Partial resistance of tomatoes against Phytophthora infestans, the late blight fungus

L. J. Turkenste L.J. Turkensteen

Partial resistance oftomatoe s against Phytophthora infestans, the late blight fungus

Proefschrift terverkrijgin g vand egraa dva n doctori nd elandbouwwetenschappe n opgeza gva nd eRecto rMagnificus ,dr.ir .H.A . Leniger hoogleraari nd elevensmiddelentechnologie , inhe t openbaart everdedige n opvrijda g9 novembe r 1973de snamiddag st evie ruu r ind eaul ava nd eLandbouwhogeschoo lt eWageninge n

Centre for Agricultural Publishing and Documentation

Wageningen - 1973 Stellingen

1 Het onderscheid tussen fysiospecifieke resistentie (overgevoeligheidsresis- tentie) en andere typen van resistentie dient bij afwezigheid van kompatibele waard-pathogeenrelaties niet gemaakt te worden aan de hand van fenomenolo- gische, maar op grond van biochemische criteria.

2 Alsi naanmerkin gword tgenomen ,da td eveiligheidstermij nvoo rd etoepassin g vanhe tcarbamaa t 'Zineb'o pandijvi e6 weken ,o psla ,spinazi ee nradij s 4weke ne no ptomate nslecht s3 dage nis ,da ndien td econsumpti eva ngrot e hoeveelhedentomate nontrade nt eworden .

Gidsvoo rziekten -e nonkruidbestrijdin gi nland -e ntuinbouw ,1973 : 174-175.

3 Hetpathotyp e2,4-p/1- tva n Phytophthora infestans, dati n197 0gevonde n werdo ptomaa ti nee nproefvel dme taardappel se ntomaten ,waari nd epatho - typen1,2,3-p ,1,3,4- pe n1,4,10- pware ngexntroduceerd ,i sontstaa nui tee n heterokaryonva ntwe eva nd edri egeintroduceerd epathotypen .

Ditproefschrift .

4 Dewaargenome nvariati etusse nmonozoosporeculture sgemaak tva nee nmono - zoospore-uitgangscultureva nPhytophthora infestans (Caten,1969 )behoef t nietuitsluiten do pcytoplasmatisch efactore nt eberusten ,aangezie n P. in­ festans eendiploi dvegetatie fmyceliu mbezi t (Sansome& Brasier ,1973 )e n dientengevolgezoospore nnie tvri jzij nva ngenetisch evariabiliteit .

C.E.Caten ,1969 .Spontaneou svariabilit yo fsingl eisolate so f Phytophthora infestans. Can.J .Bot. ,48 : 897-905. E.Sansom e& CM .Brasier ,1973 .Diploid yan dchromosoma l structuralhybridit y in Phytophthora infestans. Nature,Lond .241 : 344-345. 5 De veelgehoorde mening, dat het niveau van partiele resistentie in landrassen voldoende hoog zou zijn, is ongegrond, omdat dit niveau mede bepaald wordt door de selectiedruk uitgeoefend door het pathogeen.

6 De vrees, dat de oprichting van grote internationale landbouwkundige insti- tuten, zoals CIMMYT, AVRDCe n CIP, de beoefening van de plantenziektenkunde in de ontwikkelingslanden nadelig zal beinvloeden (Buddenhagen, 1973), is ongegrond.

I.W.Buddenhagen , 1973.Th e international research institutes in relation to future development of tropical plant pathology. Second International Congress ofPlan t Pathology -Minneapolis ,Minnesot a - September 5-12, 1973. Abstract ofpapers :N o 0805.

7 Tervermijdin gva nafwijkinge nbi jaardappel -e ntomateplante ni nkasse ne n fytotrons,zoal swaargenome ntijden speriode nme twindsti lwee ri nd ewinter - halfjaren 1971/1972e n1972/1973 ,diene nkweekruimte nvoorzie nt eworde nva n ethyleenfilters.

8 Adaptatie van Phytophthora infestans aan andere gewassen dan gekweekte aard- appeld en tomaten is een mogelijkheid die niet uitgesloten kan worden.

9 In die gebieden van Europa waar 's zomers tomaten buiten worden geteeld, is de invoering van de jaarrondteelt van tomaten de voomaamste oorzaak van de toename van de frequentie en hevigheid waarmee de epidemieen van Phytophthora •infestans bij tomaten optreden.

Dit proefschrift.

10 De conclusie van Gallegly (1964), dat de partiele resistentie van de tomate- lijn W.Va 700 tegen 1-t-pathotypen van Phytophthora infestans polygeen be­ paald is, is onjuist; de bevinding dat partiele resistentie kwantitatief vererft, mag niet zonder meer tot een dergelijke conclusie leiden. Dit proefschrift. 11 Bijd eintroducti eva nnieuw etomatecultivar sdi eresisten t zijntege n Cladosporium fulvum, dientd etele rrekenin gt ehoude nme tee ntoenam e vand ekan so paantastin gva ndez ecultivar sdoo r Phytophthora infestans .

12 Inverban dme td esymmetri ei nd eterminologi eva nRobinso n (1969)word t voorgesteldd eter mpathotyp et evervange ndoo rhospideme .

R.A.Robinson ,1969 .Diseas eresistanc e terminology.Rev .appl .Mycol. , 48:593-605 .

13

Deintroducti evan ,e nhe twerke nme the tparingstyp eA 2va n Phytophthora infestans inEurop ai sonverantwoordelijk .

E. Sansome& CM . Brasier,1973 .Diploid y andchromosoma lstructura l hybridityi n Phytophthora infestans. Nature,Lond .241 :344-345 .

14

Derecent eintroducti eva nhe tparingstyp eA 2 vanPhytophthora infestans inEurop ageef taanleidin gto tbezinnin go pd ebestri jdingsmethode ntege nd e "aardappelziekte"o paardappe le ntomaat ,di ti nverban dme td et everwachte n veranderingeni nd eepidemiologi c (overblijvenva nd eschimme li nd egrond ) end epathogenitei t (versnelde recombinatieva npathogeniteitsfactoren ) vand eschimmel .

Proefschriftva nL.J .Turkenstee n Wageningen,9 novembe r197 3 Abstract

Turkensteen,L.J . (1973)Partia lresistanc eo ftomatoe sagains t Phytophthora infestans, thelat ebligh tfungus .Doctora lthesis ,Wageningen .ISB N9 022 0 04988 ,(xv )+ 8 8p. ,2 3tbs ,1 5figs , 103refs ,Eng .an dDutc hsummaries . Also:Agric .Res .Rep .(Versl .landbouwk .Onderz. )81 0an dMeded .Inst . Phytopath.Res. ,Wageninge n633 .

Inth eNetherlands ,th esourc eo finoculu mo fth elat ebligh t funguso n tomatoes isth elat ebligh t funguso npotat ocrops .I nregion so fEurop e mentioned,wher etomatoe sar egrow ni nth eopen ,P . infestans ontomatoe si s themai nsourc eo finoculum .Especiall yi nBulgari aan dHungary ,th eeffec t ofyear-roun d croppingo ftomatoe so nprevalenc ean d severityo fth ediseas e isobvious . Infiel dan dlaborator yexperiments ,th epathogenicit yt otomatoe so f P. infestans originating frompotatoe s canb eincrease db yseria lpassage s throughtomat ofoliage .I nEurope ,n oothe rhost stha npotatoe s andtomatoe s playeda rol ei nth etomat olat ebligh tepidemidogy . Phacelia tanaoetifolia isadde dt oth ehos t listo fth efungus .Fo rth epurpos eo fgenetic a classification,tw olaborator ymethods ,probi t- lo gdosag eanalysi san d componentsanalysis ,an don efiel dmetho dhav ebee ndevelope d toasses sth e resistanceo ftomatoe sagains tP . infestans. Inparticula rth ecomponent s infectionratio ,lesio nextensio nan d sporulationintensit ywer estudied .I nth efiel dmethod ,th e'apparen t infectionrate 'r wa sused .Fo rbreedin gpurposes ,a simplifie d fieldmetho d wasindicated .A compariso no fth ethre emethod swa smade . Theresistanc eo ftomatoe s increasedconsiderabl yunti la tleas t8 week s afterseeding .Partia lresistanc eo fW.V a70 0agains tth e 1-t-pathotypeo f P. infestans isbase do na singl egene ,her ename dPti2 . Voorwoord

Mijnoprecht e dank gaatui tnaa r aliendi eo penigerle iwijz ehebbe nmee - gewerktbi jhe tto t standkome nva ndi tproefschrift . Inhe tbijzonde rwi li kmeldin gmake nva nd evel ewaardevoll e suggesties vanmij npromotor ,dr .J.C .Zadoks . Het onderzoek isverrich t aanhe t Instituutvoo r Plantenziektenkundig Qnderzoek (IPO)t eWageningen .D e gelegenheid diemij daargebode nwer d in ditwer kmij neige hwe g tegaan ,i sdoo rmi j zeergewaardeerd .He tLaborato - riumvoo rFytopathologi e vand e Landbouwhogeschool stelderuimt ebeschikbaa r ind eklimaatkamers .Beid e instellingenwi l ikgaarn emij nerkentelijkhei d betuigenvoo rhu nmedewerking . Vand emedewerker sva nhe t IPOwi li k inhe tbijzonde rnoeme ndr .J.C . Mooie nd ehee rD .Looyen ,di emij deaardappelziekt e leerdenkenne ne nme t wie iko pplezierig e wijzemoch t samenwerken.D ehee rC.F .Scheffe lbe ni k zeerdankbaa rvoo rhe tmake nva nd etekeninge ne nmevrou w0 .Krechting - Janssenvoo rhe t typewerk. Eenreissubsidi e vanhe t Landbouwhogeschoolfondsmaakt ehe tm emogelij k omd eprobleme nbetreffend ed e aardappelziektebij tomaati nenkel e Europese landenva nnabi j te lerenkennen ,wa the tonderzoe k zeer tengoed e isgekomen . Dehee rW .Hoogkame rverleend emi j zijntechnisch ebijstand ,di ei k zeer opprij she b gesteld. Tenslott ebedan k ikmij nvrou we nmij nschoonvade rvoo rhu n onbaatzuch- tigesteu ne nvoo rhe ti nmi j gesteldevertrouwen . Curriculum vitae

Deauteu rwer do p4 Septembe r 1936t eNijmege ngeboren .Hi j volgdeee nMULO - opleidingt eArnhe me ndoorlie pd eLandbouwwinterschoo lt eZutphen .N azij n militairedienstplicht ,volgd ehi jd ebeid ehoogst eklasse nva nd eLorentz - HBSt eArnhem .Hi jstudeerd evana f196 0aa nd eLandbouwhogeschoo lt e Wageningene nverkree gi n197 1he tdiplom ava nlandbouwkundi gingenieur .Van ­ afme i 1970werkt ed eauteu ral spromotie-assisten tbi jhe tInstituu tvoo r PlantenziektenkundigOnderzoe kt eWageninge nte rvoorbereidin gva nzij nproef - schrift. Samenvatting

Hoofdstuk 7 Ind ejare n 1965to te nme t 1968kwa mP . infestans, deverwekke r vand eaardappelziekte ,i nNederlan depidemisc hvoo ro ptomate ni nkoud ee n lichtgestookt ekassen .Voo rdez etij dwa sd eziekt ei nkastomate nbetrekke - lijkonbekend .He tonverwacht e optredenva ndez e ziektewer dbehalv edoo rd e vooraardappelziekt egunstig eklimatologisch eomstandighede ni ndez eperiod e medebelnvloe ddoo rd eteel tva ntomate ndi eresisten tware ntege n Cladospovium fulvum. Deresistenti etege n C. fulvum hadto tgevol gda tchemisch ebestrij - dingva ndez e ziekteachterweg ebleef ,waardoo rP . infestans dekan skree g zichongehinder dt eontwikkelen . Qmdatbehandelin gme tfungicide nnie taltij dafdoend ei se nresidue nva n fungicide-middelenhe taanzie ne nd econsumpti ekwalitei tva ntomate nnadeli g kunnenbelnvloeden ,i sresistenti edringen dgewenst .E rzij nweini gbronne nva n resistentiebeken de nhe tnivea uva nd egevonde npartiSl eresistenti ei son - toereikendc md egewasse nzonde rd etoepassin gva nfungicide ntege nP . infestans afdoendet ebeschermen .Accumulati eva nresistenti edoo rmidde lva ngenetisch e techniekenlijk thie ree noplossing ,maa rd everedelin gme tbehul pva ndi t typeva nresistenti ei smoeilijk ,omda td egenetisch e grondslagva npartiel e resistentie onvoldoendebeken dis ,e nhe teffec tva npartiel eresistenti ei n sterkemat ebepaal dword tdoo rmilieu-invloeden .

Hoofdstuk 2 Indi edele nva nEuropa ,waa rd etomaa ti nd eope n luchtword t geteeld,i sd eaardappelziekt ed ebelangrijkst eziekt eva nd ebuiten-tomate n end eo pdri en abelangrijkst e ziekteva nd ekas-tomaten .D ealgemen emenin g is,da td eziekt ei nd elaatst etie njare nveelvuldige re nme tgroter ehevig - heidoptreedt .D eoorzaa kword tgezoch ti nd ejaarrond-teel ti ndez egebieden , diealleng si nbetekeni stoenam .D einvloe dva nd ejaarrond-teeI to phe top ­ tredenva naardappelziekt ebi jtomaa tka nhe tbes ttoegelich tworde naa nhe t voorbeeldva nBulgarij ee nHongarije ,waa rn ad eintroducti eo pgrot e schaal vanvoorjaars -e nnajaarsteel tva ntomate ni nkasse nomstreek s 1965P . infestans eenenor mproblee mgeworde nis . Eoofdstuk 3 Ond eherkoms tva nd e aardappelziekteo ptomaa tn a tegaan ,wer d studiegemaak tva nd ewaardplantenreek s vanp . infestans. Eennieuw ewaardplant , Phaaelia tanacetifolia, konaa nd elijs tworde ntoegevoegd . InNederlan d bestaat allerede n CM aant eneme nda the t inoculum vand e aardappelziekte optomate n afkomstigi sva naardappel .Ui tonderzoekinge nme therhaald e passages overver - ouderdtomatebla de njong e zaailingenva n tomaat isgebleken ,da td eaanvanke - lijkegering epathogenitei tvoo r tomaatva nP . infestans isolaten afkomstig van aardappelaanzienlij kko nworde nverhoogd .Overeenkomstig eervaringe n werden opgedaanme toorspronkelij kvoo r tomaatniet-pathogen e P. -in/estons-isolaten inee nveldproe fme t tomaten.

Eoofdstuk 4 Hetonderzoe k richtte zicho ppartie'l eresistentie .D eaanpa kwa s tweeledig.Enerzijd swerde n laboratoriummethodenontwikkel d voorhe tm ete nva n partieleresistenti e aanenkele ,speciaa lvoo rdi t doelgekoze n 'resistentie- componenten',zoal s 'infectie resistentie', 'uitbreidingsresistentie'e n'spo - rulatie resistentie'.Anderzijd swer d getracht totee nmethod eva nveldtoet - singt ekome nwaarme everschille n inpartiSl e resistentie bepaaldkunne nwor ­ den.He tmete nva nresistentie-conponente nvereis t de ontwikkeling vantechnie - kenvoo rnauwkeurig e doseringva nhe t inoculum.C mhe t effectva n verschillen doormilieuinvloede nz ovee lmogelij k tebeperken ,werde n deplante nopge - kweekte ngetoets t ind eklimaatkamer sva nhe t Laboratoriumvoo r Fytopathologie vand eLandbouwhogeschool .

Hoofdstuk 5 en 6 Demeest eonderzoekinge nwerde nverrich t aanplante nva nd e vatbare cv.Moneymake re naa nplante nva nd e lijnW.V a 700,di e eenhoo gni ­ veauva npartiel eresistenti ebezi t endaaro mwaardevo lword t geachtvoo rd e resistentieveredeling.D eresistenti e vanbeid e tomatelijnen,gemete naa nhe t aantalgeslaagd e inoculaties,d euitbreidin g vand e lesies end e sporulatie mtensxteit,neem tme td e leeftijdva nd eplante n toe tot tenminst e8 weke n nahe tzaaien .

Eoofdstuk 7 Deproefresultate nva n6 weke noud eplante nva nd eF 2:Money ­ makerx W.V a70 0e nd eeerst eterugkruisin gMoneymake r* (Moneymake rx W.V a

700)Z1J nme t endi eva n8 weke noud eplante n zijnwe lgeschik tvoo r eenge - net1Sche analyseva nd eresistenti e vanW.V a 700.Zowe ld eveldproeve nal sd e laboratorxumproevenleidde n totd econclusi e datd e resistentieva nW.V a 700 tegenhe t 1-t-pathotypeva nP . infestan8 TOrdt bepaalddoo r^ ^ volledlgebeschermin gbied t tegenstengelaantastin g enpartiel ebeschermin g tegenhladaantasting .Di tge nword the tPh 2-gengenoemd .

Hoofdstuk 8 Geblekeni sda tva nd edri e toegepastemethode nd eprobi t -lo g dosisanalys ed emees tnauwkeurige ,maa roo kd emeest bewerkelijk ee nduur - stemethod e is.D eander e laboratoriummethode,waarme ed egroe iva nd e lesies end e sporulatie-intensiteit werdbepaald ,wa saanmerkelij kminde rbewerke - lijkmaa rwe lkostbaar .D eopze tva nd eveldproeven ,waari nn a inoculatieva n debesmett erije nieder ewee kd eplante ngedurend e 40uu rwerde nna t gehouden dooree nautomatisch eberegeningsinstallatie ,blee kgeschik tvoo rhe tbepale n vanverschille n inparti&l eresistentie .D emaa tvoo rresistenti ewa sd e 'schijnbare infectiesnelheid1 r,i nlogi teenhede npe rdag .Dez emethod e is aanmerkelijk goedkopere neenvoudige r dand ebeid e laboratoriummethodene ni s bruikbaarvoo rd epraktisch e veredeling opresistenti e tegenP . infestans. Daarbijmoe tda nwe lrekenin gworde ngehoude nme the tfeit ,da tbepaald e ty- penva nresistentie ,zoal sd esporulati ebeperkend e resistentieva nd ecv . Atom,i ndi tsoor tproeve n (continuemonocyclisch etoetsingen )nie tto thu n rechtkomen .Voo ree npraktisch euitwerkin gva nd e resultaten,verkrege nme t develdproeven ,i see nvereenvoudigd emethod e aanbevolen. Contents

1 Introduction

3 2 The disease 3 2.1 Introduction 3 2.2 Symptomatology 3 2.2.1 Symptomso nleave s 2.2.2 Symptomso nstem s 4 2.2.3 Symptomso nfruit s 4 2.3 Epidemiology 5' 2.3.1 Epidemiologicparameter s 5 2.3.2 Rateo fepidemic s 5 2.3.3 Epidemicsan dchemica lcontro l •> 2.3.4 Epidemicsan dth eway so ftomat ogrowin g 6 2.3.5 Relationsbetwee nepidemic so ntomatoe san do npotatoe s 8 2.4 Economicimportanc eo fth ediseas e 9 2.4.1 Inth eNetherland s 9 2.4.2 Inothe rEuropea ncountrie s 10

The pathogen H 3.1 Biology H 3.2 Hostrang e 12 3.2.1 Introduction 12 3.2.2 Thetomat o 14 3.2.3 Thepotat o 14 3.2.4 Solanum dulcamaraL . 14 3.2.5 Solanaceoushosts ,excludin gpotatoe san dtomatoes ,wit h 15 potentialsurviva lvalu et oth efungu si ncrop-fre eperiod s 3.2.6 PhaaeKa tanaaetifolia, ane whos tspecie so fP . infestans 16 3.2.7 Developmento fP . infestans on Datura stramonium, 16 Mirabilis jalapa, and Schizanthus pinnatus 3.3 Methodso fisolatio nan dcultur e 18 3.4 Variabilityi npathogenicit y 19 3.4.1 Terminology 19 3.4.2 Differentialpathogenicit yt ovariou shost s 21 3.4.3 Variabilityi npathogenicit yt opotatoe san dtomatoe s 22 3.4.4 Theappearanc eo fa pathotyp e highlypathogeni ct o 24 tomatoesi nth efiel d 3.4.5 Afiel dexperimen tt odemonstrat eth eincreas ei n 26 pathogenicityt otomatoe s 3.4.6 The'logistic 'increas ei npathogenicit yt otomatoe s 30 ina fiel d experiment 3.4.7 Alaborator y experimentt odemonstrat eth eincreas ei n 32 pathogenicityt otomatoe sb yseria lpassage s 3.4.8 Alaborator yexperimen tt odemonstrat eth eincreas ei n 37 pathogenicityt o Solanum dulcamara byseria l passages 3.5 Conclusions 38

4 The assessment of resistance 40 4.1 Monocyclic andpolycycli c tests 40 4.2 Partialresistanc e 40 4.3 Numericalvalu eo fpartia lresistanc e 41 4.4 Componentso fpartia l resistance 42 4.5 Mathematical techniques 44 4.5.1 Probit- lo gdosag e analysis 44 4.5.2 Variance analysis 45 4.6 Materials andmethod s 45 4.6.1 Introduc tio n 45 .4.6.2 Thegrowin go fth etes tplant s 46 4.6.3 Thepreparatio no fth e test leaves 46 4.6.4 Thepreparatio no fth e zoospore suspension 46 4.6.5 Theapplicatio no fth einoculu m 47 4.6.6 Theincubatio no fth e inoculated leaves 48

5 Results: monocyelie tests in the laboratory 49 5.1 Introduction 49 5.2 Withinplant svariabilit yo flesio nextensio n 49 5.2.1 Sixweek sol d'Moneymaker 'plant s 49 5.2.2 Eightweek sol d'Moneymaker 'plant s 50 5.3 Theeffec to fdosag eo nlesio nextensio nan dsporulatio n 51 intensityo fsom etomat o lines 5.4 The 'resistancet oinfection 'o f6 ,7 ,an d8 week sol d 52 W.Va70 0plant s

5.5 The'resistanc et oinfection 'o f6 an d9 week sol d 55 'Moneymaker'plant s

5.6 The 'resistancet oinfection 'o f9 an d1 1week sol d 56 'Moneymaker'plant s

5.7 The'resistanc et oinfection 'o f6 ,7 ,an d8 week sol d 58 'Moneymaker'plant s

5.8 Theresistanc eo f6 an d8 week sol dplant so fth e 59 F2:Moneymake rx W.V a70 0 5.9 Resistanceo f6 an d8 week sol dplant so fth efirs tbac k 61 crossBj :Moneymake rx (Moneymakerx W.V a700 ) 5.10 Conclusions 62

6 Results: Continuous monocyclic tests in the field 64 6.1 Introduction 64 6.2 Partialresistanc eo fth ecv .Moneymaker ,th elin eW.V a 64

700,thei rF xan dthei rF 2i na fiel dexperiment ,197 2

6.3 Partialresistanc eo fth ecv .Ato man do fth eF 2:Ato mx 67 W.Va70 0

6.4 Thepartia lresistanc eo fsom etomat oline si nth e197 2 67 fieldexperimen t 6.5 Conclusions 69

Genetic analysis of resistance against Phytophthora infestans 70 7•1 Introduction 70 7.2 Resistanceo fth estem s 70 7.2.1 Fieldtes tdat ao fth ecros sMoneymake rx W.V a70 0 70 7.3 Resistanceo fth eleave s 71 7.3.1 Statisticalanalysi s 71 7.3.2 Environmentalvarianc eo fpartia lresistanc e 72 7.3.3 Fielddat ao fth ecros sMoneymake rx W.V a70 0 73 7-3.4 Geneticanalysi so fth elaborator ydat ao fth ecros s 74 Moneymakerx w.V a70 0 7.3.5 Fielddat ao fth ecros sAto mx W.V a70 0 „ 7-4 The 'Atom'parado x 7.5 Discussion 7S 7.6 Conclusion 76 77 8 Comparison of testing methods with special reference to 78 their application in commercial breeding programs 8.1 A comparisono ftestin gmethod s 78 8.2 A simplifiedrecip efo rth ebreede r 79

Summary 81

References 84

Appendix 1 Introduction

Inth eyear s 1965til l 1968, Phytophthora infestans wasepidemi co nto ­ matoesi nsom eglasshous edistrict so fth e Netherlands (Anonymous, 1968) Thediseas e caused seriouslosse si nunheate do rslightl yheate dglasshouses . Before 1965,lat ebligh to ntomatoe si nglasshouse swa shardl ya proble mi n theNetherlands .Grower suse dcultivar s susceptiblet o Cladosporium fulvum, andtreate d theircrop swit hfungicide st ocontro lthi sdisease .Afte rth e introductiono fcultivar sresistan tt o C. fulvum, growerscease dt ous e fun­ gicides,thu sgivin gP . infestans anopportunit yt odevelo pwithou thindranc e (deBoer , 1966).I nth eyear safte r 1968,lat ebligh to ntomatoe swa sfoun d sporadically.I nth eNetherlands ,n otomatoe sar egrow ni nth eopen ,becaus e ofth elat eripenin go fth ecro pan dth ehig hris ko flat ebligh t infection. Incountrie s likeBelgium ,France ,Italy ,Bulgari aan dHungary ,lat e blighti sconsidere dt ob eth emos t importantdiseas eo foutdoo rtomatoes . Inglasshouse san di nplasti c tunnels,p . infestans takesth ethir dplac e among thedisease so ftomatoe si nth esouther npar to fFrance ,i nItal yan di n Bulgaria. Inheate dglasshouses ,lat ebligh tca nb esuccessfull yprevente db ymean s ofclimati c control.I nunheate dglasshouse san di nth efield ,th ediseas e canonl yb econtrolle db yfungicides .Thi smetho do fcontro li sno talway s effective.Weathe ran dsoi lcondition sca nb eto oba dt oente rth ecrop , and therei sa relativel y longharves tperiod ,durin gwhic hth ecro pcan,no tb e treatedi norde rt oavoi dundesirabl eresidues .Th esusceptibilit yo fmoder n cultivarsi ssuc htha ta dela yi ntreatmen ti sa grea tproductio nrisk ;there ­ foreresistanc ei sdesirable . Unfortunately,resistanc e againstP . infestans israr ei nth egenu s Lycopersioon (Richards& Barratt , 1946).Attempt st outiliz ea gen e forhyper ­ sensitivityhav ebee nmade ,bu tthes ehav ebee nunsuccessfu la sa compatibl e raceo fth efungu sappeare dalread ybefor eth ebreedin gprogra mwa s completed (Conover& Walter , 1953).Othe rgene sfo rhypersensitivit y areno tye tknown . Theexperience sgaine dwit hthi styp eo fresistanc e againstP . infestans in potatoesar eunfavourabl ebecaus eo fth efrequen tappearanc eo fcompatibl e

1 races (Schick, 1932; Niederhauser &Cobb , 1959; Malcolmson, 1969). Nowadays, the attention is directed towards partial resistance (see Chapter 4). The life- expectancy of this type of resistance is possibly longer than that of resistance based on hypersensitivity. Breeding for partial resistance is complicated as the genetic background of partial resistance is usually unknown. Many problems about this type of resistance have to be solved, and therefore more research is needed. The success of breeding for partial resistance against P. infestans depends partly on the stability of the pathogenicity of the fungus (see sec­ tion 3.4). The unexpected appearance of epidemics of P. infestans in the Dutch glasshouses gave rise to questions like: 'Which other hosts are important?', 'What is the relation of late blight on tomatoes to late blight on potatoes?' and 'How do tomato races of late blight overcome unfavourable periods?' These questions will be discussed in sections 2.3 and 3.2 and answered in section 3.5. 2 The disease

2.1 INTRODUCTION

Thediseas eha sbee nknow nt ooccu ro ntomatoe s since 1847 (Payen, 1847), butver y fewdat ao ncro p lossesi nth e19t hCentur yar eavailable .A tth e endo fth e19t hCentury ,th egrowt ho ftomatoe so na large r scalebecam epop ­ ular.Concurrently ,th edifficultie s causedb ylat ebligh t increased (Howitt, 1917; Reed, 1912;Wiltshire , 1915).Th ehistor yo flat ebligh ti npotatoe si s wellknow nan dwil lno tb ediscusse d (Hanni,1949 ;Leac h& Rich , 1969; Umaerus,1968 ;Upshall , 1969).A tpresent ,th ediseas ei sa permanen t threat tooutdoo r tomatoesi nEurope ,an di ti sa nimportan t diseaseo fglasshous e crops.

2.2 SYMPTOMATOLOGY

2.2.1 Symptoms on leaves

Symptomscause db yraces ,whic har eweakl ypathogeni ct otomatoe smus t bedistinguishe d from symptomsdu et oraces ,whic har e stronglypathogeni c totomatoe s (seesectio n3.4) . Theweakl ypathogeni c racesfor m slowlyextending ,brow nt oblac k col­ oured lesions,whic hbecom e angularo fshape ,a sth evein s limitth egrowt h ofth epathogen .Th erat eo fgrowt hi su pt o1m ma day .Th elesions ,usual ­ ly, shown o sporulation,bu tsometime sa sligh t sporulationca nb efoun do n dead tissue.Whe ncondition sfo rlat ebligh tar efavourable ,th efungu sma y traverseth esmalle rveins ,an dth esporulatio nma yb emor e intense.Normal ­ ly, lesionsceas et ogro wi na nearl ystage ;th emajorit yo flesion sdoe sno t exceed2 t o3m mi ndiameter .Senescen t leaves,however ,ma yb einvade d com­ pletely,an dthes e leavesca nsho wa nabundan t sporulation. The stronglypathogeni crace sfor mwater^soake dlesion so fabou t4 m m indiameter ,whe nkep tunde rfavourabl e conditionsunti ltw oday safte rin ­ fection.Thre ean da hal fda yafte r infectionth elesion shav ea diamete ro f 10t o1 5mm ,an dsporulatio nbegin si na smal l zoneo fapparentl yunaffecte d tissue,tha t surroundsth ewater-soake d tissue.Th elinea r growthrat eo fa lesioni su pt o5m ma day .Whe ncondition sar eles sfavourable ,a typica l pigmentationo fth einfecte dtissu eappears ,whic hresult si na dendritica l patternspreadin gi nal ldirection sfro mth e siteo finfection .Withi na fe w daysth ecentr eo fth e lesionturn sligh tgre yt oblack .

