Fungicide resistance incro p protection

Editors J. Dekker and S.G. Georgopoulos

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Centre for Agricultural Publishing and Documentation Wageningen 1982

O •*, i^o- PLANTEZIEKTENKUNDIG GENTA. Binnenhaven8 - Postbus812 2 6700 ER WAGENINGEN

ISBN9 022 0079 79

© Centrefo rAgricultura l Publishingan dDocumentation ,Wageningen ,198 2

Nopar to fthi sboo kma yb ereproduce d andpublishe d inan yform ,b y print,photoprint ,microfil mo ran yothe rmean swithou twritte nper ­ mission fromth epublishers . Preface

Inrecen tyear s awareness ofth e importance of fungicide resistance in cropprotectio n hasbee ngrowing . Inresponse ,a n internationalpost-grad ­ uatecours e onthi s topicwa shel d inWageningen ,Th eNetherlands ,fro m 13t o 20Augus t1980 . Because ofth egrea tinteres t and largenumbe r ofapplication s toat ­ tend thecourse , itwa s repeated in198 1 (22Jul y -5 August) .Th eai m of thecours ewa s to informphytopathologist s whoma yb e confronted withfun ­ gicide resistance inthei rwor k aboutth e origins andnatur e ofth ephenom ­ enon andt o discusspractica lmeasure s thatmigh tprov ehelpfu l incom ­ batting theproblem . Thecours ewa s organized by theFoundatio n ofPost-graduat e Studies of theAgricultura l University atWageninge n onth e initiative ofth eChemi ­ calContro l Committee ofth e International Society ofPlan tPathology , andwit h the supporto fth e Food andAgricultura l Organization ofth eUni ­ tedNation s and the chemical industry.Th ecours eprogramm ewa s chosenb y a scientific course committee,whos emember swer eR.J.W . Byrde (U.K.), L.Chiarapp a (FAO), J.Dekke r (TheNetherlands) , J.W. Eckert (U.S.A.), S.G. Georgopoulos (Greece), F.J. Schwinn (Switzerland)an dH.D . Sisler (U.S.A.). Therehav ebee n requests fromman y sidest omak e theinformatio npro ­ vided inth ecours e availablet oa broade rpublic .Thi sha s ledt oth e publication ofthi sbook ,whic h isbase d onth e courselectures . We should liket otak ethi sopportunit y tothan kM r I.Cressi eo fPudo c forhi s assistance ineditoria lmatters ,M rW.C.T .Middelplaat s fordraw ­ ingth e illustrationo nth ecove ran dth e figures,an dM rF.W . deVrie s forth ecompilatio n ofth eindex .

J. Dekker S.G. Georgopoulos Contents

Introduction (J. Dekker) 1

Chemical controlo ffunga l diseases:importanc ean d problems (F. J. Schwinn) 1

Socio-economic impacto ffungicid e resistance (F. J. Schwinn) 16

Detectionan dmeasuremen to ffungicid e resistance

CS. G. Georgopoulos) 24

Mechanismo factio no ffungicide s (A. Kaars Sijpesteijn) 32

Genetical andbiochemica l backgroundo ffungicid e resistance (S. G. Georgopoulos) 46

Cross-resistance (S.G. Georgopoulos) 53

Benzimidazolecompounds :selectivit yan dresistanc e (L.C. Davidse) 60 Resistancet oergosterol-biosynthesi s inhibitors. I.Chemistr yan dphenomenologica l aspects (A. Fuchs & M.A. de Waard) 71 Resistancet oergosterol-biosynthesi s inhibitors. II. Genetican dphysiologica l aspects (M. A. de Waard & A. Fuchs) 87

Resistancet oth edicarboximid e fungicides (R. E. Beever & R.J.W. Byrde) 101 Acylalanines: resistance indown ymildews ,Pythiu man d Phytophthoraspp . (L.C. Davidse) 118

Canw eestimat eth efungicide-resistanc e hazard inth e field from laboratory andgreenhous e tests? (J. Dekker) 128

Dynamics ofth epathoge npopulatio n inrelatio tn o fungicideresistanc e (M.S. Wolfe) . 139

Canw eus emodel s describing thepopulatio n dynamics of fungicide-resistant strains? (J.C. Zadoks) 149

Fungicide resistance andmicrobia l balance (G.J. Bollen) 161

Countermeasuresfo ravoidin g fungicide resistance (J. Dekker) 177 Case study1 :Cercospor a beticolao fsugar-beet s (S.G. Georgopoulos) 187

Case study2 :Venturi ao fpom e fruits andMonilini ao f stonefruit s (J.D. Gilpatrick) 195

Case study3 :Pyriculari a oryzaeo fric e (y. Uesugi) 207

Case study4 :Powder y mildewso fbarle y andcucumbe r (K.J. Brent) 219 Case study5 :Pénicilliu m decayo fcitru s fruits (J.W. Eckert) 231

Participants ofth e198 0an d198 1internationa l post-graduate courseso nFungicid e Resistancei n Crop Protection 251

Sponsors 256

Index 257 Introduction

J.Dekke r Laboratory ofPhytopathology ,Agricultura l University,Wageningen , TheNetherland s

General

Agricultural crops areunde r constantattac kb ynoxiou s organisms.Th e total crop losses thatresul t areestimate d at25-4 5% (Cramer, 1967) ofproduction .T o safeguardworl d foodproduction ,cro pprotectio nmea ­ sures are indispensable.Whe n theus eo fresistan tvarieties ,cro p rotation andothe r culturalpractices ,o r alteration ofth eenvironmen t areinade ­ quate to suppresspathogen s sufficiently, theus e ofchemical sbecome s essential.Nowaday s chemicalspla y apredominan t role inplan tprotection . Withoutpesticide s active againstinsects ,nematodes , fungi,bacteri a and weeds,moder n agriculture wouldno tb epossibl e andhunge r andmiser y in theworl dwoul d increase dramatically. Butcontro l ofdisease s andpest swit h chemicalsma y also encounter problems, forexampl ewhe nth ecausa l organism becomes resistant toth e toxicant.Thi s resistance involves oneo fth emos t fundamental properties oflivin gmatter ,namely , theabilit y oforganism s to adaptt o changing environmental conditions and to survive undernew ,ofte nadverse ,circum ­ stances.Th eevolutio n oforganism swoul d havebee n impossible without thisproperty , asillustrate d by thenatura l history oflif e onearth . The application ofa pesticid e alsoconstitute s anadvers e change inth e environment for anorganis m thati s sensitive tosuc h acompound : itma y adaptt oth ene w situationb ybecomin gresistant . There arevariou s examples ofdevelopmen t ofresistanc eb y organisms tobiocida l chemicals usedb yma nt ocontro l those organisms,suc ha s thedevelopmen to fresistanc eb ybacteri a toantibiotic s and insectst o insecticides.Unti l ashor ttim e agother ewere ,surprisingly , hardly anyproblem s ofresistanc e to fungicides,eve nthoug h someo fthe mha d beenuse d ona larg e scale forcontro l of fungal diseases foralmos ta century.Howeve r afterth e introduction ofsystemi c fungicides -abou t a decade ago -severa lproblem swit h fungicide resistance occurred in practice. The concept resistance

Ina funga lpopulatio n that isoriginall y sensitive to afungicide , formsma y arise or already existtha t areles s sensitive to thefungi ­ cide. Suc h adecreas e insensitivit yma yb ecause db y genetic ornon - genetic changes inth e fungal cell.Th e latter typeo fchang e isno tsta ­ ble andusuall y disappears rapidly inabsenc e ofth etoxicant .A s iti s therefore hardly ofan y importance inpractice ,i ti sno tdiscusse d in thisbook .Decreas e insensitivit y duet ogeneti c changes inth epathoge n ismor e serious,however .Recentl y itwa s recommendedb y theFA OPane l of Experts onPes tResistanc e toPesticide s (1979)tha tth e term resistance should apply tothes ehereditabl e changes insensitivit y in fungi and bacteria,an d thatth ewor d tolerance should notb euse d inthi s sensea s iti s ambiguous.Tha trecommendatio n is followed inthi sbook .

History

Thebeginnin g of large-scale application of fungicides tocomba tfun ­ galplan tdisease s ismarke db y the discovery ofBordeau xmixtur eb y Millardeti n188 2 inBordeaux .Thi spreparation , amixtur e ofcoppe r sul­ phate and lime,remaine d themos tuse d fungicide formor e than fiftyyears . Hardly anyproblem swit h resistance tothi s fungicide inpractic e have been reported. To acertai nextent ,thi s is alsotru e forth e organic mercury compounds,introduce d around 1914,th edithiocarbamates ,in ­ troduced inth e 1930s,an dvariou s otherorgani c fungicides developed later.Al l thesecompound s have incommo ntha tthe yprovid e onlyprotec ­ tion atth e surface ofth eplant . Inspit e ofth e results obtainedwit h theseprotectant ,o rconventional , fungicides,thei r shortcomingswer e obvious.Therefor e after the SecondWorl dWar ,th e searchstarte d for fungicides thatcoul dpenetrat e theplant ,an dthu s eradicate pathogens evenafte r infection, orprotec tplan tpart sno ti ndirec tcontac twit h the fungicide.Suc hcompounds ,whic h aretake nu pb y theplan tan d transported throughoutth eplan tsyste m arecalle d systemic fungicides. Various researchgroup s inth eU.S.A. , U.K., theNetherland s and other countriesbega nresearc ho nthes e fungicides.Th echemica l industry also played animportan trol e inth e search forsystemi c fungicides,test ­ ingthousand s ofavailabl e andnewl y synthesizedchemicals . Various fungicides thathav e systemic actionwer e discovered inthi s way.Mos to fthe mwer eno t fitfo rpractica luse ,however ,sinc ethe y gavephytotoxi c side-effects. Itwa s difficult to find compounds selec­ tiveenoug h tokil l the fungus (alowe rplant )withou tharmin g thecro p (ahighe r plant). Itals obecam e cleartha tther emigh tb e otherprob ­ lems.Whil eworkin gwit h 6-azauracil, anexperimenta l systemic fungicide. Dekker (1967)foun d strains of Cladosporium cucumerinum thatsuddenl y had become resistant toth echemical .Moreover , inlaborator y experiments with some fungicidal antibiotics,suc ha santimyci n (Lebene t al., 1955)an d cycloheximide (Hsu,1963 )i nth eU.S.A. , andkasugamyci ni nJapa n (Ohmori, 1967), resistant fungal strainswer e found.Thes eoccurrences ,observe d before the erao fsystemi c fungicidesha dbegu nwer e awarnin g thatfun ­ gicides,too ,migh tencounte r resistanceproblems . Indeed, thishappene d soon after the introduction ofsystemi c fungi­ cides intopractica l agriculture, alittl emor e than adecad e ago.Noto ­ riouscase s ofresistanc e haveoccurre dwit hbenzimidazol e fungicides and thiophanates inmos to fth e originally sensitivepathogeni c fungi,dime - thirimol incucumbe rpowder ymildew ,kasugamyci n in Pyricularia oryzae, polyoxin in Alternaria kikuchiana (Dekker, 1977), andrecentl y metalaxyl inpathogen s belonging toth eOomycete s (Georgopoulos &Grigoriu , 1981). However,resistanc eproblem s areno tconfine d to systemic fungicides,a s demonstrated byth epenta -an d tetrachloronitrobenzenes (Georgopoulos, 1962)an d organic tincompound s (Giannopolitis, 1978). These compounds areconsidere d tob e specific-site inhibitors,i ncontras tt omos tcon ­ ventional fungicides,whic h acta tman y sites inth e fungal cell.Ther e appears tob e lessris ko fdevelopmen t ofresistanc e with this latter typeo ffungicide . Sinceth e 1960smor e than fifty fungicideswit h specific actionhav e become available,an dproblem swit h resistance have appeared inman yin ­ stances.

Importance

The sudden appearance ofresistanc e in apathoge npopulatio nmay ,whe n iti sno trecognize d ata nearl y stage,resul ti n failure of diseasecon ­ trol and,consequently , serious crop losses.A s examples,th e resistance tocarbendazi m of Venturia inaequalis inth e apple-growing area around Hannover inWes tGerman y in197 4an dth e suddenappearanc e ofresistanc e tometalaxy l in Phytophthora infestans onpotatoe s inth eNetherland s in 1980 (Davidsee t al., 1981)ma yb ementioned . Sometimes theus eo fa noriginall y effective compound hasb erestric ­ tedo reve nabandoned . Sucha situatio nma ypu tgrower s ina difficul t position, ifn o adequate substitutes are available,an d itma y further reduceth e financial returns ofth ecompan y thatdevelope d -invariabl y athig hcos t -th e fungicide.Shoul d this occurrepeatedly , itmigh t make theagrochemica l industryhesitan tt oinves ti nth edevelopmen to f new systemic fungicides,especiall ywhe n thecompanie s runth e risko f costly lawsuits ;i nth elon gru nthi scoul dmea nth elac ko fpowerfu l newchemical s toth edisadvantag e ofth e grower.Ther ema y alsob e legal consequences forthos ewh o have soldth ecompound , and for farmadvisers . Serious crop losses andmanagemen tproblem s could alsob edisadvanta - geous forworl d foodproduction .Problem s ofresistanc e areexpecte d to increasewhe n selective fungicideswil lb euse d to aconsiderabl e extent inth edevelopin gcountries . The importance ofth eproble m ofresistanc eprompte d theFoo dan d Agriculture Organization ofth eUnite dNations ,whic h already in196 5ha d begun tostud yproblem s causedb y insecticide resistance,t o organize meetings inRom e in197 5e n197 8 fora pane lo fexpert s on fungicidere ­ sistance.Thos emeeting s resulted ina repor ttha tth eFA OPane l ofEx ­ perts onResistanc e toPesticide s (1979)presente d toth e organization's director-general.Th eCommitte e onChemica l Controlo fth e International Society ofPlan tPatholog y (1979), too,issue d aspecia l reporto npro ­ blems andprospect s ofchemica l control. Intha trepor tclos e attention ispai d toth eproblem s of fungicide resistance.Thi srepor tan dtha to f theFAO' spane l contain recommendations for furtherresearc hneede d to fillth egap s inou rknowledge .

