drosophila and Esche­ richia coli, be identified anddevelope d fordetaile d investigations of tree-pestinteractions , 13. Genetic consequences ofpes tcontro l strategiesb e consideredb y forestmanager s sincechemica l andbiologica l controlmeasure s acta s agents ofselectio n onpes tpopulations . 14. Forestresearc h agenciesb e encouraged toemplo y scientists equipped tostud y the genetics of arthropod-treeinteractions .

Recommendations Committee Wageningen 21 September,198 0

497 Index of hosts and parasites

Abies 95,99 ,169 ,254 ,25 5 - tabescens 257 - amabilis 97 Armillariella mellea 255 - balsamea 290 Aspergillus - grandis 97,174 ,26 9 - amstelodami 216-217 Acer - nidulans 217 - rubrum 105,10 6 Aureobasidium pullulans 56 - saccharinum 106 Avena - saccharum 108 - sativa 377-378,382 ,38 4 Adel ges - sterilis 361-369,372-37 9 - cooleyi 37 Beta vulgaris38 3 - laricis 466-471 Betula 22,25 3 - piceae 95-97 - alleghaniensis 105,10 8 - prelli 37 - papyrifera 105,10 8 Adelgidae 27,3 7 -puiescen s28-29 ,29 1 Agrobacterium 48,53-5 4 Blissus leucopterus 73 - radioAacter53-5 4 Botrytis cinerea 155,39 0 - -var . tumefaciens 49 Brassica oleracea 16 - tumefaciens 53 Brevicoryne brassicae 16,2 7 AInus25 3 Bupalus piniarius 13,2 4 - glutinosa 68 Bursaphelenchus lignicolus 19, 49, - ruAra 67,27 6 153-160, 387-395 Amaryllis 285 Caliunavulgari s27 ,25 3 AmpAorapftora Cardiaspina densitexta 23 - agathonica 73,8 1 Carga 72,7 9 - rubi 73,8 1 - illinoensis 74,78-83 ,28 5 Anasa tristis 74 - - 'Burkett* 81 Anthonosiis grandis 73 - - 'Caddo'8 1 Aphis gossypii 74 - - 'Caspiana'8 1 Aradus cinnamomeus 448 - - 'Cheyenne'8 1 Araucaria 285 - - 'EasternSchley '8 1 Armillaria 40-44 - - 'Ideal'8 1 - mellea 65,106 ,251-252 ,255-259 , - - 'Mahan'8 1 279, 333 - - 'SanSab a Improved'8 1

498 - - 'Schley'80 ,8 1 - -f.sp . virginianae 238 - - 'Sioux'8 1 - ribicola x, 12,16 ,119-129 ,173 , - - 'Stuart'80 ,8 1 236-238, 344,404-411 ,412-414 , - - 'Success'80 ,8 1 415-416 - - 'TexasProlific '80 ,8 1 - -f.sp . ribicola 237 - - 'Western Schley'8 1 - -f.sp . pedicularis 237 Castanea Cryptococcus fagi 16 - crenata 396-403 Cucurbita 74 - dentata 329,339 ,344 ,39 6 Cupressus 5 - sativa 396-403 - lusitanica 5 Castilleja miniata 237 Curculio caryae 79-82 Cephus cine tus 73 Cytospora chrysosperma 65,6 6 . Cerambycidae 14 Datana integerrima 78,8 1 Ceratocystis 171 Dendroctonus frontalis 30 - fagacearum 329 Dendrolimus spectabilis 388 - minor 171 Dendromyza carbonaria 484 - ulmi x, 15,105 ,143-152 ,171 , Diaprotica vittata 74 220, 224-235,329 ,332 ,33 9 Dioryctria zimmermani 166/ Choristoneura 8,290 ,46 0 Dipcadi erythraeum 368 - fumiferana 24,28 7 Diplodia pinea 170,171 ,17 3 - occidentalis 460-465 Diprion 472,47 4 Chrysomela scripta 63 - similis 474 Citrus 49,5 2 Dothichiza populea 66 Claviceps purpurea 383 Dothistroma 4-5,7 Coffea arabica 77 -pin i 182-183 Coleosporium 119 - septosporum 13 Compositae 169 Drechslera turcica 88 Corylus 49 DysapAis Corynebacterium halepensoïdes 49 -cfevect a7 3 Coryneum cardinale 5 - plantaginia 73 Cotoneaster 48-50 Elatobium abietinum 27,32-3 8 Crataegus 48-50 Empoasca 73-74 Cronartium 111, 127,236-24 2 Endocronartium 111 - flaccidum 123,125 ,238 ,433 , - harknessii 123,450-45 1 435-440 Endothia parasitica 12,218-219 , - fusiforme, see - quercuum f.sp. 329, 339,396-40 3 fusiforme Eriosoma lanigerum 73 - quercuum 238 Erwinia 48 - -f.sp . banksianae 238 - ajnyiovora 49-54 - -f.sp . echinatae 238 - herbicola 53 - -f.sp . fusiforme x,2 ,6-7 , - nigrifluens 49 110-118, 119-120,123 ,125-127 , - Salicis 49 164-165, 172,238-241 ,243-250 , ffrysipAe 280, 417-426,427-43 4 - graminis 91-92, 215, 373, 376