2.2.2 Symptomson stems

Raceso fp . infestans, thatar eweakl ypathogeni ct otomatoe s causesmal l blackspot so fles stha n1 m mdiamete r (rarelyu pt o4 m m<(>) .Race s strongly pathogenict otomatoe scaus elarg e lesionso fa typica l sepiacolour .Whe n theweathe rcondition sar efavourable ,ther ema yb ea nabundan t sporulation. Often,concentri cring so fmor ean dles sdarkene d tissuear e present.Afte r abouta week ,th ecolou ro fth elesion sfade san dbecome spalis h grey.Durin g windyweathe rth estal kma ybreak ,a sth eaffecte d tissue losesit s elastici­ ty.

2.2.3 Symptomson fruits

Greenfruit sinfecte db yP . infestans first showa reddis hbrown ,feath ­ erlikepatter no nth eskin .Withi na fe wdays ,th eski nturn sdar kbrown , and becomeslumpy ,a sth eunderlyin gtissu edrie sup .O nlarg efruits ,concentri c ringso fmor ean dles sdarkl ycoloure dtissu eca nb efound .Th erat eo fsymp ­ tomdevelopmen tvarie saccordin gt oth epathogenicit yo fth erace s tested. Racesstrongl ypathogeni ct otomatoe s invadeth efruit s systemically,whil e weaklypathogeni crace susuall ycaus e locallesions .Th einfecte d tissue driesan dbecome shard .Infecte dfruit sca nsporulat e abundantly whenever theweathe rcondition sar efavourable ,th efruit sthe n lookingwhit ean d woolly.R lpe fruitsan dcolourin gfruit sar ever yresistan tt oP . infestans. Fordifference sbetwee nsymptom scause db yP . infestans andP . niootianae. var. mootianae (buckey erot) , seeWeststeijn , 1973. 2.3 EPIDEMIOLOGY

2.3.7 Epidemiologic parameters

Themathematic so fepidemic sha sbee nelaborate db yva nde rPlan k (1963). An epidemicca nb echaracterize db yth eapparen t infectionrate ,whic hi sth e regression coefficiento fth efractio ndisease dfoliage ,expresse di nlogits , ontime :

*2 - *i a;i(,1- X2)

r =apparen t infectionrat e t =tim e x =fractio no fdisease dfoliag ea ttim et * e nl = log= log e Theapparen t infectionrat e r willb eexpresse di nlogi tunit spe rday ;it s dimensioni s t~l

2.3.2 Rate of epidemics

Themos t surprisingfac to flat ebligh t epidemiologyi sth erapidity , withwhic hth eepidemi cca nru nit scourse .Withi na fortnigh tafte rth eob ­ servationo fth efirs t lesionsth ecro pca nb ecompletel yruined .Thi si sth e consequenceo fth eshor tlaten tperiod ,th ehig hgrowt hrat eo fth efungus , theabundan tsporulation ,an dth ecrowdin go fman ysusceptibl eplant stogether . Vande rPlan k (1963,p.184 )foun d r =0.4 2fo rth esusceptibl epotat oculti - varBintj eunde rcondition sfavourabl efo rlat eblight. I na fiel dtrial , theautho rfoun da valu eo fr =0.4 3fo rth esusceptibl etomat ocultiva r Moneymaker,r = 0.4 3 implies,tha twithi na fortnigh tth ediseas eseverit y cang ofro m1 t o7 5pe r cent.

2.3.3 Epidemics and chemical control

Chemicalcontro lreduce sth ediseas eincidence .Th ediseas eincidenc e neverbecome szero ,a sth eapplicatio no ffungicide si sneve rperfect .Ther e alwaysremain sa sourc eo finoculu msomewher ei na crop .Thi si sa permanen t threat,whic hbecome smanifes twhe ncondition sar efavourabl e for P. infeatans. Theminimu m intervalbetwee nth elas tapplicatio no fzine bo rmane ban dth e pickingo ffruit si sthre eday si nth eNetherlands .Th epickin gperio di s ratherextended ,a stomat ofruit sripe ni nsuccessio ndurin ga four-mont hpe ­ riod.Thi ssituatio ncause sth egrowe rman ydifficulties .Eithe rh ecanno t spray,o rh ecanno tharves tfo rsom edays ;i nbot hcases ,cro p lossesca n oc­ cur.I naddition ,problem so fplannin gan dlabou r arrangementarise .

2.3.4 Epidemics and the ways of tomato growing

InBulgaria ,Hungary ,an dItaly ,grower snotice d thati nrecen tyear s theattack so fP . infestans havebee nmor e severetha nbefore .I nBulgari a andHungary ,th eyea ro fchang eca nb eidentified .Befor e 1965,lat ebligh t ontomatoe swa sknow na sa mil ddisease ,appearin goccasionall yo ntomatoe s inautumn .I n1965 ,th ediseas e causedseriou slosses ,an dfro m 1965u pt o 1972considerabl e lossesoccurre dnearl yever yyear .I nHungary ,tomatoe si n theope nar edamage di nlat esumme ran dautumn .I nBulgaria ,lat ebligh ti s alsosever ei nglasshouse s inMay ,June ,September ,an dOctober .

Bulgaria InBulgaria ,tomatoe shav ebee ncultivate do nlarg efield s since thecollectivizatio ni nth eperio dfro m 1947u pt o1956 .Abou t 1965,tomat o culturei nglasshouse stwic ea yea rwa s initiatedo na larg e scalei n Bulgarianglasshouses .Befor e 1965,lat ebligh tattack so npotatoe san dtoma ­ toesoccurre d simultaneously.Ther ewa sa nattac ko ftomatoe sonly ,i fther e wasa nepidemi co flat ebligh to npotatoes .Afte r 1965,epidemic so flat e blighto ntomatoe ssee mt ob eindependen to fepidemic so npotatoe s (DrH . Helenkove,1972 ,pers .commun.) .Th emutua ldependenc eo fP . infestans epi­ demicso ntomatoe san dpotatoe si sa nepidemiologica lproble mpe rs e(se eSec ­ tion 2.3.5).Informant sassum etha tth ediseas eha sbecom es oseriou sbecaus e ofth eyear-roun dcultivatio no ftomatoes ,thu s giving tomato raceso fP . infestans anopportunit yt ooversumme ri nth efiel dcrops ,an dt ooverwinte r inth eglasshous ecrops .

Hungary InHungary ,th ecultivatio no ftomatoe so nlarg efield swa sintrodu ­ cedabou t 1965;th ecultivatio no ftomatoe si nglasshouse s twicea yea rwa s beguni nth esam eperiod .Befor ethi stime ,tomatoes ,potatoes ,an dothe r cropswer egrow ni nsmal lfields .N oreport so fseriou slat ebligh t attacks

fromth eperio du pt o19 65ar e known (S.Hodosy , 1972,pers . camnun.).I n Hungary,wher e latebligh ti sno t i^ortanti nglasshouses ,colleague ssup ­ posetha tth eris ei nprevalenc ean dseverit yo fth edisease si sdu et oth e growing oftomatoe so nlarg efields .

Italy In Italy,lik ei nth eothe r south-westEuropea ncountries ,a sudden rise inth e severityo fattac kdi dno t occur.Th eexplanatio n issough t in amor egradua lchang eo fgrowin gmethods .Her etoo ,tomat orace so fP . infestans may overwinter inglasshous etomat ocrops .A nexceptio nha st ob e madefo rth e southernpar to fItaly ,wher ewinter sar es omild ,tha tP . infestans canoverwinte ro nplan tremnant san dseedlings .T oexplai nth ein ­ creasedseverit yo flat eblight ,Italia ngrower ssuppor tth ehypothesi stha t modernhig hyieldin gtomat ocultivar sar emor e susceptibletha nth eol dones .

New cultivation methods InBulgari aan dHungary ,a sudde nris e inprevalenc e and severity oflat ebligh tattack so ntomatoe scoincide dwit hth e largescal e introduction ofne wmethod so ftomat ogrowing .Tw oepidemiologicall y impor­ tantfactors ,cultivatio no nlarg efield san dyear-roun d cultivation,hav e been introduced. InBulgaria ,tomatoe shav ebee ngrow no nlarg efield swithou t serious damageb y lateblight .Therefore ,th ehypothesi stha tcultivatio no ftomatoe s on largefield spromote s epidemicsi sno t sufficientt oexplai nth e increased attacki n 1965an dfollowin gyears .Th eeffec to flarg efield so nepidemic s istwofold ;th elarge rth efield ,th emor e likelyth einoculu mwil lcom efro m withinth efiel di fi ti spresen t (Vande rPlank ,1968 ,p . 132);.thelarge r thefields ,th egreate rth edistanc ebetwee nth efields ;an dth egreate rth e distancesbetwee nfields ,th esmalle rth echance ,tha ton efiel dinfect sth e other (Vande rPlank , 1960). Anotherhypothesi sis ,tha tth eyear-roun dcultivatio no ftomatoe sin ­ creasesth ediseas e incidence.I fthi shypothesi si strue ,ther emus tb ea specificreaso nwh y latebligh t isno t seriousi nHungaria nglasshouses .Th e fact,tha tlat ebligh t inHungaria nglasshouse s iso fn o importance,ca nb e explained intw oways .First ,Hungaria ngrower sfac eattac kb y CladospoHum fulvvm inthei rglasshouses ,jus ta si nth eNetherlands .Consequently ,the y treatthei rcrop swit hfungicides ,therewit hcontrollin gP . infestans aswel l as C. fulvum. Second,th eclimat ei nHungar y iscoole rtha ni nBulgaria .There ­ fore,th eheatin g seasonfo rglasshouse si nHungar y islonge rtha ni nBulgaria . Heatingreduce sth ehumidit yo fth eair ,an dthi si sknow nt ob ea nexcellen t methodo f latebligh tcontro li nheate dglasshouses ,a tleas ti nth eNether ­ lands. (Anonymous,1968a) .Th eyear-roun dcultivatio no ftomatoe s seemst ob e thedecisiv efeatur ei nth edevelopmen to fseriou slat ebligh tepidemic so n tomatoes,thu sgivin gtomat orace so flat ebligh tth eopportunit yt oover ­ winter andt ooversumme ri ntomat ocrops .Th ecultivatio no ftomatoe s over large areaswit ha hig hfrequenc yo ftomat o cropsma yb eimportan ttoo ,be ^ causeth e opportunityfo roverwinterin gan doversummerin go fth epathoge ni s improved. Therelatio nbetwee nyear-roun d cultivationan dincrease d incidenceo f diseaseha sbee nobserve d forothe rhost-pathoge n combinations {Chrysanthemum morifolium and Asooehyta chrysanthemi, Sauthof,1963 ; C. morifolium and Puaoinia horiana, Zadoks,1967) .

2.3.5 Relations between epidemics on tomatoes and on potatoes

Inth eNetherlands ,n olat ebligh tattac ko ftomatoe shav e been reported whenther ewa sn oepidemi co flat ebligh to npotatoes .Frequently ,however , therear eepidemic so npotatoe swithou tnoticabl e attacko ftomatoes .I n1968 , whenlat ebligh twa s seriouso npotatoe san dtomatoes ,th ediseas e hardly occurredo ntomatoe sgrow ni nth eWestlan dglasshous e district,wher en opo ­ tatoesar egrow n (Weststeijn, 1973).I nth eNetherlands ,lat ebligh t epidemics oftomatoe sevidentl ydepen do nlat ebligh t epidemicso npotatoes . InBulgaria ,i fther ewa sn oseriou sattac ko npotatoes ,ther ewa sn o epidemico flat ebligh to ntomatoes ,befor e 196S.Th esituatio nwa sth esam e asi nth eNetherlands .Afte r 1965,epidemic so ntomatoe s occurred independent­ lyo fepidemic so npotatoe s (H.Helenkove ,pers . commun.).I tseem spossible , thattomat ocrop si nwinte r aremor e importanta ssourc eo flat ebligh tin ­ oculumfo rpotatoe stha npotat otubers .Th erelatio nbetwee n epidemicso npo ­ tatoesan dtomatoe safte r 1965seem st ob eth e inverseo ftha tbefor e 1965. InIsrael ,lat ebligh ti sa seriou sproble mo npotatoe san dtomatoe s excepti nsummertime .I nth eho tan drainles s summern opotatoe sar egrown ; inthi sperio dtomatoe sar erelativel y freeo finfectio n (Palti& Netzer , 1963). Kedare tal . (1959)teste d2 5isolate scollecte d frompotatoe so nto ­ matodifferentia lhosts .Sixtee no fthes e isolateswer e pathogenict otoma ­ toes,thu sgivin ga nimpressio no fth eimportanc eo flat ebligh to ntomatoe s as*a sourc eo flat ebligh t inoculumfo rpotatoes .I nth eNetherlands ,wher e therei sn oevidenc etha ttcmatoe sar eimportan ta sa lat ebligh t sourcefo r potatoes,isolate so flat ebligh tcollecte do npotatoe sneve rwer e markedly pathogenict otomatoes .I nIsrael ,lat ebligh tprobabl yoversummer si nth e tomatocrop: . InItaly ,tomat orace scoul db ecollecte dfro mpotatoe s (Ciccaronee tal . 8 1961). Here,man y regionshav e suchmil dwinter stha tplan tremnant san d seedlings areno tkille db yfrost ,thu sofferin ga nopportunit yt oP . infestans tooverwinte ro ntomatoe si nth efields . Inth eMediterranea n regiono fFrance ,i ti scustomar yt opic kth e last greenfruit si nth efield sa tth een do fth e season,an dt ostor ethe mi n glasshouseso rplasti c tunnelsfo rripening .Som eo fthes e fruitsma yb ein ­ fectedb yP . infestans. Whena ne wcro pi sgrow ni nth e same room,th eyoun g plants,whic har ever y susceptible,ma yb einfecte db yspore sforme do ndisea ­ sedfruits .Thi smod eo foverwinterin gmigh tb ea nexplanatio no fth eoccasion ­ allyoccurrin g severe attacko fyoun gplant si nglasshouse so rplasti c tunnels inearl y spring.

Inregar dt oth emutua ldependenc eo flat ebligh tepidemic so ntomatoe s and latebligh t epidemicso npotatoes ,severa ltype so finterdependenc yca n bedifferentiated .Wher eP . infestans overwintersi npotat otuber smainly , latebligh tattack so ntomatoe sdepen do nth edevelopmen to flat ebligh t epidemicso npotatoes .Thi styp eo fone-directiona l relationi sknow nt oexis t inth eNetherland s (Weststeijn, 1973),an di ssuppose dt ohav e existedi n Bulgariabefor e 1965.I nregions ,wher etomat ocrop salternat ewit hpotat o cropsi na nannua lrhythm ,th einterrelatio no fth etw olat ebligh t epidemics isexpecte dt ob etwo-directional .Thi styp eo finterrelatio nma y existi n Israelan di npart so fJapan .I nregions ,wher eth eclimat e allowsth e toma­ tohos tt opersis t continuously eithera scrop so ra splan tremnants ,lat e blight epidemicso ftomatoe sar eexpecte dt ob eindependen to fepidemic so n potatoes.I nthi s case,lat ebligh t epidemicso npotatoe smigh tdepen do nlat e blightepidemic so ftomatoes ,agai na one-directiona lrelation .Thi styp eo f interrelationi ssuppose dt oexis ti nsom eMediterranea n areas,an di n Bulgariaafte r1965 .

2.4 ECONOMIC IMPORTANCEO FTH EDISEAS E

2.4,1 In the Netherlands

Inth eNetherlands ,th e diseasei simportan tmainl yi nunheate d glass­ houses.Ther ei sa gradua lshif tfro munheate dt oheate dglasshouse s (Table 1).Mor e andmore ,th ecultivatio no ftomatoe si nunheate d glasshousesbecome s economicallymarginal .Consequentl ya nattac kb ylat ebligh t canhav ea deci ­ siveeffec to nth efinancia loutcom eo fa nindividua lgrower . Interestingly, Table 1.Are ao ftomatoe si nheate dan dunheate d glasshouses in the Netherlandsi n196 3an d1972 .

1963 1972 Heated glasshouses 1430h a 2200h a Unheated glasshouses 1400h a 1000h a

thediseas ei sfoun dwit hth e occasional tomatogrowe rwh oi seithe runexpe ­ rienced,o rspeculatin g insofara sh etreat shi s crops accordingt ohi sex ­ pectationso fmarke tprices . Theeconomi cimportanc eo flat ebligh t depends,too ,o nth eus eo fcul - tivarsresistan tt o Cladosporium fulvum. Atpresent ,th egene sfo rresistanc e to C. fulvum inth erelease d cultivarshav ebee nmad e ineffectiveb yth eap ­ pearanceo fcompatibl erace so fthi s fungus (Koopmans& Strijbosch , 1969). Therefore,thi sdiseas eha s againt ob econtrolle d with fungicides.Whe n these fungicidesar eals oeffectiv e against lateblight ,th eeconomi c importance of P. infestans willb ereduce dt ozero .I n1974 ,ne wtomat o cultivars resist­ antt oC. fulvum willb erelease dagain .

2.4.2 In other European countries

Nodat aeconomi caspect so flosse scause db yth e diseasear eavailable . Duringa tri pthroug hFrance ,Italy ,Bulgaria ,an dHungary , informants stated thatlat ebligh twa sth emos t important diseaseo foutdoo r tomatoes,respon ­ siblefo r crop1losse sestimate da t5 t o3 0pe rcen tyear ,unde r conditionso f regular treatmentwit hfungicides .Fo rglasshous e crops,th ediseas e was said totak eth e thirdplac ei nimportanc eamon gdisease so ftomatoes .

10 3 The pathogen

3.1 BIOLOGY

Phytophthora infestans isa heterothalli cspecie s (Smoote t al., 19S8;

Savagee tal. ,1968) . InMexico ,bot hmatin gtype sA jan dA 2 canb efoun di n aone-to-on erati o (Gallegly& Galindo , 1958). InEurope ,onl yth eAi-typ ei s known;accordingly ,sexua lreproductio nno rth eresultin goospore shav ebee n foundi nEurope .Th eoospor ei sth eonl yrestin g stageo fP , infestans. The asexualreproduction ,however ,i sver yimportant .P . infestans forms zoosporangia,whic hca nb etransporte db yai ran dwate r (rainsplash) (Uhlrich, 19S7;Hirst ,1958 ;D eWeille ,1964) .Th eviabilit y of'thesesporangi ai so f shortduration .I ndr yweather ,th e zoosporangialoos ethei rabilit yt oger ­ minatewithi na fe whour s (Crosier, 1934).Th eabundan tproductio no f zoo­ sporangiaunde rfavourabl econdition scompensate sfo rth epoo rabilit yt o survive. Zoosporangiao fP . infestans cangerminat e intw oways .I nth ecas eo f directgermination ,th esporangiu m formsa hypha ,an dfunction sa sa singl e infectiveparticle .Direc tgerminatio ni sfavoure db ytemperature sabou t24°C , buti tneve rexceed s 201o fth esporangi a (Melhus,1916 ;Crosier , 1934), In thecas eo findirec tgermination ,th ezoosporangi agerminat eb y the formation ofzoospores ,o fwhic hu pt o3 8hav ebee nobserve doriginatin gfro ma single zoosporangium (Laviola, 1968).Th eoptimu mtemperatur e forindirec tsporu - lationi sabou t 12°C.Th ezoospore sar epropelle db ytw oflagellae' .Th edu ­ rationo fth eswarmin gstag edepend so nenvironmenta lfactor samon gwhic h temperaturean davailabilit y ofnutritiona lsubstances .A t 21°C,mos to fth e zoospores stopmovin gwithi n3 h (Crosier, 1934).A t 3°C,th e zoosporesre ­ mainmotil e over 18h (McKee, 1964).Whe nth eswarmin gstag eends ,th efla ­ gellaedisappear ,an dth eformatio no fa germ-tub ebegins .Eac hzoospor e functionsa sa single infectiveparticle .Th eformatio no fgermtube sb y zoo­ sporesi sno tmuc h affectedb ytemperature sbetwee n3 an d21° C (Crosier, 1934).A ttemperature sbetwee n 15an d 18°C,th efirs tpenetratio no flea f tissueo fa susceptibl ehos ti seffectiv ewithi n2 h afte rinoculatio nwit h

11 motile zoospores (Crosier, 1934; Pristou & Gallegly, 1954).

On good evidence, Mexico is considered as the centre of origin of P. infestans. First, it is the only region in the world, where both mating types of P. infestans are known to exist (Smoot et al., 1958), and where the sexual stage of P. infestans is supposed to be functional (Gallegly &Galindo , 1958). Second, there is much diversity in pathotypes of the fungus (Niederhauser & Mills, 1953), and great diversity in resistance in the Solanum species indi­ genous in this area. This diversity on both sides can only be explained by the prolonged presence of both partners in this region (Reddick & Crosier, 1933; Mills & Niederhauser, 1953).

3.2 HOST RANGE

3.2.7 Introduction

The host range of p. infestans includes a number of species belonging to the Solanaceae and closely related genera, and a few species belonging to rather distantly related genera. These species are food crops, ornamentals, and wild plants. It is important to know, whether some of these species apart from Solanum tuberosum and Lyoopersicon esoulentum can function as a source of late blight inoculum. Enumerations of late blight host species have been made by several authors (Table 2). In some species the foliage was not susceptible, but the fungus was spo- rulating well'on infected tissues of petals, fruits, tubers, or roots. Success­ ful inoculations have been made on petals of Brassiaa oleracea L., Phaseolus multiflorus Lam. (Muller, 1950), Verium oleander L., and Vinaa rosea L. (Sztejnberg & Wahl, 1966), on fruits of P. multiflorus Lam., Solanum aapsicastri-

aumLink . (Muller, 1950), and Phaseolus vulgaris L. (Sztejnberg & Wahl, 1966),

and on tuber tissues o£ ^ ^^ u> ^ ^ . ^ (Muller, 1950). A few species showed more or less clear lesions after infection, but without spoliation. These species are: Dahlia variabilis Hort., Anemona O^oniea Hort., ^otuoa sativa L., Phaseolus multiflorus ^, Brassica eraoea (Muller, 1950), Lyceumeuropem L., Nerium oleander L., and Vinaa rosea (Sztejnberg & Wahl, 1966).

12 Table2 .Hos trang eo f Phytophthora infestans

Host Author

Solanum L. S. antipoviazii Buk. Reddick,193 0 S. avioulare Forst Driver,195 7 S. boreale (A.Gray )Bitt . Niederhauser& Mills , 1953 S. aardiophyllum Lindl. Niederhauser &Mills , 1953 S. aapsiaastriaum Linke xSchaue r Mailer,K.O. ,195 0 S. arispum Ruize tPav . Moore,194 3 S. demission Lindl. Niederhauser& Mills , 1953 S, dulaamare L. DeBary , 1876;Hirs t& Stedman , 1960 S. inaanum L. Nattrass& Ryan ,195 1 S. indiewi L. Nattrass& Ryan ,195 1 S. iopetalum (Bitt.)Hawke s Niederhauser& Mills , 1953 S. jamesii Torr. Niederhauser& Mills , 1953 S. laaineatum Ait. Driver,195 7 S. melongena L. Haskel,192 1 S. nigrum L. Hirst& Stedman ,196 0 S. panduraeforme Drege Nattrass& Ryan ,195 1 5. pinnatiseotum Dun. Niederhauser& Mills , 1953 S. pyraoanthum Jacq. Sfctejnberg& Wahl ,196 6 S. rostratvm Dun. Kotila,194 9 S. simile F.Muell . Driver,195 7 S. sambuainum Rydb. Niederhauser& Mills ,195 3 S, stolonifevwn Schlechte tBeh e Niederhauser& Mills , 1953 S. tuberosum L. Montagne,184 5 S. villosum (L.)Lam . Sztejnberg& Wahl ,196 6 S. xanthaoarpum Schrad.& Wend . Russel,196 9

Lyoopersioon Mill. L. chilense Dun. Richards& Barratt ,194 6 L. esoutentum Mill. Payen, 1847 L. hirsutum Humb. Richards& Barratt ,194 6 L. peruvianum (L.)Mill . Richards& Barratt ,194 6 L. pimpinellifolium (Jusl.)Mill . Richards& Barratt ,194 6 Solanaceous speciesno tbelongin gt oth egener a Solanum and Lyaopersicon

Anthocerais visaosa R.Br. Berkeley, 1846(cf rD eBary ,1876 ) Datura metel L. Sztejnberg& Wahl ,196 6 Datura stramonium L. Sztejnberg& Wahl , 1966 Hyosayamus aureus L. Sztejnberg& Wahl , 1966 Hyoayamus niger L. Reddick,192 8 Lyoium halimifolium Mill. Reddick,192 8 Lyoium turoomanniaum Turcz. Reddick,192 8 Mandragora offioinarum L. Sztejnberg& Wahl ,196 6 Mirabilis jalapa L. Romero& Fourton ,196 3 Nicotiana glauaa Grah. Sztejnberg& Wahl , 1966 Petunia hybrida Hort. Hirst& Moore , 1957;Sztejnber g& Wahl , 1966 Physalis alkengi L. Reddick,192 8 Physalis angulata L. Peterson,194 7 Salpiglossis sp. Peterson,194 7 Schizanthus grahami N.H. Reddick, 1928;D eBary ,187 6 Sahizanthus pinnatus R.& P . Peterson,194 7 Withiana somnifera L. Sztejnberg& Wahl , 1966 13 3.2.2 The tomato

Thegenu s Lyaopersiaon Mill,i sclosel yrelate dt oth egenu s Solanum L. (Rick,1960) .Th ecentr eo forigi no fth egenu s Lyaopersiaon issough ti nth e coastalregio no fSout hAmeric abetwee nth eAnde smountain san dth ePacifi c Ocean,fro mth eequato rt oabou t30 °S.L. ,an di nth eGalapago s Islands (Luckwill,1943 ;Rick ,1960) .A secon dcentr ei ssuppose dt ob ei nMexico , inth eVeracru zPuebl oare ai nparticula r (Jenkins,1947) .Th etw o Lyaopersi­ aon speciesi nthi scentre ,whic hi sprobabl ya secondar ycentre ,ar e L. esoulentum Mill,(cultivate dtomatoes )an d L. pvmpinellifolium (Jusl.)Mill , (curranttomatoes) .Th elatte ri ssometime sconsidere dt ob esubspecie so f L. esaulentum. Allspecie so f Lyaopersiaon arerathe rsusceptibl et oP . in- festans (Richards& Barratt ,1946 ;Alexande r& Hoover , 1955). Inth eSout hAmerica ncentr eo forigi no ftomatoes ,lat ebligh twa sno t knownbefor e194 7(Co x& Large ,1960) ,an dther eha sbee nn oselectio npres ­ surefo rresistanc et oP . infestans untiltha ttime .Th efirs tencounte rbe ­ tweenth egenu s Lyaopersiaon andP . infestans musthav etake nplac ei nMexico , whereth etomat owa simporte di npre-Columbia ntime s (Jenkins, 1947),Mexic o supposedlybein ga centr eo forigi no fP . infestans (seeSectio n 3.1).I nev ­ olutionaryperspective ,th eperio do fconfrontatio ni sshort .Thi s shortness maya tleas tpartiall yexplai nth erarit yo fresistanc et oP . infestans in thegenu s Lyaopersiaon. Theline so fL. pimpinellifoliwn withlat ebligh tre ­ sistanceofte nsee mt ob eo fMexica norigi n (Gallegly, 1952).

2.2.3 The potato

The potato is the most common host of P. infestans in Europe. There is much literature available on this subject, and it will not be discussed here Some aspects of the relations between the potato and P. infestans, relevant to the subject of late blight on tomatoes, are mentioned in 2.3.4, 2.3.5, 3.4.2, and 3.4.3.

3.2.4 Solanum dulcamara L.

rt»JU !Tl-!° S' duloamara Plants were grown from cuttings taken from 12 H TT l0Cati0ilS ^ ^ M**N»«*ood of Wageningen. They were iT " : Pl0t SitUatSd * m e*P--ntal field used for the test­ ing of potatoes for resistance to P. infestans. ^ potatQ plQts were ^ 14 latedwit hth epathotype s 1,2,3-p, 1,3,4-p,an d1,4,10- p (seeSectio n 3.4.1). Thoughth eweathe rcondition swer eunfavourabl efo rlat ebligh tdevelopment , thefoliag eo fsusceptibl e cultivars like 'Bintje*an d'Eersteling 1 werede ­ stroyedcompletel yb ylat ebligh twithi na mont hafte rinoculation .Onl ya fewlesion swer e foundo nplant so f S. duloamara. Theinfecte d leavesdroppe d withina fe wday s afterth efirs tobservatio no fth e lesions. In1972 ,6 S. dulcamara plantswer egrow ni na nexperimenta lfiel duse d totes ttomatoe san dpotatoe sfo rresistanc et oP . infestans. Thedevelopmen t oflat ebligh twa sfavoure db ysprinkle r irrigation (seeSectio n 5.3.3).Fo r inoculation,th epathotype s 1,2,3-p, 1,4,10-p,an d2,4-p/1- twer eused .Th e conditionsfo rlat ebligh t developmentwer equit efavourable ,an da larg ea - mounto finoculu mwa savailabl e duringth eepidemic so npotatoe san dtomatoes , butonl ya fe wlesion swer efoun do nth eleave so fS . duloamara. Theleaves , onwhic hlesion swer e found,droppe dwithi na fe wday safte rth efirs tobser ­ vationo fth elesions .N osporulatio nwa sobserve do nth elesions . Apparently,th e S. duloamara plantsteste dwer ever y resistantt oth e pathotypesuse d (seeals oSectio n 3.4.8).

3.2.5 Solanaoeous hosts, excluding potatoes and tomatoes, with potential survival value to the fungus in crop-free periods

Someperennia l solanaceousplant sprovid eP . infestans witha nopportuni ­ tyt osurviv eperiod swithou tpotatoe so rtomatoe si nth efield .IXirin gcrop - freeperiods ,P . infestans has beenfoun do nS. aviculare Forst.an d S. lacineatum Ait(Driver , 1957).I nKenya ,th ega pbetwee ntw osuccessiv e crops isa shor tone ,a stw ocrop spe ryea rar egrown .I nNe wZealan d (Auckland), thesituatio ni ssomewha tmor e complicated,a sn ocrop-fre eperio di spresent . Bothcountrie sar echaracterize db ya climat ewithou tfrost . InIsrael ,wil dSolanacea ean dcultivate dplant so fthi sfamily ,excep t potatoesan dtomatoes ,d ono tsuppor tth epropagatio nan ddisseminatio no fth e pathogeneithe ri nwinte ro ri nsumme r (Sztejnberg& Wahl , 1966). Peterson (1947)concluded ,tha tther ei sn oevidenc eo foverwinterin go fP . infestans onperennia lSolanacea ei nLon g Island,U.S.A .Accordin gt oCo x& Larg e (1960), therei sn oevidenc efo rth eoverwinterin go fP . infestans inan ysolanaceou s planti npart so fth eUnite dState smarke db ycol dwinters .Accordingly ,i ti s unlikely thatP . infestans overwintersi nwil dsolanaceou splant si nthos e partso fEurope ,wher efrequen tand/o rprolonge dperiod swit h temperatures below0 QCoccur .