Research needed

There isstil lmuc ht o learnabou tth edevelopmen t of fungicidere ­ sistance, forno tonl y isi ta rathe rrecen tproblem ,bu t also aver y complexone ,ful lo fapparen tcontradictions . Inparticular ,genetic , biochemical and epidemiological studies arerequire d togai nne wunder ­ standing ofth ephenomeno n ofresistanc e and itsdevelopment . Genetic studieso f fungicide resistancehav ebee ncarrie dou tb yvar ­ ious researchworker s (Georgopoulos, 1977). Forthes e studies fungi with aperfec t stage areused ,whic h allows genetic analysis forth ere ­ cognitiono fgene sconferrin gresistance . Studieshav ebee ncarrie d out withnon-pathogens ,suc ha s Emericella nidulans (imperf. Aspergillus ni- dulans), Neurospora crassa and Saccharomuces cerevisiae, andwit hpatho ­ gens,suc ha s Venturia inaequalis, Vstilago maydis and V. hordei, and Nectria haematococca. Genetic aspects of fungicide resistance inimper ­ fect fungima yb e studiedwhe nth e fungihav e apara-sexua l cycle andth e techniques exist forexperiment sbase d onthi s cycle.Detaile d genetic studieshav ebee ncarrie d outwit h somemember s ofth e aromatic-hydro­ carbon group,benzimidazol e fungicides andcarboxamides .Thi styp e of study isofte ndifficult ,especiall y for fungihavin gpolykaryoti c cells orwit h obligateparasites . Inman y cases,resistanc e to specific-site inhibitors emergesb y single-gene mutation.Conventiona l fungicides,how ­ ever, acta tman y sites inth e fungal cell and areconsidere d tob egene ­ ralplasmatoxicants . Itseem splausibl e thatth epossibilit y ofa patho ­ genadaptin gt oth echemica l atman y sites isver y limited. Iti simpor ­ tanttha tgeneti c andbiochemica l studies arecontinued , to determine thebasi s ofresistanc e tone w compounds andexten d knowledge aboutcom ­ pounds alreadyused . Progress inclarifyin g thebiochemica lmechanis m ofresistanc eha s beenmad ewit h some systemic fungicides,o fwhic h the so-called carben- dazimgenerator s (benomyl,thiophanates) , thecarboxamide s (carboxin, oxycarboxin)an d someorganophosphoru s fungicides canb ementione d (Georgopoulos, 1977). Theoccurrenc e ofresistan t strains has evenpro ­ moted studies onth emechanis m ofactio no fne w fungicides,a si tallow s comparison ofsensitiv e and resistant individuals ofon epathogen .Th e developmento fne w fungicidesb y thechemica l industry andth e complexity ofth eproble m of fungicide resistancemea n thatmuc hresearc h stillha s tob e doneo nth emechanis m of actiono f fungicides andth e resistance mechanism inpathogens .Suc hstudie shav e directpractica l importance, since fungima yb e cross-resistant to other fungicideswit h asimila rme ­ chanism ofaction . Inadditio nt o research ongeneti c andbiochemica l aspects ofresis ­ tance, the study ofth eecologica l aspects concerned isindispensabl e for aninsigh t intoth edevelopmen to f fungicideresistanc e inth e field.Re ­ sistancema y develop notonl y inpathogens ,bu t also inantagonisti c fun­ gi, whichcoul dbrin g aboutchange s inth emicrobia lbalanc e inth e soil. Little isknow nye t aboutth eepidemiologica l sideo f fungicideresistan ­ ce. Emergence ofresistan t individuals bymutatio no r otherprocesse s doesno t alwaysmea ntha ta fungicide-resistan t pathogenpopulatio nwil l buildu p inth e field.Thi sma yb e influencedb y several factors,i npar ­ ticular therelativ e fitness ofth eresistan t strains.Studie s onth edy ­ namics ofth epathoge npopulatio n inrelatio nt o fungicide resistance are urgentlyneeded . Information aboutal lth e genetic,biochemica l andecologica l aspects of fungicide resistancewil lb e thebasi s fordevisin g countermeasure s to avoid ordela yth edevelopmen to f fungicide resistance inpractica l agriculture.Ther e isstil lmuc hconfusio n anduncertaint y aboutsuc h countermeasures . Itwoul db eworthwhil e to investigatewha trol ecom ­ putermodellin g canpla yhere .

Dissemination of information

Mosto ftoday' splan tpathologist s have little orn oexperienc ewit h theproble m of fungicide resistance.A s aresul tresistanc e is seldomre ­ cognized ata nearl y stage,an dwhe n iti srecognize d iti softe nno t described accurately. Further,lac ko f informationo nho w resistance developsmake s itdifficul t todevis e effective countermeasure s tocop e withth eproblems . Iti simportant ,therefore ,tha tth eknowledg eob ­ tainedb y research and fromexperienc e ismad e available topractica l agriculture.Thi s ison eo fth ereason sbehin d theorganizatio n oftw o courses on fungicide resistance incro pprotectio nhel d in198 0 and1981 , and forth epublicatio n ofthi sbook ,whic h isbase d onlecture s givena t thesecourses . References

Cramer,H.H. ,1967 .Pflanzenschut zun dWelternte .Pflanzenschutz-Nachrichte n 'Bayer' 20 (1).p .523 . Committeeo nChemica lControl ,1979 .Problem san dprospect so fchemica lcontro lo f plantdiseases .Specia lRepor tInternationa lSociet yo fPlan tPatholog yNo .1 , p.18 . Davidse,L.C. ,D .Looyen ,L.J .Turkenstee n& D .va nde rWal ,1981 .Occurrenc eo f metalaxyl-resistant strainso fPhytophthor a infestans inDutc hpotat ofields . NetherlandsJourna lo fPlan tPatholog y87 :65-68 . Dekker,J. , 1967.Conversio no f6-azauraci li nsensitiv ean d resistantstrain so f Cladosporiumcucumerinum .In :Wirkungsmechanisme nvo nFungizide nun dAntibiotica , ProceedingsSymposiu mReinhardsbrunn ,D.D.R. ,Ma i 1966.p .333-339 . Dekker,J. , 1977.Resistance .In :R.W .Mars h (Ed.):Systemi cFungicides .Longman ,Londo n & NewYork .p .176-197 . F.A.0.pane lo fexpert so npes tresistanc et opesticides ,1979 .Repor tmeetin g 28Augus t- 1 Septembe r 1978AGP :1979/M/2 .p .41 . Georgopoulos,S.G. , 1962.Geneti cnatur eo ftoleranc eo fHypomyce ssolan if .Cucurbita e topenta -an dtetrachloronitrobenzene .Natur e194 :148-149 . Georgopoulos,S.G. , 1977.Pathogen sbecom eresistan tt ochemicals .In :J.G .Horsfal l& E.B.Cowlin g (Eds.): PlantDisease .Academi cPress ,Ne wYor k 1:327-345 . Georgopoulos,S.G .& A.C .Grigoriu ,1981 .Metalaxy lresistan tstrain so fPseudoperonos - poracubensi s incucumbe rgreenhouse s inSouther nGreece .Plan tDisease .I npress . Giannopolitis,C.N. ,1978 .Occurrenc eo fstrain so fCercospor abeticol a resistant totripheny lti nfungicide s inGreece .Plan tDiseas eReporte r62 :205-208 . Hsu, K.S.,1963 .Th egeneti cbasi so factidion eresistanc e inNeurospora .Journa l GeneralMicrobiolog y32 :341-347 . Leben,C , D.M. Boone& G.W .Keitt ,1955 .Venturi a inaequalis (Cke)Wint .IX . Searchfo rmutant s resistantt ofungicides .Phytopatholog y45 :467-472 . Ohmori,K. ,1967 .Studie so ncharacter so fPiriculari a oryzaemad eresistan tt o kasugamycin.Journa lAntibiotic s (Tokyo)A20 :109 . Chemical control of fungal diseases:importanc e and problems

F.J. Schwinn SectionBiology , Research andDevelopmen tDepartment ,Agricultura lDivi ­ sion, Ciba-Geigy Ltd,Basel , Switzerland

Abstract

Incompariso n toherbicide s and insecticides,fungicide s areth e smal­ lestgrou p ofpesticides ,bu tthe ysho w agoo dgrowt h rate and future potential.Thei rmai nmarket s are inWester nEurop e and theFa rEast .Th e maintarge tcrop s aregrape-vines ,rice ,vegetables ,deciduou s fruit, potatoes and small grains.Th eclassica lprotectiv e organic fungicides areth emos tcommonl y used, althoughus eo fth ehighl y active,systemi c compounds withcurativ e action isincreasing . Fungicides will remainth e mainpilla r ofdiseas e control forth e foreseeable future.The y control themajorit y ofdisease s satisfactorily.Howeve r themajorit y offungi ­ cides areprotectants ,whic hhav e tob e applied repeatedly, afac to fin ­ creasing importance invie w ofth erisin gcos to fenergy .Moreove r their utility in forecasting systems of IPMprogramme s islimited .Th e systemic fungicides aremuc hbette r suited to such systems,bu tthe yma y create resistanceproblems ,a ris kwhic hshow sth enee d formor e appropriate use concepts, onth eon ehand , and alarge rvariet y ofchemicall y different products, onth e other.Th epresen t applicationmethod s for fungicides arewasteful :researc ho nbette r targetting of applications isneeded .

Keywords: agrochemicalmarke tstatistics ,pesticid e use, fungicideuse , economics, roleo f fungicides,limitation s of fungicides,recommend ­ ations.

Introduction

Chemicals areundoubtedl y themain ,i nman y cases,eve nth eonl y use­ fulan deconomicall y acceptable means foreffectiv e and reliable control of fungalplan tdiseases .Thi s incontras tt oth econtro l of insects,fo r which, insom ecrop s atleast ,alternative s areavailabl e -vira l orbac ­ terialpreperations ,biologica l control agents -an d forwhic h strongef ­ forts forth edevelopmen to fintegrate d control strategies havebee ngo - Table 1.Worl dpesticid eusers 'marke t (salesi nU S$ 1 00 0millions) .

Salest ouser s Proportion pesti- Increase 1980comp ­ cide types intota l aredt o197 8(% ) 1978 1980 1984 1980 market: a) Herbicides 3.71 4.23 4.79 43.5 13.4 Insecticides 3.02 3.35 3.82 34.5 13.8 Fungicides 1.53 1.69 1.97 17.4 16.6 SoilFumigant s 0.19 0.20 0.23 2.1 14.5 Others 0.22 0.26 0.30 2.6 14.9 Total 8.67 9.73- 11.11 14.2

Source:Anonymou s (1979b).

ingo n forsevera lyears .Eve ntakin g into accounttha t similar efforts for fungal-disease controlhav e alsobee nbegu nrecently , inal lprobabi ­ lity theywil lno tpla y amajo rpractica l role inagricultur ebefor e the turno fthi s century.Thus , fungicideswil lb ewit hu s forquit e awhile , and iti stherefor euseful ,a sa novertur et othi sbook ,t obriefl yre ­ view their role indiseas e control andth eproblem s theypose . Inparti ­ cular,economi c andpractica l aspects ofchemica l control of fungal plant diseases arediscusse d inthi scontribution .

Economic importance of fungicides

Insightint oth eeconomi c importance of fungicides canb egaine d throughth e analysis ofth eworl dpesticid e usermarket , fori ti si n suchmarket s thatuse r attitudes,preferences ,prioritie s andpractice s arereflected . Indeed, applied science,whic h isoriente d topractica l application,ma y findmuc h ofinteres t from suchmarke tanalysis . Ofth ethre emajo r groups ofpesticides ,namely ,herbicides ,insecti ­ cides and fungicides,th e latter isth esmalles t interm s ofworld-wid e sales touser s (Table1) . Itsgrowt h rate isslightl y higher thanthos eo fth eothe rproduc t groups,probabl y due toth e recentintroductio n offungicide s in field crops thatwer eno ttreate dwit hthe mbefor e (e.g.wheat ,barley ,peanut s and soybeans).Th edistributio n ofth e agrochemical users'marke tb y geo­ graphicregio n isshow n inFigur e1 . TheNort hAmerica n subcontinenti sclearl yth e leadingpesticid euser , followedb yWester nEurope .Thes e twoaccoun t foralmos t6 0% ofth eto ­ talworl dusers 'market .Thi s indicates thedominanc eo fth e developed countries overth edevelopin gcountrie s inth econsumptio no fagrochemi - cals.However , ifon econsider s theproduc tgroup sherbicides ,insectici ­ des and fungicides,th ecompariso n ofdevelope d withdevelopin g countries looks different (Table2) . Whereasbot hherbicide s and fungicides aremostl yuse d indevelope d Mid+ Nea r East 2% Africa 3%

Figure1 .Distributio no fth e197 8worl dpesticid eusers 'marke tb ygeographi cregion ; totalworl d saleso fpesticide s= U S$ 9 70 0million .(Source :Ciba-Geigy ,1979) . countries,ther e is amajo r outlet for insecticides indevelopin gcoun ­ tries,namel y 40% ofth e total insecticide consumption.Th e distribution ofworl d agrochemical salest ouser sb y crop (Figure 2)indicate swh y this is so.Figur e 2 showstha tth ecro pplan t forwhic hmos t agrochemi- calsar euse d iscotton ,mos to fth eproducer s ofwhic har e developing nations;maize ,deciduou s fruit,an dvegetable s follow intha torder . Seventy-threepercen to fal lpesticide s are applied onthes e fourcrops . The importance ofth eherbicide , insecticide and fungicide groups for pestcontro lvarie swidel y from crop tocro p (Table3) . Incotto ngrowing , forexample ,insecticide s areth edominan tpesticid e used. Incontrast ,herbicide s hold aleadin gpositio n amongth epesti ­ cides used ingrowin g corn,soy a and small-grain cereals.Onl y forgrowin g fruitan dvegetable s are fungicides themos t frequentlyuse d agrochemi- cals. Inparticula r for fungicides.Wester nEurop e isth edominan tuse r followedb y theFa rEas t andEaster nEurop e (Figure 3). Figure 3show s clearly how small the importance of fungicides isi nNort hAmerica ,i n contrastt oth e largeproportio n (34% )o fth etota l agrochemical users' market accounted forb y this subcontinent; iti sth e largestuse ro f herbicides andth e second largestuse ro finsecticide s (Table 4). In

Table2 .Us eo fpesticide si ndevelope dan ddevelopin gcountrie si n 1979.

Proportiono fpesti ­ Proportiono fpesti ­ cideuse di nde ­ cideuse di ndevelop ­ velopedcountrie s ingcountrie (%s )

Herbicides 80 20 Insecticides 60 40 Fungicides 85 15 Total 75 25 Source:Anonymou s(1980b) . soya4°/ o

Figure2 .Distributio n(% ) of197 9worl dagrochemica lsale st ouser sb ycrop . (Source:Anonymous ,1977) .

Western Europe, Japan and Eastern Europe the fungicide market is larger than the herbicide or insecticide markets. Analysis of the fungicide market by crop (Table 5) shows vines ranked first, followed by rice, vegetables, deciduous fruit, potatoes and small-grain cereals. Around 70 % of the total amount of fungicides sold is used on these six crops, almost exclusively against foliar diseases. The high ranking of Western Europe in the geographical distribution of fungicide sales reflects the importance of viticulture, wheat and deciduous fruit, vegetable and potato growing on this subcontinent. The high ranking of the Far East is largely due to the importance of the Japanese rice-fungicide market. Note also the evenly distributed use of fungicides in vegetable production around the world, and the strongly varying importance of the small-grain-cereal fungicide market. Western Europe is the leading user of fungicides on small-grain cereals, as about ten years ago the economic importance of foliar diseases of wheat and barley became evident. In contrast, the US, the largest wheat producer in the world, uses very little fungicides on this crop. On a world-wide basis, rice and small-grain cereals may be considered the two crops with the highest potential for increase in the use of fungicides.

Table3 .Proportio n(% ) ofherbicides ,insecticide san dfungicide suse d inselecte d cropsi n1979 .

Cotton Corn Rice Soya Wheat Fruit& Total Vegetables

Herbicides 26 78 35 92 75 10 43 Insecticides 71 20 47 6 10 38 37 Fungicides 3 2 18 2 15 52 20 Total 100 100 100 100 100 100 100

Source:Anonymou s (1980a).

10 Mid+ Near East1 % Africa 27 »

Figure 3. Distribution (%) of 1978 world sales of fungicides by geographic region; total sales of fungicides to users = US $ 1 700 million, or 18 % of total pesticide users' market. (Source: Ciba-Geigy, 1979).

The distribution of the fungicide market by chemical group is shown in Table 6. Unfortunately, these figures are estimates for 1980 made in 1975. I have been unable to find more recent figures. From my knowledge of the new types of fungicides introduced in the past five years, I am certain that since 1975 the proportions have changed in favor of the systemic fungicides, although the dithiocarbamates still hold their leading position.

Practical importance and problems of fungicides

To discuss technical aspects of fungicides clearly, one must distin­ guish the terms used to describe them. The terms commonly used are pre­ sented in Table 7. As stated in the introduction, chemicals are the only effective and reliable means of directly controlling plant diseases. Thus to the person faced in his daily work with the immediate problems of disease - be he farmer, adviser or researcher - chemicals will remain his main weapon

Table 4. Distribution (%) of 1979 world sales to users of herbicides, insecticides, fungicides and other pesticides by geographic region.

Herbi­ Insecti­ Fungi­ Others Total cides cides cides

NorthAmeric a 52 23 15 44 34 WesternEurop e 21 13 37 38 22 Japan 8 13 20 7 12 EasternEurop e 12 12 19 6 13 Resto fworl d 7 39 9 5 19 Wordtota l 100 100 100 100 100

Source: Anonymous (1980a).