499 - - hordei 361-364 Larix 62,64,-47 2 Eucalyptus 3-5, 11,13 ,290 ,49 2 - decidua 43,131 ,466-471 ,47 6 - fasciculosa 24 - eurolepis 476 Euchlaena mexicana 88 - gmelini 476 Eucosma gloriola 165-166 - laricina 62,47 5 Exobasidium vexans 15 - leptolepis 466-471,47 6 Fagus 329,33 9 - occidentalis 461,47 6 Fomes annosus 6, 7,17 ,40-44 ,68 , - sibirica 476 106, 170-174,194-197 ,198-205 , lauraceae16 9 251-259, 260-267,268-274 ,326 , Lentinus lepideus -171 329 Lenzitus saepiaria 171 Fraxinus 48-49 Leopoldia eburnea 368 Fusarium 148 Ligustrum 255 - lateritium f.sp. pini 283 Liliaceae 169,28 5 - moniliforme 283 Loasa 435 - -var . subglutinans 283-286 Lophodermella sulcigena 441-447 - oxysporum f.sp. lycopersici 148 LopAoderflïiusi Gentiana 435 - austraie1 3 Gladiolus 285 - pinastri 13,178-185 ,326 , Gmelina arborea 77 333-338, 388,448-44 9 Gossypium 73 - seditiosum 13 Gramineae, see Poaceae Lymantria dispar 12,1 3 Grammatocarpus 435 Magicicada septendecim 65 Gremmeniella abietina x, 15,18 2 Waiacosoffla disstria 287 Heliothis virescens 73 Maius49-51 , 73,7 7 HeimintAosporiuffl Marssonina brunnea 62, 64 - maydis 371,38 2 Watsucoccus38 8 - victoriae 382 Mayetiola destructor 16,73 ,,8 1 Hemileia vastatrix 15 Medicago sativa 74-75,8 1 Heterobasidion annosum, see Fomes Melampsora ix,6 6 annosus - larici-populina x, 15,130-142 , Hevea brasiliensis 77,32 8 331 tforcfeiHn - lifli22 0 - spoiitaneuffl 361-369 - medusae 61-64, 331 - vuigare 73, 215, 220, 318, Nelilotus 74 321-324, 383 Monochamus alternatus 153-155,38 8 Hylobius radieis 166 «usa 77,14 8 Hypoxylon mammatum 65-66 Myzus persicae 34 Illinoia pisi 74,8 1 Nectria Impatiens 435 - cinnaAarina 326,33 1 Juglandaceae 78 - ditissima 329,33 9 Juglans 48,49 ,5 2 Nemesia 435 Juniperus communis 253 Neodiprion Lamiaceae 169 - lecontei 474