15 3.2.6 Phaaelia tanaaetifolia, anew host species of P. infestans

In 1972, Phaaelia tanaaetifolia Benth (fam. Hydrophyllaceae) was grown for apicultural purposes on a strip of land adjacent to an experimental plot, where potato and tomato cultivars were tested for late blight resistance. Overhead irrigation by sprinkling was applied to stimulate the development of P. infestans. When the weather conditions were favourable for late blight development, the late blight fungus was repeatedly found to sporulate on leaves and inflorescences of P. tanaaetifolia. Late blight infection was mark­ ed by necrotic reactions, and the infected leaves turned darkly red. The late blight isolates obtained from the infected tissues were only slightly patho­ genic to leaves of P. tanaaetifolia taken from field grown plants, and sub­ sequently tested in the laboratory by inoculating and incubating the leaves as described in 4.4. Though the leaves were covered with small necrotic spots (up to 2 mm), no sporulation was observed. The disease was never seriousi n the field, and, when the plants grew older, late blight was only sporadical­ ly found.

3.2.7 Development ofP. infestans on Datura stramonium, Mirabilis jalapa, and Schizanthus pinnatus

Introduction In 1971, an experiment was designed to investigate the possibi­ lity of stimulating the development oflat e blight epidemics by overhead irri­ gation. There were three fields with different sprinkling regimes, and on each fleld there were plots with tomatoes, potatoes, and the ornamentals

Datura stramonium L. (fam. Solanaceae), Mirabilis dalapa L. (fam. Nyctagina- ceae>, and Schizanthus pinnatus R. &p . (fam. Scrophulariaceae). The reaction of these ornamentals to late blight under the regime with the most frequent sprinkling is discussed below.

Its^ZTrT8 ^ £leld Pl0t ^ °mamentals rained two rows of r ws ofT 7 DatUm PlantS' °ne r°W o£ 8 ««««* Pl-ts andthre e L 1 ThUS Pl3ntS (2° Pl3ntS Per m)' *» SP- between the rows was h io and 21 Hnlen8th ^ ' *' ^^ t0°k Place at night between La 22 ^ °neSPrlnklin g ^^ °f 1S0 » P" * »*. *« ^ 710 .6* 6 T T°n "I night> *" P°tat0eS ^ ^ *«*** ™ - -toes were ;S;Pdt;n; ? Z ^7'°^ " *" ^^ /KU6*23- The day ofinoculatio n will be andi- 16 cateda sth ezer oda yo fthi sexperiment .Th eornamental swer e inspecteddail y forlat ebligh t symptoms.Lesion ssuspecte dt ob ecause db yP . infestans were collectedfo rreisolatio no fth efungu so npotat otube rslice s (seeSectio n3.3.2 )

Mirabilis jalopa Manybrow ncoloure d spotswit hdiameter su pt o2 c mhav e beenfoun do nth eleave so fth eMirabili splants ,bu tattempt st oreisolat e P. infestans fromthes e spotswer eunsuccessful .

Datura stramonium On Datura, thelesion scause db yP . infestans werefe w but large.Th efirs ttw olesion swer e observedo nda y34 ,an dda y50 ,respec ­ tively.Whe nth eweathe r conditionswer e favourablefo rlat ebligh tdevelop ­ ment,th elesion sexpande dquickly ,an dabundan tsporulatio nwa svisibl eo n apparentlyhealth y tissue surroundingth elesions .P . infestans wasfrequent ­ lyisolate d from infectedtissue .Whe nth eweathe rwa sto odr yfo rth ede ­ velopmento flat eblight ,th einfecte dtissu edried ,becam ebrittle ,an d des- integrated.Ultimately ,onl ya nangula rhol ewa sleft ,an dn ofungu scoul db e isolatedfro mth esurroundin g tissue.Th ediseas e severityneve r exceeded 51.

Sohizanthus pinnatus Onda y26 ,th efirs tlesion sdu et olat ebligh twer e foundo n Sohizanthus .O nda y56 ,95 1o fth efoliag ewa sdestroye db yP . in­ festans (Fig. 1).A heav y infectiono fth e stalkscause dlodgin go fth e plants. Sporulationwa s abundanto nleave san dstalks ,almos twithou tpreceedin g dis­ colorationo fth ehos t tissues.Th eapparen t infectionrat ewa s0.23 .Althoug h theprimar y inoculummus thav ecom efro mth elat ebligh tdevelopin go nth e potatoplots ,th emajo rpar to fth einoculu m seemst ohav eoriginate dfro m latebligh tdevelopin gwithi nth ecrop ,a sevidence db yth e presenceo fabun ­ dantsporulatio no n Sohizanthus.

fraction of foliage affected in logit units IT

Fig. 1.Sohizanthus pinnatus, Wageningen. Progresso fa Phytophthora infestans epidemic -I- ina fiel d experimentwit hartificia linocu ­ 30 40 50 60 days after field inoculation lationan doverhea dirrigation .

17 Conclusions Themateria lo f Mirabilis jalapa used inthi s testwa sresist ­ antt oth eP . infestans pathotypeused .Th e Datura stramonium plants tested hereshowe da considerabl e degreeo fresistanc e toth e latebligh t pathotype used.I n Datura stramonium, tworesistanc emechanism s seem to operate:re ­ sistancet oinfection ,an dresistanc eb y eliminationo f the fungusunde rcon ­ ditionsunfavourabl et oth elat ebligh tfungus . Sehizanthus pinnatus showed tob esusceptible .

3.3 METHODSO F ISOLATIONAN DCULTUR E

Artificial media Manyartificia lmedi asuitabl efo rth ecultivatio n ofP . infestans areknown .Som ear ecompletel y synthetic (Wilde, 1961), othersar e partiallycompose do fnatura lmaterial s likerye ,oat so rlim abean s (Snieszko etal. ,194 7;• Hodgso n& Grainger ,1964) . Onmos t ofthes eP . infestans grows well.Th efungu sloose sit spathogenicit y afterprolonge d cultureo n artifi­ cialmedi a (Jinks& Grindle ,1963 )partl yo rcompletely .Th eus eo f artifi­ cialmedi at ogro wP . infestans forth epurpos eo fpathogenicit y tests should bediscouraged .

Natural substrates Mosto fth elat ebligh t isolateswer e cultivated accord­ ingt oa metho ddevelope do nth e Institutefo rPhytopathologica l Research (IPO),Wageningen .Ever yweek ,non-sterilize d sliceso fwashe d tuberso f the potatocv .Bintj ewer einoculated ,b ypickin gu pwit ha needl e sememyceliu m fromslice sinoculate dth ewee kbefore ,an dpassin g thisove r the surface offres hslices .Th einoculate dslices ,store di n9 c mpetri-dishes ,wer e kepta t15° Ci nth edark .Som eisolate spathogeni ct otomatoe swer e growing poorlyo npotat oslices ,an ddi dno tsporulat ea tall .Thes e isolateswer e growno ntomat oleaflet sstore di ntranslucen t shoe cases.Th e leafletswer e irradiatedb ya singl e4 0W fluorescen ttub e (PhilipsT L4 0W/33 ) during 16h perday .Th eleaflet swer emounte di nsmal l 'bricks-o fa spong y substance onurea-formaldehy dbas emanufacture d forth epurpos eo fflora l arrangement (trademark 'Oasis',commercialize db yV.L .Smither sA/S , Ganlrfse- MaLmtfv e- Danmark).Ever ywee ka fres hleafle twa splace d inclos e contactwit h theol d one,whereupo nth efungu sinvade dth ene wleaflet .Th eus e ofver yyoun gleaf ­ letso rleaflet so ftomat oplant syounge rtha n6 week s isno t recommended, becauseo fthei rsusceptibilit yt obacteria lrot .

isolation Theisolatio no fP . infestans frQminfecte d ^^ ^ ^.^

18 byplacin g excisedpart so fth einfecte d tissueunde rpotat o slicesi npetri - dishes.Th epetri-dishe swer e storeda t15° Ci nth edark .Th efungu s grewint o thetube r tissue,an dwithi na wee ki tsporulate do nth euppe r surfaceo fth e slices.Whe nth einoculate d fungus grewpoorl yo npotat o slices,a plu go fth e infected tuber tissueo ra par to fth eorigina ltissu e (if still available) wasuse dt oinoculat ea gree ntomat ofruit .Fo rtha tpurpose ,th einfecte d tissuewa stransferre dt oa dro po fdeionize dwater, whic hwa splace do nth e fracture resulting from the removalo fth ecalyx .Afte r2 o r3 days ,a smal l stripo fth eepidermi swa s removed from thefrui tb ymean so fa razor ,t o facilitate fructification.Accordin gt oth e pathogenicityo fth eisolat et o tomatoes,sporulatio n couldb eobserve dwithi n5 t o1 2days .

3.4 VARIABILITY INPATHOGENICIT Y

3.4,1 Terminology

An attempti smad et oappl y and/ort oadjus tth e terminologyo fRobinso n (1969)t oth eavailabl e datao npathogenicit yo fP. infestans tovariou s host species.

The deme system Thedem e system,devise db yGilmou r& Heslop-Harriso n (1954), isa syste mo fclassificatio nt odenot ea se to findividual swithi na speci ­ fied taxonhavin ga tleas ton echaracteristi ci nccmon .Th esuffi xdem emus t beuse dwit ha prefi xt odescrib e thisparticula r characteristic.Fo rth epur ­ poseo fdiseas e resistance terminology,Robinso n (1969)propose st oreserv e thesuffi x demefo rth ehost ,an dt ous ea paralle l systemwit hth esuffi x typefo rth epathogen .Bot hsuffixe smus tb ecombinate dwit hth eprefi x pathj. InRobinson' s terminology,a pathodem ei sa populatio no fa hos to nwhic hal l individuals havea particula r charactero fresistanc ei nccxnmon . Unfortunately,th eter m 'host'ma ycaus e confusion,becaus e thxster m sometimes referst oa susceptibl e individualo rsusceptibl e cultivar and / ,i»c CPTIUS orhighe runi to fclassification) , sometimest oa plan t taxon (species,genus ,o riu- g ., ^i w> rnnfusinei nth ecombinatio n 'hostrang e, Furthermore,th eter m 'host'ca nb econtusin g m uic asa hos t rangei slittl emor e thana se to fcultivar so ra se to fspecie s Placed together accordingt oa phytopathologica lcriterium .Therefor e iti s convenientt oadher e closelyt oth eorigina ldem edefinition ,an dt odefin e apathodem ea sa se to findividual swithi na specifie d taxonhavin ga tleas t one charactero fresistanc ei ncorcnon .Tn epathodem ean dcorrespondin g patho-

19 Table 3. Phytophthora infestans on potatoes and tomatoes. Skeleton survey of pathodemes and pathotypes. Three theoretically possible pathodemes and corre­ sponding pathotypes are represented. A late blight pathotype only pathogenic to tomatoes is not yet known.

Hosts Taxon Solanaceae P. infestans

S, tuberosum p-pathodeme p-pathotype L. esaulentum t-pathodeme t-pathotype S, tuberosum and L. esaulentum p/t-pathodeme p/t-pathotype p = potato t= tomato

typeca nb edescribe db ya lette ro rcombinatio no fletters ,denotin gth e hostrang e (Table 3). Anydesirabl e subdivision canb emad e for everypatho - demeo rpathotype ,an dindicate db ya nappropriat e symbol (Table 4).Fo rthi s purpose,th efollowin g termsar e introduced:

Uniform (U) A 'uniformpathodeme 'i scharacterize db y'unifor m resistance'. •Uniformresistance 'i sresistance ,tha tact sequall yt oal lpathotype s test­ ed.Th eter m 'uniform resistance'ha sbee npropose db yVa nde rPlan k (1969) toreplac e theter m 'horizontal resistance' introduced earlier (Vande rPlank , 1963). ^A 'uniformpathotype 'i sa pathotyp e characterizedb y'unifor m pathoge­ nicity'. 'Unifor mpathogenicity 'act sequall yt oal lpathodemes , characterized by 'uniformresistance' .Th eter m 'uniform pathogenicity' replacesth eter m •horizontalpathogenicity 'introduce db yRobinso n (1969).

Differential (D) A 'differentialpathodeme 'i scharacterize db y'differen ­ tialresistance' . -Differentialresistance 'i sresistance , that doesno tac t equallyt oal lpathotype s tested,bu tshow sdifferentia l interaction.Th e term 'differentialresistance 'i sintroduce d (Vande rPlank , 1969)t oreplac e theter m 'verticalresistance ' (Vande rPlank , 1963). A 'differentialpathotype 'i scharacterize db y'differentia lpathogeni ­ city. 'Differentialpathogenicity 'i spathogenicity , that doesno tac te - quallyt opathodeme s characterizedb y'differentia lresistance' .Th eter m differentialpathogenicity - replacesth eter m 'verticalpathogenicity 'in ­ troducedb yRobinso n (1969).

20 Table4 . Phytophthora infestans ontomatoes .Theoretica lschem eo fpathotype s characterized according tohos trang ean dtyp eo fresistanc epe rhost .Th e symbol+ stand sfo rpathogenicit y toth ehos tresistanc etyp eindicate di n therespectiv ecolum nheads .No tal lo fthes etheoreticall ypossibl epatho ­ typeshav ebee nfound .

Hosts Pathotypes S. tuberosum L. esculentum D U D U

+ D.p-pathotype U.p-pathotype + Two-dim.p-pathotype + + + + D,p/D.t-pathotyp e D.p/U.t-pathotyp e + + D.p/Two-dim.t-pathotype + + + U.p/D.t-pathotype + + U.p/U.t-pathotyp e + + U.p/Two-dim.t-pathotype + + + Two-dim.p/D.t-pathotype + + + Two-dim.p/U.t-pathotype + + + Two-dim.p/two-dim.t-pathotype + + + +

D - Differential U - Uniform

Two-dimensional (Two-dun.) A -two-dimensionalpathodeme 'i scharacterize d by -two-dimensionalresistance' .Th eter m -two-dimensional,resistance'wa s introducedb yZadok s (1972)t oindicat eth esituatio nwhe ndifferentia lan d uniformresistanc ebot har epresen ti na singl ehos tgenotype . A -two-dimensionalpathotype 'i scharacterize db y -two-dimensionalpa ­ thogenicity'.Ther ei sa unifor mactio nagains tunifor mresistance ,an da differentialactio nagains tdifferentia lresistance .

3.4.2 Differential pathogenicity to various hosts

Potato Themos tstrikin gobservatio no nth evariabilit yi npathogenicit yo f P. infestans concernsth esuccessiv eappearanc eo fpathotype scapabl et oover ­ cometh eresistanc eo fnewl yintroduce dpotat ocultivars .Thi sresistance-i s usuallycharacterize db ya hypersensitiv ereaction ,controlle db ysingl edo ­ minantgene soriginatin gfro m Solans demissum. Thesegene sar ecalle d Rgenes . J* „^ii Pthi stvo eo fresistance .I n1932 , In1920 ,Germa nbreeder sstarte dt outiliz etm 7styp e , J • „i .«tha tcoul dattac ka cultiva rcarry - thefirs trepor twa spublishe do nisolate cstna !lttcoux u

21 ing an R gene (Schick, 1932). From 1932 till now, many cultivars originally unaffected because of the presence of one or more R genes became attacked by P. infestans in the long run. To-day, 11 R genes have been identified. Patho- types matching these 11 R genes singly or in combinations have been found (Malcolmson & Black, 1966). 211 differential late blight pathotypes are pos­ sible, and the 1,2,3,4,5,6,7,8,9,10,11-p-pathotype can match all R genes known. This pathotype has not yet been found, but Malcolmson (1969) identi­ fied two late blight pathotypes with differential pathogenicity, 1,2,3,4,5, 6,7,8,9,10-p and 1,2,3,4,5,6,7,10,11-p respectively. Apparently, the contin­ ued use of R gene resistance is not the ultimate solution of the problem of late blight control.

Tomato Tomato breeders met with a similar problem after the introduction of the Phi gene for hypersensitivity, that has been found in an ornamental toma­ to of the species Lyoopersioon pimpinelli folium. This gene, originally called TRi (Gallegly & Marvel, 1955), provided resistance to late blight at the time of introduction into the breeding program. During the execution of the pro­ gram, a new and compatible late blight pathotype appeared, and caused consi­ derable damage (Conover & Walter, 1953; Gallegly & Marvel, 1955).

Tomato& potato Differential pathogenicity to tomatoes has been found to be independent of differential pathogenicity to potatoes as determined by means

of pathodemes carrying the genes Phu Ru R2, R3} and i?4. (Wilson & Gallegly, 1955; Kedar et al., 1959; Kishy, 1962).

Other hosts There have been no investigations into the differential patho­ genicity to other solanaceous species. A few observations are reported, how­ ever, on the existence of late blight isolates, collected from potatoes, which differed in the ability to infect plants of some species like Solanwn incanum (Nattrass & Ryan, 1951), S. villoswn and Withiana sormifera (Sztejn- berg &Wahl , 1966).

3.4.3 Variability in pathogenicity to potatoes and tomatoes

In early publications, no reference has been made to a special relation between isolates of P. infestans from potatoes and those from tomatoes (Payen, 1847; Thaxter, 1891; Clinton, 1903; Smith, 1906). In later papers, references to a kind of biological specialization can be found (Wiltshire, 1915; Giddings

22 &Berg ,1919 ;Berg , 1926;Small ,1938) . Isolateso fP . infestans collected frompotatoe swer e saidt oattac ktomat oleave smildl y atmost .Isolate so fP . infestans collected fromtomatoes ,o nth econtrary ,severel y infectedtomat o leaves.Thi sdifferenc ebetwee npotat o andtomat oisolate swit hrespec tt o thepathogenicit y totomatoe swa s foundt ob establ edurin ga tleas ton eyear , whenisolate so fP . infestans (pathogenict otomatoes )wer e growncontinuous ­ lyi npotat otuber s (Berg,1926 ;Rtider ,193S ;Small ,1938) . In1940 ,Mill sdescribe d aphenomeno nwhic hh ecalle dadaptativ epara ­ sitism.Startin gwit hmonozoospor e cultureso fP . infestans originating from potatoes,h emad e severalpassage s throughtomat ofoliage .H enotice dtha t thepathogenicit y ofisolate sgraduall y increaseddurin ga sequenc eo fpas ­ sages.Afte r 7passages ,n odifference s couldb esee nbetwee nth e 'transfers' andth eisolate s ofP . infestans originatingfro mtomatoes .Th echang em pa­ thogenicityprove d tob e stabledurin ghal fa yea ro fculturin g inpotat otu ­ bers.Th epathogenicit y topotat ofoliag ewa sno taffecte db y thesuccessiv e passagesthroug htomat o leaves.Graha me t al. (1961)obtaine dsimila rre ­ sults.D eBruy n (1952),too ,mad epassage sthroug htomat ofoliag ewit hmono ­ zoosporeculture so f isolatesoriginatin g frompotatoes .Sh efoun dtha tth e acquiredpathogenicit y totomatoe swa sstable ,whe nsh emad epassage swit hth e changedisolate sthroug hleave so rtuber so fclone so f Solanm tuberose Men shemad epassage s throughleave so rtuber so fclone so fresistan tderivative s fromth ecros s S. demises * 8. tuberosum orfro mthei rsuccessiv eback - crossest o S. tuberosum, shenotice d thatth eacquire dpathogenicit y toto ­ matoescoul db e lost.Conversely ,th e acquiredpathogenicit yt o R genere ­ sistantpotat o clonescoul db e lostb y successivepassage sthroug htomat o

foliage. Kishy (1962)collecte d isolateso fP . infestans frompotat ocrop si n thefields .H e subdivided theisolate sint othre egroup saccordin gt othei r pathogenicity toth etomat o cv.Ponderosa :-potat otype' ,'intermediat etyp e, , •,• v. * mrvWate andsever eattack .B y and -tomatotype 'wit hrespective! ,slight , "*£££ whichwer emoderate - l -atingpassage s throughtomatoleave s he ^ evivale„t lypathogeni c totomatoe s atth estart ,a leve l pa s ' totha to fth eto,at otype .Kish ydi dno t succeedi nchang e P" ° ™"

into tM0 types,a sth epotat otyp eha ddecke d afterfou rpa« , • It isevfdent ,tha t thepathogenicit y totomatoe so f, »'" ° lates,originatin g ft. potatoes,ca nb echange db y«an s of £"£<£* tonatofoliage .Mos t reportso nthi sphenomeno nagre e rntw or pect the «. c; qstable ,an dth epathogenicit y topotatoe s acquiredpathogenicit y totomatoe si sstaDie , spores.The yinoculate dwit hpopulation so fth efungus ,population s thatwer e intendedt ob egeneticall yuniform ,bu t asmutation s canoccu ri nth eperio d neededt oproduc eth emas sinoculum ,ther e isn o certainty that thesepopu ­ lationswer estil lunifor m atth emomen to fmas sinoculation . Consequently, theobserve dchange si npathogenicit ycoul dhav ebee n changesa tth epopula ­ tionlevel .A sn odetaile dinvestigation shav ebee nreporte dnothin g isknow n ofth efrequenc yno ro fth enatur eo fchange .

3.4.4 The appearance of a pathotype highly pathogenic to tomatoes in the field

Introduction In1970 ,a fiel dexperimen twa sdesigne dwit hth epurpos et o collectlat ebligh tisolate spathogeni ct otomatoes .Thes epathotype swer e neededfo rinvestigation so nresistanc e oftomatoe sagains t lateblight .

Materials and methods Foursmal ltomat oplot swer eplante d sideb ysid et o potatoplot sm anexperimenta lfiel ddesigne dfo rth epurpos e oftestin g the tied resistanc eo fpotat Qcult .carsagains tp > ^ Thnje 4-0 wereplanted ,eac hwit h2 row so f8 Moneymake rplants .Tn e fourth plot Pl3nte d 16 M neymakePlantSr 8 lantoSf lin eW Va 700700,an 2d4 plant T,so feac *h *o fth e°followin g lines':Ottaw P a30 *, Ottaw » 'a 31,Ottaw a 24 33,Ne wHampshir e Surecrop,Jamaic aCherry ,Rockingham ,an dNort hCarolin a 1951-315-N.Th eline swer e distributeda trando move rth eplot .Apar tfro m Moneymaker,al lthes e lineswer e characterizedb yresistanc et oO-t-pathotype s ofP . infestans. On70.07.0 7 (day zero),th epotat oplot san dal l1 6plant so fon eo fth e 3smalle rtomat oplot swer e inoculatedwit ha mixtur eo fth epathotype s 1,2, 3-p,1,3,4-p ,an d1,4,10- pb ysprayin ga suspensio no f100 0zoospore spe rm l ofeac hpathotype .Isolate swer e collected fromtomatoes ,wheneve r lesions suspectedt ob ecause db yP . infestans werepresent .Th eisolate swer eteste d forpathogenicit yt otomatoe sb ysprayin ga suspensio no fzoosporangi a (10,000 perml )ove r leaveso f8 t o1 0week sol dMoneymake rplants .Th eleave swer e inoculateda sdescribe di n4.4 .Th ecriterio nfo rpathogenicit yP wa sth e per- centageaffecte d leaf areao nda y7 .

Results Inth eperio d fromda y6t o74 ,onl ysmal lan dnecroti c lesionswer e foundo nth eleave so fth ecv .Moneymaker ;th eothe rtomatoe swer e apparently freefro mattack .Fruit so fal ltomat oplant swer eseriousl yaffected .Iso ­ latescollecte d from fruitsan dleave swer e onlyslightl ypathogen yt oto ­ matoes,P<10 .O nda y75 ,3 water-soake d lesionswer efoun do nleave so fa Moneymakerplan ti nth elarges t tomatoplot ;th enex tda yth elesion san dap ­ parentlyunaffecte d surrounding tissueswer e coveredwit hsporophore s Ref­ latestake nfro mon eo fthes e lesionsan dteste di nth elaborator yproduce d

P- 100 .Thes e isolatesbelonge dt oth e2,4-p/1-t- Pathotype.O nda y8 two focio fheav y latebligh t attackwer epresen ti nth e largesttomat oplot .an d onda y112,l e severityo fattac ko ncv .Moneymake rwa sS>95 .Fro mth eothe r linesi nthi splot ,Jamaic aCherry ,Ne wHampshir eSurecrop ,an d ^^ allcarryin gth e* , genefo rresistanc et olat eblight ,wer enearl y - 1» T 4-uro-rni-in awer e themos tresistant , lyaffecte da sMoneymaker .H.V a70 0an dNort hCarolin a»er e •ada y „2, S<50. rTthe othert-at oplots ,n oseriou sattac k ««£•£»• Oneisolate ,Electe d fromW.V ,70 0 (isolateI 7023 ,an dbelon g to* .p a

thotype 2,4-P/,-tha sbee nuse dfo ral lresistanc etest sdescrrbe do nthr s paper.

• - * T-PIsolat e was collected from a to- Conclusion On day 112 of the experiment a reisol ^^ ^ ^ «o plant, which was ^ *^^~ **„,, differed frcm the gene. This reisolate, belonging to the 2,4 p/ P ^ ^^ ^

initial pathotypes not only in pathogenicity ^iti3Lny introduced in differential pathogenicity to potatoes from all three init

25 pathotypes. It was remarkable that this new pathotype had not spread to the other plots of the experimental field.

Discussion The origin of the 2,4-p/1-t-pathotype is not clear. There are two possibilities: it came from elsewhere, or it originated in the experimental field. Concerning the first possibility, it must be remarked that (1), the 2,4-pTpathotype is always rare in the Netherlands, (2), in 1970 the weather conditions were unfavourable for late blight attack, and (3), no late blight on tomatoes was reported in 1970. Consequently, the chance, that the new pa­ thotype came from elsewhere, is considered to be extremely small. The only reasonable conclusion is that the new pathotype originated in the field from the initial inoculum.

3.4.S A field experiment to demonstrate the increase in pathogenicity to tomatoes

Introduction In laboratory tests, a gradual increase of the pathogenicity to tomatoes during serial passages experiments has been reported (Mills, 1940; de Bruyn, 1952; Graham et al., 1961; Kishy, 1962). It was supposed that the same gradual increase of pathogenicity takes place in the field. This hypo­ thesis was tested in a field experiment.

Materials and methods In 1971, 3-tomato plots of 30 m2 were grown next to 3 potato plots of 150 m*. Cn 71.06.16, 80 plants per potato plot were inocu­ lated with a suspension of 500 zoospores per ml of the 1,2,3-p-pathotype of P. infestans. In total 6500 ml suspension was applied to 240 plants by means of a knapsack mistblower. The pathotype used is seldom found in the Nether­ lands, and is supposed to have been virtually absent in 1971 because the weather conditions were unfavourable to late blight. Consequently, all re- isolates collected from this experimental field and reacting like the 1,2,3- p-pathotype to potatoes are considered to have descended from the original inoculum.

After the first observation of sporophores on potato leaves, the tomato Plots were examined daily for the presence of late blight. Once the late blight had appeared on the tomato plants, reisolates from tomato plants were collected at regular intervals. The pathotypes of the reisolation were de­

termined ln the laboratory using a differential set consisting of the potato

itterentials Bu R2l i?3j md ^ ^ ^ tmatQ ^ Moneymaker> ^ ^

26 test,tw oleaflet s from eacho fth epotat odifferential san dtw oleave so f6 weeksol dtomat oplant swer eused .Thes e detached leaveswer e inoculatedb y sprayinga suspensio no f500 0zoosporangi ape rm ltil la continuou sfil mo f moisturewa svisible .Th epathogenicit yt otomatoes ,P ,o fth ereisolate swa s evaluatedb yestimatin gth epercentag e diseased leafare ao nda y7 afte rin ­ oculation.

Results Onda y7 afte r inoculationo fth epotat oplots ,th efirs t sporophores werenotice do nth epotat oplant si nth einoculate d rows.O nda y43 ,th elat e blightepidemi co npotatoe s camet oa sto pa shardl yan ypotat ofoliag ewa s left.O nda y37 ,th efirs t reisolateso fP . infestans couldb ecollecte d from thetomat oplots ,an do nda y5 1a smal lfocu so flat ebligh twa snotice di n oneo fth etomat oplots .O nda y63 ,a secon dfocu swa sfound ,and ,fro mtha t dayonward ,lat ebligh t spreadmor eo rles sgenerall y throughth etomat oplots . Thirtyo fth ereisolate s collected fromth etomat oplot swer eteste dfo r differentialpathogenicit yt opotatoes ;2 9belonge dt oth e1,2,3-p-pathotype , andon et oth e0-p-pathotype .Thirt ythre eresiolate s collectedfro mth eto ­ matoplot swer e testedfo rpathogenicit yt otomatoes .Wit hth eadvanc eo f timeth epathogenicit yt otomatoe s increased (Fig. 2).Th evarianc ei npatho ­ genicityt otomatoe s also increasedwit htime .A nisolat ewit hP = 10 0wa s founda tabou tda y100 .Th esingl e isolateo fth e0-p-pathotyp e collectedo n day9 4wa sonl yweakl ypathogeni ct otomatoe s (P= 25) , andmoderatel ypatho ­ genict opotatoe s (P=60) . Thereproducibilit y ofth elaborator y testswa schecked .Fo rthi spur ­ pose,5 isolate s collectedo ndifferen t datawer eteste dtwic ewit ha one - veekinterva lbetwee nth efirs tan dsecon dtes tfo reac hisolate .Thi styp e

o „• ' 9 P infestans ontomatoe s cv.Money -

! mcity o» «" h. int „val b.tmen field mocuj.^ inoculationi s •! ,(abSCiST Sh dotrepresent son ereisolat e E « dayzero b ). k*f *" enPt reisolates fromlea f -j—j—-S-TT-T. . Th?:seblac uerkdo thr:pnt represend:rt s reisolate^ s fro^.m fruits^ . days of reisolation tlSSUeS, tue.«F«- 27 Table5 .Assessmen t ofth epathogenicit y oflat ebligh t isolates, collected from tomatoplant si nth efield ,t otomat o leaves inlaboratory .Fo rmethod s see text.Th epathogenicit yP i sexpresse d asth epercentag e oflea f area affectedb ylat ebligh t7 day s after inoculation.Longitudina l replication.