11 Table 5. Fungicide sales (US $ millions) to users by crop in geographic regions in 1978.

North Western Japan& Resto f Total Proportion America Europe FarEas t World oftota l market(%)

Corn 5.0 4.4 4.0 8.4 21.8 1.6 Cotton 12.7 2.1 4.5 14.0 33.3 2.6 Wheat 5.8 62.7 5.4 28.2 102.1 8.0 Rice 2.7 1.5 144.6 33.2 182.0 14.1 Soya 13.1 1.8 5.2 7.4 27.5 2.2 Tobacco 2.3 3.6 10.5 9.4 25.8 2.0 Peanuts (Groundnuts) 26.7 - 6.9 4.7 38.3 3.0 Sugar-beets 4.3 8.1 9.9 7.8 30.1 2.3 Coffee Cocoa Plantation - - 33.8 68.0 101.8 7.9 Tea Crops Rubber Citrus 13.9 8.1 16.2 9.5 47.7 3.7 Apples 19.6 56.6 22.2 25.4 123.8 9.6 Vines 11.6 200.5 11.3 63.3 286.7 22.4 Potatoes 13.6 45.0 10.8 38.9 108.3 8.5 Vegetables 18.1 47.3 44.0 45.2 154.6 12.1

Total 149.4 441.7 329.3 363.4 1283.8 100.0

Source: Anonymous (1979b).

against plant diseases. Therefore they are for him of great practical im­ portance (Anonymous, 1979a). In spite of the continuous efforts by the chemical industry to develop highly active and safe products, the chemi­ cals in the arsenal available to the farmer in his struggle with disease vary in their effectiveness. Table 8 gives a general picture of the pre­ sent state of fungal-disease control. For several types of disease, chem­ ical control is still weak or not possible at all. Even for those diseases that can be controlled, one has to realize that the major fungicides used to do that job are technically old-fashioned: most of them (Table 6) are residual products suited for a prophylactic use only. In other words, they are neither systemic nor curative. The progress during the past 15 years in the development and introduction of systemic and curative fungicides

Table 6. Projected 1980 world fungicide market (sales in US $ millions) by fungicide group.

Sales

Copper compounds 180 Dithiocarbamates 600 Phthalimides 515 Mercurials 40 Systemics 46

Source: Anonymous (1975).

12 Table7 .Terminolog yuse dt odescrib etechnica lattribute so ffungicides .

Fungicidalattribut e Complementary terminology forattribut e

Mobility inth eplan t residual systemic (aero-, basipetal, two-way translocation) Rolei nprotectio n protective curative Timeo fapplicatio n prophylactic duringdiseas ecycl e eradicative is certainly considerable - and encouraging. However, it has led to the appearance of a new problem too, namely, resistance. Resistance is caused by the exclusive and continuous use of a fungicide. Therefore, a larger variety of technically high-grade fungicides needs to be made available to farmers. Chemicals are the foundation of the most manageable of the disease con­ trol tactics at our disposal. However, the present range of fungicides available have a number of limitations: - Most of the fungicides available are protectants. This means that the farmer has to apply them following a prophylactic schedule, i.e. whenever the probability of infection has reached a certain threshold value. Other issues aside, the increasing energy costs of this method are causing a major problem. But even these chemicals could be used in a more sophisti­ cated way if the forecasting methods were better. Currently, efforts are being made to improve these, as part of research on integrated disease management. Even so, because of their inflexibility in use, the value of

Table 8. State of chemical control of major fungal diseases.

State of con- Pathogens attacking leaves, Pathogens attacking trol twigs and fruits roots and stems good downy mildews, late blight, powde­ Pythium, Phytophthora ry mildews, rusts, smuts, leaf- spot pathogens (Venturia, Pyricu- laria, Septoria, Helminthosporium), fruit rots. fair Botrytis, leaf-spot pathogens Rhizoctonia, Pseudocer- (Alternaria, Cercospora, Stem- cosporella phylium) weak vascular wilts, (Phoma trachei- Fusarium, Verticillium phila, Ceratocystis), Nectria, Geotrichum

Chondrostereum Phymatotrichum, Armil- laria, Gaeumannomyces, Plasmodiophora.

13 protectants inintegrate d controlprogramme s is limited. -Th ene w systemic compounds,eve nthoug hthe yhav e asignifican t cura­ tive action,ar ewidel y used according to thestandar dpattern , i.e.a s ifthe ywer eprotectants . Inothe rwords ,farmer shav eno tye t learntt o make fullus eo fthei rparticula r features:thei r systemicmod e of action and their 'kick-back' (curative)activity .N o doubt,the ynee d tob eedu ­ cated tomak e amor e intelligentus eo fthes e fungicides.Mor e research isneede d thatwil l demonstrate thatthe yca nchang e thewa y theyus e fungicideswithou trunnin gth eris k of fallingprofits .O fcourse ,th e degree ofchang epossibl evarie s from crop tocrop .Here ,too ,mor eap ­ plied researchneed s tob e done.Systemi c fungicides dosee m tob emuc h better suited for integrated controlprogramme s -anothe r area inwhic h more research isneeded . -Al lplant-protectio n chemicals,an d thus fungicides too,ar eapplie d wastefully. Iti swel l knowntha t irrespective ofth e applicationmetho d used, themajo rportio n ofth echemica l doesno treac h itstarget , i.e. theplan tpathogen .O fth ethre emos tcommo n applicationmethods ,namel y foliar, soil and seed application, the latter gives thebes trati oo f utilization, although iti sstil l far frombein g satisfactory (Graham- Bryce &Hartley , 1979). Thus therei s ample space forimprovemen t inap ­ plication technology,whic hwil l eventually leadt o amor eprecise ,jud ­ icious andeconomi c use ofexistin g fungicides.Thei rbiologica l potential isgreatl y underutilized atpresent . -A limitationo fincreasin g importance isth e restrictive policies of many national registration authorities:i ti sbecomin gmor e andmor edif ­ ficultt oregiste rproduc tmixture s andproduct s forspecia lcrops . -A further,bu tonl yminor ,limitatio no f fungicidespresentl yuse d is their toxicity tocertai ncrops .O fcourse ,non e ofthe mwoul db emarke ­ tedunles s theyme tth eneed s ofa tleas ton eparticula r cropplan to r crop group,bu twide r usewoul db epossibl e ifthe ywer ebette r tolerated by othercro pplants .Example s ofsuc h limitations areth ecoppe rsensit ­ ivity ofman y row crops and fruittrees ,th e zineb sensitivity ofpears , and thegenera lphytotoxicit y ofdinoca p and sulphur inho tweather . - Thephysico-chemica l incompatibility ofcertai n fungicides limits their combination inmixture s thatwoul db e desirable fordiseas e control,in ­ cluding thereductio n ofth eris ko fresistance , andth e saving ofenerg y through jointapplication . -Huma n and environmental hazards limitth epresen tus eo ffungicides , although onemus t admittha tn omajo rproblem s have so fararise nwhe n the instructions forus ehav ebee n followed.Ther ehav ebee n some severe cases ofacut epoisoning , forexampl e in Iraq inth e 1970s following the consumption ofmercury-treate d seed,but ,thi s andmos tothe rknow n cases canb epu tdow nclearl y tomisuse .Ther e islittl e documented evidence of adverse environmental effects duet ous eo f fungicides.Bu ttha tsitua -

14 tion has changed recently in so far as both the ethylenebisdithiocarba- mates and some of the phthalimide fungicides have come under RPAR (rebutt­ able presumption against registration) by the Environmental Protection Agency in the USA because of reported chronic toxicity. Since these cases are still under investigation, it would be premature to comment further here.

Conclusions

From the information presented in this contribution, there can be little doubt about the leading role of chemicals in the continuous fight against plant diseases. Albeit the present range of fungicides looks fairly broad and satisfactory, it has great limitations in view of the needs of the integrated approaches to disease control that are attracting more and more attention in applied research. The dominating conventional fungicides do not have the flexibility of use needed for such approaches, and the range of systemic, curative products is still too small. In addition, little experience has been gained to date in coping with resistance to these prod­ ucts, a phenomenon that is seriously threatening their usefulness. Thus, more effort by academia and industry is needed to understand the various aspects of resistance and to develop viable use strategies. Beyond this, the search for new types of fungicidal molecules, to broaden our spectrum of weapons, is needed. A further area that calls for more attention in basic and applied research programmes is application techniques. The high percentage of the product quantities applied that never reaches its final target clearly indicates the need for improvement. Recent advances in seed treatment technology and electrostatic spraying systems should stimulate this avenue of research.

References

Anonymous, 1975. World Pesticide Markets. Farm Chemicals Special Report, Farm Chemi­ cals, September, p. 45-48. Anonymous, 1977. Agrochemical Review. Wood, McKenzie & Co., Edinburgh. Anonymous, 1979a. Contemporary control of plant diseases with chemicals. Present sta­ tus, future prospects and proposals for action. American Phytopathological Socie­ ty, 170 p. Anonymous, 1979b. A Look at World Pesticide Markets. Farm Chemicals, September, p. 6l-6i Anonymous, 1980a. Agrochemical Overview. Size of the Agrochemical Market in 1979. Wood, McKenzie & Co., Edinburgh. Anonymous, 1980b. News from GIFAP. Industry Facts. GIFAP Bulletin, 6 (2): 1-2. Ciba-Geigy, 1979. Market of agrochemicals 1978. Ciba-Geigy Market Research, Basel. Graham-Bryce, I.J. & G.S. Hartley, 1979. The scope for improving pesticidal effi­ ciency through formulation. In: H. Geissbühler, G.J. Brooks and P.C. Kearney (Eds): Advances in Pesticide Science, part 3. Pergamon Press, Oxford, p.710-725.

15 Socio-economic impact of fungicide resistance

F.J. Schwinn SectionBiology , Research andDevelopmen tDepartment ,Agricultura l Divi­ sion, Ciba-Geigy Ltd,Basel , Switzerland

Abstract

The impacto fdevelopmen t of fungicide resistance onvariou s groups in society,namely , farmers,consumers ,manufacturers ,extensio n officers and regulatory authorities, isdiscussed . Special attention ispai d to thepositio n ofth e agrochemical industry onth eproble m of fungicidere ­ sistance.

Keywords: fungicides,resistance ,registratio npolicy , industry-adviser co-operation, resistance strategies,agrochemica l industry.

Introduction

Whendevelopmen t ofresistanc e to fungicidesbecam e aproble m inprac ­ tical agriculture -afte r the introduction of systemic organicmolecule s - discussionbega n aboutstrategie s thatcoul db euse d tocop ewit h this phenomenon. Ifth e strategies thathav ebee ndevelope d prove successful inpractice ,resistanc ema yhav en o socio-economic impact.However , since they are stillunprove n and controversial, and sincemos to fth estrat ­ egies developed byma n inhi s continuous fightagains tharmfu l organisms haveturne d outt ohav e shortcomings and limitations,i ti s certainly worthwhile to look atth e socio-economic impacto f fungicide resistance inmor e detail.Yet ,thi s is afairl yne w topic,abou twhic hver y little information isavailable .Thu s Ia m forced tophilosophiz e onth e subject rather thanpresen t agrea tdea l of facts. The titleo fthi s contribution refers totw omajo r aspects:socia l im­ pact and economic impact.Bu tsinc e they are strongly linked, theywil l be treated asone .T obegin , Iwil lpu t aquestion . 'Ifresistanc earises , who isgoin g tosuffe r from it,or ,i nothe rwords ,upo nwhic h groups in society isi tgoin g tohav e animpact? 'Th e groups thatma yb eaffecte d are: - theuse r (farmer)

16 - the consumer - the manufacturer - the adviser (extension officer) - thenationa l regulatory authorities - the agrochemical industry. Before discussing the impacto f fungicide resistance onthes egroups , attentionwil lb epai d topes tcontrol ,whic hha sha d toconten dwit hth e problem ofresistanc e to synthetic insecticides.

Experience with insecticide resistance

Experience with insecticide resistance,whic hha sbee nknow n forsev ­ eral decades (Brown, 1977), has somevaluabl e lessons foru so nth e impact ofresistance .Resistanc e isa commo nphenomeno n inal l themajo rchemi ­ cal classes ofinsecticide s introduced to date,primaril ywit h insects thatar eeithe rvector s ofhuma nmicrobia l pathogens oraffec tpubli chy ­ giene. Inthi s area certainly, resistance hasha d astron g social impact inth epas t and itcontinue s to dos otoday . Itno w seemstha tvecto rre ­ sistance topesticide s isth ebigges t single obstacle inth e struggle againstvector-born e diseases,an d iti slargel y responsible forpreven ­ tingth e successful completion of somecontro lprogrammes ,mainl y inde ­ veloping countries (Pal,1976) . Inth e case ofmalaria , forexample ,re ­ sistance inmosquitoe s hasdevelope d in1 4countries ,an d thetota lpopu ­ lationo f areas affectedb y iti sestimate d tob e 250million ,whic hre ­ presents abouton e third ofth e totalpopulatio n ofth emalariou s areas ofth eworld . Inthi s case resistance has amanifol dimpact : - itexpose s thepopulatio n to ahighe r disease risk,thu s affectinghu ­ manhealt h directly - the change-over, duet oresistance , fromDD Tt oothe r insecticides has led to asubstantia l increase inth etreatmen tcosts ,causin g severe hardship indevelopin g countries,whic h areofte nunabl e todefe r funds from otherurgen tneed s - itha s forcedWH O andothe r international bodies to develop newstrat ­ egies forth eeffectiv e control ofmalari avectors . Clearly, iti si nth econtro l ofhuma ndiseas e that insecticideresis ­ tanceha sha d its strongest socio-economic impactt odate .Bu t also in insectcontro l inagricultura l crops,resistanc e has caused socio-econom­ icproblems .Cotton ,th ecro p uponwhic hmor e insecticides are applied world-wide than allothe r crops combined,ma yb euse d asa nexample .Eve n though resistance is awide-sprea dphenomeno n inthi s crop,a sa neconom ­ icproble m iti s still restricted torelativel y small areas formos t pestspecies ,a s arecen tsurve y conductedb y Ciba-Geigy has shown.Proba ­ bly,th eproble m causedb y thecotto nbollworm s (Heliothis virescens, H. zeae) onth eAmerica ncontinen t inth e 1960s ison eo fth e fewexample s

17 ofheav y economic losses due toth e development ofresistanc e (Reynolds, 1976). Notonl y farmers suffered,b y losingprofi t andb ybein g forcedt o switcht oothe r crops,bu t also the economy of somecountrie swa sbadl y damaged. InNicaragua , acountr y thatrelie s oncotto n for 20% ofit s total exchange earnings,productio n decreased annually by 16% ,whic h almostresulte d inth e country goingbankrupt .Howeve r eveni nsuc hse ­ vere casesproblem swer e overcomeb y theus eo f integrated controlstrat ­ egies,whic h involved inparticula r themor e intelligent use ofne wpes ­ ticides (Vaughan& Leon , 1976). Inspit eo f allth e experience gained in insecticide resistance.Brow n (1976)wa srigh ti nstatin gtha t 'wemus t agreetha tou rpes tmanagemen t in face ofth e insecticide resistance fac­ torove rth epas t3 0year s leaves considerable room for improvement'.I would add tothi s statement that since the question isn o longer 'if're ­ sistance againstinsecticide s develops,bu t rather 'howsoon '(Reynolds , 1976), such improvements areurgentl yneeded .

Impact of fungicide resistance

Onth e farmer

Several caseso fseriou sresistanc e haveoccurre d inpractice .Firs t resistance toth epyrimidines ,late rt oth ebenzimidazoles , and,mos tre ­ cently,t oth e acylalanine metalaxyl.Wher e resistance occurred, itcer ­ tainly had animpac to nth e farmer,a sh e lostpar to fhi sprofi tbecaus e ofth e attacko nhi s cropb yresistan t strains.Thi swa s the case innor ­ thernGerman ywhe nth e applesca b (Venturia inaequalis) developed resistan­ cet obenzimidazol e fungicides,a sdi d Cercospora beticola on sugar-beets innorther nGreece ,an d inWester nEurop ewhe n latebligh t inpotatoe s (Phytophthora infestans) developed resistance tometalaxy l (Staub & Schwinn, 1980). However,befor emor e canb e said aboutth e economic im­ pacto fresistanc e onth e farmer,exac t figures oflosse s areneeded .T o my knowledge,the yd ono texis t forfungicides .