500 - sertifer 165-166,47 4 armandii 121-126,412-41 3 - swainei 474 balfouriana 412 Neurospora intermedia 214 banksiana 238,472-47 4 Nicotiana 53 brutia 435 Nuculaspis californica 16,2 6 caribaea 3-4,7 7 Operophtera brumata 12,16 6 cembra 236,412-413 ,41 5 Ophiostoma ulmi, see Ceratocystis ciausa 284,42 4 ulmi contorta 3,24 ,209 ,450-45 1 Oporinia autumnata 28-29,29 1 densiflora 49,153-160 ,387-39 5 Ornithogalum 361-369 echinata 43,238 ,284 ,417-418 , Orygia pseudotsugata 287 424 Oryza sativa 17,73 ,75 ,88 ,9 1 elliottii 123,169 ,17 2 Ostrinia nubilalis 73 -var . densa 283 Oulema melanopus 73 -var . elliottii 110-118,125 , Peaonia 435 164-165, 238-240,283-286 , Pales 3 418-426, 427-434 Panolis flammea 24-25 flexilis 412-413,41 5 Pedicularis 237,43 5 griffithii 412-413,41 5 Pemphigus 27 halepensis 49,43 5 Peniophora Candida 171 koraiensis 412-413,41 5 Peridermium 111,23 7 lambertiana 120-127,236-238 , -pin i 125 412-414 Petrova cristata 388 monticola 121-126,173 ,236-23 8 Phacidium infestans 446 344,404-411 ,412-414 ,47 3 PAellinus mugo 435 -pin i 171 nigra 123,182 ,435-43 7 - tremulae 187-193 - laricio 206-212,435-43 7 - weirii 67,275-28 2 - nigricans 206-212,435-43 7 Phomopsis macrospora 66 - pallasiana 206-212 Phyllosticta 64 occidentalis 284 Phylloxera 74 oocarpa 3 - devastatrix80-8 2 palustris 284,41 8 Phytophtora infestans 88-91,126 , parviflora 412-413 319, 384-385 peuce 413,41 5 Picea 107,163-164 ,182 ,254 ,47 4 pinaster 123,238 ,433 ,435-44 0 - aiu'es11 ,35 ,43-44 , 105,173-174 , pinea 123,433 ,435-44 0 185, 253,260-267 ,268-274 , ponderosa 24,26 ,123 ,253 ,436 , 472-475,49 2 461 - pungens 35 pumila 237 - sitchensis 27,32-37 , 194-197 pungens 284 Pinus 2-3, 7, 11-13, 17-18,163 , radiata 3-7,24 ,182-183 ,252 , 252-255,47 4 284,49 2 - albicaulis 412-413 resinosa 106,472-47 4 - aristata412-41 3 rigida 182,284 ,39 2