Isolatenumbe r Sampling dates P A B

7108 71.08.06 3 0 7116 71.09.13 80 75 7122 71.09.16 5 5 7135 71.09.23 30 50 7143 71.10.11 100 100

A= Firs t replication. B= Secon d replication,on ewee k after thefirs t replication.

ofreplicatio nmigh tb ecalle d longitudinal replication,a scontraste d toth e usual simultaneouso rcross-sectiona l replication (Zadoks, 1972). Thediffer ­ encebetwee nth eresult so fth etw otes t serieswa ssmal lan dno tsignifi ­ cant (p;>0.20;Table s5 an d 6).

Conclusion Inspit eo fth elarg e amounto finoculu mbuildin g upi nth epo ­ tatoplot sfro mda y7 t oda y43 ,n oisolate so flat ebligh t couldb ecollec ­ tedfro mth etomat oplot sbefor eda y37 .Apparently ,th e1,2,3-p-pathotyp e used forfiel dinoculation ,whic h originated frompotatoes ,wa snon-pathogeni co r weaklypathogeni ct otomatoe sunde r fieldconditions . As (1)th eorigina l 1,2,3-p-pathotype wasa tmos tweakl ypathogeni ct o tomatoes, (2)

Table6 .Analysi so fvarianc e ofth edat a from Table5 .

Sourceo f variance Sun1 o f squares df Variance Variance ratio Probabi Isolates 15, 196 4 .001 Betweentest s 3,779 83 p<0, 15 1 Residual 15 <1 p>0 .25 182 4 45 Total 15,,39 4 9

28 From day 37 onwards,ther ewa sa 6 5day sperio ddurin gwhic ha nincreas e ofth epathogenicit y totomatoe s ofth e funguspopulatio ncoul db edemonstra ­ ted.Lik e inth e laboratory experiments thisincreas e inpathogenicit ywa s gradual.Th emea n pathogenicity ofth epopulatio nP increasedwit ha rat eo f 0.07 logitunit spe rda y (seeSectio n 3.4.6).

Discussion In 1971,n o latebligh t ontomatoe sha sbee nreporte d inth e Netherlands.Therefore ,i tca nb e concludedtha tth e latebligh t reisolates collected from the fieldexperimen t andpathogeni ct otomatoes ,coul dno t havecom e from sourceswithi n aradiu s ofa tleas ta 100km .Thes e isolates musthav e originated from the inoculum introduced intothi sexperiment ,un ­ lessa hithert ounknow n long distancedispersa l ofP . infestans overmor e thana 100k m had occurred.Betwee nth ever yfirs tcollectio no fa lat ebligh t reisolate from tomatoplants ,a reisolat e thatwa sweakl ypathogeni ct oto ­ matoes, and the collection ofa latebligh treisolate ,tha twa shighl ypatho ­ genict otomatoes ,ther ewa s aperio do f6 4days . Ifth elaten tperio d inth e field isestimate d at 7t o 10days ,a 64days 'perio dallow sfo ra numbe ro f 6 to9 passage s atmost .Thi snumbe r isi naccordanc ewit hth enumbe rneede d inlaborator y experiments to gofro m an isolateweakl ypathogeni ct o tomatoes toa nisolate ,tha t ishighl ypathogeni c totomatoes . Ifth e increase inpathogenicit y ofth epopulatio nwoul dhav ebee ndu e toa singlemutation ,and ,accordingly , thegradua l increase inpathogenicit y wouldhav ebee n due toth e increase infrequenc y ofth emutant ,th e following couldb e expected.Afte r themutatio nha stake nplace ,a rapi dmultiplicatio n ofth emutan t on thetomat oplant s canb eexpected .Th ereisolate s fromtoma ­ toes shouldhav e either lowo rhig hpathogenicit y totomatoe sbu tn o inter­ mediatevalues .Ther e shouldb e little orn ovarianc e inth e pathogenicity ofth ehighl ypathogeni cpathotypes .Th erelativ e frequencyo fth emutan t should increase strongly atth eexpens e ofth eorigina lpathotype ,an d con­ sequently,th eproportio n ofth eweakl ypathogeni creisolate spe r sampling day shoulddecrease .Thi sreasonabl e expectationha sno tbee ncorroborate d by the facts.Th e abovehypothesi s ofth esingl emutatio nca nb e «*«*£, -d theorigina l hypothesis of agradua l increase ofth epath°*-« ^ *>" •natoesca nb e accepted. Theexperimen t doesno tprovid e aclu ea st o theun ­ derlying geneticmechanis m ofgradua l increase inpathogenicity .

29 3.4.6 The 'logistic' increase in pathogenicity to tomatoes in a field experiment

Inth eexperimen treporte di nSectio n3.4.5 ,th etes tresult spe r sampl­ ingda yca nb eaverage d intoa valu eP ,an dth efunctio n logit (P) canb ede ­ termined (Vande rPlank , 1963).Th elineai rregressio no f logit (P) ontim e t (indays )i sgive nb yth eequatio nP = 0.07 *- 6.4 0(Fig .3) . Fora singl emutatio ncausin g increasedpathogenicity ,th e'logistic ' increaseo fP ca nb edemonstrated ,whe nth efollowin g conditions aremet : 1.th elesion sar eo fshor tduration ; 2.al llesion so fon epathotyp e formth e samequantit yo feffectiv e spores per lesionunde ral lconditions ; 3.th erati oo fth enumbe ro feffectiv e sporespe rlesio no fon epathotyp e totha to fanothe rpathotyp ei sconstan t (here,effectiv emean s causinga lesion). Thefrequenc yo fth epathotyp ea ttim e t isproportiona lt oth efrequenc y attim e t-p (p= laten tperiod ) (vande rPlank , 1963). Thefrequenc yo fth e'wild 'pathotyp ei sFw, thato fth e'mutant 'patho ­ typei ft;.s A ttim e t theamoun to fth ewil dpathotyp ei sxw. , andtha to f themutan tpathotyp ei s xm+.

Xm. 1 Fm xm,+ xw, xw, (D z t 1__+ t xmt XWr. Definea a ttim e t =0 :a= — - xmQ (2)

logit p = 0.07 f-6.40

t50 = 91day s

t' 3-P " ^nfesi'ans on tomatoes cv.Money ­ makeri na fiel dexperiment .Th eregressio n ontim eo fth emea npathogenicit yP o freiso - iatescollecte do nvariou sday safte rfiel d inoculation.Fo rth elaborator yassessmen to f Pse etext . Abscissa:mea npathogenicit y P ofreisolate s 70 90 „o "lay of ^isolation onvariou scollectio ndays ,a sassesse d ina laboratorytest ,an dexpresse di nlogi tunits . 30 Define b in the logarithmic phase of the epidemic, where rm and rq are the 'logarithmic infection rates' of the mutant pathotype and the original patho­ type, respectively:

b = rm - rq | rm > rw (3)

In the logarithmic phase of the epidemic

xa, xu„ x erw * fi b -* = — = a.e"e * (4) xm+t XTTIQ x er m t

Combine (1) and (4):

Fm = L_ = ] (S)

1 +f^t 1 • a-e""* mt

Equation (5) is a logistic equation, which after differentiation reads,:

f2 = b-fla (1 - a,) (6)

The mean pathogenicity of the population is P. When Pw is the pathogenicity of the 'wild' pathotype, and Pm is the pathogenicity of the 'mutant' patho­ type,

P = **•«•> + ^Vm = p„ (1 . a,) + Rn.fte =. Pw + Fm

=P w+ (Pm- Pw ) -Fm ^)

WhenP m increases logistically with time,P als oincrease s logistically from itsstartin g levelP wt oit sfina l levelPm . Strictly spoken,th eabov e reasoningi svali d onlyi nth elogarithmi c Phaseo fth eepidemic .Th eargumen t onlyserve st odemonstrat e thatther ei s noconflic tbetwee nepidemiologica l theoryan dth eobserve dsigmoida lincreas e inpathogenicit y with time.Thi s conclusion alsoseem st ohol d true,whe n moremutant s appeara tdifferen ttimes .

31 3.4.7 A laboratory experiment to demonstrate the increase in pathogenicity to tomatoes by serial passages

Introduction Theseria lpassage sexperimen twa sdesigne dt oinvestigat ewhe n andt owha texten tchange si npathogenicit y couldb eobserve do nth eleve lo f smallpopulation soriginatin g fromsingl e lesions.I ti ssuppose d thatmos t ofthes e singlelesion swer eth eresul to fmonozoospor e infections.

Materials and methods Single zoospore isolatesmad e from the 1,4-p-pathotype of P. infestans originatingfro mpotatoe swer e testedfo rthei rpathogenicit y topotatoes .On eisolate ,tha twa sa spathogeni ca sth eorigina lculture , was selectedfo rus ei nthi sexperiment .Thi sisolat ewa scalle dth e PQ-isolate. Allisolate swer egrow no npotat otube rdisks . Senescentleave so fth etomat ocv .Moneymake rwer euse dfo rth e passages. Thesenescen t leaveswer etake nfro m1 0week sol dplant so fwhic h2 0wer e growni na singl e1 0c mdiam .po ti na glasshouse .Tomat o leaveso f6 week s old 'Moneymaker'plant swer euse dfo rth etest so npathogenicit yt otomatoes ; thetes tplant swer egrow ni na walk-i ngrowt hchambe ri norde rt oprovid e testleave so fconstan tquality .Onl y leaveso fth e 4th,5th ,an d6t hposi ­ tionabov eth ecotyledone swer eused .Th eclimati ccondition swere :tempera ­ ture20°C ;relativ ehumidit y 80%;da ylengt h1 8h ; light intensity 27.5J m-2s" 1.Reisolate smad e duringthi sexperimen twer e testedfo rpathogenicit y onleaflet so fpotat oplant swit han dwithou tth egene sR lt R2,R 3,an dR 4. Theseria lpassage sexperimen tconsiste do f7 successiv epassag ecycles . Eachpassag e cycleha d4 steps .

Step1 :Fiv edetache dsenescen t leaveso fth etomat o cv.Moneymake rwer ein ­ oculatedwxt ha zoospor esuspensio n (2000spore spe rml )o fth eP^isolat e Cn- 0 ,1 ,2 ,3 ,4 ,5 ,6 ,7) ,an dwer e incubateda sdescribe di nSectio n4.4 . Thesuspensio nwa sapplie db ysprayin gwit ha natomize runti lrun-off .

Step2 :Afte ra week ,^isolation so fth efungu swer emad e fromth esenescen t leaves.Thes eisolate s were calledP ^isolates,0 < n < 7 bein gth enumbe ro f thecycl epasse dthrough ,an d1< m <1 2 beingth eseria lnumbe ro fth e isolate. Forisolation ,9 t o1 2o fth elarges tlesion swer e selected,an dth e affected tissuewa streate da sdescribe di nSectio n4.4 .

Step3 :Fo rcomparativ etest so fpathogenicit yt otomatoe so fth eP -isolates n,m 32 collected in step 2, leaflets of 6 weeks old tomato plants of the cv. Money­ maker were inoculated by immersing them in a suspension of sporangia (2000 spores per ml), and incubated as described in 4.6.6. The criterion for pa­ thogenicity to tomatoes was the percentage diseased leaf area, see 3.4.5. The isolate, which caused the highest percentage diseased leaf area, was used as P -inoculum in the next passage cycle.

Step 4: Simultaneously with the next passage cycle, the isolate selected in step 3 was tested alone or together with another P-isolate for pathogenicity to tomato leaves of 6 weeks old 'Moneymaker' plants by means of a probit - log dosage analysis.

The first passage cycle differed from the later ones, because the PQ- isolate was tested for pathogenicity to tomatoes by means of a probit - log dosage analysis without being selected in a preceding step on the base of pathogenicity to tomatoes.

Results In step 3 of the first passage cycle, the 12 reisolates tested were not pathogenic to tomatoes. In step 3 of the second passage cycle, the reiso­

lates P2 g and P, „ showed to be somewhat more pathogenic to tomato leaves than the'other 7 reisolates tested. In step 2 of the third passage cycle, af­

ter inoculation with isolate ?2 g, 12 P3-reisolates were collected of which 5 were not pathogenic to tomato'leaves, 5 were slightly pathogenic to tomato leaves, and 2 were moderately pathogenic to tomato leaves according to the test results of step 3 of the third passage cycle. In the passages 4 to 7, all reisolates tested for pathogenicity to tomatoes in step 3 were moderately pathogenic. The results of the tests for pathogenicity- to tomato leaves of 6 weeks old plants of the cv. Moneymaker by means of probit - log dosage analyses executed in step 4 of the passage cycles are represented in Table 7 and Fig.

3. The calculated estimates for the ED5[)-values derived from these probit ana­ lyses serve as a measure of pathogenicity to tomatoes.

^ ED50-values of the following reisolates or groups of reisolates differed significantly (p<0.05): ]' P2,9 Versus P2,8' ' P2,8 and P2 9 versus pn an^L P1" 3 - P2>9 and its dependents P3 to P? versus PQ, ?v and ?2>&. 4 - Isolate 7023 (see Section 3.4.4) versus all reisolates PQ to Py.

33 Table7 .A seria lpassage s experiment inth elaboratory . Reisolates collected ina seria lpassage s experimentwer e tested forpathogenicit y totomat o leaves of6 week sol d'Moneymaker 'plant sb ymean so fa probi t- lo gdosag e analysis. The regression equationsar ecompute db ymean so fth emetho do fFinne y (1952). TheEDsQ-value srepresen tth emea n concentrationo fzoospore spe rul , atwhic h 50%o fth einoculation s resulti nnoticeabl e attack.Th elowe rth eED 5o,th e higherth epathogenicit y ofth e(re-)isolate . Testswit hth esam e serial test numberwer eperforme d simultaneously.

Serial Isolate Linear ED50 Fiducial Variance ofth e test regression limits regression number equation p4 0.05 coefficient

1 Po V = 2.5a; + 2.2 13.4 10.7 - 16.6 0.1244 2 Pi V " 2.5a; + 2.2 13.3 11.1 - 15.8 0.0790 3 P2.8 y = 3.3a: + 2.2 7.0 6.5 - 7.8 0.0432 3 P2.9 y = 3.3a; + 2.8 4.6 4.1 - 5.0 0.0421 4 *3,H y = 2.2x + 3.3 6.3 5.4 - 7.3 0.0290 4 P3.5 y = 2.2a; + 3.5 4.9 4.1 - 5.6 0.0353 5 PQ y = 1.7a: + 2.8 18.3 14.6- 23.9 0.0396 5 *3,5 y = 2.1a + 3.6 4.5 3.8 - 5.3 0.0382 6 P3.5 y = 2.4a: + 3.6 3.8 3.2 - 4.7 0.0704 6 Pt y = 2.4a; + 3.4 4.4 3.7 - 5.7 0.0565 7 P7 y = 1.9a: + 4.0 3.2 3.0 - 3.5 0.0379 7023 y= 2.5a: + 6.4 0.25 0.2 - 0.3 0.0724

Theregressio ncoefficient so fth eprobi t linesd ono tdiffe rsignifi ­ cantlyfro meac hothe r accordingt oth eStuden t test,excep tfo rth e regres­ sioncoefficient so fth ereisolate sP „g an dP 2g ,whic hhav ea significantl y highervalu etha nth eothe rregressio n coefficients.Reisolat e P7 reactedt o potatoes liketh e1,'4-p-pathotype ;P „di dno tdiffe rnotabl y fromP Qi nit s pathogenicityt opotatoes .

Conclusion Apathotyp emarkedl ymor epathogeni ct otomat o leaveso f6 week s old 'Moneymaker'plant stha nth eorigina lP Q-isolatewa sreisolate di nth e secondpassag e cycle.I nth efollowin gpassag e cyclesn ofurthe r increasei n pathogenicityt otomatoe scoul db edemonstrated .Th epathogenicit yo fth ene w pathotype,whic hwa smarkedl y lesspathogeni ct otomatoe s thanth e2,4-p/1-t - pathotype,isolat e 7023,seeme dt ob estable .

Discussion Inth ecours eo fseria lpassage sexperiment swit hP . infestans onpotatoe sreporte du pt ono w(Mills ,1940 ;d eBruyn ,1952 ; Grahame tal. , 1961;Kishy , 1962),a gradua lincreas ei npathogenicit yt otomatoe swa sob ­ served,whic hresulte di nstabl epathotype sa spathogeni ct otomatoe sa st - 34 probits Stn 1 Stn 2

5+Pn 5-HP. n =60 nllOO 4

3

2H J_ "I -I 16 32 8 16 32

8 16 32 16 32

Fig. 4. Graphical representation of the results ob­ tainetamed bbyv testintesting isolatesisolates, collectecollected durinduring aa seriseria. l passages experiment, for pathogenicity to leaves o "eeks old tomato plants of the cv. Moneymaker by means °f probit - log dosage analyses. Ordinates: Fraction °f inoculations resulting in noticeable attack. Pressed in probit units. Abscissae: Concentrations or the zoospore suspensions used for inoculations rep se nted on a logarithmic scale. The numbers of tne graphs correspond with the serial test numbers or 7 n = number of inoculations made per dosage. 35 pathotypescollecte d fromtomat o fieldcrop swithi n 7t o 12passage s through tomatofoliage .I nth e above-mentionedexperiments ,th e inoculations andre - isolationswer emarke db y largepopulations ,an dbeside s selection due toth e selectivemediu m tomatofoliage ,n o other selectionwa s operating. Theabove - mentioned results couldb e confirmedb y the author insom epreliminar y expe­ rimentsexecute d according toth e 'classic'design . Inth epresen texperiment ,th e increase inpathogenicit y was abrupt,an d didno treac hth e levelo fa t-pathotyp e collected from a tomato field crop. The deviant resultma yb erelate d toa devian t designo f the experiment. Con­ trary toth eexperiment s reported earlier,a nartificia l selection pressure was appliedo nth ebas e ofon ecriterio n forpathogenicity , lesion extension. The selection oflesion s implied the selection ofsub-populations ,whic hha d originated from single orfe wzoospore s causing the selected lesions.Both , the selection criterionuse d andth e smallpopulation spromot e genetic fixa­ tion.A t the same time,however ,smal lpopulation s contain little geneticva ­ riability, andthu s counteract that accumulation ofmutation sneede d toat ­ tainhig h levelso fpathogenicit y totomatoes . The increase inpathogenicit y totomatoe swa s limited intim e anddegree , andwa seas y tostabilize .Therefore ,i ti s supposed that this increase in pathogenicity totomatoe swa sdu et oa singlemutatio nwit h a considerable effect onpathogenicit y totomatoes .N ogeneti cproo f for thishypothesi s can begiven . According toPet o (1953),th eslop eo fth eprobi t - log dosage curvemus t have thevalu e 2a tth eED 5Q-point (y= 5probi tunits ) inabsenc ebot h of variabilityof'resistanc ean dsynergism .Variatio n inresistanc e reduces this value;synergis m increases thisvalue .Th e interaction ofbot hmigh t result inan yvalu ebetwee n zeroan d infinity.Th e regression coefficients of the probit linesfo r the isolatesP 2)8 and ?2 Q arehigh ,thu sgivin g theimpres ­

siono fth epresenc e ofsynergism . Incontras twit hth e isolatesP , toP ?,

whichha dmuc h lowervalue sfo rthei r regression coefficients,th eP ,g -re-

isolate segregrated inth edescenden tP 3population s forpathogenicit y toto ­ matoes.On eexplanatio n is,tha t ?2 g is amixtur e ofth e original pathotype anda ne wone ,an dtha tther ewa s synergismbetwee n zoospores of the old and thene wtype.

36 3.4.8 A laboratory experiment to demonstrate the increase in pathogenicity to Solanwn dulcamara by serial passages

Introduction P. infestans is reported to sporulate on foliage of S. dulcamara plants growing in the neighbourhood of affected potato crops during late blight epidemics (de Bary, 1876; Hirst &Stedman , 1960). In a laboratory test, inocu­ lation of leaves of S. dulcamara by the 1,4,10-p-pathotype failed to induce symptoms. As the pathogenicity of late blight isolates, initially non-pathogenic to tomatoes was increased by passages through either senescent tomato leaves or leaves of young tomato plants (Mills, 1940; de Bruyn, 1952; Graham et al., 1961; Kishy, 1962), two serial passages experiments were designed to find out wether the pathogenicity to S. dulcamara of the 1,4,10-p-pathotype of P. infestans could be increased by passages through senescent leaves or through foliage of S. dulcamara.

Materials and methods Plants of S. dulcamara were grown in a glasshouse from cuttings taken from a plant found in the neighbourhood of Wageningen. Senes­ cent leaves were selected, and mounted on a piece of 'Oasis1. The leaves were inoculated by spraying a zoospore suspension (2000 spores per ml), placed in a closed translucent plastic box, and incubated by putting them during one day at 15°C in dark, and thereafter in a walk-in growth chamber with 18 h light per day and a temperature of 15°C by day and 12°C at night. When pre­ sent, lesions were excised on day 8 after inoculation, placed under potato slices in petri dishes, and stored at 15°C in dark. After a week, the spo­ rangia formed at the surface of the potato slices were collected by washing,

and a new passage cycle was started. When there was spoliation on the leaves, the next passage cycle was started with sporangia washed from the leaves. Simultaneously with the inoculation of the senescent leaves, fresh leaves were inoculated with the same suspension to test the pathogenicity of the re- isolates. Young leaves were obtained by cutting the tops with the upper leaves from the plants. These young top leaves were treated in the same way as the senescent leaves.

Results: senescent leaves On day 8 after inoculation, lesions with diameters ^ging from 1 to 4 •* were present on the senescent leaves. Tne lesion were necrotic, and no spoliation was observed. The second !-»«*;<"* »* * ** ^th a mixture of reisolates originating from 4 lesions, resulted in sUght sponsion on the senescent leaves on day 7 after inoculation; no reactwn wasnoticeabl e onth eyoun gleaves .Th ethir dpassage ,starte dwit hth e spo­ rangiacollecte dfro mth e senescent leaves,resulte d ina complet e infection ofbot hth esenescen tan dyoun g leaves,followe db ysporulatio no nda y 5af ­ terinoculation .N ofurthe rincreas e inpathogenicit yt o S. dulcamara wasob ­ serveddurin g thefollowin g3 passages .

Results: Xoung leaves Threesuccessiv e inoculationsdi dho t succeed instart ­ ingth efirs tpassag ecycle ;n oinfectio nwa sobserved .A t thefourt hattempt , one leafwit ha broke nmidri bwa sals oinoculated .O nda y 11afte rinocula ­ tion,som esporulatio nwa sobserve do nth edista lpar to fth e leaf.A second passage,starte dwit ha zoospor e suspension (500pe rml )originatin g fromth e broken leaf,resulte di nman yslowl yextendin gwater-soake d lesions.O nda y 5 afterinoculatio nsem esporulatio nwa sobserve do nth enecrotizin g centres ofth e lesions,an do nda y 11sporulatio noccurre do nth e chlorotictissu e surroundingth ewater-soake dtissue .Th ethir dpassag eresulte d inabundan t sporulationo nth eda y5 afte rinoculatio nbot ho nyoun g foliage and full- grown leaves.I nth efollowin gthre epassage sn ofurthe r increase inpatho ­ genicitywa sobserved .

Conclusion Solanum dulcamara wasno t susceptiblet oth e original isolate of P. infestans, butth epathogenicit ywa sincrease db ypassage s throughsenes ­ cento rdamage dleaves .Wheneve r P. infestans hasa nopportunit y to infect thehos ttissue ,th e increasei npathogenicit y iseffectuate d inonl y afe w passages.Thes eexperiment sgiv en oclu ea st oth eunderlyin g geneticalan d physiologicalphenomena .

3.5 CONCLUSIONS

Chapter3 discusse sa.o .th esubject s 'hostrange' ,i nparticula rhost s withsurviva lvalu et oth elat ebligh t fungus,an d 'variabilityi npathogeni ­ city',especiall yth erelatio nbetwee n 'tomatoraces *an d 'potatoraces' .Th e resultspresente di nthi schapte rwil lb euse dher e toanswe r thequestion s mentionedi nth elas tparagrap ho fth eintroductio n (Chapter 1). The import ofthes equestion san do fth eanswers ,i srestricte dt oth e situationi nth e Netherlands. 1 Which other hosts are important? Therei sn oevidence ,tha tapar tfro mpo ­ tatoesan dtomatoes ,othe rhost sar eimportan tt olat ebligh tdevelopmen t on tomatoes.

38 2 Whatis the relation of late blight on tomatoes to late blight on potatoes? There are three relations. The first relation is a climatic one: there will be a late blight epidemic on potatoes and tomatoes only, when the weather con­ ditions are favourable to the development of a severe epidemic of late blight on potatoes. The second relation is a epidemiological one: late blight on potatoes is a permanent source of inoculum to tomato crops. The third relation is a fysiological one: late blight pathotypes highly pathogenic to tomatoes and potatoes (p/t-pathotypes) originate from pathotypes only pathogenic to potatoes (p-pathotypes), as can be demonstrated by means of serial passage experiments in the laboratory, and in field experiments. 3 How do tomato races of late blight overoome unfavourable periods? There is no evidence, that tomato races of late blight overwinter in glasshouse crops. An alternative could be the overwintering of tomato races in potato tuber tissues, with subsequently infection tomato crops. When present, this mode must be rare, as late blight on tomatoes is a disease found in late simmer and autumn mainly and the growth of potato seed, the next year's crop, comes to an end before August. The normal situation seems to be a permanent reser­ voir of p-pathotypes, overwintering in potatoes, from which p/t-pathotypes originate under favourable conditions by a proces not unlike that seen in the serial passages experiments.

39 4 The assessment of resistance

4.1 MONOCYCLICAN DPOLYCYCLI C TESTS

Testso nresistanc e canb eclassifie da seithe r 'monocyclic'o r'poly - cyclic* tests (Zadoks, 1972).Cycl e standsfo rinfectio n cycle,whic hi s the sequenceo fprocesse s from one generationo fspore s through infection, latency etc.t oth e nextgeneratio no fspores .I n'monocyclic ' tests,onl y one infec­ tioncycl ei sinvolved ,an dmostl y this typeo ftes ti sexecute di nth e labo­ ratory.I n'polycyclic * tests,mor e thanon e infectioncycl ei sinvolved ;man - y fieldexperiment sbelon gt othi s typeo ftest .I nth epolycycli c test, the inoculumfo rth enex t infection cyclei sproduce db yth eplan to rcro p tested. The testpermit st oestimat eth eeffec to fpartia l resistanceo nth egrowt h rateo fepidemics .When ,however ,mos to fth e inoculum originates from out­ side theplan to rcro ptested ,e.g . fromnearb y fieldso rfro m spreaders plantedt otha tpurpose ,th eeffec to fpartia l resistance willb eunder-esti ­ mated.Thi s typeo ftes tha dbee n calleda 'continuousmonocyclic ' test (Zadoks, 1972).

4.2 PARTIAL RESISTANCE

A typeo fresistanc ewell-know nt obreeder si sresistanc e basedo nhyper ­ sensitivity,whic hprotect sth ecro p completely.I nsegregratin g offspringo f crossesbetwee nsusceptibl e andresistan t parents,th eindividual sar eeasil y classifieda seithe r resistanto rsusceptible .N ospecia lmethod sfo rth e measuremento fresistanc e areneeded .Th erati oo fresistan tt osusceptibl e individualsi nsegregratin g offspring agreeswit hexpectation s basedo nth e 'Mendeleanwa yo finheritance 'o fon eo ra fe wgene s causing resistance.Thi s typeo finheritanc ei scalle dqualitativ e inheritance;th eresistanc e involved isconsidere dt ob ea qualitativel y inheriting character,an di sshortl y called qualitative resistance.Th ehope ,tha t this typeo fresistanc ewil l ever lead toa permanen t solutiono fth e latebligh tproble mo fpotatoe san dtomatoe s (seeSectio n 3.4.2),i sdecreasin gnowadays .Attentio ni sbein g focussed more

40 andmor eo npartia l resistance.Th e philosophy behind thisgrowin g interest inpartia l resistancei sth e expectation thatpartia l resistancewil l last longertha nresistanc e basedo nhypersensitivity .Also ,ther ei sa fea rtha t thepoo lo fgene s forhypersensitivit ymigh tbecom e exhausted.I nsom ein ­ stances,gene s forhypersensitivit y are rare,a si sth ecas ei nth e genus Lyaopersiaonwit hrespec tt oP . infestans. Partial resistancei sno ts oeas yt ohandl ei nplan tbreeding .Th ein ­ heritanceo fpartia l resistancei sno t alwaysi naccordanc ewit h expectations basedo nth e segregration ofon eo ra fe w genes causing resistancea si nth e 'Mendeleanwa yo finheritance' .I nsegregratin g offspring,a nindividua lma y havean yleve lo fresistanc e within the range limitedb yth etw oparents , and -whe nther ei stransgressio n- eve n outside thisrange .N oclearl y dis­ tinctclasse so fresistanc e canb eseen ,an d specialmethod s forth eassess ­ mento fresistanc e are needed. This typeo finheritanc ei scalle d quantitative inheritance;th eresistanc e involvedi sa quantitativel y inheriting resistance, orshortl yquantitativ e resistance. Inthi s section various aspectso fpartia l resistancewil lb ediscussed , usingth eter m 'partial resistance' onlya sa phenomenologica ldescriptio n withoutan y genetical connotation.A genetica l interpretationo fth edat a presentedwil lb egive ni nChapte r 7.

4.3 NUMERICAL VALUEO FPARTIA L RESISTANCE

Thenumerica l valueo fpartia l resistance RESo fa tes tplan tca nhav e anyvalu e from zerot oone :0 < RE S<1 (Zadoks , 1972).Whe nRE S= 0 ,th etes t Planti sa sdisease d asth emos t susceptible standard.Whe nRE S= 1 ,i » dis­ easei snoticeable ,an dresistanc ei scomplete .So ,partia l resistance is thatresistanc e which hasa numerica l valuebetwee n zeroan done :. 0< RE S< 1 . A«ually,no tth eresistanc ebu tth ediseas e ratingDIS(T )o fth e testplan t andth ediseas e rating DIS(S)o fth emos t susceptible standard are assessed. ^ resistanceo fth e test plant canb erelate dt otha to fth e susceptible standardb ymean so fth e equation (Zadoks, 1972):

RES= 1 - DISC O DISTS) DIS(T)car .hav e any values from zerot oone ,relativ et oth evalu eo fth emos t susceptible standardwhic hi sgive n the disease rating 1. feWS(T )< DIS(S) ,RE Si sagai na valu ebetwee n zeroan done .