Onth e consumer

Inshar p contrastt oth e farmer andth e othergroup smentione d inth e introduction, theconsume r isno t immediately affectedb y fungicidere ­ sistance.H ewil l onlyrealiz e thatresistanc e hasbecom e aproble m in practicewhen ,becaus e ofsever e losses causedb y resistantpathogens , either foodcost s increase orcertai n commodities,lik e flowerso r fruits, are locally and temporarily unavailable.Therefor e inth edeve ­ loped countries,a tleast ,wit h theirbroa d international base of food supply,th e consumerwil lno t sufferbecaus e ofresistance . Indevelopin g countries however, ifresistanc e occurred ina stapl ecro p or ahigh -

18 value exportcrop ,i tmigh thav e immediate impacto nthei r populations andnationa l economies.

Onth emanufacture r

Nowadaysmanufacturer s areconfronte d with ane w situation ofproduc t liability asresistanc e tonewl y introduced fungicides has developed. It isknow ntha t farmerswh o have lostmone y through the occurrence ofre ­ sistantstrain s inthei rcrop shav e filed suits againstchemica lcompa ­ nies,wh o the farmers hold liable forproduc t failures.Thi s isa ne wde ­ velopment asth eproduc t liability ofth emanufacture r waspreviousl y lim­ itedt oth e technical quality ofth eproduct .A numbe r ofth e law suits havebee nwo nb y thecompanies ,bu tsinc eothe rsuit s areno tye t settled, Iwil l refrain fromcommentin g onthe m further.Howeve r thepossibl econ ­ sequences forth emanufacturer s of fungicidesma yb e considerable,an d theyma y influence them inthei r sales and research activities inth efu ­ ture. For example,i fresistanc e occurs ina specialit y crop growno na small area (e.g.onion s orcucumbers )th e financial consequences ofresist ­ ance for acompan yma yb emuc hhighe r thanpossibl e sales revenues over theyears .Thu s acompan yma y decide nott oregiste r resistance-prone products forsuc hcrops .Legalitie s aside,th e farmer isi nprincipl een ­ titled totechnica l guidance from industry -an dhi sextensio n or advi­ sory service,thu s resistance has animmediat e impacto nthes etw ogroups , too.

Onth e adviser

Themai ndut y ofa nextensio n officer ist oadvis e the farmerwhic h fungicides he should use atwha ttim ewit hwha tsprayin g schedule,t oob ­ tain anoptima lyield .Th e adviser collects orgenerate shi s knowledge from technical information from themanufacture r and fromhi sow nexperi ­ mental work.Further ,h erelie s onth e findings ofworker s active infun ­ gicide research.Nobod y canblam e advisers forno thavin g foreseenth e risk ofresistanc e toth e firstsystemi c fungicides appearing onth emar ­ ket, thebenzimidazole s andpyrimidines ,whic hwer ewidel y usedb y farmers because ofthei r attractive qualities as fungicides. Inth e lighto fthi s practical experience, andno w thatther e isevidenc e from research and practical experience,tha tothe r groups ofmoder n fungicides are also subjected toresistance ,adviser s are,wit h good reason,extremel y keen to learn asearl y aspossibl ewhethe r sucha ris k existswit h anyne w type of fungicide.Th e farmer,too ,no w expects advisers tob e awareo f these risks and tokno wth e strategies thatmus tb euse d tominimiz e the potential damage tohi s crops.Ther e is,therefore , aclea rnee d forth e evaluation ofth eris k ofresistanc e tobecom e apar to fth eroutin e pro­ duct-developmentprocedur e -a direc t impacto nth e chemical industry. 19 On regulatory authorities

So far,whe n filingpetition s forth e registration of ane wpesticide , the chemical industry hasha d todemonstrat e thepesticide' s biological effectiveness,it susefulnes s for agriculture and itsbiochemica l andto - xicological harmlessness.Wit h the increasing awareness ofresistanc e as apotentia l threatt oth eefficac y of anagricultura l chemical,th eau ­ thoritiesma ywel l askth emanufacture r for astatemen t aboutth epoten ­ tial risko fresistanc e developing toth echemical .Bu ta knowledg e of thepotentia l risk should alsoencourag e the regulatory authorities tob e more cautious inrestrictin gth eus eo folde r fungicides,lik eth edithio - carbamates orth ephthalimides , that so farhav eno tbee n subjected to the risk ofresistance .Thei r availability iscrucia l tocurren tstrateg ­ ies forcopin gwit h resistance, for iti sthes eproduct s that areth eal ­ ternatives -alon e or inmixture s -t ochemical s thathav eme tresistance . Thus the appearance ofnove l andpoten tproduct s fordiseas e control shouldb yn omean s lead tocancellatio n ofol dbu t still usefulprotec ­ tiveproduct sb y theregistratio n authorities.Th ebroade r our arsenal of weapons againstth epathogen s is,th ebette r areou rchance s of success againstresistance ,especiall y using strategies ofapplicatio n thatcom ­ binebiochemicall y different fungicides.Actually ,th e losso f effective fungicides duet oregulatio nwoul d increase thepossibilit y ofresistance , by increasing thedeman d forne wchemical s (mostlikel y systemic).A tth e sametim estrategie s forth epreventio no fresistanc ewoul db e limited in theirvalu e ordiversity .Therefor e iti swit hmixe d feelings that Iawai t theoutcom e ofth ependin g RPAR investigations byth eEnvironmen tProtect ­ ionAgenc y (EPA)o fvariou smajo rprotectiv e fungicides inth eUSA . Iti s inth einteres to fal lgroup s involved inplan tprotectio n thatthes e im­ portanttool s forth e control ofpathogen s remain atou rdisposa l (Anonym­ ous, 1979).

Onth e agrochemical industry

The agrochemical industry hasbee nexpose d to resistance toinsecti ­ cides forsevera ldecades .Durin gmos to fthi stim edevelopmen to fresis ­ tancema yhav emean ta shor tcommercia l life for ane wproduc t ina cer ­ tainmarke t segment.However ,t om y knowledge itneve r ledt oth ecomple ­ tedisappearanc e ofa ninsecticid e from themarket . Indeed, someobser ­ versbeliev e thatresistanc ewa s one ofth e driving forces ofth edevelop ­ mento fne w insecticides.Beside s this, increasing research efforts have beenmad e over thepas tyear s tounderstan d thevariou s aspects ofresis ­ tance asa biologica l phenomenon and todevelo p strategies tocop ewit h it (Plapp, 1979;Dittrich , 1979;Georghiou , 1980). Thus industry could adopt asimpl e 'waitan d see' attitude on fungicideresistance .

20 But, inth emeantime ,th esituatio nha schange d soradicall y thatsuc h apolic y isn o longervalid . First,th e search forne w chemicals forin ­ sect anddiseas e control,an d their development and introduction into the market, isgettin gmor e costly allth e time,becaus e the actualdevelop ­ mentcost s arerisin g continually andbecaus e thechance s of finding new products aredecreasing .Nowadays ,th e development costs fora ne wfungi ­ cide are inth eorde r ofU S $2 0 -3 0million , and there areno tman y market segments thatca nsuppor tsuc ha n investment.Therefore , thepros ­ pects forth e size ofth e envisagedmarke t andth e life-time of aproduc t mustb epromising . Iti scertainl yno trealisti c toexpec tchemica l com­ panies todevelo p systemic fungicideswit h apotentia l risk of resistance foremergenc yus eonly , i.e.upo nth eoutbrea k ofheav y epidemics orwhe n multi-site-inhibiting fungicides areno teffective .Thus , the losso fa newly introduced fungicidebecaus e ofth e development ofresistanc eha s such strong financial repercussions thatn ocompan y cantak e asimpl e 'waitan d see'position .Onc e aproduc tha sbee n introduced intoth emar ­ ket, atth e costo fmuc h time,expens e and effort,th e development ofre ­ sistancemus tb ehel d atba y for aslon g aspossible . What can acompan y do?Wha tar eth echance s ofreasonabl y predicting the risk? Checking the risk ofresistanc emus tbecom e aregula r element ofth edevelopmen tproces s ofa ne w fungicide,a proces swhic h comprises several steps ofbiologica l testingunde r anincreasin g rangeo fclimati c conditions overa perio do fsevera lyears .Tha tcheckin gprocedur e could easily includemode l studies,i nvitr o and invivo .On e could evenenvi ­ sage special fieldtrial s totes t forth ebuild-u p ofresistan tstrains . However,th eproble m remains ofho wt o judge the findings of suchexperi ­ ments,provide d theyyiel dmeaningfu l results. Ifthe y are favourable, i.e. ifn oresistanc e emerges,doe s itmea ntha t itwil lno toccu r after theproducts ' introduction intoth emarket ,especiall y ifi ti shighl y attractive, and thereforewil lb ewidely ,probabl y exclusively used? If they areunfavourable , i.e. ifresistanc e emerges,doe s itmea ntha tth e compound shouldb e dropped for further development? Iti sth e company researcherwh o is inth edecisiv epositio nhere ,be ­ cause iti sh ewh o advises hiscompany' smanagement ,whic h intur nha st o setprioritie s and allocate funds forproduc tdevelopment .Eve n ifh e can convince hismanagemen tt og o aheadwit h development, andeve ni fth e compound surmounts thehurdl e ofregistration , thequestio nwhic h still remains iswhethe r to introduce ane w fungicide aggressively andexten ­ sivelywit h theris k of shortening its life ort opromot e itmoderatel y and speculate ona possibl y longerlife-time . Competition inth emarke tplac e andth enee d torecou p investmentan d reach thezon eo fprofi ta searl y aspossibl e during the limitedlife ­ spano fa produc tusuall y dictate aggressiveness.However ,eve n ifth e company decides to follow acarefu lpolicy , the farmerma y overturn itb y

21 simplypreferin g tous eth ene w fungicide.Becaus e ofspecifi c features, such asit s systemic translocation and curative effectiveness, forexam ­ ple, itma yb e soattractiv e tohi m thath e tends tous e it exclusively and continually; inothe rword s ina wa ywhich , ifth e risk inherently exists, almostinevitabl y leadst oresistance .Marketin g experts knowho w hard iti st oconvinc e the farmert o temporarily refrain fromusin g anef ­ fectiveproduc tunles sh eunderstand s thereason s forsuc hrestrictions . Moreover,a slon g ashi sneighbou r continues tous e it,h e islikel y tod o the same.Strategie s forus emus ttherefor eb e enforceable, otherwise they are inevitably unsuccessful.This , inturn ,mean s thatth ebes t theoretical strategyma yno tnecessaril y beth emos t successful one in practice. Chemical companies need the collaboration ofagricultura l adviserst o encourage and enforcepromisin g strategies in farmingpractice s andt o educate the farmeri nne wus econcepts . Since thechemica l industryan d advisers aredirectl y affectedb y failure duet oresistance ,the y are bound to findway s andmean s ofcooperatio n inthi smatter .Beyon d this, thechemica l industry mayb e forced tomak e somechange s init smarketin g philosophy,whic h todat e aims formaximu m sales inth eshortes tpossibl e time. Further,th eunderstandin g ofresistanc e needs tob e improved in themarketin g organization, and inm y opinion iti sno w time forcompa ­ nieswh o sell fungicides ofsimila rmod e of action,t odevelo p jointly strategies orrule s tob e followed inorde r tokee p theris ko fresis ­ tance low.

Conclusions

Resistance certainly hasth epotentia l to increase the farmers costo f production and decrease hisprofits ,unles s he canpas s onth e increased costs toth econsume r orth emanufacturer .Resistanc e also complicates the life ofth e agricultural adviser.But ,abov e all,i tadd st oth eal ­ ready considerable uncertaintieswhic hth e agrochemical industry faces when developing andmarketin g fungicides,o rmor egenerally ,pesticides . Nevertheless,th e facttha tresistanc e has developed hascertainl yha d thepositiv e andbeneficia l effecto f forcing allgroup s tothin kmor e carefully aboutho wt ous epesticide s inth ebes tan dmos teconomica lway . And itha s openedu p ane w andbroa d discipline ofbot h fundamental and applied research. Itma ywel lb e thatsom eo fth e integrated pestmanage ­ ment (IPM)strategies ,whic h arebein g developed invariou s countries and forcertai ncrop s (e.g.apples ,potatoes ,banana s and horticultural crops), forwhic hdiseas e controlha s reached ahig h level oftechnology , will turnou tt ob eusefu l forpreventin g thebuild-u p ofresistance . Ifal lthos e involved canreac h agenera l understanding asdescribe d inthi spape r and,especially , ifindustry , researchers and advisersco -

22 operate closer in defining the resistance risk, in developing adequate realistic strategies to prevent or delay it, and in educating the farmer to use pesticides in an intelligent way, there may still be a chance that the socio-economic impact of fungicide resistance will remain small. But given the many uncertainties, it is unrealistic to expect the immediate appearance of a magic formula for long-term success. It remains to be seen whether there is enough time left for us to develop and introduce new concepts of use that are urgently needed for the new fungicides, those we have and those to come.

References

Anonymous, 1979. Contemporary control of plant diseases with chemicals: present sta­ tus, future prospects and proposals for action. American Phytopathological Society, 170 p. Brown, A.W.A., 1976. Epilogue: Resistance as a factor in pesticide management. Pro­ ceedings of the XV International Congress of Entomology, Washington, U.S.A., p. 816-824. Brown, A.W.A., 1977. The progression of resistance mechanisms developed against in­ secticides. In: J.R. Plimmer (Ed.): Pesticide chemistry in the 20th century. ACS Symposium Series. American Chemical Society, 37: 21-34. Dittrich, V., 1979. The role of industry in coping with insecticide resistance. Proceedings of the IX International Congress of Plant Protection, Washington, U.S.A., p. 249-253. Georghioü, G.P., 1980. Insecticide resistance and prospects for its management. Resi­ due Revue, 76: 130-145. Pal, R. , 1976. Problems of insecticide resistance in insect vectors of human dis­ eases. Proceedings of the XV International Congress of Entomology, Washington, U.S.A., p. 800-811. Plapp, F.W., jr., 1979. Ways and means of avoiding or ameliorating resistance to insecticides. Proceedings of the IX International Congress of Plant Protection, Washington, U.S.A., p. 244-249. Reynolds, H.T., 1976. Problems of resistance in pests of field crops. Proceedings of the XV International Congress of Entomology, Washington, U.S.A., p. 794-799. Staub, T., & F.J. Schwinn, 1980. Fungicidal properties of metalaxyl and its use strategies. Proceedings of the 5th Congress of the Mediterrannean Phytopatho­ logical Society, p. 154-156. Vaughan, M.A. & G. Leon Q., 1976. Pesticide management on a major crop with severe resistance problems. Proceedings of the XV International Congress of Entomology, Washington, U.S.A., p. 812-815.

23 Detection and measurement of fungicide resistance

S.G. Georgopoulos Laboratory ofPhytopathology ,Athen s College ofAgricultura l Sciences, Votanikos,Athens, Greec e

Abstract

The generalprinciple s andmethod susefu l forth edetectio n andth e measurement of fungicide resistance aredescribed . Some ofth epossibl e errors indeterminin g responset ovariou s concentrations and insamplin g the fungalmateria l are indicated.

Keywords:base-lin e sensitivity, extento fresistance ,degre e ofresis ­ tance, fungicide solubility, dosage response,monitoring .