501 - serotina 284,41 8 - - 'Italica'55-5 7 - sibirica 412-413 - 'Oxford'479' ,48 5 - strobiformis 412-413 - x petrowskyana 134-140 - strobus 122-124,182 ,237 ,284 , - 'Rap'483-48 5 412-413,415 ,472-47 6 - 'Raspalje'47 9 - sylvestris 41,43 ,68 ,111 ,123 , - 'Rochester'47 9 125, 161-167,169 ,178-185 ,194 , - tacamahaca 134-136 198-205, 253,284 ,333-338 , - tremuloides 61,65 ,187-19 3 435-437,441-447 ,448-44 9 - trichocarpa 134-140,480-48 2 - taeda 2, 111, 119,123 ,126-127 , - - 'Blom'48 5 164-165,238-242 ,243-250 , - - 'FritziPauley '48 5 252-253, 283-286,387-393 , - - hastata 482 417-426,431-43 2 Poria weirii, see Phellinus weirii - thunbergii 49,153-160 ,387-39 5 • Porthetria dispar 25 - virginiana 238,283-285 ,418 ,42 4 Pristiphora erichsonii All, Alb-Alb Pissodes strobi 472-476 Prunus 48,49 ,5 2 Platanus 285 - avium 49 Platypodidae 14 - persica.52-53, 55,25 2 Poaceae 169,36 8 Pseudomonas 48,52-5 7 Polyporus brumalis 171 - fluorescens 56 Populus 3,27 ,48-57 , 60-68, - mors-prunorum 49, 52 105-106, 130-142,327 ,331 ,355 , - - f.sp. persicae 52,5 5 478-485,492 ,49 3 - savastanoï f.sp. fraxini 49 - alba 53 - syringae 49,52-53 ,55-5 6 - 'Androscoggin'48 5 - viridiflave 56 - balsamifera 64,140 ,16 7 Pseudotsuga - 'Barn'48 5 - menziesii 3,43 ,67 ,275-281 , - 'Boelare'47 9 460-465 - deltoïdes 61,134-140 ,481 ,482 , - -var . glauca 460 485 Psylla pyricola 74 - -ssp . angulata 135-140 Puccinia - 'Donk'48 5 - coronata 371-380,38 4 - x euramericana 61,66-67 , 134-136 - - avenae 361-369 - - 'Blancd ePoitou '48 3 - graminis 236,37 3 - - 'Dorskamp'48 5 - - avenae 65,361 ,363 ,37 3 - - 'Regenerata'48 3 - - tritici 218,37 3 - - 'Robusta'53 ,479-481 ,48 5 - hordei 361-369 - - 'Serotina' 135,48 3 - sorghi 385 - - 'Serotinad ePoitou '134-137 , - striiformis 373,38 5 140 Pyracantha 49-50 - grandidentata 53,18 8 Pyricularia oryzae 88 - 'Hunnegem'47 9 Pyrus 49-53,7 4 - maximowiczii 52,137-14 0 Quercus 166,255 ,339 ,417 ,419 , - nigra 52,135-137 ,140 ,480-48 1 424, 428

502 - nigra 424 Spodoptera frugiperda 73 - pubescens 208 Stereum 261 - rubra 428 - sanguinulentum 171 Rhamnus 363-364 Synchytrium endobioticum 381,38 4 - palaestina 364 Syringa 49 Rhizobium 53 Thecodiplosis japonensis 388 Rhizosphaera kalkhoffii 182 TAerioapAis Rhopalosiphum maidis 73 -maculat a 74 Rhyacionia - riehmi 74 - buoliana 206-212 Trichoderma vi ride 171 - duplana 448 Tripsacum 88 RiZ>es 236-237, 405-408, 415-416 Triticum aestivum 8,73 ,75 ,81 , - alpinum 415 91-92, 373,383 ,38 5 - hirtellum 237 Tropaeoluffl43 5 - lacustre40 6 Tsuga heterophylla 97,99 ,280-28 1 - nigrum 412,415-41 6 ylrousx , 105,143-152 ,224-235 ,49 3 - petiolare 237 - americana 105,146 ,147 ,22 8 - petraeum 415 - carpinifolia 332-333,^339 - viscossissimum 406 - glabra 232-233 i?osa25 5 - x hollandica 'Belgica'14 8 Rubus idaeus 73,75 ,8 1 - japonica 228 i?utaceae16 9 - laevis 233,332-333 ,33 9 Salix48-4 9 - procera 227-233 - dasyclada 49 - pumila 225,22 8 Saperda inornata 65 ystiiago Schizaphis graminum 73 - hordei 220,318 ,321-32 4 Schi zophy Hum commune 171,21 4 - maydis 218 Scirrhia pini 13 VerAena43 5 Scleroderris lagerbergii, see Vincetoxicua? officinale 435-440 Gremmeniella abietina ^antAofflonas48 ,5 7 Scolytidae 3,6 , 14,24 ,27 ,143-14 4 - corylina 49 Scrophulariaceae 237 - juglandis 49 Selidosema suavis 24 - populi 48,49 ,54 ,55-57 ,478-48 6 Senecio vernalis 416 - populi ssp. Salicis 49 Septoria musiva 64,6 5 Zeamay s73 ,283 ,285 ,286 ,372 , Septotinia populiperda 64 382, 385 Sirex 5 ZeirapAera diniana 287 Siricidae 14 Solanum 90 - demissum 88,90-9 1 - tuberosum 91,126 ,255 ,319 , 381-385 Sorbus 48-50 Sorghum vulgare 73,75 ,38 3

503