41 4.4 COMPONENTSO FPARTIA L RESISTANCE

Partialresistanc ei ssubdivide di ncomponents ,whic hca nb eassesse db y typologicaland/o rquantitativ emethods .Th etypologica lmetho duse sa descrip ­ tiveke yt ocharacteriz etw oo rmor e classeso freaction s denotedb yfigure s orsymbols ;th equantitativ emetho duse s characteristics,whic hca nb emeas ­ uredo rcounte d (Zadoks,1972) .Th eresistanc e componentsdescribe di nthi s sectionhav ebee nstudie dextensivel yi npotatoe s infectedb ylat eblight . Differencesi nresistanc et oinfectio nhav ebee nnotice db yLapwoo d& McKe e (1966)an dUmaeru s (1969a,b),difference si nresistanc eb yprolongatio no f thelaten tperio dhav ebee nfoun db yLapwoo d (1961),an ddifference si nre ­ sistancet osporulatio nar eobviou s (vande rZaag ,1956 ;Lapwood , 1961b). Forlat ebligh to ntomatoes ,onl ystudie so nspor eproductio nar eknow n Grummer& Eggert ,1968) .Th efollowin g characterswer echosen : 1.Percentage successful inoculations, S thenumbe ro fresultin glesion s expresseda sa percentag eo fth enumbe ro fdro pinoculations ,usin g dropso f a specifiedvolum ean do fa specifie d zoosporesuspension . 2. Lesion extension, E thelinea r lesiongrowt hwithi na specifie d timein ­ terval. 3. Sporulation intensity, I thedensit yo fsporophores ,classifie db ymean s ofa nin eclas sscal e (Table &X* Thefirs t2 character sar eassesse db ymean so fquantitativ emethods ,th e thirdi sassesse db ymean so fa typologica lmethod . Foreac hcharacte rth ecorrespondin g 'componento fresistance 'ca nb e defined: a. Resistance to infection theabilit yo fth eplan tt oreduc eth e'percentag e successful inoculations'.Th e measureuse di sth enumbe ro fzoospor epe ry lo f thatdosage ,tha tcause s 504successfu l inoculationswhe n4. 2u ldroplet sar e applied (seeSectio n4.5.1) .Thi sdosage ,th eso-calle d effectivedosag e5 0

(ED50),i sestimate db ymean so fa probi t- lo gdosag e analysis (see Section 4.4.1).Th e'resistanc et oinfection 'o fa tes tplan ti srelate dt otha to f thesusceptibl e standardb yth eequation :

RES(S)= 1 - SSsJ l ED50l

inwhic hED 50(S)i sth eED 50 ofth esusceptibl e standard,an dED 50(T)i sth e ED50o fth etes tplant . b. Resistance to extension theabilit yo fth eplan tt oimped e lesiongrowth . Themeasur euse di sth emea nvalu ei nm mo fth elonges tdiameter so flesion s

42 Table 8. Sporulation density. The estimate used is the number of sporophores per unit area, relative to the number of sporophores per unit area of sporu- lating lesions on the susceptible standard 'Moneymaker'. The sporophore den­ sity on the susceptible standard is taken to be 100%. For convenience, a 9 class subdivision has been made.

Sporulation intensity (sporophore 0 5 10 25 50 75 90 95 100 density) in % Sporulation classification number 0 1 2345678 measured parallel to the large veins originating from the midribs. The 're­ sistance to extension' of a test plant is related to that of the susceptible standard by the equation RES(E) = 1- f§ inwhic hE(T )i sth elesio n extensiono nth etes tplant ,an dE(S )i sth ele ­ sionextensio no nth esusceptibl e standard. c. Resistance to sporulation theabilit yo fth eplan tt oreduc e sporophore density.Th emeasur e usedi sth e numbero fsporophore spe runi tarea ,rela ­ tivet oth e numbero fsporophore s peruni t areao fsporulatin g lesionso na susceptible standard (Table 8), applying standardized techniques (seeSectio n 4.6).Th e'resistanc et osporulation 'o fa tes tplan ti srelate dt otha to f thesusceptibl e standardb yth e equation

RES(I) = i-im in which I(T) is the number of the sporophore density class of the test plant, 311(1 I(S) is that of the susceptible standard. The resistance components thus assessed can be combined into a combined index of relative resistance (Zadoks, 1972). In the experiments described in this chapter the combined index for partial resistance RES(E.I) = 1 - E(T)/E(S) * * I(T)/I(S) is sometimes used. As the ratios E(T)/E(S) and I(T)/I(S) both are < 1, the product of both ratios also will be 4 1, and consequently 0 ^RES^I) <, 1- Note, that a distinction has. been made between the following concepts: character, measure, and 'component of resistance'. A character is a phytopathological or epidemiological aspect of the disease, that can be indicated by a technical tern like percentage successful inocu- iations, lesion extension, or sporulation intensity. Measure is a specific property of a character, that is actually counted, measured, or ranked, like the ED50, the longest lesion diameter, or the spo- 43 rulationclassificatio nnumber . A 'component of resistance' isth ecomplemen to fth echaracte rsee nfro mth e breeder'spoin to fview .I tca nb ecalculate dusin gth emeasur eo fa character . Thecorrespondin g 'componentso fresistance " are: 'resistancet oinfection' , 'resistancet oextension' ,an d'resistanc et osporulation' .

4.S MATHEMATICALTECHNIQUE S

4.5.7 Probit - log dosage analysis

The inoculationwit ha drople to fa zoospor e suspensioni scalle d 'drop inoculation'.A dro pinoculatio ni ssuccessful ,whe ni tresult si na lesion . Successdepend sa.o .o nth enumbe ro finfectiv e zoosporespe rdrop .Th enum ­ bero fzoospore spe rdro pi sa functio no fth econcentratio n (morecorrectly : density)o fth e zoospore suspensionan dth e sizeo fth edrop .Th einfectivit y ofth e zoosporesi sa functio no fth epathogenicit yo fth eisolate ,th e treat­ mento fth ezoospores ,an dth eresistanc eo fth etes tplant .A dro pinoculatio n iseithe rsuccessfu lo rnot .Thi styp eo freactio ni scalle da quanta lresponse . Forth estatistica lanalysi so fquanta lresponse si nbio-assays ,variou smeth ­ odshav ebee nintroduced ,al lo fwhic hmak eus eo ftransformation so fth eob ­ serveddata .Th emos t commontransformation sar eth e probit transformation (Bliss, 1934),th elogisti ctransformatio n (Berkson, 1944),an dth e angle transformation (Knudsen& Curtis ,1947) .Here ,th eprobi t transformation,a linearmetri ctransformatio no fth enorma ldistribution ,ha sbee nchosen .Thi s choisei sbase' do nth eassumption ,tha tth etoleranc eo fth e inoculated leafare a tolo gdosag eha sa norma ldistribution .Finney' s (1952)metho do fprobi t- logdosag eanalysi swil lb eused .Th emetho d impliesth eestimatio no fth e linearregressio no fth eprobi tvalu ecorrespondin gwit hth epercentag esuccess ­ fulinoculation so nlo gdosage .Th eregressio nline ,represente db yth eequa ­ tion y= h e- a ,i scalle d 'probitline' .Th efiducia llimit so fth elinea r regressioncoefficien tca nb edetermined .I naddition ,estimate sfo rth e ED50 (seeSectio n4.4 )an dit sfiducia l limitsca nb ecalculated .Whe nth eprobi t lineso ftw o (groupsof )plant sar eparallel ,th eestimat efo rth erelativ epo ­ tencyca nb ecalculated .Th eantilo go fth e relativepotenc yAR Pi sth edis ­ tancebetwee nthes eprobi tlines ,measure dparalle lt oth eabscissa .Th e ARP- value indicatesho wman ytime son eplant (orgrou po fplants )i smor eo rles s resistanttha nth eothe rplan t (orgrou po fplants )o fth epai runde rcomparison . Theexperimenta ldat apresente di nthi spape rsho wtha tth enumbe ro f

44 zoospores per drop was low (1 - 256), especially when the susceptible tomato cv. Moneymaker was inoculated with the 2,4-p/1-t-pathotype. Nevertheless, Finney's probit - log dosage method appeared to be fully applicable to results from experiments executed with low dosage drop inoculations.

4.5.2 Variance analysis

Apart from variance analysis with equally filled classes of data, vari­ ance analysis had to be executed with unequally filled classes of data. Un­ equal classes of test data originated from experiments using drop inoculations with different dosages, when lesion diameters had to be measured or sporula- tion intensities had to be estimated on resulting lesions. When the differences between and within inoculated leaves are small, and the variance is only due to a normal distribution of the variables observed, the pooled within dosage variance can be considered as an estimate of the residual variance. An estimate of the variance due to dosage effects can be obtained by substracting the pooled within dosage variance from the total variance (over all dosages). The significance of between dosages effects can be determined by means of a Fisher test.

4.6 MATERIALS AND METHODS

4-e>7 Introduction

In testing resistance of the hypersensitive type, small variations in environmental conditions or dosage of inoculum do not markedly affect the test results. Results of tests for partial resistance depend, however, to a large extent on environmental conditions and inoculum dosage. Therefore, tests of Partial resistance require environmental control and high precision of dosage. :« order to reduce environmental variance to a minimum, most of the test plants w*e grown and all tests were performed in the walk-in growth chambers of the Apartment of Phytopathology, State Agricultural University, Wageningen. Much Mention was paid to the experimental design with a view to the statistical ^alysis of the test results.

45 4.6.2 The growing of the test plants

Thetes tplant swer e growni na walk-i ngrowt hchambe runde r fluorescent tubes (PhilipsTL-M F 40W/33RS)wit ha radiatio nintensit yo f27. 5J m -2 s"1 (9500lx )a tsoi llevel .Th eothe rclimati c conditionswere :temperatur e 20°C + 0.2;relativ ehumidit y 904+ 34 ;da ylengt h1 6h an dnigh t length8 h .Whe n the lampswer e on,th etemperatur ewithi nth ecanop ywa sraise db yca .1.5°C . Eight seedlingswer e growni n1 3c mdiamete rpot so fsterilize d compost (Trio special 17),an dtransplante d after4 week st oa wate r culture system.Thre e watercultur eunit swer e available.Eac hconsiste do fa 10 01 tank ,tw otrough s inwhic hth eplant swer egrown ,an da circulatio n systemwit ha pump .Eac h trough (120x 1 4x 1 4cm )wa scovere db ya boar dwit hhole st oaccomodat eth e plants.I neac hunit ,a hundre d literso fa nutrien t solutionwa s circulating. Thenutrien t solutionha sbee nprepare d afterrecipe sfo rmacro-element sb y Steiner (1968), spore-elementsb ySteine r (1970),an d'iron' ,applie di nth e formo fFeEDTA ,b ySteine r& va nWinde n (1970).Th erecommende d qualityo f FeEDTAha dt ob edouble di norde rt opreven tth echlorosi s observedi npre ­ liminaryexperiments .

4.6.3 The preparation of the test leaves

The test leaveswer edetached ,an dth epetiole swer e fitted intoa piec e ofwater-soake d foamo nurea-formaldehy dbas emanufacture dfo rth e purposeo f floralarrangemen t (trademark 'Oasis',commercialize db yV.L .Smither sA/S , GanlsSse- MalmcSv e- Danmark) .Thes e leaveswit h' Oasis 'wer eplace di nplas ­ tictrays. Th etrays ,measurin g3 6x 2 6x 5. 5cm ,wer e linedwit ha shee to f thoroughlywette dfilte rpaper .A small-mes hpv cnettin gwa smounte di na woodenframe ,an dplace di nth e trays0. 5c mabov eth efilte rpaper ,thi s nettingpreventin gth eleave sfro mcontac twit hth ewe tpaper .On etra ygav e roomt o2 leave swit h5 leaflet seach ,o rt oon elea fwit h7 leaflets .

4.6.4 The preparation of the zoospore suspension

Sporangiao fP. infestans weregrow no nleaflet so fth etomat ocv .Money ­ maker (seeSectio n3.3.2 )an dwer eharveste db yimmersin g3 leaflet s during halfa minut ei n5 0m ldeionize dwater .Th esuspensio nwa s sieved through polyamidfilterin gtissu e (polyamid 1383,manufactere db yVereinigte nSeiden - webereienAG ,Krefeld ,Germany) .Smal l sporangia andbacteri apasse d through

46 thistissue .Th efilte r was rinsedS time swit h2 0m ldeionize dwate r tomak e suretha tmos to fth e bacteriaha d gone throughth e sieve.Th e sieve residue includingth e large zoosporangia was resuspendedi nS Om ldeionize dwater ,t o which1 0m lpotat o tuber extract per literwa sadde d (McKee, 1964).Thi s sus­

pensionwa skep ta t1 0oCfo r2. Sh t oinduc e zoospore liberation.Subsequent ­ ly,th esuspensio n was filtered again,t oseparat e thesmalle r zoosporesfro m emptyan dnon-germinate d sporangia,whic hd ono tpas sth efilterin gtissue . Thezoospor e suspension was storeda tS° Cfo r2 h a tmost .Te nm lo fth e sus­ pensionwa smixe d with 10m lformo l 81fo rkillin g andfixatio no fth e zoo­ spores.Th e concentrationo fth e zoosporesi nth esuspensio nwa sdetermine db y meanso fa haemocytomete r (Fuchs-Rosenthal,0.20 0mm) . Countswer erepeate d untilth etota lnumbe ro fzoospore s counted surpassed 1000,o runti l2 0count s weremade .Th econcentration s required for thetest swer eprepare db ydilutio n ofth eorigina l zoospore suspensions using deionizedwate rwit hpotat o tuber extract.

4-6.5 Theapplication of the inoculum

Theinoculu m was appliedi nth e formo fdroplet so fa zoospor esuspen - wn (Section 4.S.4). Reproducibilityo fth edrople t sizewa sassure db yus - lnga re Peating dispenser, constructed aftera desig no fDr .J.W .Seinhors t ( p0.unpublished) .Th edispense r consistso fa 2 m lsyring e (Socorex,standar d >> providedwit ha mechanis mt odischarg e dropletso f4. 2 yla tth epres s abutton .Th edroplet s were placedo nth e abaxial sideo fth eleaflets . 0Preven tdamag eo fth e leaf tissues,a 3 0c mteflo nneedl e (Hamilton,KF30TF ,

°>033m m$ )Wa s used. When4 dosage s were used,th e2 lowe rdosage swer e appliedt oth eright - 311(1leaf letso fth e leaf,an dth e2 highe r dosagest oth e left-handleaflets . SUally,th eapica l leaflet wasno t inoculated,bu twhe nthi swa sdone , the °West dosage was appliedt oth e right-hand side andth ehighes tdosag et o eieft-han dside .O neac h leaflet,5 droplet so fth e lowestdosag ewer e aPPliedt oth e right-hand side,an dS droplet so fth eothe rdosag et oth eleft - handside . When 8 dosages hadt o be applied, numbered 1 through 8 from the lowest ^ the highest dosage, the dosages 1, 3, 5, and 8 were applied to leaves with ^ even leaf position number. The remaining dosages were applied to the leaves t ^ an odd leaf position number. Leaf positions were numbered from bottom to ' ^ cotyledones being indicated by the number 0 (zero).

47 Foreac hgrou po fdosages ,th esam edistributio no fth edosage s over theleaflet spe rlea fwa suse da sdescribe d abovefo rinoculatio nwit h4 dosages. When6 dosage swer e applied,th edosage s1 ,2 ,5 ,an d6 wer e appliedt o leaveswit ha neve nlea fpositio nnumber ,an dth edosage s1 ,3 ,4 ,an d6 wer e appliedt oleave swit ha nod dlea fpositio nnumber .

Whenth edifferentia lpathogenicit yo flat ebligh t isolatest oa se to f differentialsha dt ob edetermined ,th einoculatio nwa sperforme db yspray ­ inga spor esuspensio nove rth eabaxia lsid eo fth eleave sunti lrun-off .

4.6.6 The incubation of the inoculated leaves

After inoculation,th etray swer e coveredwit hanothe rpv cnetting , againmounte di na woode nframe ,t oprotec tth einoculate d leaves from contact witha nenclosin gbag .A colourles spolythen eba gwa spu taroun deac h tray andclosed ,i norde rt oguarante ea hig hmoistur e leveli nth etray s conducive toth edevelopmen to fsymptoms .The nth e trayswer e storedfo r2 4h a t 15°C inth edark .Afte rthi sinitia ldar kphase ,th etray swer eplace di na growt h chambera t18°C .Th eleave swer e irradiatedb ymean so ffluorescen t tubes (PhilipsTL-M F 40W/33RS),th eligh t intensitya tlea fleve lwa s 10.6J m" 2 s-1 (3,650 lx),th eligh tperio d lasted 16h an dth edar kperio d8 h pe rday . Irradiationb yth e lamps raisedth e temperaturei nth etray sc a1.5°C .Thre e days after inoculation,th esheet so ffilte rpape ran dth ebrick so f'Oasis ' werewette dagain .

48 5Results : Monocyclic tests in the laboratory

5.1 INTRODUCTION

The monocyclic tests in the laboratory were designed to develop suitable techniques for measuring partial resistance, making use of detached leaves of plants grown in growth-chambers or greenhouses. Within-plant variances have been studied. Special attention has been given to the effect of age of the host plant on the expression of partial resistance. The possibility of a dos­ age dependent response to inoculation has also been considered.

5.2 WITHIN PLANTS VARIABILITY OF LESIONEXTENSIO N

5-2.7 Six weeks old 'Moneymaker' plants

Materials and methods Four 'Moneymaker' plants were grown as described in 4-6.2. At the age of 6 weeks after seeding^ the leaves with positions 4 to 8 were harvested, inoculated, and incubated as described in 4.6.3 to 4.6.6. Four drops per leaflet were applied of a suspension of 10 zoospores per pi of the 2>4-p/1-t-pathotype of P. infestans. Five days after inoculation, the lesion extension was measured.

Table 9. Tomato cv. Moneymaker. 'Resistance to extension' of 6 weeks old plants against a 2,4-p/I-t-pathotype of P. infestans. Analysis of variance of the w ithin-plant variability of the character lesion extension. s°urce of variance Sum of squares df Mean squares Variance ratio Probability

05 Plants 838 3 279 '4.83 P = °' j*af position 272 4 68 1.17 p > 0.2 Wlthin leaflets 694 12 58 T°tal 1804 19

49 Table 10.Tomat ocv .Moneymaker . 'Resistancet oextension 1 of8 week sol d plantsagains ta 2,4-p/l-t-pathotyp eo fP . infestems. Analysiso fvarianc eo f thewithin-plan tvariabilit yo fth echaracte rlesio nextension .

Sourceo fvarianc e Sumo fsquare s df Meansquare s Variancerati o Probability

Plants 660 1 660 6.7 p4 0.05 Leafposition s 3445 8 431 4.4 p4 0.05 Leafletsposition s 49 3 16 <1 p> 0. 2 Residual 5863 59 99 Total 10017 71

Results and conclusions Thetes tresult shav ebee nuse di na nanalysi so f variance (Table9) .Th emea nlesio nextensio nm = 31. 4mm ,wit ha standar d deviations = 3. 1mm ,an dth ecoefficien to fvariatio nm/ s= 0.10 .Ther ei s asmal lbu tsignifican teffec tbetwee nplant sa tp 4 0.0 5 (thecoefficien to f variationm/ s= 0.06) .Ther ewa sn osignifican teffec to flea fpositio no n lesionextension ,an dconsequentl yo n 'resistancet oextension' .Th ewithi n leafletvarianc einclude sth evarianc edu et oth edifference si nleafle tpo ­ sitionspe rleaf ,an dth evarianc edu et odifferen tlocation so flesion spe r leaflet.A sal lo fthe mwer eno tsignifican ta tp > 0.2 ,the yar epoole dwit h theresidua lvarianc et oobtai nth ewithi nleaflet svariance .

5.2,2 Eight weeks old 'Moneymaker' plants

From2 'Moneymaker'plants ,treate da sth eplant sdescribe di n5.2.1 ,th e leaveswit hposition s4 t o1 2wer einoculated . Thetes tresult shav ebee nuse di na varianc eanalysi s (Table 10).Th e meanlesio nextensio nm = 26. 8m mwit ha standar ddeviatio ns = 1. 8mm ,an da coefficiento fvarianc em/ s= 0.07 .Ther ei sa smal leffec to flea fpositio n onlesio nextension ,an dconsequentl yo n 'resistancet oextension' .Fig .4 suggests,tha tyounge ran dolde rleave sar emor e 'resistantt oextention 'tha n theleave so fth eposition s6 t o8 .Fo rexperimenta lpurposes ,th eobserve d differencesar enegligibl ea slon ga sleave sfro mth eoptimu mposition sar e used.Ther ei sn odemonstrabl eeffec to fleafle tposition . Theinfluenc eo flea fpositio no n 'resistancet oinfection 'ha sals obee n studied.I na nexperimen twit h6 week sol d 'Moneymaker'plants ,o fwhic hth e leafposition s2 t o9 wer einoculate dwit hdrop so fsuspensio nwit h1 zoospor e perp iresultin gi n76 1successfu linoculations ,n osignifican teffec tbetwee n leaveswithi nplant scoul db edemonstrated .Th ecoefficien to fvariatio nwa s 0.05.A smal lbu tsignifican tbetwee nplant seffec ta tp 4 0.05wa spresent . 50 probit S

T- J_L '/4 '/2 4 8 dosage

Fig. 5. Tomato cv. Moneymaker. Fig. 6. TomatoW.V a 700.Th e effect of The effect of dosage on spol­ age on 'resistance to infection1.Leave s iation intensity I and lesion of 6, 7,an d 8week s old plantswer e extension E, after inoculation tested for 'resistancet oinfection ' with a 2,4-p/l-t-pathotype of against a2,4-p/l-t-pathotyp e of p P. - infestana. The dosage is ex­ infeatans. Thetes t results arepresen ­ pressed as the number of zoo­ ted in the form of probit S (ordinate) spores per ul on a log scale. " on log dosage,expresse d asnumbe ro f I = sporulation classification zoospores per yl (abscissa). number (Table 9) S = 'percentage successful inoculations' E = lesion extension in mm O = test results of 6week s old plants •k = test results of 7week s old plants • = test results of 8week s old plants

5.3 THEEFFEC TO FDOSAG EO NLESIO NEXTENSIO NAN DSPORULATIO NINTENSIT YO F SOMETOMAT O LINES

ateHala and methods Twoplant so fth ecv .Moneymake rwer egrown ,harvested , Moculated,an dincubate da sth eW.V a 700plant suse di nth etes tdescribe di n •4.Al lplant swer e8 week s old.Fo r thistest ,leave so fth eposition sS t o wereinoculate dwit h4 suspension so fa 2,4-p/l-t-pathotyp e ofP . infestans w"h respectively 1, \, 1,an d2 zoospore spe ryl .

Results and conclusions Theresult s arepresente di nTabl e 11an dFig .S . ithinth erang eo fdosag e tested, lesionextensio nincrease dwit h increasing os age accordingt oth eregressio nequatio n y= 3. 2log 10 x+ 13.6 ; yi s expressedi nmm , and x asth enumbe ro fzoospore spe ryl .Whe nth edosag ewa s doubled,th elesio n extensionwa s increasedwit h1 mm .Th esporulatio ninten - SltyI increase d with increasing dosage accordingt oth eequatio n y= 2. 5 lo Sioa ;+ 2.8 ; y is themea n sporulation classificationnumbe r (seeTabl e9 ) x lsth enumbe ro fzoospore spe ryl .Whe n thedosag ewa sdoubled ,th e sporu- lationclassificatio n number increasedwit h0.7 5 point.A nanalysi so fvarianc e

51 Table 11.Tomat ocv .Moneymaker .Th eeffec to fdosag eo nsporulatio nintensit y andlesio nextension ,afte rinoculatio nwit ha 2,4-p/l-t-pathotyp eo fP . infestans. Thetes tresult sar eexpresse da smea nvalue spe rtreatment .Dosag e isexpresse d asth enumbe ro fzoospore spe ryl .

Dosage i i 1 2 Lesionextensio n E 11.5 12.8 13.9 14.3 Sporulationintensit y I 1.1 2.3 2.9 3.4

(seeSectio n4.5.2 )wa suse dt oestimat eth ewithi ndosage svarianc efro mth e variancepe rdosage .Accordin gt othi sanalysis ,th evarianc edu et odosage s wassignifican ta tp 4 0.0 1 (Table12) . Inth esusceptibl ecv .Moneymaker ,lesio nextensio nan dsporulatio nin ­ tensityshowe dt ob edosag edependent .Whe nth ecv .Moneymake r isuse da sth e susceptiblestandard ,th eeffec to fdosag edependenc ymus tb etake nint ocon ­ sideration.

5.4 THE-RESISTANC ET OINFECTION 'O F6 ,7 ,AN D8 WEEK SOL DW.V A70 0PLANT S

Introduction Wilson& Gallegl y (1962)noticed ,tha tth eresistanc eo ftomat o seedlingsbelongin gt oa susceptibl ecultivar ,tw oaccession swit h'dominan t resistance',an dtw oaccession swit h 'multigenicresistance' ,increase dwit h agefro m7 t o3 9day safte rtransplanting .I twa simportan tt ofin dou tt o whatag ean dt owha texten tresistanc eincreases.T othi spurpose ,experiment s havebee ndon eusin gth eresistan ttomat oaccessio nW.V a70 0an dth esuscep ­ tiblecv .Moneymaker .Th ecriterio nfo r 'resistancet oinfection 'chose ni n theseexperiment swa sth e 'percentagesuccessfu linoculations ' (seeSectio n 4.4).

Table 12.Tomat ocv .Moneymaker .Analysi so fvarianc eo flesio nextensio nE .

Sourceo fvarianc e Sumo fsquare s df Meansquare s Variancerati o Probability

Withindosage s 2145 190 II.1 Betweendosage s 164 3 54.7 4.93 p4 0.0 1 Total 2309 193

52 Table 13. Tomato accession W.Va 700. Results of tests for 'resistance to in­ fection' against a 2,4-p/l-t-pathotype of P. infestans in 6, 7, and 8 weeks old plants. The measure of 'resistance to infection1 is S, the 'percentage successful inoculations'. The results are analysed by the probit - log dosage method.

Linearregressio n ED50 Fiduciallimit s Varianceo fth e probit- lo gdosag e (p4 0.001 ) regressioncoefficien t y6 =2.0a ;+ 4.8 1.25 1.1- 1.4 0.018 #7= 1.9a :+ 4. 6 1.70 1.5- 1. 9 0.010 2/8= 1.4a :+ 3. 9 6.12 4.3- 7. 4 0*028 y = response in probits of the tested plants; the subscript indicates the plant age in weeks after seeding x - logjo dosage (dosage is measured as number of zoospores per ul) ED50 = dosage, expressed in number of zoospores per ul, causing 50% success­ ful inoculations

Materials and methods Plants of the accession West Virginia 700 (W.Va 700) were grown on water cultures (see Section 4.6.2). On 72.07.06, leaves of 6, 7, and 8 weeks old plants were harvested, inoculated, and incubated with the 2i4-p/1-t-pathotype of P. infestans as described in 4.6.3 to 4.6.6. Suspensions with concentrations of 4, 2, 2 and 8 zoospores per yl were prepared, and 110 drops per suspension were placed on leaves with an even leaf position number (4.6.S). 110 drops of suspension with concentrations of \, 1, 4, and 8 zoo­ spores per ul were applied to leaves with an odd number. For each of the 3 age groups 2 plants were used, and from every plant the leaves of the posi­ tions 4 to 8 were inoculated. On day 4 and S after inoculation, the numbers of successful inoculations were scored for all treatments.

Results The results, expressed as the 'percentage successful inoculations' s> were transformed into probits, and analysed according to Finney's (19S2) method of probit - log dosage analysis as described in 4.S.1 (Table 13, Fig. 6 )- The ED50, this is the estimated dosage effectuating 501 successful inocu- la tions, and its fiducial limits were calculated. The ED50-values of different age groups differed significantly at p < 0.001; the value of 6 weeks old plants Was 1-3 times, and the value of 8 weeks old plants 4.9 times the value of 6 weeks old plants. The regression coefficients decreased with increasing plant age from 6 to 8 weeks after seeding. The regression coefficients of 6 and 7 *el« old plants did not differ significantly, but both differed significantly at P < 0.05 from the regression coefficient of the 8 weeks old plants (Table 14)- The probit values have been subjected to an analysis of variance, to test

53 Table 14.Tomat oaccessio nW.V a700 .Th eregressio nline so fTabl e 11ar e testedfo rparallelis mb ymean so fStudent' stest .

Probability Pairso fregressio ncoefficient s dh ""n /Va rd , b bgan d by t10 = 0.60 p> 0.6 0 bgan db g t9 =2.6 5 p< 0.0 5 b7an db g tg = 2.43 p< 0.0 5 b =regressio ncoefficient ;th esubscrip t indicatesth eag eo fth eplant s inweek s d,• *differenc ebetwee nth eregressio ncoefficient so fan ypai r n =numbe ro fdegree so ffreedo m

Table 15.Tomat oaccessio nW.V a700 .Result so ftest sfo r 'resistancet oin ­ fection1 againsta 2,4-p/l-t-pathotyp eo fP . infestans of6 an d 7week sol d plants.Th etes tresults ,score da sth epercentag esuccessfu l inoculationsS , aretransforme dint oprobits ,an dtabulated .

Agei n Plant Dosage:numbe ro fzoospore spe ru l weeks number i \ 1 I

3.9 4.3 4.6 5.7 6.3 6.9 31.7 61.7 3.5 4.3 4.5 5.2 6.2 6.3 30.0 3.5 3.8 4.4 5.2 5.8 6.2 28.9 58.3 3.7 3.8 4.2 5.3 6.2 6.2 29.4 14.6 16.2 17.7 21.4 24.5 25.6 120.0

Table 16.Tomat oaccessio nW.V a700 .Analysi so fvarianc eo fth edat afro m Table15 .

Sourceo fvarianc e Sumo f df Variance Variance Probability squares ratio Dosage 25.62 5 5.12 206.6 p< 0.00 1 Age 0.48 1 0.48 19.4 p <0.00 1 Plantswithi nag egroup s 0.25 2 0.13 5.1 p< 0.0 5 Residual 0.37 15 0.025 Total 26.72 23

54 thesignificanc e of differences betweenplant swithi nag egroup so f6 an d7 weeks (Table 15, Table 16).Th edifference swer e smallbu t significant at p< ,0.05 .

Conclusions The resistance to infectiono fth eW.V a 700plant steste d increases withth eag e of theplants ,i nth e test situationdescribed .Eigh tweek sol d plantsar e about 5time s as resistant toinfectio nagains tth e 2,4-p/1-t- pathotypeuse d as 6week s oldplants .Th eregressio n coefficient ofth eprobi t- logdosag e relation decreaseswit h increasing ageo fth etes tplants .I nothe r words,th evarianc e of thepercentag e successfulinoculations ,va rS ,increase s withincreasin g age of theplants .Ther e islittl e reasont oexpec t thatth e increasedvarianc e was of experimental origin.Th e increase inva rS coul d indicatetha t the resistance to infectiono fth evariou spart so fth e leaves doesno t increase in the samewa y ort oth e sameextent .