Introduction

Fungicide resistance mayb edetecte d andmeasure d invariou sways ,de ­ pending onth e fungus-fungicide combination; some examples ofagricultu ­ ral significance willb e examined inth ecas e studiespresente d later in thisbook .Th egenera lprinciple s areth e same,however .A swit h other organisms,th erecognitio n ofresistan t strains of fungimus tb emad eb y comparisonwit hdat aobtaine dwit h sensitive strains.Accordingly , iti s essential thatth e 'base-linesensitivity ' forth e fungus-fungicidecom ­ bination inquestio nb e established eitherb y appropriate experiments with strains thatar eunquestionabl y wild typewit hrespec tt osensitivi ­ tyt o the fungicide involved orb yth eus eo fdat a from theliterature . Itmus tb e remembered thattoxicit yma yvar yno tonl ywit h fungicidecon ­ centrationbu tals owit h inoculum size,compositio n ofmedium ,temperature , pH, etc.Fo r this reasonth e suspected resistant strainsmus tb estu ­ diedb yth emetho duse dt oestablis hth ebase-lin e sensitivity, under exactly the sameconditions . After resistance hasbee ndetecte d there aretw oparameter s that shouldb emeasured : theexten to fresistance , i.e.th eproportio n ofth e population thatn olonge rexhibi tth enorma l sensitivity ofth especies ; and thedegre e ofresistance , i.e. themagnitud e ofth edifferenc e in sensitivity.Wit h some selective fungicides,e.g . thebenzimidazoles ,th e

24 carboxamides, and the acylalanines,grea tdifference s insensitivit ybe ­ tweenwil d type and resistant strains exist. Insuc hcase s the detection and themeasuremen t ofresistanc e is fairlyeasy .Wit h some otherfungi ­ cides, e.g. dodine andmember s of the aromatic hydrocarbon group,th edif ­ ferences are small,althoug h oftenver y significant inpractica lagricul ­ ture, and detection andmeasuremen t require careful testing.

Methods in general use

Sinceth edetectio n ofresistanc e isessentiall y amatte r ofrecogni ­ tiono fa differenc e insensitivity , iti simportan t that ineac hcas e themos t appropriate method for anaccurat e determination of sensitivity ischosen .Fo r some arthropod pests,standardize d testmethod s havebee n prepared by the FAOPane l ofExpert s onPes tResistanc e toPesticides ; these are accepted internationally. Iti sexpecte d that inth enea r future such standardized testmethod swil lb e approved for someo fth emos tim ­ portant fungus-fungicide combinations. Descriptions of severalmethod s forth emeasuremen t offungitoxicit y canb e found inth e literature (AmericanPhytopathologica l Society,1947 ; Horsfall, 1956;Peletier , 1978;America nPhytopathologica l Society and Society ofNematologists , 1978). Suchmethod shav ebee n adoptedb ysever ­ al investigators forth erecognitio n ofresistan t strains and themeasu ­ remento fth e degree ofresistance . The rateo f increase ofcolon y diameter ontreate d agarmediu m appears tob eth emos t frequently used criterion formeasurin g fungitoxicity. Usually agarblock s of afe wmillimetre s diameter aretake n from theperi ­ phery ofcolonie s growno ncontro lmediu m tob euse d asinoculum .Afte r incubationth ediameter s ofcolonie sproduce d on fungicide-containing and controlmedi a aremeasure d and from this the diameter ofinoculu m should be subtracted. Thismetho dha sbee nuse d forth edetectio n and themea ­ suremento fresistanc e toorganomercurial s in Pyrenophora avenae (Green- away& Cowan , 1970), to diphenyl and sodium 2-phenylphenolatei n Nectria haematococca (Vomvoyanni& Georgopoulos , 1966), to dyrene in Sclerotinia homoeocarpa (Nicholson et al., 1971)t ododin e in N. haematococca (Kappas & Georgopoulos,1968 )t oorganotin s in Cercospora beticola (Giannopolitis, 1978), tobenzimidazole si n Botrytis cinerea (Bollen& Scholten,1971 ; Miller& Fletcher , 1974), in Fusarium oxysporum (Sozzi& Gessler , 1980), and in Sclerotinia fructicola (Whan,1976 ;Penros e et al., 1979), todi - carboximides in Botrytis cinerea (Gullino& Garibaldi , 1979), topolyoxi n in Alternaria kikuchiana (Nishimura et al., 1973)an d tokasugamyci n in Pyricularia oryzae (Tagae t al., 1978). Therat eo fdr yweigh tincreas e inliqui dmedi acontainin g thefungi ­ cide isuse d less frequently formeasurin g fungitoxicitybecaus e itsmea ­ surementrequire smor e time.I tis ,however , amor e accurate criterion

25 thancolon y diameter. Fororganism swit hyeast-lik e growthth e increase inturbidit y canb euse d asa criterio n ofgrowth .Liqui dmedi ahav ebee n used forth emeasuremen t ofcycloheximid eresistanc e in Saccharomyces ce- revisiae (Wilkie& Lee , 1965), ofresistanc e tobenzimidazole s in Asper­ gillus nidulans (Hastie& Georgopoulos , 1971)an d C. beticola (Georgopou- los& Dovas ,1973 )an do fresistanc e tocarboxamide s (Georgopoulos et al., 1972)an dchlorone b (Tillman& Sisler, 1972)i n Ustilago maydis. Plating spores orothe rpropagule s onaga rmedi a containing afungi ­ cideconcentratio n thatcompletel yprevent s thegrowt ho fth ewil dtyp e isth eusua lmetho d for firstdetectin g resistance and its subsequentmo ­ nitoring. Similarplatin g onvaryin g concentrations ofth e fungicide and colony countshav ebee nuse d less frequently tocompar e resistant andsen ­ sitive strains.Example s include resistance tododin e in Venturia inaegva- lis (MacNei l & Schooley, 1973;Polach , 1973)t obenzimidazole s in A. nidulans (Hastie& Georgopoulos , 1971), Cercospora arachidicola (Littrell, 1974), Pseudocercosporella herpotrichoides and Septoria nodorum (Horsten & Fehrmann, 1980), andt odicarboximide s in B. cinerea (Maraite et al., 1980). Amodificatio n ofthi smetho dwa suse db y Jones& Walker , (1976), who, insteado f incorporating dodine intoth eaga rmedium ,use d dodine solutions to impregnate assaydiscs ,eac ho fwhic hwa splace d inth ecen ­ treo fa plat e onwhic hconidi a of V. inaegualis hadbee nplated .Sensi ­ tivitywa s thenmeasure d byth ediamete r of inhibitionzones . Germination ofspore s infungicid e solutions or onmedi a containing fungicideha sbee nuse d todetec t andmeasur e resistance todiphenyl , sodium 2-phenylphenolate (Vomvoyanni &Georgopoulos ,1966 )an ddodin e (Kappas& Georgopoulos ,1968 )i n H. haematococca, tobenzimidazole s in V. inaegualis (Wicks,1974 ;Jone s& Walker ,1976 )an d to fentinderiva ­ tives in C. beticola (Giannopolitis, 1978). Iti softe nnecessar y inthi s typeo ftes tt oconside rno tonl ygerminatio nbu tals ogerm-tub eelon ­ gation andmorphology . For fungiwit hyeast-lik e growth,bu d formation rather thanspor egerminatio n isth ecriterio nuse d (Wilkie& Lee , 1965). For fungicideswit h knownpronounce d effects ona cellula rprocess , e.g. respiration orprotei n synthesis,th e sensitivity ofvariou s strains isver y appropriatelymeasure db y theeffec to fvaryin g toxicantconcen ­ trations onth eprocess .Thes eexperiment s canb edon eeithe rwit hwhol e cells (invivo )o rsubcellula r preparations (invitro) . Examples areth e studieso ncarboxamid e resistance in V. maydis (Georgopoulos et al.,1972 , 1975; White et al., 1978)an d A. nidulans (Gunatilleke et al., 1976;Whit e et al., 1978)an dcycloheximid e resistance in S. cerevisiae (Coopere t al., 1967)an d Neurospora crassa (Vomvoyanni, 1974). Resistance canals ob e detected andmeasure d onliving , appropriately treated,plant s ofplan tparts .Thi smetho d ispracticall y indispensable inth ecas eo fobligat eparasites ,e.g . thepowder ymildew s (Schroeder &

26 Prowidenti, 1969;Ben te tal. ,1971 ;Vargas ,1973 ;Shephar d etal. ,1975 ; Dekker &Gielink , 1979)an d the downymildew s (Georgopoulos &Grigoriu , 1981; Katan& Bashi ,1981 ;thi sbook ,p .118-127) .Detail s on experiments with thesetw o groups of fungi aregive no np.219-23 0 andp.118-127 .Th e method isals o applicable inth ecas e ofnon-obligat eparasites ,e.g . the smuts (Kuiper,1967 ;Georgopoulo s et al., 1975).

Adjusting fungicide concentration

Formeasurin g sensitivity inartificia lmedi a iti sadvisabl e tous ea formo fth e fungicide that isa spur e aspossible . Ifonl yth e commercial product isavailabl e there isalway s thepossibilit y thatothe r components ofth e formulationwil l affectth eresults ,particularl y inth e caseo f resistant strains,fo rwhic hhighe r amountshav et ob eused .Recrystaliza - tion from anappropriat e solventca nb euse d to separate the activeingre ­ dients from somebu tno t allothe rcomponents . Ifblin d formulations are available they shouldb euse d ascontrols . The lowwate r solubility ofmos tfungicide s ison eo fth e commonest sources oferro rwhe nmeasurin g resistance.Wit h somecompounds ,particu ­ larly the structurally non-specific,Ferguson-typ e toxicants,suc ha sth e chlorinated nitrobenzenes (Eckert, 1962), activityma y increasebeyon d saturation.However ,i ngeneral ,materia l thati sno t insolutio n isinac ­ tive, sotha tincreas e ofth e amounto ffungicid e ina mediu mbeyon d solu­ bility doesno tgiv e anincreas e ineffect . Insoluble reserves ofcom ­ pounds thatten d tob e accumulatedb y fungal cellsma y servet o replenish the amounts thatar etake nup ,bu twha tportio nbecome s active inthi s way isno teas yt o determine in allcases .Solubilit y increases ifa mix ­ turewit h anorgani c solvent isused ,bu tmos t solvents aretoxi c andon ­ lysmal l amounts,usuall yno texceedin g 2.0 %,ca nb eused . Iti sbes tt o firstprepar e stock solutions ofth e fungicide ina suitabl e solventan d thenad d small amounts toth emedium ,makin g sure thatn oprecipitat e is formed;th e same amounto fsolven tmus tb e addedt oth econtrols . After therespons e ofth e strains studied toth evariou s concentrations ofth e fungicideha sbee ndetermined , thedegre e ofresistanc e isusuall y measured by acompariso nof :th eresponse s to the sameconcentration ; theminima l growth inhibiting concentrations (MIC); or the concentrations thatproduc e the sameeffec t (e.g.ED 50 or ED95). Depending onth e slope ofth edosage-respons e curves,th e firstmetho dma yoverestimat e the degree ofresistance .Th e secondmetho d isals ono tver y dependable,par ­ ticularly for fungicides oflo wwate r solubility. Inwor kwit h sensitive strains, solubility (orvolatilit y inth ecas eo fvapor-phas e fungicides) limitationsma yb e oflittl e concern.Resistan t strains,however ,ar e oftenno tcompletel y inhibited atsaturatio n andthu s theMI C cannotb e

27 determined ina nacceptabl y accurateway . Since themetho d doesno tcon ­ siderth eeffec to fconcentration s causing lesstha ntota l inhibition, resistant strains are frequently considered tototall y lack sensitivity. Ifaccurac y isimportant ,i ti sbes tt ous eth ethir dmethod , i.e.t oob ­ tainth edosage-respons e curvesb yplottin g response againstonl yaccu ­ rately determined concentrations.Th eline s arethe ncompare d onthei r position and slope,an d thedegre e ofresistanc e isgive nb y the ratio of concentrations thatgiv e the samepercentag e of inhibition inth ewil d type andth emutan tstrain .

Fungal material

The objective ofth e testdetermine s the amountan d the typeo ffunga l material used.Durin g thedevelopmen to fa ne w fungicide,informatio nma y be required on its ability to selectou tresistan t strains ifi ti sap ­ plied commercially forplant-diseas e control. Inthi s situation iti sim ­ probable that resistancema yb e readily detected innatura l populations ofth epotentia l targetorganisms ,unles s thesepopulation s havepre ­ viouslybee nexpose d to arelate d (interm s ofcross-resistance )chemi ­ cal.Th eusua l test ist oexpos e severalmillion s ofcell s treatedwit ha mutagenic agentt oa concentratio n ofth e fungicideunde r developmenttha t completely inhibits thewild-typ e fungus.Th emutageni c treatment should be sufficient tokil l alarg eproportio n ofcells .Th emutage n usually delays theonse to fgrowt ho fsurvivors ,whic h sometimes necessitates long incubation. Ifonl y afe wo fth e survivors eventually grow inth epres ­ enceo fth e fungicide,thes eprobabl y represent resistantmutants .Bu ti f many colonies grow oneac hplate ,i ti slikel y thatth e long incubation caused abreakdow n ofth e fungicide andth egrowt ho fnon-mutan tcells . After commercial applicationbegins, i ti sver y desirable tob e able todetec tchange s insensitivit y inth epathoge npopulatio n ata nearl y stage,befor e suchchange s result in failure ofth e fungicide andexten ­ sivecro p losses.Thi s requires testingo f aver y largenumbe r ofisola ­ tes, eacho fwhic h should represent anindependen tinfection .Testin gman y spores from a fewlesion s (orfro m a fewplant s inth ecas eo f systemic infections)doe sno tincreas eth esampl e size. Inaddition , iti simpor ­ tantt ous erando m samples thatrepresen tth ewhol e ofth epopulatio nun ­ derconsideration . Ifth e fungicide failst ocontro l the disease,tha t failure cannotb e ascribedt oth edevelopmen to fresistanc e unlessresistan t strainso fth e pathogen represent aver y considerableportio n ofth epopulatio n inth e area andthos e strains areabl et ocaus e diseaseo ntreate dplants .De ­ tection ofresistanc ewhe ndiseas e control failsrequire s testingo fa rather smallnumbe r ofisolates .However ,i ti simportan tt ocollec tsam - pies from the well treated areas with the severest infection. If resis­ tance is shown to be involved and the fungicide is withdrawn, monitoring is necessary to follow changes in the pathogen population as to the ex­ tent of resistance. This requires more-or-less random samples. If possi­ ble, each sampling should examine a new infection cycle of the pathogen. For this reason old infections should be avoided, e.g. by avoiding older leaves. When transferring from the diseased material care must be exercised to avoid residues of the fungicides with which the plants have been treated in the field. In the case of non-systemic fungicides this is made easier by a thorough washing of the samples, which also removes old spores, which, perhaps, have low germinability. Then the material can be kept under conditions allowing renewed sporulation. An accurate description of resistance requires fungal material that is as genetically homogeneous as possible. Pure cultures of the strains to be compared must be established, preferably by transferring uninucleate, haploid spores.

References

American Phytopathological Society, Committee of Standardization of Fungicidal Tests, 1947. Test tube dilution technique for use with the slide-germination method of evaluating protective fungicides. Phytopathology 37: 354-356. American Phytopathological Society and Society of Nematologists, 1978. Methods for evaluating plant fungicides, nematicides and bactericides. American Phytopathologi­ cal Society, Saint Paul, Minnesota. 141 p. Bent, K.J., A.M. Cole, J.A.W. Turner & M. Woolner, 1971. Resistance of cucumber mildew to dimethirimol. Proceedings of the 6th British Insecticide Fungicide Conference, p. 274-282. Bollen, G.J. & G. Scholten, 1971. Acquired resistance to benomyl and some other sys­ temic fungicides in a strain of Botrytis cinerea in cyclamen. Netherlands Journal of Plant Pathology 77: 83-90. Cooper, D. , D.V. Banthorpe Si D. Wilkie, 1967. Modified ribosomes conferring resistance to cycloheximide in mutants of Saccharomyces cerevisiae. Journal of Molecular Biology 26: 347-350. Dekker, J. & A.J. Gielink, 1979. Decreased sensitivity to pyrazophos of cucumber and gherkin powdery mildew. Netherlands Journal of Plant Pathology 85: 137-142. Eckert, J.W., 1962. Fungistatic and phytotoxic properties of some derivatives of nitro­ benzene. Phytopathology 52: 642-649. Georgopoulos, S.G., E. Alexandri & M. Chrysayi, 1972. Genetic evidence for the action of oxathiin and triazole derivatives on the succinic dehydrogenase system of Ustilago maydis mitochondria. Journal of Bacteriology 110: 809-817. Georgopoulos, S.G., M. Chrysayi & G.A. White, 1975. Carboxin resistance in the haploid, the heterozygous diploid, and the plant parasitic dicaryotic phase of Ustilago maydis. Pesticide Biochemistry and Physiology 5: 543-551. Georgopoulos, S.G. & C. Dovas, 1973. A serious outbreak of strains of Cercospora beti- cola resistant to benzimidazole fungicides in Northern Greece. Plant Disease Repor­ ter 57: 321-324. Georgopoulos, S.G. & A. Grigoriou, 1981. Metalaxyl resistant strains of Pseudoperonos- pora cubensis in cucumber greenhouses of Southern Greece. Plant Disease 65: 729-731. Giannopolitis, C.N., 1978. Occurence of strains of Cercospora beticola resistant to triphenyltin fungicides in Greece. Plant Disease Reporter 62: 205-208. Greenaway, W. & J.W. Cowan, 1970. The stability of mercury resistance in Pyrenophora avenae. Transactions of the British Mycological Society 54: 127-138. Gullino, M.L. & A. Garibaldi, 1979. Osservazioni sperimentali sulla resistenza di isolamenti italiani di Botrytis cinerea a vinclozolin. La Difesa délie Piante 6: 341-350.