5.5 THE 'RESISTANCE TO INFECTION* OF 6AN D 9WEEK SOL D 'MONEYMAKER1 PLANTS

Introduction Thisexperimen t was designed tofin dou t towha texten tth e resistanceo f the susceptible tomato cv.Moneymake r increaseddurin g thein ­ tervalfro mwee k 6t owee k 9 after seeding.

Materials and methods Three plants frombot h theag egroup so f6 an d9 week s oldtomat oplant s of the cv.Moneymake rwer e grown,harveste d and incubated inth esam ewa y as theW.V a 700plant suse d inth etes tdescribe d in5.4 .Fo r thistest ,leave s of theposition s 4t o7 abov e thecotyledone s ofth e9 week s °ldplant swer e used. Four suspensionswit hrespectivel y I, 1,2 ,an d4 zoo ­ sporeso fth e 2,4-p/1-t-pathotypepe ry lwer eapplie d asdescribe d in4.6.5 .

Results and conclusions The results areanalyse d statistically asi n 5.4

(Table17 ,Fig . 7).Th e ED50-values andth erelativ epotenc yhav ebee ncalcu ­ lated.Th erelativ e potency is the ratioo fth e 2dosage s causingequa leffects . Here> ^ relative potency isa measur e for therelativ e increasei nresist - m ™ inth e interval from 6t o 9week s after seeding.Th eED 50-valueso fth e tw°probi t lines differ significantly atp <0.001 ;th eantilo g ofthei rrela ­ te potencyi s 1.7 with fiducial limits 1.15 and 2.40 (p< 0.001) .Consequent - ly> itca nb e concluded that the 'resistancet oinfection 'b ya 2,4-p/l-t -

Pathotypei ncreases significantly inth einterva l from 6t o9 week s after Ceding.

55 5.6 THE 'RESISTANCET OINFECTION *O F9 AN D1 1WEEK SOL D 'M3NEYMAKER'PLANT S

Introduction Thisexperimen ti sa follow-u po fth eexperimen tdescribe di n 5.5,designe dt otes tth echang ei n 'resistancet oinfection 'o f'Moneymaker ' plantsfro m9 t o1 1week safte rseeding .

Materials and methods Threeplant so feac hag egrou po f9 t o1 1week sol d tomatoplant so fth ecv .Moneymake rwer etreate da sth e9 week sol d'Money ­ maker'plant sdescribe di n5.5 .Si xsuspension swit hrespectivel y I, I, 1,2 , 4,an d8 zoospore spe ry lo fth e2,4-p/1-t-pathotyp e wereapplie da sdescribe d in4.6.5 .Fo rth estatistica lanalysi so fth eresult sse e4.5.1 .

Results and conclusions Thedifference sbetwee nth eED 50-valuesan dbetwee n theslope so fth etw oline srepresentin gth etw oage sar eno tsignifican ta t p4 0.05 .ARP ,th eantilo go fth erelativ epotenc yi s1.0 0 (Table18 ,Fig . 8). Consequently,ther eha sbee nn onoticabl eincreas ei n 'resistancet oinfection ' inth einterva lbetwee n9 an d1 1week safte rseeding .

probitS probitS 7-i ageo fplant s inweek s 7- ageo fplant s • inweek s

6-

5+- •

4H

I ±L r '/2 4 1/4 dosage V2 8 dosage

Fig.7 .Tomat ocv .Moneymaker .Th eef ­ Fig.8 .Tomat o cv.Moneymaker .Th eef ­ fecto fag eo n'resistanc et oinfec ­ fecto fag eo n 'resistancet oinfec ­ tion'agains ta 2,4-p/l-t-pathotyp eo f tion'agains t a2,4-p/l-t-pathotyp e of P. infestans. Thetes tresult sar epre ­ P. infestans. Thetes tresult sar epre ­ sented inth efor mo fregressio nline s sented inth efor mo fregressio nline s ofprobi tS (ordinate),o nlo gdosage , ofprobi tS (ordinate),o nlo gdosage , expresseda snumbe ro fzoospore spe r ul (abscissa) expresseda snumbe ro fzoospore spe r ul (abscissa) S= percentag e successfulinoculation s S= percentag e successful inoculations "fr= tes tresult so f6 week sol dplant s O =tes tresult so f9 week sol dplant s O =tes tresult so f9 week sol dplant s * =tes tresult so f 11week sol dplant s

56 Table 17. Tomato cv. Moneymaker. Results of tests for 'resistance to infection' against a 2,4-p/l-t-pathotype of P. infestans in 6 to 9 weeks old plants. The measure of resistance is S, the 'percentage successful inoculations'. The re­ sults are analysed by the probit - log dosage method.

Linear regression ED5Q Fiducial limits Chi square tests df Probability probit - log dosage (p <_ 0.001)

z/6 = 2.2a; + 5.6 0.53 0.45 - 0.63 par. 0.01 1 p > 0.9 2/9 = 2.2a; + 5.1 0.90 0.80 - 1.01 lin. 4.42 4 p > 0.9 ARP = 1.66 (Fiducial limits: 1.15 - 2.40, p 4 0.001)

y = response in probits of the tested plants; the subscript indicates the age of the plants in weeks x = logio dosage (dosage is measured as number of zoospores per pi) par. = parallelism lin. = linearity df = degrees of freedom ED50 = dosage, expressed in mean number of zoospores per pi, causing 50% success­ ful inoculations ARP = antilog of the relative potency indicating how many times the 'resist­ ance to infection' increased

e 18. Tomato cv. Moneymaker. Results of tests for 'resistance to infection' gainst a 2,4-p/l-t-pathotype of P. infestans in 9 to 11 weeks old 'Moneymaker' • The measure of resistance is S, the 'percentage successful inoculations', e results are analysed by the probit - log dosage method.

n r J f? Agression ED50 Fiducial limits Chi square tests df Probability Pr°oit - log dosage (p ^ 0.05)

^9= 1.7a;+ 5. 4 0.60 0.53- 0.6 8 par. 0.01 1 p> 0. 9 y\l- 1.7a:+ 5. 3 0.68 0.61- 0.7 6 lin.0.5 5 8 p> 0. 9 ARP = 1.00(Fiducia l limits:1.0 0- 1.00,p < 0.05 ) V response inprobit so fth eteste d plants;th esubscrip t indicatesth e age ofth eplant s inweek s x ~ loglO dosage (dosage ismeasure d asnumbe ro fzoospore spe rpi ) P«- "Parallelis m ln Jf -- linearit y ED Ide §rees offreedo m 5Q= dosage ,expresse d inmea n number ofzoospore spe rpi , causing50 % AKp successful inoculations -antilo g ofth erelativ e potency indicatingho wman y times 'resistance to infection' increased

57 5.7 THE'RESISTANC ET OINFECTION 'O F6 ,7 ,AN D8 WEEK SOL D 'MONEYMAKER'PLANT S

Introduction Thissection ,wher eth eeffec to fag eo n 'resistancet oinfec ­ tion'o f 'Moneymaker'plant si sestimated ,differ sfro mth etw oprecedin g sectionsbecaus eth etes tresult shav eno tbee nobtaine dfro ma singl eexperi ­ mentdesigne dfo rthi spurpose ,bu thav eoriginate dfro mvariou sexperiments , inwhic hplant so fth ecv .Moneymake rwer euse da sa susceptibl estandard .

Materials and methods Invariou sexperiments ,leave so fth etomat ocv .Money ­ makerwer eharvested ,inoculated ,an dincubate di nth ewa ydescribe d in4.6 . Onlyth etes tresult sfo rth edosage so f \, \, 1,an d2 zoospore spe ry lwil l beconsidered ,becaus ethes edosage swer eapplie dt oal lplant stested .Th e resultso f6 week sol dplant sar efro ma singl etes twit h3 plants ;th ere ­ sultso fth e7 an d8 week sol dplant sar efro m3 experiment sfo reac hag e group,i nwhic h2 plant spe rexperimen twer etested .Th eoveral lpercentag e successfulinoculation sS wa scalculated ,fo reac hdosag ean dag egroup ,an d transformedint oprobi tS .

Table 19.Tomat ocv .Moneymaker .Result so ftest sfo r 'resistancet oinfectio n againsta 2,4-p/l-t-pathotyp eo fP . infestans in6 ,7 ,an d8 week sol dplants . Themeasur efo rresistanc e isth e 'percentage successfulinoculations' .Th e resultsar eanalyse db yth eprobi t- lo gdosag emethod .

Linear regression ED5o Fiducial limits Chi square tests df Probability probit - log dosage (p 4 0.001)

j/6 = 2.5a: + 6.4 0.25 0.23 T 0.29 par. 3.53 1 p 4 0.05 j/7 = 2.5a; + 5.9 0.45 0.42 - 0.47 lin. 15.16 8 p <. 0.05 j/8 = 2.5a; + 5.5 0.61 0.58 - 0.65

ARP6>7 = 1.6 (Fiducial limits: 1.4-2, ,0, p < 0.05) ARP6#8 = 2.3 (Fiducial limits: 1.9-2, ,7, p < 0.05) ARP7-8 = 1.4 (Fiducial limits: 1.2-1, .5, p < 0.05)

y responsei nprobit so fth eteste dplants ;th esubscrip t indicatesth e plantag ei nweek safte rseedin g x =logj odosag e (dosagei smeasure da snumbe ro fzoospore spe rpi ) par.= parallelis m lin.= linearit y df =degree so ffreedo m ED50 =dosage ,expresse di nnumbe ro fzoospore spe ryl ,causin g50 %success ­ fulinoculation s ASP =antilo go fth erelativ epotenc yindicatin gho wman y timesth e'resist ­ ancet oinfection 'increase dwit hage ;th esubscript sindicat e theag e ofth epair so fag egroup scompare d

58 probit S age of plants 7-1 in weeks/. Fig. 9. Tomato cv. Moneymaker. The effect of age • -. j~.u»*.«.• me client ui age b/0 on 'resistance toinfection 'agains ta 2,4-p/l-t - 6^ ys. pathotypeo fP . infestans. Thetes t resultsar e '/ presented inth efor mo fregressio n lineso fpro - bit S (ordinate),o nlo gdosage ,expresse da snum ­ ber ofzoospore spe ry l (abscissa) S= percentag e successful inoculations 0 = test resultso f6 week sol dplant s G = test resultso f7 week sol dplant s "Ar= test resultso f8 week sol dplant s 2 dosage

Results and conclusions Theresult sar eanalyse d statisticallya si n5. 6an d

.5(Tabl e 19,Fig .9) .Th eED 5Q-valuesan dth erelativ epotencie shav ebee n

calculated.Th e ED50-valueso fal lag egroup sdiffe rsignificantl ya tp < _0.00 1 Consequently,i ti sconclude d thatth e'resistanc et oinfection 'increase di n theinterval s fromwee k6 t o7 an dfro mwee k7 t o8 afte rseeding .Accordin g toth erelativ e potencies,th e'resistanc et oinfection ' increased about1. 6 timesfro mwee k6 t o7 afte r seeding,an dabou t1. 4time sfro mwee k7 t o8 . Intota lth e 'resistancet oinfection ' increased about2. 3times .

S.8 THERESISTANC EO F6 AN D8 WEEK SOL DPLANT SO FTH EF, :MONEYMAKE Rx W.V A 700

ntroduotion This experimentwa sdesigne dt odetermin eth eexten to fth e

changei nresistanc eo fF 2-individualsfro mth ecros sMoneymake r* W.V a70 0 m tneperio d from6 t o8 week s after seeding,an dth eeffec to fthi schang e

onth efrequenc y distributiono fpartia l resistancei nth eF 2-population. Testso n'resistanc et oinfection *b ymean so fth eprobi t- lo gdosag e analysisusin ga nadequat e numbero fplant s requiredto omuc hspac ei nth e growthchamber . Instead,th echaracter s lesionextensio nE an dsporulatio n

mtensityI wer e assessed.I twa sassume d thatth ewithin-plan tdifference s etween leaveso fth e positions4 an d5ar esmal li ncompariso nwit hdiffer ­ encesbetwee n leaveso fdifferen t plantso rdifference sdu et oag eo fth e Pla*ts(se eSectio n5.2) .

Materials and methods Three plantso fth ecv .Moneymake ran d2 7plant so fth e F2: Moneymaker* W.V a70 0wer e growni na glasshous eo ncompos t (Trio1 7 spe- cial)i n2 lite rpots .Th etemperatur ewa s2 0+ 2°C ;th eplant swer e irradiated

59 by fluorescent tubes (PhilipsTL-M F 140W/33RS)durin g 18h per day.Th eirra ­ diationintensit y atsoi l levelwa s 40.6J m -2 s_1. The leaveso fth eposition s 4an d 5wer eharvested , inoculated, andin ­ cubated asdescribe d in 4.6 inth e 6than d 8thwee k after seeding, respectively. Each leafwa s inoculatedwit hte ndrop s ofa suspensio n of 20zoospore spe ry l ofth e 2,4-p/l-t-pathotypeo f P. infestans. Onda y 5afte r inoculation,th e lesionextensio nE wa smeasured , andth e sporulation intensity Iestimated .

Results and aonolusions Foreac h individual F2-plant,th e test resultswer e expressed asit sresistanc e relative tocv .Moneymake rusin g the equationfo r relativeresistanc e (see4.4) : RESfE.I)= 1- |g j x Ig}

E = lesionextensio n inm m I = sporulation intensity;sporulatio n classificationnumbe r (Table 9) T =tes tplan t S = susceptible standard. TheRES(E,I)-value sar epresente d inFig . 10.Th emea nextensio nE ofth e 3 'Moneymaker'plant s 6an d8 week sol dwa s30. 6an d 19.6m m respectively.Th e mean sporulation intensity Iwa s 8an d 6respectively . In all F2-plants,re ­ sistance increased considerably inth eperio d from 6t o8 week s after seeding. The 6week sol d 'population'di dno t showa noticabl e differentiation ofth e resistance distributionpattern ;al l classeswer emor e or lessequall y filled (x2= 2.06 ;p <0.8) .Th e same 'populations',bu t 8week s old,showe d adiffe - rentiation intoa 'resistant'grou pan d a 'moderately susceptible' group;a 2 testo nequall y filledclasse sgav ex = 19.85;p <0.01 ,Ther e isa centrifu- galshif twit hag eint oa 'susceptible'an da 'resistant'grou p in a 1 :3 ratio.

InFig . 10,th erankin gnumbe ro fth eF 2-plantsar e shown inorde r ofin ­ creasingRES(E,I)-valu eassigne dwhe nth eplant swer e 6week s old.Th e 9 plants,whic hwer emos t susceptible atth eag e of6 weeks ,wer e still the most susceptible atth eag e of8 weeks ,wit honl yon eexceptio n (plant6) .

F2-plants,tha t showeda resistan t reaction inth e 6week s test,als o showed aresistan treactio ni nth e 8week stest ,bu t thenumbe r ofresistan tindivi ­ dualsincrease d atth eexpens eo fth e intermediate group. Thegenetica l implications ofthes e testresult swil lb ediscusse d in, 7.3.4.

60 number of individuals per class

8- 6 weeks old slants 6- 27 26

14H 27 Fig. 10.Tomat oF2 SMoneymake r xW.V a 12 A 8 weeks old plants 26 700. Frequency distributions ofrela ­ 25 tive resistance against P. infestans 10 A 24 in aF 2 population, tested at theage s 23 of 6week s (upperdiagram )an d 8week s *A 22 21 (lower diagram). The relative resist­ 20 anceRES(E,I ) isa combined estimate 9 19 of 'resistance toextension 1 and 're­ 4H 8 18 sistance to sporulation'.Fo r details 5 16 17 see text 4.4 and 5.8.Th efigure s in 3 13 24 15 the columns represent ranking numbers 2 7 11 14 1 4 10 6 13 assigned toth eF2~plant s in the order .0 .2 .4 .6 .8 U0 of increasing RES(E,I)-valuewhe n the .1 .3 .5 .7 .9 - plantswer e 6week sold . classes of F IES (E.I -va ues

RESISTANCEO F6 AN D8 WEEK SOL DPLANT SO FTH EFIRS TBAC KCROS SB 1: MONEYMAKERx (MONEYMAKERx W.V A 700)

a trials and methods Fourplant so fMoneymaker ,W.V a700 ,an d Fj:Money - 7°°>an d1 6plant so fth efirs tbac kcros sBj :Moneymake rx eymakerx w.V a 700)wer e growni na glasshous eo ncompos t (Trio1 7special ) itrepots .Th etemperatur e was2 4+ 2° Cb yday ,an d2 0+ 2° Ca tnight . eplant swer e irradiatedb yfluorescen t tubes (PhilipsTL-M F 140W/33RS) lng1 8h Per day.Th eradiatio n intensitywa s40. 6J m -2 s"1.Th eleave s rvested,inoculate dan dincubate da si nth eexperimen tdescribe di n ' day4 afte r inoculation,th elesion swer emeasure dan dth esporulatio n Wasestimated .

8 and conclusions Thetes t results,analyse di nth esam ewa ya si n5.8 , are Presente d m Fig. 11. The mean lesion extension E of the 6 weeks old 'Mon, eymaker» plants was 19.0 mm, and of the 8 weeks old plants 14.5 mm.Th e

61 number of individuals per class 6 weeks old plants Parents and Fi 4

2

0

'1 5 4 4 3 8 2 13 2 7 10 12 16 0 1 6 9 11 14 15 .2 .4 .6 .8 1.0 .3 .5 .7 .9 -

Fig. 11.Tomat oBi :Moneymake rx (Moneymakerx W.V a 700)an dparents . Frequencydistribution so frelativ e 8 weeks old plants 6 parents andF resistanceagains tP . -infestans RES (E,I)i na B jpopulatio nan di nth e 4H parents,teste da tth eage so f6 week s (upper2 diagrams )an d 8week s (lower 2 diagrams).Th erelativ eresistanc eRE S o-J (E,I)i sa combine destimat eo f 're­ sistancet oextension 'an d 'resistance tosporulation' .Fo rdetail sse etex t 4.4 and5.9 .Th efigure s inth ecolumn s representth erankin gnumber sassigne d 7 toth eB jplant si nth eorde ro fin ­ 8 B1 creasingRES(E,I)-valu ewhe nth eplant s 6 4H 16 were6 week sold . 4 15 2 12 Parentsan d Fj: 2H 1 11 Whitecolumn s - 'Moneymaker'plant s 3 9 I 10 1 13 I 14 5 Blackcolumn s -W.V a70 0 .0 .2 .4 .6 .8 1.0 .1 .3 .5 .7 .9 Shadedcolumn s- F :Moneymake rx clas ses of RES(E,I)-V! ilues W.Va70 0

mean sporulationintensitie sI wer e6. 3an d4. 5respectively .Th ecombine d valueso f'resistanc et oextension 'an d'resistanc et osporulation * RES(E.I) of6 week sol d¥ 1-plants isintermediat ebetwee n thato fbot hparent sa tth e sameage .Th e8 week sol dF^plant sar eabou ta sresistan ta sth eW.V a70 0 plantsa tth esam eage .Th epopulatio no f6 week sol d Bx plantsdi dno t show anoticabl e differentiationbetwee n susceptible andresistan t groups,bu tth e populationo f8 week sol dB x plants suggestsa differentiatio n into2 abou t equallysize dgroups ,on egrou pa ssusceptibl ea s'Moneymaker* ,th eothe r groupa sresistan ta sW.V a 700.Th erankin g ordero fth e Bx plantswa sno t

62 ™ch affected,plan t 5excepted . Like inth eF 2populatio ndescribe d in5 8 theshif t inresistanc e is restricted largely toplant swit ha nintermediat e

5.10 CONCLUSIONS

Itwa s demonstrated, that the -resistancet oinfection - ofth e susceptible Moneymaker- and of the resistant W.Va 700increase sconsiderabl y inth epe ­ riodfro m 6t o 8week s after seeding.Afte r thisperiod ,th eincreas e inre ­ sistanceo f the -Moneymaker'wa s small andno t significant.Th eresistanc e of plantso fW.V a 700 older than 8week sha dno tbee n assessed,bu tth elarg e increasei n resistance in the 8thwee k suggeststha tth eresistanc e toinfec ­ tionmigh t still increase after the 8thweek . The 'resistance to extension- and -resistancet osporulation - ofparents ,

h, andF 2 of the cross Moneymaker *W.V a 700,an do fth eBj : Moneymakerx (toneymakerx W.V a 700), also increased inth eperio d from 6t o8 week safte r

seeding.Th e rate and amount of increase differed forindividua lF 2an d Bj-

Plants.Si xweek s old plants of theF 2 andB 2 couldno tb edifferentiate d in istinctRES(E,I ) groups.Eigh t weeks oldplant s showeddifferentiatio n intw o istmct groups;on e about as susceptible as -Moneymaker',th eothe rabou ta s resistant asW.V a 700.Th e resistance ofF x plants increasedfro mintermedi - etweenbot h parents to about as resistant asth eW.V a 700plant sdurin g tteperio d from 6t o 8week s after seeding. t isconclude d that plants younger than8 week sshoul dno tb euse di n ieso nth e genetics ofpartia l resistance.Both ,lesio nextensio nan d m ation t intensity were found tob e dosage dependent inth e susceptible Moneymaker'•

63 6 Results:Continuou s monocyclic tests in the field

6.1 INTRODUCTION

Theeffec to fpartia l resistanceo nth egrowt ho fa nepidemi ci sa 'slow ­ ingdown 1 ofth erat ea twhic hth eepidemi c grows.Th ehighe rth eresistance , the lowerth egrowt hrate .I nreverse ,th egrowt hrat eo fa nepidemi cca nb e considereda sa measur e forpartia l resistance.Th erat eo fgrowt ho fa nepi ­ demicca nb ecalculate d fromdiseas e assessmentsmad ea tregula rintervals , e.g.b ycalculatin gth eregressio n coefficiento fth efractio ndisease dfoli ­ ageo ntime .Va nde rPlan k (1963)introduce dth eter m 'apparent infection rate'r (se eSectio n 2.3.1),whic hi sth eth eregressio ncoefficien to fth e fractiondisease d foliage,expresse di nlogits ,o ntime .Th evalu er ca nb e determinedfo rindividua lplant sa swel la sfo rpopulation so fplants .Whe n individualplant swithi na populatio ndiffe ri nresistance ,a sca nb eth ecas e inF 2-populations,th eeffec to fpartia l resistance canb eassesse dfo reac h plant separately,whe n theseplant sar euniforml yexpose dt oa nepidemic . Allfiel dtest sreporte di nthi ssectio nwer e artificially infected.Th e initialinoculu mwa sapplie dt ospreade rrows ,whic hi nthei rtur n infected thetes tplants .Th etes tplant sdiffere dmarkedl yi nresistanc e level.There ­ fore,th etes tsituatio ni sconsidere dt ob etha to fa 'continuousmonocycli c test' (seeSectio n 4.1). Themor e resistantth etes tplant ,th emor e likelyi t istha tmos to fth einoculu mfo rth enex t infectioncycl e comes from surround­ ingan dmor e susceptible plants,an dth emor e likelyi ti stha tth eresistanc e ofth etes tplan twil lb eunder-estimated .I nthi scontext ,referenc e should bemad et oVa nde rPlank' s (1963) 'crypticerro ri nfiel dexperiments' .

6.2 PARTIALRESISTANC EO FTH ECV .MONEYMAKER ,TH ELIN EW.V A700 ,THEI RF ,

ANDTHEI RF 2I NA FIEL DEXPERIMENT , 1972

Introduction Thisexperimen twa sdesigne dt otr yametho do ftestin gindivi ­ dualplant si nth efield ,an dt odetermin e their levelo fresistanc e against latebligh tfo rth epurpos eo fgenetica l analysis.Th emeasur eo fresistanc e

64 wasth eapparen t infectionrat e r.

Materials and methods Twoeight-plan tplot so feac ho fth ecv .Moneymaker , thelin eW.V a 700,an dth eFi :Moneymake rx W.V a700 ,an d 2sixteen-plan t

plotso fth eF 2:Moneymake r xW.V a 700wer egrow ni na nexperimenta lfield , thatwa s situated ina cor nfield .A pat hdivide d thisfiel d (20x 16m ) into 2equa lparts ,an deac hpar tconsiste do f9 plot s (7x 1.5 m). Eachplo tcon ­ tained2 row so f 8plant sa ta ro wdistanc e of0. 5m anda plan tspacin gwith ­ inth erow so f 75cm .Th e 9plot s aton e sideo fth epat hwer esubdivide d into 18eight-plan tplots .Thi sexperimenta l fieldwa s surroundedb ya ro wo f9 8 'Moneymaker'plants ,servin g asspreaders ,whic hha dbee ninoculate do n 72.08.08b yplacin g oneac hplan ton e 10y ldro po fa suspensio ncontainin g 4 zoosporangiape ry lo fth e 2,4-p/1-t-pathotype ofP . infestans. Thefiel dwa s irrigatedb y asprinklin g installationprovide dwit ha n irrigationcontro ldevice .Th edevic econsiste do fa micro-switc hactivate d byth ereactio no fa 1.10m hem pkite-line .Th ethrea d shrunkwhe nwetted ,an d turnedbac k tonorma l lengthwhe ndrying .B ymean so fthi sdevic ean d amanual , switch,th eplant swer ekep twe tdurin gon e4 0h perio dpe rwee ka ta minimu m ofwate rusage .Th eplant swer eprune dweekly ,an dthe ywer e toppedafte r having reached aheigh to f1.2 0m .I nthi sway ,excessiv egrowt ho fth efoli ­ agewa sprevented .Ever y 4thda yafte rinoculatio no fth espreade rrows ,th e latebligh t severityo fever yplan twa sassesse dseparatel ywit hth ehel po f aspecia ldiseas e assessmentke y (seeFig .14) .

Results The 'Moneymaker'plant s testedshowe dlarg elesion so nth estems ,som e ofwhic hexceede d 15c m inlength .Th eW.V a 700plant sdi dno tsho wsuc hlarg e stemlesion s at all;a fe wnecroti c lesionswer efoun dwit ha * 4 4mm .Th e plantso fth eF x:Moneymake r xW.V a 700onl yshowe da fe wste mlesion swit h a i,< 4 mm . Fromth e 32F 2plants ,ther ewer e 7plant so nwhic hth etypica l large lesionswer e found.Th eothe rF 2plant swer efre efro mlesion sexceed ­ inga (j, of 4mm . The 'apparentinfectio nrate' ,average dove r4 'Moneymaker'plants ,wa s r = 0.43.Fo r 7W.V a 700plant san d8 F :plant sth er-value scalculate dwer e

0.15 and0.18 ,respectively .Th e 32plant so fth eF 2:Moneymake rx W.V a70 0 variedi nresistance ,an dha dr-value sbetwee n0.1 5 and0.4 2 (seeFig .12) .

The 7F 2 plantswit hth e large stemlesion swer ea tth esam etim eth e7 mos t susceptibleplant s dfth eF 2population .

65 number of individuals per class

parents

4H

rrr 14 17 20 23 26 29 32 35 38 41 44 47 50

14 17 20 23 26 29 32 35 38 41 44 47 50

Fig. 12.Parents ,Fj .an dF 2o fth e crossMoneymake r xW.V a700 .Frequenc y distributions ofr ,th eapparen tinfec ­ tionrate ,expresse d inlogi tunit spe r dayfo rth e2 parent s (upperdiagram) , theF j (middlediagram) ,an dth eF 2 (lowerdiagram) .Th eblac kdot sindicat e nxn theplant smarke db y largeste mlesions ; 20 23 26 29 32 35 38 41 44 47 50 inth euppe rdiagra mthes ear eth e classes of r—values (rxioo) 'Moneymaker'plants .

Conclusions W.Va 700showe da considerabl e degreeo fpartia l resistance against the latebligh tpathotyp eused ,an dth eFi :Moneymake r xW.V a 700wa sabou t asresistan ta sW.V a 700.Th eplant so fth eF 2:Moneymake r xW.V a 700varie d inresistanc e fromth ehighes tt oth elowes tparenta lvalue ;n otransgressio n wasobserved . Conclusions aboutth egenetic so fth epartia l resistance carriedb y W.Va 700,whia hca nb edraw nfro mth e resultso fthi sexperiment ,wil lb e presentedi nSectio n 7.3.3.

number of individuals per class

Fig. 13.Parent san dF 2o fth e

11 14 17 20 23 26 29 32 35 38 41 44 47 50 53 56 cross 'Atom'x W.V a700 .Frequenc y distributionso fr ,th eapparen t infectionrate ,expresse d inlogi t unitspe rda yfo rth e2 parent s F2 (upperdiagram) ,an dth eF 2 (lower D diagram).Th eD' s inth ecolumn s D cp_rz D D indicateth edeterminan tplants ; I I I I inth euppe rdiagra mthes ear eth e 20 23 26 29 32 35 38 41 44 47 50 53 56 classes of r-values (rx 100) 'Atom'plants .

66 6.3 PARTIALRESISTANC EO FTH ECV .ATO MAN DO FTH EF 2:ATO Mx W.V A 700

Materials and methods Thisexperimen twa sdesigne d forth esam epurpose ,an d executed inth e samewa ya sth eexperimen tdescribe d in6.2 .Th ecv .Ato m is adeterminat e type (bushy type),whic hdi dno texcee da plan theigh to f4 0c m inth e fieldexperiments .Th e cv.Ato mha sa noutstandin g 'resistance tospo - rulation' (Grummer& Eggert ,1968 ;author ,unpublished) .Tw oplot swit h4

plantso fth e cv.Ato m and 2plot swit h 16plant so fth eF 2:Ato m xW.V a 700 weregrown .

Results and conclusions OneF 2plan tdie dbefor eth e inoculation ofth e spreader rows.Th er-value scalculate d asusua lar epresente d inFig .13 .I n spiteo f itscharacteristi c 'resistancet osporulation' ,th ecv .Ato m reacted

'susceptible' inth e fieldexperiment .Th e 31F 2plant sshowe da broa dspec ­ trumo fr-values .Eigh t ofthes e3 1plant sbelonge d toth edeterminan ttype ,

andthes e 8wer e among the 12mos t susceptibleplants .On eF 2plan twa smor e resistant than theresistan tparent ;al lothe rplant swer e intermediatebetwee n the 2parents . The genetic interpretation ofth etes tresult swil lb ediscusse di n Section 7.3.4.

Discussion Thever y susceptible reactiono fth ecv .Ato m isprobabl y duet o itsdeterminat e and compactwa yo fgrowing ,an dt oth efac ttha tth edesig n ofth e test,a continuou smonocycli c test,weigh s 'resistancet oinfection ' and 'resistancet oextension 'muc hheavie rtha n 'resistancet osporulation' .

6.4 THEPARTIA L RESISTANCEO FSOM ETOMAT OLINE SI NTH E 1972FIEL DEXPERIMEN T

Materials and methods The tomato linesdiscusse d inthi ssectio nwer eal l growno nth eexperimenta l fielddescribe d in6.2 ,an dtreate d asindicate d there.Tw ofour-plan t plotso feac ho fth ecultivar sAtom ,Moneymaker ,Vercla - brid,an dW.V a 63,an d 2eight-plan tplot so fth etomat oline sP I198674 ,

PI 224675,W.V a 700,F x:Moneymake r xW.V a 700,an dF lS PI 224675* W.V a70 0 hadbee nplanted .Al l ofthes eplant shav e atleas tsom epartia lresistanc e tolat eblight , 'Moneymaker'excepted .

Results and conclusions InTabl e 20,th e tomatoline sar eranke daccordin g tothei r levelo fpartia l resistance asassesse d inth efiel dexperiment .Th e

67 Table20 .Tomat oline steste dfo rpartia lresistanc ei na fiel dexperiment , Wageningen.Th emeasur eo fdiseas ewa sth e'apparen tinfectio nrate 1 r, expressed inlogi tunit spe rday .T i sth eperio do ftim edurin gwhic hth e fractiondisease dfoliag eincrease dfro m0.0 1 to0.50 .RES(r )i sth epartia l resistancerelativ et oth esusceptibl e standard 'Moneymaker'.

Tomatoline s n r T EES(r) S:Lgn .

Atom 4 0.58 8 - a Moneymaker 4 0.43 11 0.00 a Verclabrid 2 0.28 16 0.34 a b W.Va6 3 4 0.24 19 0.44 b F].:Moneymake rx W.V a70 0 8 0.21 23 0.51 bc PI19867 4 2 0.17 27 0.59 cd W.Va70 0 7 0.17 27 0.59 cd PI22467 5 4 0.16 29 0.62 cd Fi:P I22467 5x W.V a70 0 4 0.13 35 0.69 d n =th enumbe ro fplant s assessed Sign.= line swit hdifferen t letters differ significantlyi nr atp 4 0.05

partialresistanc e relativet oth ecv .Moneymake r hasbee n calculatedb y meanso fth eequation :

RES(r)= 1 7(51 r(T)i sth e'apparen t infection rate'o fth etes tplan t r(S)i sth e'apparen t infectionrate 'o fth esusceptibl e standard 'Money­ maker' Ther-value so fth eFi :Moneymake r xW.V a 700,P I198674, P I224675 ,an dF ^ PI 224675 xw.V a70 0di dno t differ significantlya tp 4 0.05 fromW.V a 700. The lineP I19867 4an dW.V a70 0differe dneithe ri ntyp ean dleve lo fresist ­ ance,no ri nmorphology .P I224675 ,thoug habou ta sresistan ta sW.V a 700, wasmarke db yth epresenc eo fsporulatin g lesions,whic hwer e seldom found onW.V a 700.I ti sconcluded ,tha tW.V a70 0an dP I19867 4ar eidentica l lines, andtha tth e resistanceo fP I22467 5i sdifferen t fromtha to fW.V a700 .

Discussion W.Va6 3i sclosel yrelate dt oW.V a 700,bu twa s foundt ob esigni ­ ficantly (p4 0.05) lessresistan t thanW.V a 700i nthi stest .I ncontras tt o W.Va700 ,th ecv .W.V a6 3ha da compac tgrowt htype ,no texceedin ga heigh to f 80cm .Bot hcompactnes san dlo wheigh tmigh tb ehel dresponsibl efo ra compar ­ ativelyhig hr-value ,becaus e compactness leadst oa micro-climat e withinth e plantcanopy ,tha ti srelativel y favourablet olat eblight ;moreover , themicro ­ climatei smor e favourablet olat ebligh ta tlowe r levelstha ntha ta thighe r

68 levelso f thecrop .

6.5 CONCLUSIONS

Themetho d described above,whic h implies theassessmen t ofth e fraction diseased foliage at regularintervals ,th e calculation ofth e 'apparentinfec ­ tionrate ' r, and theus eo fr a sa measur e ofresistance ,ha s givenusefu l results. The tomato linesW.V a70 0an dF ^ Moneymaker* W.V a70 0showe da consider ­ able level ofresistanc e inthi sexperiment ,bu t 'Atom',characterize db ybush y growthan d 'resistance tosporulation 'wa s foundt ob ehighl y susceptibleac ­ cording tothi stest .

TheF 2:Moneymake r xW.V a 700 andF 2:Ato m xW.V a 700showe da spectrum ofr-value swithi n the ranges limitedb y the correspondingparents . The continuousmonocycli c test isa goo d testfo rth eevaluatio n ofdif ­ ferencesi npartia l resistance,whe nthi si sdu et o -resistancet oinfection ' and/or 'resistance toextension' .Th econtinuou smonocycli c testcanno tb e used for the evaluation of 'resistance tosporulation' .

69 7 Genetic analysis of resistance against Phytophthora infestans

7.1 INTRODUCTION

Mosto fth eexperiment sreporte di nthi spape rhav ebee ndon ewit hth e tomatoaccessio nWes tVirgini a 700 (L. pimpinellifolium). Accordingt o Gallegly (1964),W.V a70 0bear sth ePhi-gen efo rresistanc eb yhypersensitivi ­ tyagains tpathotyp e0- to fP . infestans, inadditio nt oa hig h levelo f 'multiplegen eresistance 'agains tpathotyp e 1-t.I nvie wo fit shig h level ofsupposedl ypolygeni cresistance ,W.V a70 0ha sbee nchose nfo rth egeneti c analysiso fpartia lresistanc e againsta nisolat eo fpathotyp e 2,4-p/1-to f P. infestans. Thetomat ocv .Atom ,characterize db ya hig h levelo f'resist ­ ancet osporulation ' (Grummer& Eggert , 1968),an dbush ygrowt htype , and theF 2:Ato m xW.V a70 0hav e alsobee ntested .Unfortunately ,n o Fiplan t ofth ecros sAto m* W.V a70 0coul db etested ,becaus eo fth efailur et oob ­ tainenoug hF jseeds .

7.2 RESISTANCEO FTH ESTEM S

7.2.7 Field test data of the aross Moneymaker x W.Va 700

Introduction Allplant so f'Moneymaker 'grow ni nth efiel dexperimen tdescrib ­ edi nSectio n6. 2reacte dt olat ebligh twit hcharacteristi c large lesionso n thestems .Lesion s> 4m mwer eneve robserve do nstem so fplant so fW.V a70 0 orFi :Moneymake r xw.V a 700.Th eF 2 populationo fth ecros sMoneymake rx W.Va 700containe d7 plant swit han d2 5plant swithou tlarg e stemlesions , causedb yth elat ebligh tfungus .Thes eresult s suggestth esegregatio no f onedominan tgen epreventin g largeste mlesions . Thehypothesis ,tha ta singl edominan tgen eprevent s large stemlesions , willb eteste di nth efollowin gexperiment .

Materials and methods F3 seedswer eharveste d fromal l1 6F 2plant so fth e crossMoneymake rx W.V a70 0grow no non e ploto fth efiel dexperimen tde -

70 scribedi n6.2 .Thre eo fthes eF 2plant swer echaracterize db yth epresenc e oflarg e stem lesions causedb y2,4-p/1-t-pathotyp e ofP . infestans; theothe r

13F 2plant swer efre eo fste m lesionswit ha $ > 4mm .Fro meac hF 3 line1 2 to1 6plant swer egrow no na nexperimenta l field (18x 1 4m )withou tirriga ­ tion.Th eline swer e growni nrow sa ta distanc eo f1 m an daplan t spacing inth erow so f0.7 5m .Th efiel dwa ssurrounde db ya ro wo f6 6'Moneymaker ' plants.O n73.07.27 ,al lplant swer e inoculatedb ymean so fa knapsac kmist ; blower.Tw olite ro fa suspensio no f2 zoosporangi ape ry lo fa nisolat eo f the 2,4-p/1-t-pathotypewer e applied;th eag eo fth eplant swa s 12weeks .O n

73.08.03,th eF 3plant swer e inspectedfo rste mlesions .

Results and conclusions Large stem lesionswer epresen to nal l4 6plant so f the3 F 3 lines,whic hdescende d from the3 F 2plant scharacterize db ylarg e

stem lesionsi nth eexperimen t describedi n6.2 .Seve no fth e1 3F 3 lines descending fromF 2plant s freeo fste m lesionsi ntha texperiment ,wer emarke d byth epresenc eo f1 t o6 plant swit h largeste m lesions.Th eplant so fth e otherF 3 lineswer e freeo fste m lesions.I nth eF 3,th eobserve drati oagree s 2 witha 1:2: 1 ratio( x = 1.38 ;p > 0.5 )base do nth eexpecte dsegregratio no f 2 a single,dominan tgene .

The segregratingF 3 linescontaine d9 8plants ,o fwhic h2 3wer echarac ­ terizedb ylarg e stem lesionsan d7 5wer efre eo fste mlesions .Th eobserve d ratiobetwee nbot htype so fplant s agreeswit ha 1: 3rati o( x*- 0.12 ; p> 0.7 )a scause db yth e segregrationo fa single ,dominan tgene .Al levidenc e supportsth ehypothesis ,tha tW.V a 700carrie sa dominan tgen efo rresistanc e ofth e stemst oth e1-t-pathotyp eo fP . infestans.

Discussion Thegene ,tha tprotect sth estem sagains tpathotyp e1- to fP . infestans isdifferen t fromth egen ePh lf becausetomatoe sresistan tt oth e 0-t-pathotypeonl y showlarg este mlesion swhe nattacke db yth e 2,4-p/1-t- pathotypeo fP . infestans. Iti spropose dt onam ethi sne wgen ePh 2.

7.3 RESISTANCEO FTH ELEAVE S

7.3.1 Statistical analysis

Ingeneral ,th ephenotypi cexpressio no fpartia lresistanc edepend smor e onenvironmenta l conditions thantha to fresistanc eb yhypersensitivity . Neglecting interaction,phenotypi cvarianc e canb erepresente dby .

71 VAR(P)= VAR(G )+ VAR(E ) inwhic hVAR(G )i svarianc edu et ogenotypi csource san dVAR(E )varianc edu e toenvironmenta lsources .Whe nVAR(G )i sabsent ,a si sth ecas ei npur eline s andF ihybrid so fthes elines ,th eobserve dvarianc eo fan ymeasur eo fre ­ sistancewil lb eo fenvironmenta lorigin .Conversely ,pur eline san dhybrid s canb euse dt oobtai nestimate so fenvironmenta lvariance . Ascultivate dan dcurran ttomatoe sar emainl yinbreedin gcrops ,tomat o linesan dF xhybrid susuall yconsis to fgeneticall yidentica lindividuals . Bysubjectin gth etes tresult so fthes eline st oa nanalysi so fvariance ,th e withinlin evarianc eca nb ecalculated ,an dthi svarianc e isa nestimat eo f theenvironmenta lvariance .

7.3.2 Environmental variance of partial resistance

Materials and methods Thefiel dresult so f4 plant so feac ho fth etomat o lines 'Moneymaker',W.V a700 ,P I224675 ,Fi :Moneymake r* w.V a700 ,an dFi : PI22467 5x W.V a70 0wer euse di na varianc eanalysis .Th eplant swer echose n insuc ha way ,tha to feac h4 plant stested ,2 wer egrow ni na borde rro wan d theothe r2 somewher ei nth ecentr eo fth efield ,t ose ei fther ewa sa nef ­ fectdu et oth epositio no fth eplan ti nth efield .Plant si nborde rrow sar e supposedt obelon gt obloc kA ,an dth eothe rone st obloc kB .

Results and conclusions Theresult sar eshow ni nTabl e2 1an d22 .Varianc e betweenline si shighl ysignifican t (p4 0.001),bu tvarianc edu et oblock s orblock sx line si sno tsignificant .Apparently ,ther ei sn osignifican t

Table21 .Partia lresistanc eo ftomat oagains tP . infestans. Linesteste di n afiel dexperimen t (see4.7.4) .Th etes tresult sar epresente d inth efor m ofvalue so fr ,'th eapparen tinfectio nrate 'expresse d inlogi tunit spe r day,fo rth epurpos eo fa varianc eanalysis .Th eentrie st oth etabl ear er x 100.

Lines (genotypes) Block A Block B S

Moneymaker 46.1 50.5 38.0 35.4 170.0 W.Va70 0 13.9 16.5 18.3 19.7 68.4 PI22467 5 16.2 19.1 12.2 18.0 65.5 "S\i Moneymakerx W .Va 700 21.2 20.3 20.6 19.4 81.5 F1: PI22467 5x w . Va 700 10.4 10.2 13.9 17.1 51.6 1 224.4 212.. 6 437.0

72 Table22 .Analysi so fvarianc eo fth edat ai nTabl e21 .

Sourceo fvarianc e Sumo fsquare s df Variance Variance ratio Probability lines 2241.3 4 560.0 43.4 Pi0.00 1 blocks 7.1 1 7.1 <1 p> 0. 2 blocksx line s 92.2 4 23.1 1.79 p >0. 2 residual 128.6 10 12.9 total 2469.2 withinline s 227.9 15 15.2

Thewithi n linesvarianc ei sth eperio dvarianc eo fblocks ,block sx lines , andresidua l

difference betweenplant s growni nth eborde rrow san dthos ei nth e centre ofth e field.A sth evariance sdu et oth e sourcesblock s andblock s* line s dono t differ significantly from theresidua lvariance ,the yca nb epoole d withth eresidua lvarianc et oobtai nth ewithi n linesvariance .Th ewithi n linesvariance ,whic hi nthi sexperimen ti sa nestimat eo fth e environmental variance VAR(E),ha sa valu e 15.2( xlO^.r?) ,an d1 5degree so ffreedom .Th e coefficiento fvariatio n s/mi s/15.2/21. 9= 0.18 .

7.3.3 Field data of the cross Moneymaker x W.Va 700

The field resultso fth ecros sMoneymake r xW.V a 700,describe di nsec ­ tion 6.2,wil lb einterpretate d genetically.Afte ra subdivisio no fth eF 2 plants into2 group so nth ebasi so fabsenc eo rpresenc eo fste mlesion s(se e

Section 7.2),a varianc e analysiso fth er-value so fth eF 2 groupsha sbee n made (Table 23).Th e2 groups ,whic hha dmea nr-value so f0.2 4an d0.4 1re ­ spectively,differe d significantlya tp < .0.01 .Th evariance so fth eF 2plants ,

Table23 .F 2:Moneymake rx W.V a700 .Inheritanc eo fP"'* * "J^^Sle"' P. infestans. Comparisono fvariance scalc uate d romF 2P anJ ^ "J"^.. sions (GroupA )an dwithou t stemlesion s (GroupB ; totn ee n anceVAR(E) ,calculate d in7.3.2 ,b ymean so fa nF-test . Variance ratio Probability Variance df , F 6 = 1 27 p>0.2 v Group A 14.5 6 F24 '•'" n/ v 6 = 0.96 P > °'2 Group B 11.4 24 F15 u#* T?15 =1.33 p > 0.2 Var (E) 15.2 15 F2H 73 11.4an d14. 5 (x 10~4.r2), didno tdiffe r significantly fromth eestimat eo f VAR(E)= 15. 2 (x 10"4.r2;se eSectio n7.3.2 )a tp 4 0.2.Consequently , within the2 group sn oothe r sourceso fvarianc e seemt ob epresen t thanth eenviron ­ mental source,VAR(G )bein g smallo rabsent .Th edifferenc ei npartia lresist ­ ancebetwee nth eF 2group s couldb edu et oth edominan t gene Ph2 (seeSectio n 7.2), thatprotect s stemsagains tth e1-t-pathotyp eo fP. infestans, ort oa gene linkedwit hit .Th ehypothesis ,tha tpartia lresistanc e ofW.V a70 0i s monogenic,ha sbee n testeda ta large r scalei na nexperiment ,reporte di n Appendix 1.

7.3.4 Genetic analysis of the laboratory data of the cross Moneymakerx W.Va 700

F\: Moneymaker x W.Va 700 Theresistanc eo f6 week sol dF iplant si sinterme ­ diatebetwee n theresistanc eo f'Moneymaker 'an dtha to fW.V a70 0o fth esam e age.Th eresistanc eo f8 week sol dplant si snearl yequa lt oth eresistanc e ofth eW.V a70 0plant so fth e sameag e(se eFig . 11).

Fz: Moneymaker x w.Va 700 Thedistributio no fth eRES(E,I)-value so f6 week s oldplant swa scontinuous ,an dal lclasse so fRES-value swer emor eo rles s equally filled (Fig.10) .Suc hdat acoul dlea dt oth econclusion ,tha tth e partial resistanceo fW.V a70 0inherite squantitatively . These datagiv en o cluet oth egeneti cbasi so fth eresistanc e involved. Thefrequenc y distributiono fth eRES(E,I)-value so f8 week sol dplant s suggestsa differentiatio no fth eplant s into1 grou pa sresistan ta sW.V a700 , and another groupa ssusceptibl ea s'Moneymaker' ,i na 3: 1rati o (Fig.10) . Thesedat aindicat etha tth epartia l resistanceo fth eleave so fW.V a70 0i s controlledb ya singl egene .

Bi: Moneymaker x (Moneymaker x w.Va 700) Thedat afro mth eB jtes t(se e Section 5.10) supportth econclusio n thatth eresistanc eo fW.V a70 0i sdu e toa singl e genefo rpartia lresistance .I nth e8 week sol dplant so fthi s back cross,ther ei sa differentiation ,whic h suggeststh e presenceo f2 e - qually filled classes (Fig.11) ,on ea ssusceptibl e as'Moneymaker' ,th eothe r asresistan ta sW.V a700 .

74 7.3.5 Field data of the aross Atom x W.Va 700

Inth ecros sAto m xW.V a 700,th ephenotypi cexpressio no fpartia l resist­ ancei sinfluence db y3 geneti c systems.First ,ther ei sth egen efo rpartia l resistanceo fW.V a 700name d Ph2 (seeSectio n7. 2an d7.3.3) . Second, there isth edeterminat e growthdu et oa singl e gene.Third ,ther ei sth e'resist ­ ancet osporulation 'o funknow n geneticnatur e carriedb yth e cv.Atom .Whe n theeffec to f'resistanc et osporulation 'i sconsidere dt ob esmal l(a sca nb e seenfro m the datao fcv .Atom ,Tabl e 20), twomonogeni c systemswit ha consi ­ derable effecto nth eexpressio no fresistanc ear eleft .Al ldeterminat e plants belongt oth esusceptibl e parto fth epopulatio n (Fig. 13).Whe nth edeter ­ minate plantsar eexcluded , therear e2 3plant s left,ou to fwhic h7 ar ea s susceptiblea sth e7 F 2 plantswit h largeste m lesionsdescendin g fromth e crossMoneymake r* W.V a70 0(Fig .12) .Thes e7 plant sha da n'apparen tinfec ­ 2 tionrate 'r > 0.35 .Th erati o7:1 6agree swit ha 1: 3rati oa tp > 0.5 ;x = 0.36.Whe nth esingl eplan twit hr = 0.3 2i sals oaccepte da ssusceptible , p> 0.2 ;x 2= 1.17 . Inconclusion ,ther ei sevidenc e that,apar tfro mth egen efo rdetermi ­ nancy,th ePh 2 genei sth eonl ygen ewit ha considerabl e effecto nth eex ­ pressiono fresistanc ei nF 2 plantso fth eF 2:Ato m* W.V a 700.N oproo fca n be given,becaus eth esubdivisio n intoresistan tan dsusceptibl eplant s isa n arbitraryone .

7.4 THE'ATOM ' PARADOX

Inth eF 2 populationo fth ecros sAto mx W.V a700 ,n odeterminat e plants were foundi nth e resistant group.Th echance ,tha tal l8 determinat eF 2 plantswer e susceptible becauseo fth eabsenc eo fth ePh 2 genei sver y small

(p= 15.10-6) . So,ther emus thav ebee ndeterminat eplant swit hth ePh 2 gene present.I nth etes t situationdescribe d (seeSectio n 6.3),determinat egrowt h seemedt ocaus e epistatic susceptibility overresistanc eb yth ePh 2 gene.Thi s epistaticsusceptibilit y canb edu et oabsenc eo fth eresistanc e-chanis m (genetically epistatic)o rdu et otha tfac ttha tth edeterminat eplant s gw ™.M* tnlat ebligh t development (environmentally under conditionsmor e favourablet olat eDugm . i- • j- «--™c thatth eenvironmenta l conditionsar etn e epistatic).Ther ear eindications ,tha ttn eenv± i „,nrt„,i / •• i=nth ecas ewit hth eplant so f'Atom' ,whic hreacte d decisive ones,a si sals oth ecas ewiu i w» y •* thpir 'resistancet osporulatio n (see ashighl y susceptible inspit eo fthei nfr resibtcu^ Section6.4) . 75 7.5 DISCUSSION

There isconvincin gevidenc e thatth e resistance ofth e stems inW.V a 700 against the latebligh tpathotyp e usedi scontrolle db y asingl edominan tgene , here calledPh 2 (seeSectio n 7.2). Inth e fieldtria lwit hF 2plants ,i twa s demonstrated thatth evarianc e ofpartia lresistanc e inth e leaveswa sdeter ­ minedb yth e genotypiceffec to fPh 2 togetherwit henvironmenta l effects,an d thatn o othersource so fvarianc e couldb edemonstrate d (7.3.3).Th e labora­ torytest swit hth e crossMoneymake r xW.V a 700giv eevidenc e ofth epresenc e ofa singl egene ,tha t controlspartia lresistanc e inth e leaves (seeSectio n 7.3.4).Obviously ,thi sgen efo rpartia lresistanc eo f the leaves is the same asth e genePh 2 forresistanc e ofth estems . Partialresistanc e ofW.V a70 0increase sunti l atleas t8 week s after seeding (seeSectio n S.4).Si xweek sol dplant so fth eF 2:Moneymake r xW.V a

700an do fth eB x:Moneymake r x (Moneymaker xW.V a 700)ar eno t accessible to geneticanalysis .Th eresult sfro m 6week s oldplant s canonl y leadt o comments like:partia lresistanc e ofW.V a 700ma y inheritequantitatively . Iti sno t justified tous e the term 'multigenicresistance 'o nth ebas e ofsuc hvagu e information. Forth epurpos e ofselection ,6 week s oldplant smight b emor e suitable than8 week splants .Ther ei sevidence ,tha tth ePh 2ph2 genotype ismor e dis­ tinctfro mth ePh 2Ph2 genotypea tth e ageo f 6week s thana tth e age of8 weeks,becaus e theresistanc eo fth eheterozygot eF 2 plant is intermediate atth e ageo f6 weeks ,bu tnearl ya shig h astha to fth e resistantparen t at the ageo f8 weeks . Thehig hleve lo fresistanc eo fth eFx :P I 224675x W.V a 700,an dth e fact,tha tth ereactio no fP I 224675t olat ebligh twa s different fromW.V a 700,migh t indicatetha ti nW.V a 700an dP I 224675differen t genes for late blightresistanc e arepresent ,possibl ywit hadditiv eeffects .

The 27to pleaflet so fth e leaveso fth e 27F 2 plants and 4to p leaflets of theleave so fth e 4 'Moneymaker'plant steste d at anag e of6 week s inth e experimentdescribe di n5. 8wer e inoculatedb y spraying a zoospore suspension ofth e 1,2,3-p/0-t-pathotype ofp . infestans untilrun-off .Afte r incubation (seeSectio n 4.6),th e leafletswer eobserve d for latebligh t attack.Al l 4

'Moneymaker- leaflets and 1F 2 leaflet (F2 plant 3)wer e seriously affected.

Theothe r 26F 2 leafletswer e apparently free frominfection .Th ehypothesis , thatPh 2 genedoe sno t actagains t 0-t-pathotype ofp . infestans canb ere -

76 2 jected (x = 6.45;p <0.02) , asi nthi scas e6. 7 susceptibleF 2plant swer e expected.

7.6 CONCLUSION

Theresistanc e ofth e stemsi nW.V a70 0agains tth e 2,4-p/1-t-pathotype

P. infestans used iscontrolle db y asingl edominan tgene ,whic hi sname d Ph2. Thepartia lresistanc e ofth e leavesi nW.V a70 0i sdu et oth esam egene ,o r toa gen e closely linkedwit h Ph2.

Inth e continuousmonocycli c test,th edeterminat eF 2plant so fth ecros s

Atom xW.V a 700wer eepistaticall ysusceptibl e overresistanc eb yth ePh 2 gene.

77 8 Comparison of testing methods with special reference to their application in commercial breeding programs

8.1 ACOMPARISO NO FTESTIN GMETHOD S

Introduction Duringa 3 years 'perio dmethod shav ebee ndevelope dan dteste d forth eassessmen to fpartia lresistance .A reliabl ean deas ymetho d forth e assessmento fpartia l resistance opens thewa yt oth eapplicatio no fpartia l resistancei ncommercia lbreedin gprograms .I nthi s section,th evariou s methodswil lb ecompared ,an dthei r applicabilityi ncommercia lbreedin gpro ­ gramswil lb econsidered . Ina nattemp tt oevaluat eth evariou s testingmethods ,th ecoefficien t ofvariatio no fcertai nmeasure sha sbee n chosena sa mean so fcomparison . The coefficiento fvarianc eC Vuse dher ei sth estandar ddeviatio ns expresse d asa fractio no fth eaverag em (CV= s/m) .A sth estandar ddeviatio n depends onth enumbe ro fobservations ,th eidea li st ocompar eC Vvalue sbase do ne - qualnumber so fobservations .Thi swas ,alas ,no talway spossible .

The methods Thefollowin g threemethod sar ecompare dhere : a.Probi t- lo gdosag emetho d (seeSection s4.5. 1an d5.4) . b. Components analysismetho d (seeSectio n5.8) , c.Fiel dmetho d (seeSectio n6.2) . Strictly speaking,th eprobi t- lo gdosag emetho di sonl ya complicate dvar ­ ianto fth eusua lmetho dt oestimat eth einfectio n ratio.Accordingly ,i t couldb econsidere da sa par to fth ecomponent s analysismethod ,bu tbecaus e ofit scomplexit yi ti sconsidere da sa separat emetho dhere .

Results Theprobi t- lo gdosag emetho di sth emos tprecis emethod .Th e

CV(ED50)varie sbetwee n0.0 3(se eSectio n 5.4)an d0.06 .However ,thi smetho d demandsa hig h labourinput ,an di trequire s expensive facilities like growth chambersan dawel lequipe d laboratory.Th elabou rneede di sroughl y estimated at1 man-hou rpe rplant .Consequently ,i ti sa ver ycostl ymethod . Thecomponent s analysismetho dwa s lessprecis e thanth eprobi t- lo gdos ­ agemethod .Th edat ao fth eexperimen tdescribe di nSectio n5. 9yielde d

78 CV(E)= 0.11 .Th elabou rrequire di sabou t0. 2man-hou rpe rplant .Th e dura­ tiono fa singl e experiment fromseedin gt otestin gi sabou t2 months .Othe r characters,lik e latentperio dan dsporulatio n intensityca nb eassesse dtoo , usingth esam ematerials .Th eexperiment s canb edesigne do na year-roun d base.Th e disadvantagesar eth ehig hcost so fth e laboratoryan dglasshous e facilities,an dth e peakwork-load s during inoculation andmeasuring . The fieldmetho dwa sth eleas tprecis e one.I tca nb ecalculate d that CV(r)=0.1 8 (seeSectio n 5.4).Whe nCV(r )i scalculate dfo rth e same lines andth e samenumbe ro fplant sa sth eabov ementione d CV(E),4 plant so feac h ofth e linesMoneymaker ,W.V a 700,an dF ^ Moneymaker xW.V a 700,a figur e of0.1 8i sfoun d again.Th eestimate d labourinvestmen ti s0. 1man-hou rpe r plant.Additiona l advantageso fth e fieldmetho d areit srelativ e simplicity, thepossibilit yt oobserv eothe rcharacters,o fimportanc et oth ebreeder , and the absenceo fhig hpea kwork-loads .Disadvantage so fthi s typeo ftes t (con­ tinuousmonocycli c test)ar eth eunder-estimatio no fpartia l resistancei n general,an dth efailur et oasses sth e valueo fsom e typeso fresistance , e.g. the 'resistancet osporulation * carriedb y'Atom' .Th emetho di sseaso nde ­ pendent;i nth e Netherlands only1 experimen tpe ryea rca nb edone .Th etes t hasa duratio no f4 months ,fro m seedingt oharvest .

Conclusions 1.Th eprobi t- lo gdosag emetho di sa rapi dan dprecis emethod ,bu ti sals o a costlymethod ,an di tdoe sno t satisfyth epractica lrequiremen to fappli ­ cabilityt olarg enumber so fplants . 2.Th ecomponent s analysismetho di sa rapi dan drathe rprecis emethod , applicablet olarg enumber so fplants ,bu ti sno ta chea pmethod . 3.Th efiel dmetho di sth eleas tprecis e one,bu ti ti sa nadequat emethod . Themetho di ssimpl ean dcheap ,seaso ndependent ,an dther ei sn olimitatio n toth e numberso fplant s tested.