29 Gunatilleke,I.A.U.N. ,H.N .Arst ,Jr .& C .Scazzochio ,1976 .Thre egene sdetermin eth e carboxinsensitivit y ofmitochondria l succinateoxidatio ni nAspergillu snidulans . Genetica!Researc h26 :297-305 . Hastie,A.C .& S.G . Georgopoulos,1971 .Mutationa l resistance tofungitoxi cbenzimi - dazolederivative si nAspergillu snidulans .Journa lo fGenera lMicrobiolog y67 : 371-373. Horsfall,J.G. , 1956.Principle so ffungicida laction ,Chronic aBotanic aCo. ,Waltham , Mass.,U.S.A . 279p . Horsten,J .& H .Fehrmann ,1980 .Fungicid e resistanceo fSeptori anodorur aan dPseudo - cercosporellaherpotrichoides .I .Effec to ffungicid eapplicatio no nth efrequenc y ofresistan t spores inth efield .Zeitschrif tfü rPflanzenkrankheite nun dPflanzen ­ schutz87 :439-453 . Jones,A.L .& R.J .Walker ,1976 .Toleranc e ofVenturi a inaequalis tododin ean dbenzi - midazole fungicides inMichigan .Plan tDiseas eReporte r60 :40-44 . Kappas,A .& S.G .Georgopoulos ,1968 .Radiation -induce d resistancet ododin ei n Hypomyces.Experienti a 24:181-182 . Katan,T .& E .Bashi ,1981 .Resistanc e toRidomi l inisolate s ofPseudoperonospor a cubensis,th edown ymilde wpathoge nfro mcucurbits .Plan tDiseas e65 :798-800 . Kuiper,J. , 1967.Investigation s onchemica lcontro lo fcommo nbun to fwheat .1 .Austra ­ lianJourna lo fExperimenta lAgricultur ean dAnima lHusbandr y 7:275-282 . Littrell,R.H. ,1974 .Toleranc ei nCercospor a arachidicola tobenomy lan drelate dfun ­ gicides.Phytopatholog y 64:1377-1378 . MacNeil ,B.H .& J . Schooley,1973 .Th edevelopmen to ftoleranc et ododin ei nVenturi a inaequalis.Canadia nJourna lo fBotan y51 :379-382 . Maraite,H. ,S .Meunier ,A .Pourtoi s& J.A .Meyer ,1980 .Emergenc e invitr oan dfit ­ nesso fstrain so fBotryti s cinerea resistantt odicarboximid efungicides .Medede ­ lingenFacultei tLandbouwwetenschappe nRijksuniversitei t Gent45 :159-168 . Miller,M.W .& J.T .Fletcher ,1974 .Benomy ltoleranc e inBotryti s cinerea isolatesfro m glasshouse crops.Transaction s ofth eBritis hMycologica lSociet y62 :99-103 . Nicholson,J.F. ,W.A .Meyer ,J.B .Sinclai r& J.D .Butler ,1971 .Tur fisolate so f Sclerotiniahomoeocarp atoleran tt oDyrene .Phytopathologisch e Zeitschrift72 : 169-172. Nishimura,S. ,K .Kohmot o& H .Udagawa ,1973 .Fiel d emergenceo ffungicide -toleran t strains inAlternari a kikuchianaTanaka .Report so fth eTottor iMycologica lInsti ­ tute (Japan)10 :677-686 . Peletier,E.N. ,1977 .Detectin gpotentia lprotectiv ean dsystemi cantifunga lcompounds . In:M.R .Siege lan dH.D .Sisle r (Eds):Antifunga lCompounds ,Vol .2 ,Marcel lDekke r Inc.,Ne wYork ,p .439-495 . Penrose,L.J. ,K.C .Davi s& W .Koffmann ,1979 .Australia nJourna lo fAgricultura lRe - Search30 :307-319 . Polach,F.J. , 1973.Geneti c controlo fdodin etoleranc e inVenturi a inaequalis.Phyto ­ pathology63 :1189-1190 . Schroeder,W.T .& R .Prowidenti , 1969.Resistanc et obenomy li npowder ymilde wo fcu ­ curbits.Plan tDiseas eReporte r53 :271-275 . Shephard,M.C. ,K.J .Bent ,M .Woolne r& A.M .Cole ,1975 .Sensitivit y toethirimo lo f powderymilde w fromU Kbarle y crops.Proceeding so fth e8t hBritis h Insecticidean d FungicideConference .Britis hCro pProtectio nCouncil ,London ,p .59-66 . Sozzi,D .& C .Gessler ,1980 .Fungicid e (MBC)resistan tmutant so fFusariu moxysporu m f.sp .lycopersic i andBotryti scinerea :pathogenicit y andfitness .Phytopatholo ­ gischeZeitschrif t97 :19-24 . Taga,M. ,H .Nakawa ,M .Tsud a& A .Ueyama ,1978 .Ascospor eanalysi so fkasugamyci nre ­ sistancei nth eperfec t stageo fPyriculari a oryzae.Phytopatholog y68 :815-817 . Tillman,R.W .& H.D .Sisler ,1973 .Effec to fchlorone bo nth egrowt han dmetabolis m ofUstilag omaydis .Phytopatholog y 63:219-225 . Vargas,J.M. ,Jr. ,1973 .A benzimidazol e resistant straino fErysiph egraminis .Phyto ­ pathology63 :1366-1368 . Vomvoyanni,V. ,1974 .Multigeni c controlo fribosoma lpropertie sassociate dwit hcyclo - heximide sensitivity inNeurospor a crassa.Natur e248 :508-510 . Vomvoyanni,V .& S.G .Georgopoulos ,1966 .Dosage -respons erelationship s indipheny l toleranceo fHypomyces .Phytopatholog y56 :1330-1331 . Whan,J.H. , 1976.Toleranc eo fSclerotini a fructicolat obenomyl .Plan tDiseas eRe ­ porter60 :200-201 . White,G.A. ,G.D .Thor n& S.G . Georgopoulos,1978 .Oxathii ncarboxamide shighl yactiv e againstcarboxin -resistan tsuccini cdehydrogenas e complexes fromcarboxin -selec ­ tedmutant so fUstilag omaydi san dAspergillu snidulans .Pesticid eBiochemistr yan d Physiology 9:165-182 .

30 Wicks,T. ,1974 .Toleranc eo fth eappl esca bfungu st obenzimidazol efungicides .Plan t DiseaseReporte r58 :886-889 . Wilkie,D .& B.K .Lee ,1965 .Geneti canalysi so factidion e resistance inSaccharomyce s cerevisiae.Genetica lResearc h6 :130-138 .

31 Mechanism of action of fungicides

A.Kaar s Sijpesteijn Institute forOrgani c Chemistry TNO,Utrecht ,Th eNetherland s

Abstract

A survey isgive no fth emechanis m of actiono fa variet y ofprotec ­ tant and systemic fungicides.Occurrenc e ofresistanc e inth e laboratory and inth e field is indicated formos to f thefungicides .

Keywords:protectan t fungicides,systemi c fungicides,mechanis m ofaction , inhibition ofbiosynthesis ,inhibitio no frespiration .

Introduction

For aprope r understanding ofth emechanism s ofresistanc e tofungici ­ des somegenera l knowledge ofthei rmechanis m ofactio n isessential . Therefore thiscontributio n comprises abroa d survey ofth evariou sme ­ chanisms ofactio nrecognize d so far for agricultural fungicides;clos e derivatives of several ofthes e fungicides areuse d inhuma n andvet ­ erinarymedicine .Fungicide s forwhic hu p tillno wn oresistanc e is knownhav e alsobee n included. Muchdetaile d information onmechanism s of actiono f fungicides isa - vailable invariou s chapters ofth emonograp h 'Antifungal Compounds'b y Siegel& Sisler (1977); acomprehensiv e survey ofth emod e ofactio no f systemic fungicideswa spublishe d byKaar s Sijpesteijn in1977 .Therefor e only references topublicatio n since then arecite d inthi spresen tre ­ view. Mode-of-action studies require agrea tvariet y ofexperiments .Th eob ­ served responses toth e fungicidehav et ob e critically analyzed toestab ­ lishwhic heffec t isth eprimar y causeo fgrowt h inhibition atth elo ­ westeffectiv e doseo fth e toxicant.Man y secondary effectsma y result from theprimar y effect.Moreover , forcertai n compounds additionalef ­ fectsbecom e apparenta thighe rconcentrations . Many compoundsprimaril y affectrespiratio n or,mor e generally, energy production;man yother s affectbiosyntheti c processes;an d afe wcom -

32 pounds destroy cells,b ydirectl y affectingmembran e function.Wit hth e exception of 'Dexon',carboxi n and carboxin-related compounds, fungicides affecting respiration areno t systemic. Incontrast ,thos e affectingbio - synthetic processes generally appear tohav e systemicproperties . Inth e surveymos to fth e knownmechanism s of actiono f agricultural fungicides aresummarized . Fungicides whosemode s ofactio n areunknow n orno tknow n forcertai n are also included.Not e thati fa mutan ti sre ­ sistantt odifferen t fungicides itdoe sno tnecessaril ypoin tt oth e same mode of actiono fthos e fungicides.However , itdoe s indicate thatthi s mayb e thecase .Structura l formulae ofth e toxicants discussed are given inAppendi xA tothi s contribution.

Fungicides that interfere with energy production

Specific inhibitors

There ismuc h evidence thatth edimethyldithiocarbamates ,e.g . thiram and its salts,a tlo wconcentratio n quite specifically inhibitth edehy - drogenation ofpyruvate .A t suchconcentrations , a1: 1 complex of copper anddimethyldithiocarbamat e is formed.Probabl y this combineswit hth e dithiolcompound s (lipoic acido r lipoic acid dehydrogenase)tha t arees ­ sential fordehydrogenatio no fpyruvat e (Kaars Sijpesteijn& va nde rKerk , 1965). Athighe r concentrations othermechanism s operate.Pyruvat edehy ­

drogenation is alsoth etarge to fseondar ybutylamin e (CH3CH-CHNH2CH3), which isuse d to combat Pénicillium digitatum (Yoshikawa &Eckert , 1976). Carboxin,a systemi c fungicidewit h arathe rnarro w antifungalspec ­ trum, interfereswit h respiration, causing abloc k insuccinat edehydro ­ genation.Th esit eo factio nlie sbetwee n succinate andcoenzym eQ i n the succinate-ubiquinone reductase complex ofth eelectro n transport sys­ tem.Th e actual targeto fcarboxi nma yb e thenonheme-iro nsulphu rpro ­ tein associated with thisenzym e complex.Resistan tmutant s caneasil y beobtaine d invitro .Th erelate d compoundsmebeni l andpyracarboli d act inth e samewa y and thisma y alsob eth e casewit h the later-developed carboxamides,e.g .methfuroxam . 'Dexon' actsrathe r selectively onOomycetes ; itseem s to inhibitsom e flavinenzyme s involved inth edehydrogenatio n ofreduce d nicotinamide ad­ enine dinucleotide (NADH)i nth e respiratory chain (cf.Schew e& Müller , 1979). There ismuc hreaso nt obeliev e thattriphenylti ncompounds ,suc ha s fentinacetate ,interfer ewit h fungalgrowt hb y inhibiting oxidative phos­ phorylation. Serious resistance inth e fieldha sbee ndescribe d byGeor - gopoulos inhi sdiscussio n of fungicide resistance in Cercospora beticola (p.187-194).Dinoca p andothe r dinitrophenols,ac tb y uncoupling oxidative phosphorylation from respiration.

33 Unspecific inhibitors

Agrou p of fungicides that inhibitrespiratio n quite unspecifically areth ethio lreagents ,amon gthe m zineb,maneb , chlorothalonil,captan , folpet,captafo l and dichlone.Thes e compounds,o r their transformation products, reactquit e generallywit h -SHenzyme s and other -SHcompound s involved inrespiration . Sincethe y acta tman y sites they areofte nindi ­ cated asmultisit efungicides .

Fungicides that interfere with biosynthetic processes

Inhibition ofprotei n synthesis

The antibiotic cycloheximideinhibit s growthb y interferencewit hpro ­ teinbiosynthesis .Lo wconcentration s specifically inhibitthi sproces s atth e stagewher e in acomple xwit h theribosomes , amino acids aretrans ­ ferred from tRNA intopolypeptides .Th eprecis emechanis m and siteo fac ­ tioni sstil lno tclearl y known,however .Fo rvariou s fungalspecies , strains resistant tocycloheximid e havebee nobtaine d invitro . Theprobabl e siteo f actiono fth e antibioticblasticidin- S isth efi ­ nal stepo fprotei n synthesis,whic h takesplac e onth e ribosomes.Th e antibiotic kasugamycin,whic h likeblasticidin- S isparticularl y active on Pyricularia oryzae, also affects some stageo fprotei n synthesis.Con ­ siderable resistance tothi s compoundha sdevelope d inth e field. Streptomycin isuse d againstcertai nbacteria l and fungal diseases. Its actiono nbacteri a isascribe d to interference withprotei nsynthe ­ sis, theprimar y targetbein gth e3 0S sub-uni to fth eribosomes .

Inhibition ofnucleic-aci d synthesis

Ethirimol and demithirimol actexclusivel y onpowder ymildews .Ethiri - molha sno wbee n shownt o inhibitspecificall y theenzym e adenosine deaminase inthes e fungionly . Iti s supposed thatthi s enzyme,whic h converts adenosine to inosine,i sessentia l forappressori a formation (Hollomon, 1979). In fungi insensitive toethirimol ,thi s enzymewa s also insensitive.Althoug h thesedat ama y suggest interferencewit hnu ­ cleic acid synthesis,th epictur e isstil l far fromclear . Inpractice , resistance to thesecompound s develops easily. Metalaxyl and furalaxyl areparticularl y active against avariet y of Peronosporales. There arever y strong indications thatmetalaxy l inter­ fereswit hnucleic-aci d synthesis in Pythium splendens; presumablyRN A synthesis isth eprimar y site of inhibition (Kerkenaar& Kaar sSijpe - steijn, 1981;Kerkenaar , 1981). Resistance inth e fieldha sbee nrepor ­ ted forcertai npathogens .

34 Hydroxyisoxazole inhibits avariet y of fungi. In Fusarium oxysporum, interferencewit hDN A synthesis is itsprimar y site ofactio n (Kamimura et al., 1976).