8.2 ASIMPLIFIE D RECIPEFO RTH EBREEDE R

Themetho do fdiseas e assessmentb yestimatin gth efractio ndisease d foliage,an dsubsequen t transformationo fth efractio nestimate dint ologi t units, followedb yth e calculationo fth eregressio n line,ca nb esunplifwd . Ametho di sgiven ,i nwhic hth efractio ndisease dfoliag ei sestimate db ymean s ofa 2 1clas s scale (Fig. 14).Th escal evalue shav ebee ncalle dtransforma ­ tion-valueso rshortl yTR-values .Thes evalue swer echosen ,becaus e theywer e

79 convenientt ous ei nth efield .The y caneasil yb etransforme d into logit units,a sth eTR-value sar eproportiona lt oth elogi tvalue swithi nth erang e from 0.01 to0.S9 .Withi nthi srange ,th efollowin gequatio ni svalid : y= 1.16 s- 5.80 inwhic h y standsfo rlogi tunit s and x forTR-values .Th ecalculatio no fth e 'apparentinfectio nrate 'r ca nb esijnplifie db yusin ga graphica lmetho dt o estimater .Fo rthi spurpos eth eobserve dTR-value sar eplotte d againsttim e (Fig. 14),an dth eregressio nlin ei sdraw nb yeye-fitting .Th etangen to f the anglebetwee nregressio n linean dabsciss ai sa nestimat eo fr , expressed inTR-value spe rday .A nestimat eo fth e'apparen tinfectio nrate 'r i nlogi t unitspe rda yi sobtaine db ymultiplyin gth evalu e foundb y1.16 . Asa nexample ,th eresult sfro mtw odifferen tplant sar egive n (Fig. 15). The calculatedr-value sar e0.3 6an d0.2 0 logitunit spe rday ,respectively . The r-values obtainedb yth e graphicalmetho d are0.3 2an d0.2 0logi tunit s perday ,respectively .Th eresult sobtaine db yth e2 method sar ei ngoo d agreement.

logit units 5 - TR-values 10 Moneymaker

y=1.16jr- 5.80 Fi:Moneymaker x W.Va 700

1-

2-

3

4

5 0.00 am 003 0.10 Q25 050 0.75 0.90 097 099 100 I I I 111 I I 11 1 < \ < I 11 11 11 —1 1 1 1 1 20 30 40 0 123456 789 10 days after inoculation TR- units Fig. 14.Th erelatio nbetwee na Fig. 15. P. infestans ontomatoe s ina logitunits-scal ean dth eTR - fieldexperiment .Tomatoe sassesse dfo r units,use di nth efiel dexper ­ resistanceb ymean so fth efiel dmetho d imentst oclassif yth efractio n (seetext) ,Linea rregressio no ffractio n diseasedfoliage . diseased foliage (expressed inTR-values ) Abscissa:uppe rro wo ffigure s ontime .Graphica lmetho d toestimat er presentth epercentag e 'fraction (r= a/b ,r 1= aVb1). diseasedfoliage' ;lowe rro wo f figurespresen tth eaccordin g TR-units. Ordinate:logi tunits .

80 Summary

Chapter 7 Inth eperio d from 1965t o 1968,P . infestans causedepidemic so n tomatoes inunheate d andslightl yheate dglasshouse s inth eNetherlands .Be ­ forethi speriod, latebligh twa shardl yknow na sa diseas eo ftomatoe sunde r glass.Apar tfro m theclimati ccondition s conducive toth edevelopmen t oflat e blight,th eprominenc e ofth ediseas ewa sattribute d toth egrowin go ftomat o cultivars resistant against tomato leafmoul d (Cladosporium fulvum). As a consequence of theus e of C. fulvwn resistant cultivars,treatmen tb yfungi ­ cideswa s omitted, thusgivin g P.infestans achanc e todevelo pwithou thin - drance. As treatment withfungicide s isno t alwayseffectiv e andsometime sunde ­ sirablebecaus e ofspra yresidues ,resistanc e tolat ebligh tbecam e imperative. However,source s ofresistanc e arerare ,resistanc e isofte nincomplet e or •partial',th e level ofresistanc ebein g toolo wt oprotec tth ecro pwithou t theus e offungicides .On e solutiono fth eproble m couldb eth e accumulation ofpartia lresistanc e by adequategeneti cmethods .Partia lresistance ,how ­ ever,i sdifficul t tohandl eb ybreeders ,a sit sgeneti cnatur e isofte nun ­ known.It sphenotypi c expression dependso nenvironmenta lconditions .

Chapter 2 Inth eNetherlands ,th ediseas e isa tpresen tno t aseriou sproblem , but inthos epart s ofEurop ewher e tomatoesar egrow ni nth eope n tanato latebligh t isconsidere d tob eth emos t importantdiseas e ofoutdoo rtomatoes . Theopinio nprevails ,tha tincidenc e andseverit yo fth ediseas e-rea s d during the lastdecade .Ih eyear-roun d cropping systemi s -sPonslbl^hlS increase,a sca nb edemonstrate db y theexperience s inBulgari a f***"* before andafte r 1965.Alon gwit hth e largescal eintroductio n of^ng^d autumncrop s inglasshouse s in 1965,tomat o latebligh tros em statusfro m anunimportan t disease toa ver yseriou sone .

**- , Studies»er e-ad e onth ehos t rangeo fto»at olat e"**j » * Netherlands.A newhost , *~Ua tana.eHplia, couldb e aMedtthSt. inth eNetherlands ,ther e isn oevidenc e forothe rsource so ftoeat o

81 blight thanth e latebligh to npotatoes .Laborator y experimentswit h serial passages throughtomat o leaveshav e shown,tha tth epathogenicit y to tomatoes of latebligh t isolates collected frompotatoes ,whic ha t thestar twer eno t or slightlypathogeni c totomatoes ,coul db e raisedconsiderably . Similarre ­ sultswer e obtainedi na fiel dtrial .

Chapters 4 and 5 Investigations weremad e onpartia l resistance.Th e approach was twofold,i.e .th edevelopmen t of laboratorymethod s forth e analysis of 'componentso fresistance 1,an dth e development ofa fiel dmetho d to evaluate differences inpartia l resistance ofindividua lplant soutdoors .Th eassess ­ ment of 'componentso fresistance 'require dhig hprecisio n ofth edosag e of inoculum,an d lowenvironmenta lvariance .T oreduc e environmentalvarianc e to aminimum , thetes tplant swer egrow no nwate rculture s inth ewalk-i n growth chambers ofth eDepartmen t ofPhytopathology ,Stat eAgricultura l university, Wageningen.I nmos to fth eexperiment so npartia lresistanc e 2line shav e beenused ,th esusceptibl e cv.Moneymake r andth e lineW.V a 700 characterized by ahig hleve lo fpartia lresistance . 'Resistance to infection', 'resistance toextension' ,an d 'resistance tosporulation ' increasewit h age ofth eplan t up to 8week s afterseedin g orlater .

Chapter 6 Thefiel dmethod ,i nwhic hafte r inoculation ofth e spreader rows theplant swer ekep twe tb ymean so fa nautomati c sprinkling installation during one4 0h perio deac hweek ,wa s suitable forth eevaluatio n ofdiffe ­ rencesi npartia lresistanc ebetwee n individualplants .Th e characteruse d in the fieldexperiment swa s r,th e 'apparentinfectio n rate1, expressed inlogi t unitspe rday .

Chapter 7 Resultsfro m 6week s oldplant so fth eF 2:Moneymake r xW.V a 700 andth eB i: Moneymaker x (Moneymaker «W.V a 700)wer eno t accessible tocon ­ clusive geneticanalysis ,wherea s theresult s from the sameplant s at theag e of8 week s showedMendelea nsegregation .Fro mbot hfiel dan d laboratorytests , itwa s concluded thatth epartia lresistanc e ofW.V a 700 against the1-t - pathotype ofP . infestans iscontrolle db y asingl e gene,whic hgive s complete resistance toth estem san dpartia lresistanc e toth e leaves.Thi sne w gene hasbee ncalle d thePh 2-gene.

Chapter 8 Thethre emethod suse dfo rtestin g resistance,th eprobi t log dos­ ageanalysis ,th ecomponent s analysis,an d the fieldmethod ,ar e compared and

82 their applicability topractica l tomatobreedin g areconsidered .Th eprobi t- logdosag e analysis isth emos tprecis e andmos texpensiv emethod .Th ecompo ­ nentsanalysi smetho d issomewha t lessprecise ,applicabl e to largenumber s ofplants ,bu t it isagai na costlymethod .Th efiel dmetho d isno t sopre ­ cise,bu t iti sa nacceptabl emethod .Fo rbreedin gpurpose s asimplifie dmetho d isindicated .

83 References

Alexander,L.J .& M.M .Hoover ,1955 .Diseas eresistanc ei nth eworl d species oftomatoes .Bull .Ohi oagr .Exp .St nRes .752 . Anonymous, 1968.Moeilijk etomatenziekte no.a . Phytophthora infestans inkoud e engestookt eteelten .Tuinderi j (1968):386-389 . Anonymous,1968a .Aardappelziekt ei ntomaat .Groente ne nFrui t24 :1165 . Bary,A .de ,1876 .Researche sint oth enatur eo fth epotat ofungus , Phytoph­ thora infestans. Can.J .Bot .14 :105-126 ;149-154 . Berg,A. , 1926.Tomat olat ebligh tan dit srelatio nt olat ebligh to fpotatoes . Bull.W .Va .Univ .agr .205 . Berkeley, 1846.Cfr .d eBary ,1876 . Berkson,J. , 1944.Applicatio no fth elogisti cfunctio nt obio-assay .J .Am . Statist.Ass .39 :357-365 . Bliss,C.I. ,1934 .Th emetho do fprobits .Scienc eN.Y .79 :409-410 . Boer,W .den ,1966 .Meeldau we naardappelziekt ei ntomaten .Groente ne nFrui t 22:2061 . Bruyn,H.L.G .de ,1952 .Pathogeni cdifferentiatio ni n Phytophthora infestans (Mont)d eBary .Phytopath .Z . 18:339-359 . Caten,C.E. ,1969 .Spontaneou svariabilit yo fsingl eisolate so f Phytophthora •infestans. Pathogenicvariation .Can .J .Bot .48 :897-905 . Ciccarone,A. ,W .Blac k& J.F .Malcolmson ,1961 .Identificazion ed irazz ed i Phytophthora infestans-(Mont) deBar ys upart id ipatat ae pomodor o raccoltei nquatt oregion iItaliani .Phytopath .Medit .2 :49-53 . Clinton,G.P. ,1903 .Note so nparasiti cfungi .Rep .Conn .agr .Exp .St n (1903): 365. Conover,R.A .& J.M .Walter ,1953 .Th eoccurrenc eo fa virulen trac eo f Phy­ tophthora infestans onlat ebligh tresistan t tomatostocks .Phytopatho ­ logy43 :344-345 . Cox,A.E .& E.C .Large ,1960 .Potat obligh tepidemic sthroughou t theworld . U.S.Dept .Agr. ,Agr .Handboo k 174:23 0pp . Crosier,W. , 1934.Studie si nth ebiolog yo f Phytophthora infestans (Mont.) deBary .Cornel lUniv .Agr .Exp .St nMem . 155:1-40 . Driver,CM. , 1957.Infectio no fnativ e Solanwn speciesb yth ePotat oBligh t fungus P. infestans. Nature,Lond. ,180 :1367-1368 . Finney,D.J. , 1952.Probi tanalysis .Cambridg eUniv .Press ,Londo nan dNe w York,31 8pp . Gallegly,M.E. ,1952 .Physiologi crace so fth etomat olat ebligh tfungus . Phytopathology42 :461-462 . Gallegly,M.E. ,1960 .Resistanc et oth elat ebligh tfungu si ntomato .Proc . PlantSci .Seminar ,Campbel lSou pCo. :113-135 . Gallegly,M.E. ,1964 .Wes tVirgini a63 ,a ne whome-garde ntomat oresistan tt o lateblight .Bull .W .Va .Univ .agric .Exp .St n490 :3 ,6 ,15-16 . Gallegly,M.E .& J .Galindo ,1958 .Matin gtype san doospore so f Phytophthora infestans innatur ei nMexico .Phytopatholog y 48:274-277 . Gallegly,M.E .& M.E .Marvel ,1955 .Inheritanc eo fresistanc et otomat orac e 0o f Phytophthora infestans. Phytopathology 45:103-109 . Giddings,N.J .& A .Berg ,1919 .A compariso no fth elat ebligh to ftomat oan d potato.Phytopatholog y9 :209-210 .

84 Gilmour,J.S.L .& J .Heslop-Harrison ,1954 .Th edem eterminolog yan dth eunit s ofmicro-evolutionar ychange .Genetic a27 :147-161 . Graham,K.M. ,L.A .Dionn e& W.A .Hodgson ,1961 .Mutabilit yo f Phytophthora infestans onbligh tresistan t selectionso ftomat oan dpotato .Phyto ­ pathology 51:264-265 . Grummer,G .& D .Eggert ,1968 .Di ePrufun gde r Phytophthora-Resistenz von Tomateni nBlattscheibentest .Arch .Pflanzenschut z4 :143-153 . Gunther,E .& G .Grummer ,1964 .Di e Phytophthora-'&esisteriz einigerTomaten - sortenun dihre rNachkommenschaft .Zuchte r34 s14-16 . Hanni,H. , 1949.Beitra gzu rBiologi eun dBekampfun gde rKraut -un dKnollen - faulede rKartoffel ,verursach tdurc h Phytophthora infestans (Mont.)d e Bary.Phytopath .Z .15 s209-332 . Haskell,R.J. , 192].. Phytophthora infestans oneggplan ti nth eUnite dStates . Phytopathology 11:504-505 . Hirst,J.M. , 1958.Ne wmethod sfo rstudyin gplan tepidemics .Outloo kAgric . 2:16-26 . Hirst,J.M . &W.C .Moore ,1957 . Phytophthora infestans onPetuni aan dDatura . PI.Path .6 :76 . Hirst,J.M . &O.J .Stedman ,1960 .Th eepidemiolog yo f Phytophthora infestans* II.Th esourc eo finoculum .Ann .Appl .Biol .48 :489-517 . Hodgson,W.A .& P.N .Grainger ,1964 .Cultur eo f Phytophthora infestans on artificialmedi aprepare dfro mry eseed .Can .J .PI .Sci .44 :583 . Howitt,J.E. , 1917. Phytophthora infestans causingdampin gof fo ftomatoes . Phytopathology 7:319 . Jeffrey,S.I.B. ,J.L .Jink s& M .Grindle ,1963 .Intraracia lvariatio ni n Phytophthora infestans andfiel dresistanc et opotat oblight .Genetic a 32:323-338 . Jenkins,J.A. ,1948 .Th eorigi no fth ecultivate d tomato.Econ .Bot .Vol .II : 379-392. Jinks,J.L .& M .Grindle ,1963 .Change sinduce db ytrainin gi n Phytophthora infestans. Heridity,Lond. ,18 :45-64 . Kedar,K. ,N.J .Rote m& I .Wahl ,1959 .Physiologi cspecialisatio no f Phytoph­ thora infestans inIsrael .Phytopatholog y49 :675-679 . Kishy,K. , 1962.Studie so nth ephysiologica lspecialisatio no f Phytophthora infestans ontomatoes .I .O nth etomato ,- intermediat e- ,an dpotat o typeso f Phytophthora infestans. II.O nth etomat orace so f Phytophthora infestans. Ann.phytopath .Soc .Japa n27 :4 :172-179 ;180-188 . Knudsen,L.F .& J.M .Curtis ,1947 .Th eus eo fth eangula rtransformatio ni n biologicalassays .J .Am .Statist .Ass .42 :282-296 . Koopmans,W .& Th .Strijbosch ,1969 .Meeldau wi ntomaten .All erasse nwee r vatbaar.Groente ne nFrui t25 :537 . Kotila,M.A. , 1949.Studie so nth epotat obligh tfungus , Phytophthora infestans (Mont.)d eBary .W.S .Thesi sUniv .o fMinn .3 5p . Lapwood,D.H. , 1961a.Potat ohaul mresistanc et o Phytophthora infestans. II. Lesionproductio nan dsporulation .Ann ,appl .Biol .49 :316-330 . Lapwood,D.H. , 1961b.Laborator yassessment so fth esusceptibilit yo fpotat o haulmt obligh t {Phytophthora infestans). Eur.Potat oJ .4 :1)7-128 . Lapwood,D.H .& R.K .McKee ,1966 .Dose-respons e relationshipsfo rinfectio n ofpotat oleave sb yzoospore so f Phytophthora infestans. Trans.Br .mycol . Soc.4 9 (4):679-686 . ,. . ^ Leach,Sim .S .& A.E .Rich ,1969 .Th epossibl erol eo fpansexualit yan dc yto- plasmaticvariatio ni nrac edifferentiation .Phytopatholog y59 :1360-1366 . Malcolmson,J.F. , 1969.Race so f Phytophthora infestans occurringm Great Britain.Trans .Br .mycol .Soc .5 3 (3):417-423 . _ Malcolmson,J.F. , 1970.Vegetativ ehybridit yi n Phytophthora infeetans. Nature 225:971-972 .

85 Malcolmson,J.F .& W .Black ,1966 .Ne wR-gene si n Solanum demission andthei r complementaryrace so f Phytophthora infestans (Mont.)d eBary .Euphytic a 15:199-203 . McKee,R.K. , 1964.Observation so ninfectio nb y Phytophthora infestans. Trans Br.mycol .Soc .47 :365-374 . Melhus,I.E. , 1916.Infectio nan dresistanc estudie so f Phytophthora ontoma ­ toes,Phytopatholog y6 :107 . Mills,W.R. ,1940 . Phytophthora infestans ontomato .Phytopatholog y 30:830 - 839. Mills,W.R .& J.S .Niederhauser ,1953 .Observation so nrace so f Phytophthora infestans inMexico .Phytopatholog y 43:454-455 . Moore,W.C. , 1943.Minis .Agr .Bull .126 . Mtiller,K..0. ,1950 .Affinit yan dreactivit yo fangiosperm st o Phytophthora infestans. Nature,Lond. ,166 :392-395 . Nattrass,R.M .& M .Ryan ,1951 .Ne whost so f Phytophthora infestans inKenya . Nature,Lond. , 168:85-86 . Niederhauser,J.S .& W.C .Cobb ,1959 .Th elat ebligh to fpotatoes .Sci .Am . 200 (5): 100-112. Niederhauser,J.S .& W.R .Mills ,1953 .Resistanc eo f Solanum speciest o Phytophthora infestans. Phytopathology43 :456-457 . Palti,J .& D .Netzer ,1963 .Developmen t andcontro lo f Phytophthora infestans (Mont.)d eBar yunde rsemi-ari d conditionsi nIsrael .Phytopath .medit . 2:265-274 . Payen, 1847.Vegetatio nd u Botrytis infestans al'interieu rde sfruit sd u Solanum lyoopersiaon, erythrooarpum (tomate),C .r .hebd .Seanc .Acad . Sci.,Pari s25 :521-524 . Peterson,L.C. , 1947.Th eoverwinterin go f Phytophthora infestans (Mont.)d e Baryunde rLon gIsland sconditions .Am .Potat oJ .24 :188-197 . Peto,S. ,1953 .A dose-respons eequatio nfo rth einvasio no fmicr oorganisms . Biometrics9 :320-335 . Pristou,R .& M.E .Gallegly ,1954 .Leaf-penetratio nb y Phytophthora infestans. Phytopathology 44:2 :81-85 . Reddick,D. , 1928.Bligh tresistan tpotatoes .Phytopatholog y 18:483-502 . Reddick,D. , 1930.Frost-toleran tan dblight-resistan t potatoes.Phytopatholog y 20:987-991 . Reddick,D .& W .Crosier ,1933 .Biologica l specializationi n Phytophthora infestans. Am.Potat oJ . 10:129-134 . Reed,H.S. ,1911 .Tomat obligh tan dro ti nVirginia .Bull .V aagric .Exp .St n 192. Richards,M.C .& R.W .Barrat ,1946 .A partia lsurve yo fth egenu s Lyoopersiaon forresistanc et o Phytophthora infestans. PI.Dis .Rep .30 :16-20 . Rick,CM. , I960.Hybridizatio nbetwee n Lyoopersiaon esaulentum and Solanum pennellii: phylogenetican dcytogeneti c significance.Proc .natn .Acad . Sci.U.S.A .46 :78-82 . Robinson,R.A. , 1969.Diseas eresistanc eterminology .Rev .appl .Mycol .48 : 593-605. Roder,K. , 1935.Untersuchunge niibe rdi ePhytophthorakrankhei t (Phytophthora infestans) derTomate .Unte rbesondere rBeriicksuchtigun gde rbiologische n Spezializationde sErregers .Phytop .Z .8 :589-614 . Romero,S .& H .Fourton ,1963 .Lea fbligh to f Mirabilis jalapa inMexic ocause d bya specialize dfor mo f Phytophthora infestans. Phytopathology 53:25 . Russel,T.A. , 1969.Potat obligh to nth ewee d Solanum xanthooarpum - S. surattense Buna.India nPhytop .22 :267-268 . Sauthof,W. , 1963. Didimella ligulioola (Baker,Dimoc ke tDavis )v .Ar xal s Krankheitserregera nChrysantheme ni nDeutschland .Phytopath .Z .48 : 240-250.

86 Schick,R. , 1932.Ube r dasVerhalte nvo n Solarium demission, Solarium tuberosum und ihren Bastarden gegeniiberverschiedene n Herktinftenvo n Phytophthora infestans. Der Zuchter 4:233-237 . Small, T., 1938.Th e relationbetwee n potatobligh t and tomato blight.Ann . appl. Biol.25 :271-275 . Smith,R.E. , 1906.Tomat o diseases inCalifornia .Bull .Calif , agric.Exp . Stn 175. Smoot, J.J., F.J. Gough,H.A . Lamey, J.J. Eichenmuller &M.E . Gallegly, 1958. Production and germination of oospores of Phytophthora infestans. Phyto­ pathology 48: 165-171. Snieszko, S.F., J.B. Carpenter,E.P .Low e &J.G . Jakob, 1947.Improve d methods for the cultivation and storage of Phytophthora infestans. Phytopathology 37: 635-649. Steiner,A.A. , 1968.Recep t voor eenuniversel e voedingsoplossing. Gestencild verslag 35.Centr e forPlan t Physiological Research,Wageningen , The Netherlands. Steiner,A.A. , 1970.Nieu w recept voor spore-elementen.Centr e for Plant Physiological Research,Wageningen , TheNetherlands . Steiner,A.A . &H . vanWinden , 1970.Recip e for ferric salts of ethylene- diaminetetraacetic acid. PI.Physiol .46 :862-863 . Sztejnberg,A . & I.Wahl , 1966.Studie s on thehos t range of the potato late blight fungus and its oversummering onwil d and ornamental Solanaceae plants. Proceedings of the first Congress of theMediterranea n Phyto- pathological Union,Bari ,Naples ,452-460 . Thaxter, S.P., 1891.Disease s of Tomatoes.Rept .Conn .agr .Exp . Stn (1893): 95-96. Ullrich, J., 1957.Di e Biologie und Epidemiologie von Phytophthora infestans (Mont.) de Bary.Nachr.Bl .dt .Pflschutzdienst .Braunschwei g 9: 129-138. Umaerus, V., 1968.Screenin g of Solarium species for field resistance to Phytophthora infestans. InAbstr .o f Papers Presented at the 1stInt . Congr. Plant Pathol.,London . Umaerus, V., 1969a. Studies on field resistance to Phytophthora infestans. 1.Th e infection efficiency of zoospores of P. infestans as influenced by the host genotype.Z .Pflziicht .61 :29-45 . Umaerus, V., 1969b.Studie s on field resistance to Phytophthora infestans. 2. Ametho d of screening young potato seedlings for field resistance to P. infestans. Z. Pflziicht 61: 167-194. Upshall, A., 1969.Variatio n amongmonosporou s progeny of Phytophthora infestans. Ca.J . Bot. 47:1171-1174 . Van der Plank, J.E., 1960.Analysi s of epidemics.I n J.G. Horsrall& A.H. Dimond.Plan t Pathology III:229-289 .Academi c Press,Ne wYor k andLondon. Van der Plank, J.E., 1963.Plan t diseases:Epidemic s and control.Academi c

Press,Ne w York and London. . _„„„ M„. Van der Plank, J.E., 1968.Diseas e resistance inplants .Academi c Press,Ne w York and London. . , •„,••.„ „r pz,,,+,,,,;,_ Laviola, 1968.Cf r Gallegly,M.E. , 1968.Genetic s onpathogenicit y of Phytoph thora infestans. A.Rev .Phytopatholog y 6: 375-396. vi^ht on Walter, J.M. &R.A . Conover, 1952.Hereditar y resistance to latebligh t on tomato. Phytopathology 42: 197-199. .„!.'•„•„ M,-„hf-fundu s Weille, G.A. de, 1964.Forecastin g crop infection.b yth e potatobligh tfungus .

WesJeljnlt; ^Ti^^^J^^ ^tianae ontomatoes .

Wilde!t;?9JMntltra g Stn^nis derVariabilit yvo n Phytophthora infestans (Mont.)d eBary .Arch .Mikrobiol .40 : 163-95 . Wilsonn,I.R.E .& M.E .Gallegly ,1955 .Th einterrelationshi po fpotat oan d of Phytopathology45 .47 34/o . tomatorace s Phytophthora infestans. 87 Wiltshire,S.P. ,1915 .A not eo n Phytophthora infestans occurringo ntomatoes . Rep.agric .hort .Res .St nUniv .Bristo l (1915):92-93 . Zaag,D.E .va nder ,1956 ,Overwinterin g andepidemiolog y of Phytophthora infestans and somene wpossibilitie so fcontrol .Tijdschr .PIZiekt .62 : 89-156. Zadoks,J.C. , 1967.Internationa ldispersa lo ffungi .Neth .J .PI .Path .73 : Supp.1 :61-80 . Zadoks,J.C. , 1972.Reflection so ndiseas eresistanc ei nannua lcrops .I n Bingham,R.T. ,R.J .Ho fan dG.I .McDonal d (Eds).Biolog y ofrus tresist ­ ancei nFores ttrees :Proc .o fa NATO-IUFR Oadvance dStud yInstitute . U.S.Dep .Agr .Fores tServ. ,Misc .Publ .1221 :43-63 . Appendix

Resistance of the tomato lines Moneymaker, W.Va 700, F^ Moneymaker x W.Va 700,

F2: Moneymaker * W.Va 700, and the B^ Moneymaker x (Moneymaker x W.Va 700)

INTRODUCTION

In 1973, an experiment was designed to verify the conclusion, that resistance of W.Va 700 against the 1-t-pathotype of P. infestans is controlled by a single gene which determines complete resistance of the stems and partial resistance of the leaves, a conclusion based on earlier experiments (see Section 7.3.3).

MATERIALSAN DMETHOD S

Three fieldplot s (12x 1 2m) , situatedi na cor nfield ,wer e organized asfollows : a. 5row so f1 1plant so fth eF 2:Moneymake r xW.V a 700, b. 3row so f1 3plant so fth eB ^ Moneymaker x (Moneymakerx W.V a 700), and c. 2row so f1 1plant so fa randomize dmixtur eo f2 2'Moneymaker 'plants ,

22W.V a 700plants ,an d4 4plant so fth e V::Moneymake rx W.V a700 . The rowswer e randomized.Th ero wdistance swer e0. 5an d1m i nturn , and theplan t spacingwithi n the rowswa s 0.75 m.Eac h fieldwa s surroundedb y 48 'Moneymaker'plants ,whic hwer e inoculatedo n73.07.2 8a sdescribe d in6.2 . The testplant swer e treatedan dclassifie dfo rresistanc ea sdescribe di n6.2 .

RESULTS

Resistance of the stems From the 66 plants of the mixed population (sub c), only the 'Moneymaker' plants showed stem lesions. Among the 166 F2 plants (sub a; including one extra plant), there were 39 plants with and 127 plants without stem lesions. From the 117 Bx plants (sub b) tested, there were 57 plants with and 60 plants without stem lesions. The ratios of the numbers ofplant swit h andwithou tste mlesion so fth eF 2an dB jagre ewit hth e expectedratio s 1:3an d1:1 ,base do nth esegregratio no fa singl e dominant 2 2

genefo rresistanc ea tp > 0. 5(X x= 0.20 ) andp > 0. 8(X j= 0.08) , respectively.Thes e results corroborateth econclusio ni n7.2.1 :th e resistanceo fth e lineW.V a70 0agains tth e2,4-p/1-t-pathotyp e ofP. znfes-

tans isdu et oa singl edominan tgen e (calledth ePh 2 gene). Resistance of the leaves Fromda y2 0t o2 3o fthi sexperiment ,n oincreas e inth e fractiondisease dfoliag ewa s observed,hig htemperature shavin g temporarily stoppedlat ebligh tdevelopment .A sthes eday swer emeaningles s forth e developmento fth eepidemi co nth etomatoes ,the ywer eexclude d from thecalculatio no fth e 'apparentinfectio nrate 'r .

Themea nr-value so fth eF 2plant swit han dwithou tste mlesion swer e 0.24an d0.14 ,respectively .Th emea nvalue so fth e Bi plantswit h andwithou t stemlesion swer e0.2 6an d0.16 ,respectively .Thes evalue sar econsiderabl y lowertha nth evalue s obtainedi nth e 1972fiel dexperimen t (seeSectio n6.2) . High temperaturean ddr ycondition sar eresponsibl efo rth eslo wlat ebligh t developmenti nth e197 3fiel dexperiment .Th esomewha thighe rmea n r-values

ofth eBj-plant s comparedwit hth ecorrespondin gF 2plant smigh tb edu e to the facttha ti nth eB ipopulatio ntwic ea smuc h susceptibleplant swer e

presenta si nth eF 2population ,resultin gi nmor einoculu mfo rth eneares t neighbours,an dthu si nmor elesions . Themea ndifference sbetwee n r-valueso fplant swit h andwithou t stem

lesionsar e'probabl ysignificant 'a tp < 0. 1fo rbot hth eF 2an dth e Bi population.Tn emea ndifference sbetwee nr-value so fcorrespondin g groupso f

plantso fth eF 2populatio nan dth eB x populationar eno tsignifican ta t p> 0.2 . Anestimat eo fth eenvironmenta lvarianc eVar(E )o fr i sobtaine db y poolingth evarianc e calculated foreac h linementione dsu bcpe rfiel d(se e 4 2 Section 7.3.1),it svalu ebein g 16.34( x10 ". r ). Thevarianc ewithi n fields

was calculated forbot hth eF 2plant swit han dth eF 2plant swithou t stem lesions,th eresult sbein g 18.08an d16.0 2( *10"\r 2), respectively.Th e varianceswithi nfield sfo rth eB ,plant swit han dthos ewithou t stemlesion s _4 2 were 13.07 and 18.05 (x 10 .r ), respectively. These values do not differ significantly at p > 0.2 from the estimate of the environmental variance, and they do not differ significantly from each other. The same result was found for the variance ratios per field. These results confirm the conclusion drawn in 7.3.3, that within both groups of plants, those with and those without stemlesions ,n oothe rsourc eo fvarianc eca nb edemonstrate dtha nth e environmentalsource .

CONCLUSION

Theevidenc e derived fromth emea nvalue so fth eapparen t infection rates isno t conclusive.Nevertheless ,th eresult s fromth e 1973experimen t reported inthi s appendixcorroborat e theconclusio ntha tth epartia l resistance ofth e leaveso fW.V a 700i sbase do nth egene ,whic h alsopro ­ tectsstem s against latebligh t attacko fth e 2,4-p/1-t-pathotype of P. in- festans (oro na gen eclosel ylinke dwit hit) .