Interference withnuclea r processes

Benomyl andthiophanate-methy l areknow nt o actafte rthei r conversion toth e toxic agentcarbendazim .Th eprimar yproces s inhibitedb ythi s broad-spectrum fungicide ismitosis .Carbendazi m appears to form acom ­ plexwit h sub-units ofmicrotubuli ,thu spreventin g thenorma l assembly ofmicrotubul e sub-units intospindl e fibres. Inpractice ,resistanc e to the abovecompound s develops easily incertai n fungi (seep.60-70) .Re ­ cently thiabendazole was shownt ohav e the samemod e ofactio n ascarben ­ dazim (Davidse &Flach , 1978). The dicarboximides (vinclozolin,procymidon e and iprodione)an dth e aromatic-hydrocarbon fungicides (chloroneb,hexachlorobenzene , quintozene, dicloran)ar eremarkabl y similar inthei r action.Moreover ,man y examples ofcross-resistanc e havebee nobserve d forthes ecompound s (Leroux, 1977). They allcaus emitoti c instability in Aspergillus nidulans, whichpoint s tointerferenc e withmitoti c division (Georgopoulos et al., 1979).Never ­ theless, thetru enatur e ofth eprimar y effect isno tye tclear .Inter ­ ferencewit hDN Abiosynthesi sha sbee nobserve d forchlorone b (Tillman & Sisler, 1973)an dvinclozolin ,procymidon e and dicloran (Fritze t al., 1977).N ocross-resistanc e hasbee n foundwit h carbendazim.

Inhibition ofcell-wal l synthesis

The antibiotic polyoxinD inhibits chitinsynthesis ;th e intermediate UDP-N-acetylglucosamine accumulates andprotoplast-lik e cells are formed. The fungicide shows astructura l resemblance with this intermediate sub­ strate. Itobviousl y acts asa n anti-metabolite,resultin g in inhibition ofth e enzyme chitin synthetase.Resistanc e topolyoxi nD ha sbee nobser ­ ved inpractica l agriculture.

Inhibitiono flipi d biosynthesis

Themod eo f actiono f IBPha s recentlybee n further clarified byKoda - mae t al. (1979). Thecompoun d isparticularl y active on P.oruzae and inhibits specifically the conversiono fphosphatidylethanolamin e tophos ­ phatidylcholine by transmethylation ofS-adenosylmethionine .Phosphati ­ dylcholine isa nimportan tconstituen t ofth emembranes .Sinc e IBPha s some structural resemblance with the substratephospatidylethanolamin e it maywel l acta sa nantimetabolit e ofthi s substrate.Anothe rphosphoro - thiolate fungicide,edinfenphos ,ha srecentl ybee n shownt oac ti nth e

35 samewa y as IBP (Kodamae t al., 1980). 14 Asth e incorporation of C-methionineint ophospholipid s isals oin ­ hibitedb y isoprothiolane (Kakiki &Misato , 1979), themod e ofactio no f isoprothiolane maywel lb eth esame ;cross-resistanc e with IBPha d already been observedb yKatagir i& Uesug i (1977). Many fungicides areno w knownt ob e sterol-biosynthesis inhibitors. Triarimol, abroad-spectru m fungicide thatha sbee nwithdraw n from the market,wa s the firstcompoun d recognized to acti nthi sway . Inth e years that followed severalmore-or-les srelate d fungicideswer edevel ­ oped forwhic h asimila rmod eo f actionwa s observed: fenarimol (de Waard &Ragsdale , 1979), triadimefon (Buchenauer, 1976)an d itsreduce d derivative triadimenol (Buchenauer, 1978), bitertanol (Kraus, 1979), imazalil (Lerouxe t al., 1976;Siege l &Ragsdale , 1978), andphenaproni l (Girardet& Buchenauer , 1980). Forprochlora z (cf.Lerou x &Gredt , 1978), diclobutrazol (Bent& Skidmore,1979 )an d CGA 64250 (Ciba-Geigy AG,Basel , Switzerland) asimila rmod e of action isver y likely. Iti s remarkable thattw ounrelate d fungicides triforine andbuthiobat e also acti na similarway . All inhibitors ofstero lbiosynthesi s cause arapi d alteration inth e quantity and thenatur e ofth e lipid components inth ecells .Especiall y 14 ... the incorporation of C-acetate intoergostero lwa s strongly inhibited ata ver y earlymoment , and several intermediates accumulated. Inhibition ofdemethylatio na tC-1 4appear s tob e theprincipl e site of inhibition instero lbiosynthesis .A n effecto nsynthesi s and functiono fcel lmem ­ braneswil lcertainl y resultfro m this; itma y explainth e morphological changes ofth etreate dcells . Tridemorphha sman y effects incommo nwit hthes ecompound s (Kerkenaar 14 et al-, 1979); itsexac ttarge t isno t C-demethylation,bu t aste p fol­ lowingdemethylatio n (Katoe t al., 1980;Kerkenaa r et al., 1981).Fenpropi - morphma y actsimilarly . Althoughno talway spresent , andeve nthe nno talway s toth esam ede ­ gree, invitr o cross-resistance hasbee nobserve d betweenman y inhibitors ofstero lbiosynthesis , (e.g.Baru g& Kerkenaar , 1979). Inth e field,n o resistance has so far developed. Fungicides that interfere with cell structure

Dodine affects thepermeabilit y ofth ecel lmembrane ,whic h results in leakage ofth e cell contents.Inhibitio n ofrespiratio n isa secondar y effect (Brown& Sisler, 1960). Theexac tnatur e ofth e interference with themembrane s isno tye tknown .Resistanc e inth e fieldha sbee nrepor ­ ted.

36 Fungicides whose mode of action is unknown or not known for certain

Presumably pyrazophos isitsel fno tactive . Itsmetabolit e (PP),how ­ ever, ishighl y fungitoxic tocertai n fungi inmedi a of lowpH .Thi sme ­ tabolite affects respiration,bu tresearcher s are stilluncertai n whether this is itsprimar yeffect . Prothiocarb and its derivativepropamocar b areparticularl y effective againstth e Peronosporales, Pythium and Phytophthora. Prothiocarb does not inhibitrespiration ,whic h suggests itinterfere swit h abiosyntheti c process. Cholesterol,althoug hno t acel l constituent of Pythium somewhat antagonizes the fungitoxicity (Kerkenaar& Kaar s Sijpesteijn, 1977). Similar effectswer e alsodescribe d forpropamocar b byPapaviza s et al. (1978), who,moreover ,observe d leakage ofcel l constituents following growth inth epresenc e oflo wconcentration s oftha t fungicide.Papaviza s et al. suggesttha tth emod e of actionmigh tb e related to cell-membrane function. Theactio no fprothiocar b on Achlya radiosa differs fromtha to n Py­ thium. Itca nb e ascribed toth eeffec to frelease d ethylmercaptan (Kerke- naar& Kaar s Sijpesteijn, 1977). Etridiazole affectsparticularl y Phycomycetes. Halos& Huisma n (1976) reported that itcause d inhibitiono frespiratio n of Pythium species.I n contrast,Ly re t al. (1977)sugges tthat ,i n Mucor mucedo, the compound actsb y liberationo fphospholipase s withinth emitochondria . 'Curzate' (Dupont,Wilmington ,USA )i seffectiv e inth ecomba to f Pe­ ronosporales inth e field.Littl e isknow no fit s fungicidal activity in vitro.Unpublishe d results show inhibition of Pythium onaga rmedi a at relatively highconcentrations .

Discussion

A shortsurve yha sbee ngive no fth emechanis m of actiono fman yo f the fungicides used inagricultura l practice orstudie d as experimental fungicides. Inman y cases resistant strains areknown ,especiall y forcom ­ pounds that acto nspecifi c systems only.Compound s thatprotec tth ehos t plantsb y an indirectmechanis m orar e supposed tod o so,e.g . tricycla- zole,probenazole ,CG A4910 4an d 'Aliette',hav eno tbee ndeal twit hbe ­ cause they areno t fungicides.

37 Appendix A: Structural formulae of toxicants discussed in this contribution.

(CH3)2N-C-S-S-C-N(CH3)2 (CH3)2N-C-S-Na II II S S Na- dimethyldithiocarbamate thiram

0_CH3 (CH3)2N—Ü V-N =N-S0 2-0Na Il H \=/ 0 carboxin dexon

0 Il H H 0-C-C=C-CH3

02N Sn-0-C-CH3 ÇH-C6H13

CH3

N02 fentin acetate dinocap

S S S S Il H H II Il H H II -S-C-N-CH2-CH2-N-C-S-Zn- -S-C-N-CH2-CH2-N-C-S-Mrr -"n -Ti zineb

CN 0 Ck X .CI N-S-CCI3 CI J "CN Ö Cl chlorothalonil captan

0

)\I-S-CCI2-CCI2H

0

captafol

38 CH LH3 0 3 ^ I N. ^NH J>-CH-CH2^ NH C 2 ç ,wAnA o NH2 II N NH r OAc ^0 ° C02H CH 3 H2lsT^o

cycloheximide acetate b!asticidin-S

CH3 HOOC-C-N-^X-Q

HO-i-T-V OH J OH kasugamycm

OH OH n-C«Ho 1

CHA^N^2"5 CHAV™3 H I CH ethirimol 3 dimethirimol

ÇH ,CH3 f 3 CH3 p3 C00CH3 fWS- ^< C-COOCH3 N \=< C-CH2-OCH3 CH3JI P metalaxyl furalaxyl

OH ,N CH3 O'

hydroxyisoxazole

39 o C-NH-CH2-CH2-CH2-CH3 o O II II ,pNH-C-OCH -NH-C-0-CH3 3 -N -N carbendazim benomyl

S 0 H II H II N-C-N-C-OCH3

N-C-N-C-OCH3 H II H H S 0 '*l N u—S thiophanate - methyl thiabendazole

xCH u 3 'CONHCH V CH3

/ // 'CH3

vinclozolin procymidone iprodione

OCH3

hexachlorobenzene quintozene

40 O COOH HN HOOC 0=C-N-CH I H H2N-ÇH

HÇ-OH ÓH ÓH

HO-CH I CH2-0-C-NH2 II O

polyoxin D

O x // \\ /0-CH(CH3)2 l N ^4 ) ' ^—CH2-S-PV X C2H5OF> || 0-CH(CH3)2 \

IBP 'KitazinP' edifenphos (EDDP,'Hinosan' )

,-S .C-0-CH(CH3)2

/ ^S ^C-0-CH(CH3)2

isoprothiolane

C1 OHV " / OH /r\Ajr\ Cl

II

triarimol fenarimol

41 // V / V-o-CH-CHOH-C(CH3)3 Cl—

Cl

Cl—

IT N

imazaul phenapronil

£\-\j>— 0-CH2-CH2-N-C3H7 I C=0 Cl I if N- prochloraz

N=N C I N-CH-CO-C(CH3)3 Nrrx 0-^_/ 3H7 l=NI I N-CH2-C' 7 CH2 Cl 0-« Cl

Cl CGA 64250 diclobutrazol propiconazol

42 CHO CHO I C«Hg-S-C-S-CH -^ //^C(CH ) NH NH 2 3 3 N CH-N N-CH I ^ I CCl3 CCI3 ^>N

triforine buthiobate

CH3 CH- ÇH3 Ci3H27-N .0 (CH3)3C—

CH3 CH3

tridemorph fenpropimorph

NH © n-C12H25-NH-C-NH3-CH3-COO © dodine

H3C^ ^N^^ -0-P-0C2H5

0C2H5

pyrazophos metabolite of pyrazophos

0 0 H II H II (CH3)2N(CH2)3N-C-SC2H5HCl (CH3)2N(CH2)3N-C-OC3H7- HCl

prothiocarb propamocarb

0 0 H II H II CI3C. C2H5-N-C-N-C-C-CN

N N-0-CH3 ^OC2H5

etridiazole 'Curzate' 43 References

Barug,D .& A .Kerkenaar ,1979 .Cross-resistanc eo fUV-induce dmutant so fUstilag omay - dist ovariou s fungicideswhic hinterfer ewit hergostero lbiosynthesis .Mededelinge n FaculteitLandbouwwetenschappe n Rijksuniversiteit,Gen t44 :421-427 . Bent,K.J .& A.M .Skidmore ,1979 .Diclobutrazol :a ne wsystemi c fungicide.Proceeding s ofth e197 9Britis hCro pProtectio nConference ,Brighton ,England .Britis hCro p ProtectionCouncil ,London ,p .477-484 . Brown,I.F .& H.D .Sisler ,1960 .Mechanism so ffungitoxi cactio no nn-dodecylguanidin e acetate.Phytopatholog y50 :830-839 . Buchenauer,H. ,1976 .Hemmun gde rErgosterinbiosynthes e inUstilag oavena edurc hTria - dimefonun dFluotrimazol .Zeitschrif tPflanzenkrankheite nun dPflanzenschut z83 : 363-367. Buchenauer,H. ,1978 .Inhibitio no fergostero lbiosynthesi sb ytriadimeno l inUstilag o avenae.Pesticid eScienc e9 :507-512 . Davidse,L.C .& W .Flach ,1978 .Interactio no fthiabendazol ewit hfunga ltubulin . BiochimicaBiophysic aAct a543 :82-90 . Fritz,R ., P .Lerou x& M .Gredt ,1977 .Mechanism ed el'actio n fongitoxiqued el apromi - dione (26019R Po uglycophène) ,d el avinclozolin ee td udichlora nsu rBotryti sci ­ nereaPers .Phytopathologisch e Zeitschrift90 :152-163 . Georgopoulos,S.G. ,M .Sarri s& B.N .Ziogas ,1979 .Mitoti c instability inAspergillu s nidulans causedb yth efungicide s iprodione,procymidon ean dvinclozolin .Pesticid e Science 10:389-392 . ** Girardet,F .& H .Buchenauer ,1980 .Wirkungsweis evo nPhenaproni l (Sisthane^ gegenüber Ustilagoavena eun dErisiph e graminis f.sp.hordei .Mededelinge nFacultei tLand ­ bouwwetenschappenRijksuniversitei tGen t45 :435-443 . Halos,P.M . &O.C .Huisman ,1976 .Inhibitio no frespiratio ni nPythiu mspecie sb yetha - zol.Phytopatholog y66 :158-164 . Hollomon,D.W. , 1979.Specificit y ofethirimo l inrelatio nt oinhibitio no fth eenzym e adenosinedeaminase .Proceeding so fth e 1979Britis hCro pProtectio nConference , Brighton,England .Britisc hCro pProtectio nCouncil ,London ,p .251-256 . KaarsSijpesteijn ,A .& G.J.M .va nde rKerk ,1965 .Fat eo ffungicide s inplants . AnnualRevie wPhytopatholog y3 :127-152 . KaarsSijpesteijn ,A. ,1977 .Effect so nfunga lpathogens .In :R.W .Mars h (Ed.): Systemic fungicides.Longman ,London/Ne wYork ,p .131-159 . Kakiki,K .& T .Misato ,1979 .Effec to fisoprothiolan e onfatt yaci dsynthesis . Journalo fPesticid eScienc e4 :305-313 . Kamimura,S. ,M .Nishikaw a& Y .Takahi ,1976 .Mod eo factio no fsoi lfungicid ehymexa - zol,3-hydroxy-5-methylisoxazole , onFusariu moxysporu mf .cucumerinum .Annal sPhy - topathologicalSociet yJapa n42 :242-252 . Katagiri,M .& Y .Uesugi ,1977 .Similaritie sbetwee nth efungicida lactio no fisopro ­ thiolanean dorganophosphoru s thiolate fungicides.Phytopatholog y67 :1415-1417 . Kato,T. ,M .Shoam i& Y .Kawase ,1980 .Compariso no ftridemorp hwit hbuthiobat ei n antifungalmod eo faction .Journa lo fPesticid eScienc e5 :69-79 . Kerkenaar,A. ,1981 .Antifunga lmod eo factio no fmetalaxyl ,a ninhibito ro fnuclei c acidsynthesi s inPythiu m splendens.Pesticid eBiochemistr y andPhysiolog y 16:1-13 . Kerkenaar,A .& A .Kaar sSijpesteijn ,1977 .O nth emod eo factio no fprothiocarb . NetherlandsJourna lPlan tPatholog y8 3(Supplemen t 1):145-152 . Kerkenaar,A .& A .Kaar sSijpesteijn ,1981 .Antifunga lactivit yo fmetalaxy lan dfura - laxyl.Pesticid eBiochemistr yan dPhysiolog y 15:71-81 . Kerkenaar,A. ,D .Baru gan dA .Kaar sSijpesteijn ,1979 .O nth eantifunga lmod eo fac ­ tiono ftridemorph .Pesticid eBiochemistr yan dPhysiolog y 12:195-204 . Kerkenaar,A. ,M .Uchiyam a& G.G .Versluis ,1981 .Specifi ceffect so ftridemorp ho n sterolbiosynthesi s inUstilag omaydis .Pesticid eBiochemistr yan dPhysiolog y16 : 97-104. Kodama,0. ,H .Yamad a& T .Akatsuka ,1979 .Kitazi nP ,inhibito ro fphosphatidylcholin e biosynthesis inPyriculari a oryzae.Agricultura lBiologica lChemistr y43 :1719-1726 . Kodama,0. ,K .Yamashit a& T .Akatsuka ,1980 .Edifenphos ,inhibito ro fphosphatidyl ­ cholinebiosynthesi s inPyriculari a oryzae.Agricultura lBiologica lChemistr y44 : 1015-1021. Kraus,P. , 1979.Untersuchunge nzu mWirkungsmechanismu s vonBaycor .Pflanzenschutznach ­ richtenBaye r32 :17-30 . Leroux,P .& M .Gredt ,1978 .Effet sd equelque sfongicide ssystémique ssu rl abiosyn ­ thèsed el'ergostéro l chezBotryti s cinereaPers. ,Pénicilliu mexpansu mLink ,e t Ustilagomaydi s (DC.)Cda .Annale sPhytopatholog y 10:45-60 .

44 Leroux,P. ,R .Frit z& M .Gredt ,1977 .Étude se nlaboratoir ed esouche sd eBotryti s cinereaPers. ,résistante sà l adichlozoline ,a udicloran ,a uquintozène ,à l avin - clozolinee ta u2601 9R P (ouglycophène) .Phytopathologisch e Zeitschrift89 :347-358 . Leroux,P. ,M .Gred t& R .Fritz ,1976 .Similitude se tdifférence sentr ele smode sd'ac ­ tiond el'imazalil ,d utriadimefon ,d utriarimo le td el atriforine .Phytiatrie-Phy - topharmacie25 :317-334 . Lyr,H. ,G .Casperso n& B .Lausmann ,1977 .Mod eo factio no fterrazo lwit hMuco rmuce - do.Zeitschrif tAllgemein eMikrobiologi e 17:117-129 . Papavizas,G.C. ,N.R .O'Neil l& J.A .Lewis ,1978 .Fungistati c activityo fpropyl-N - (y-dimethylaminopropyl)carbamat eo nPythiu m spp.an d itsreversa lb ysterols . Phytopathology68 :1667-1671 . Schewe,T .& W .Müller ,1979 .Zu mmolekulare nWirkungsmechanismu svo nDexon .In : Systemfungizide.V .Internationale s Symposium,Reinhardsbrunn ,1977 .Akademie-Ver ­ lag,Berli n 1979,p .317-324 . Siegel,M.R .& N.N .Ragsdale ,1978 .Antifunga lmod eo factio no fimazalil .Pesticid e Biochemistryan dPhysiolog y9 :48-56 . Siegel,M.R .& H.D .Sisler ,1977 .Antifunga l compounds.Vol .2 .Marce lDekke rInc. , NewYork ,67 4p . Tillman,R.W .& H.D .Sisler ,1973 .Effec to fchlorone bo nth egrowt han dmetabolis mo f Ustilagomaydis .Phytopatholog y63 :219-225 . Waard,M.A .d e& N.N .Ragsdale ,1979 .Fenarimol ,a ne wsystemi c fungicide.In :H .Ly r & C.Polte r (Eds):Systemfungizide .Internationale sSymposiu mReinhardsbrunn ,1977 . Akademie-Verlag,Berlin ,p .187-194 . Yoshikawa,M. ,W .Ecker t& N.T .Keen ,1976 .Th emechanis m offungistati cactio no f secondarybutylamine .I IEffect so fsecondar ybutylamin eo nth emetabolis mo nhypha e ofPénicilliu mdigitatum .Pesticid eBiochemistr yan dPhysiolog y6 :471-481 .

45 Genetical and biochemical background of fungicide resistance

S.G.Georgopoulo s Laboratory ofPhytopathology ,Athen s College ofAgricultura l Sciences, Votanikos,Athens ,Greec e

Abstract

Iti sexplaine d how sensitivity totoxi c compounds iscontrolle d by genes thatcontro l acellula rproces s important forexertio no fth etoxi c effect.The n attention ispai d toth ebiochemica l genetics of resistance to themai ngroup s of agricultural fungicides.Wit hmos t fungicides know­ ledge oneithe r genetics orbiochemistr y isincomplete . Ina fe winstan ­ ces, however,resistanc e geneshav ebee ndefinitel y identified and their action atth ebiochemica l level ismor e or less understood.

Keywords: fungicide resistance,geneti c control,gen e action,benzimida - zoles, carboxamides, sterol-biosynthesis inhibitors,aromati chydrocar ­ bons,polyoxins ,pyrazophos ,kasugamycin .

Introduction

Somechemical s aretoxi cbecaus e they interactwit h cell constituents ina wa y that adversely affects importantcellula rprocesses .Th e ability of anorganis m todevelo p resistance to sucha chemica l isdu e to its cel­ lular components being able toexis t inmor e thanon e formwithou tpreven ­ ting survival undernorma l conditions.Change s fromon e form to another that aredisadvantageou s forth e organism arequit epossible ,bu tthe y areo flittl e concern tothos e engaged inth echemica l control ofpatho ­ gens. Theproble m ofresistanc e arises from changes thatno tonl ypermi t survivalbu t alsodecreas e sensitivity toth echemica l used. Inothe r branches ofscience ,however ,problem sma y arise from anincreas e insen ­ sitivity.Ordinar y anesthesia, forexample ,ma yprov e fatal tosom e indi­ vidualswit h increased sensitivity. Such individuals arenorma l inever y other respect,bu tdu et o anunfortunat e hereditary enzyme constitution they cannotdetoxif y the anesthetic asmos tothe r individuals do,an d hence they cannotrecove r from anesthesia (Elliott, 1973). Tounderstan d achang e ofcellula r components fromon e form toanother .

46 iti simportan t torecognise :whethe r there aregeneti c determinants (genes)involve d ando fwha tkind ; andwha tcellula r components (enzymes orothers )ar ecode db y these genes and inwha twa y theyhav e changed. This contribution summarizes the available information onth e biochemical geneticso fresistanc e toagricultura l fungicides,wit hemphasi s onrecen t findings;earlie rwor kha sbee n reviewed (Georgopoulos, 1976).

Genetics

Ifa chang e insensitivit y doesno tinvolv e achang e inchromosoma l or extrachromosomal DNA, iti sthough tt ob e theresul to fphenotypi c adap­ tation. Such adaptationma y takeplac e incultur e after longexposur et o the fungicide under conditions favourable forth e organism (e.g.nutri ­ tion). Phenotypic resistance isusuall y easily lostupo ntransfe r toa toxicant-free medium.Whethe rphenotypi c adaptation to fungicides ispos ­ sibleunde r field conditions isno tknown . Instability ofresistanc e of field isolatesma y indicatephenotypi c adaptation,bu t itshoul d notb e confusedwit h regressiono fresistanc e inth e field,whic hma ywel lb e due toreduce d fitness ofgeneti cmutants . Involvement ofextrachromosoma l genes inresistanc e totoxicant s is very common inbacteria . Suchgene s areborn eo nPlasmids ,extrachromo ­ somal, circularDN Amolecules ,an d aretransmitte d more easily than chromosomal genes.Plasmid - orvirus-lik e particles havebee n recognised recently in fungi andhav ebee n associated withheritabl e changes.Th e presence of aDN Aplasmi d inyeas ti scorrelate d with thedeterminan to f onetyp e ofoligomyci n resistance (Guerineau et al., 1974). Fungi also possessmitochondria l DNAwit h adefinit e role inheredit y and inse ­ veral instances resistance totoxicant sha sbee n foundt ob eth eresul t ofmutation s ofmitochondria l genes (cf.Georgopoulos , 1977). Howevermos tgeneti c studies onresistanc e to fungicides have revealed the involvement ofchromosoma l genes.A s arul e several genescontro lsen ­ sitivity toth e same fungicide orgrou p of fungicides.Th e easiestwa yt o recognise them ist ous e aheterothalli c ascomycetewhos eperfec t stage isproduce d readily andtha tgive seasil y analyzableprogen ywithi n afe w days.Thes e advantages areoffere db yth enon-pathoge n Neurospora crassa and,mor e or less,b y somepathogens ,e.g . Nectria haematococca or Ventu- ria inaequalis. Someheterothalli c Basidiomycetes,e.g . Ustilago maydis, are alsouse d forth e recognition of fungicide-resistance genes.Th egen ­ etics ofresistanc e canb e studiedb y thismeioti c analysis also inhomo - thallicAscomycete swhe n ametho d isavailabl e todistinguis h between crossed and selfedperithecia .Mitoti c analysisma yb euse d alsot orecog ­ nisegene s for fungicideresistanc e inth eabsenc eo fth e sexual stage. Themethod s ofgeneti c analysis inth emai ngroup s of fungica nb e found ina numbe r oftexts ,e.g . Fincham etal . (1979).

47 Biochemistry

Inth e areao fgen e expression, i.e. information transfer from genet o protein,knowledg e ofresistanc e to agricultural fungicides israthe r lim­ ited.Rationally , aresistanc e genemus t firstb e transcribed tomessen ­ gerRNA ,whic h istranslate d inth eribosomes , and aprotei nmolecul e is formed.Thi sprotei nma y itself interactwit h the fungicide,i.e .b e the sensitive site,o r itma y affect someothe r cellularpropert y important interm so ftoxicity . Themai nmechanism s bywhic h resistance to atoxi c compoundma ydeve ­ lop are:modificatio n ofth e sensitive site;bypas s ofth e sitedu et o operation ofa nalternativ e pathway; decreased uptake orincrease dsecre ­ tiono fth etoxicant ;detoxification ; ordecrease d conversion of anon ­ toxic into atoxi c compound.O fthes emechanisms ,th e firsttw o areim ­ portant forth e development ofresistanc e tomainl y site-specific fungi­ cides, the remaining three tospecifi c andmulti-sit e fungicides.Not e thatwit hresistanc e to the sametoxican tdifferen tmechanism s may operate indifferen torganisms ,o r indifferen tmutant s ofth e same organism. For the study ofbiochemica lmechanism s iti susuall y necessary to examine the effecto fth e fungicide onth ewil d type andmutan t atth e cellular andth e sub-cellular level. Ifresistanc e isexpresse d inexper ­ imentswit hwhol e cellsbu tno twit h cell-free systems,the nth emecha ­ nism doesno tinvolv emodificatio n ofth e sensitive site.Comparativ eex ­ periments onth e influx and efflux ofth e toxicmolecule ,o nit smetabo ­ lism and itsbindin g tocomponent s ofmutan t andwil d type cells areals o required.Thes e studies aretime-consumin g andrequir e specially equipped laboratories.

Work with agricultural fungicides

Thebes tavailabl e example inwhic hth e actiono f aknow ngen e that controls sensitivity to agricultural fungicides hasbee n completelyelu ­ cidated istha to fth e ben-h gene forresistanc e tobenzimidazole s in Aspergillus nidulans. This gene codes forß-tubulin ,on eo fth e sub-units ofth e tubulinmolecul e (Sheir-Neiss et al., 1978). Mutations ofthi s gene affectth e electrophoretic properties of ß-tubulin and atth esam e time the ability ofth eprotei n tobin dcarbendazim ,whic h is inversely correlated tocarbendazi m resistance.Mor edetail s onth e actiono fth e ben-h gene aregive no np.60-70 . Chromosomal genes forresistanc e tobenzimidazole s have alsobee nre ­ cognised insevera l other fungi (Borck& Braymer ,1974 ;Brasie r& Gibbs , 1975; Shabi& Ben-Yephet , 1976), although their actionha s notbee n stud­ ied atth ebiochemica l level. In Mucosphaerella musicola and M. fijien- sis, inadditio n tochromosoma l factors,on e ormor e extrachromosomal

48 factors appear tob e involved inresistanc e tobenzimidazole s (Stover, 1977). This conclusion isbase d onth eobservatio n thaton e type ofre ­ sistance could notb e found inascospor eprogeny ,bu tn oresearc ho nth e nature ofextranuclea r determinants hasbee ndone .Fo r other fungi,gene s forbenzimidazol e resistance haveno tbee n identified,bu tbiochemica l mechanismsothe r thanth eon ementione d for A. nidulans havebee nsug ­ gested. Impairment of anactiv e transport system forcarbendazi mha sbee n considered responsible forresistanc e tothi s fungicide in Sporobolomyces roseus (Nachmias& Barash , 1976), while in Verticillium malthousei beno- myl resistance appeared tob e related toth e ability tolowe r thep Ho f themediu mb y theproductio n of anunidentifie d acid (Lambert& Wuest , 1976). Another instance inwhic h agen e forresistanc e hasbee n identified and its action atth ebiochemica l levelha sbecom e known isth e oxr-1gen e forresistanc e tooxathii n carboxanilides in U. maydis. These fungicides inhibitspecificall y the succinate-CoQoxidoreductas e system infungi , bacteria and animal tissues. In U. maydis, twomutation s atth e oxr-1 locusgiv e two levels ofresistanc e ofthi smitochondria l system (Com­ plex II)t ocarboxin .The yca n alsorecogniz e other carboxanilide struc­ tures,whic h arepositivel y ornegativel y correlated tocarboxin ,depen ­ ding onth emutatio n (White et al., 1978). Apparently, thegen e codes for a component ofth eenzym e complex,an dwhe n itmutate s the component's affinity fordifferen t carboxanilide structuresma y orma yno tb e altered. However thecomplexit y ofth e system hasmad e itdifficul t to identify thecomponen t thatinteract swit h the inhibitor and, therefore,ma yb e controlled by the resistance gene.Recen tresearc h (Ramsay et al., 1981) indicates thatth ebindin g site inComple x IIobtaine d from animalmito ­ chondria is formedb y two smallpeptides ,C,,_ _ andC. , ..I fthi s istru e also in V. maydis, gene actionmus t involvepeptid e components.Chromo ­ somal genes forresistanc e tocarboxamide shav e alsobee nrecognise d in A. nidulans and theyhav ebee n found to affectth emitochondria l Com­ plexII . Lancashire &Griffith s (1971)isolate d anumbe r of Saccharomyces cere- visiae mutants resistantt otrialky l andtriphenylti n derivatives,th e lattero fwhic h are important inagriculture . Inal lo fthes emutant sre ­ sistancewa s inherited ina non-Mendelia n fashion,indicatin g extrachro- mosalcontrol .Th etrisubstitute d derivatives ofti nbin d to aspecifi c site inmitochondria , inhibiting oxidativephosphorylation , and inon e class ofth e S. cerevisiae resistantmutant s amitochondria l DNA mutation appears tob e responsible fora chang e inth ebindin g site.Th e chemical nature ofth e sitewa sno tdetermined . Another A. nidulans genewhos e actionha sbee nexamine d isth e ima B gene, oneo fsevera l thatcontro l sensitivity,t oth e ergosterol-biosythe- sis-inhibiting fungicides.Thi s gene appears tocontro l theefficienc y of

49 anenergy-dependen t system forefflu x of fenarimol.Efficienc y ishighe r inth emutant ,an d this apparently results ina lowe r fungicideconcentra ­ tion atth e site.Not e thatth e single ima Bmutan t and a recombinant carrying twogene s forresistanc e (ima A and ima B)appeare d tobehav e similarly for fenarimol efflux activity (deWaar d& va nNistelrooy , 1980). The recombinantha sbee ncompare d toth ewild-typ e strain ina re ­ centstud ywit h imazalil, another ergosterol-biosynthesis inhibitor (Siegel& Solei, 1981). This study supports,i npart ,th ehypothesi s that resistance to imazalil isals obase d onreduce duptak eo fth efungicide . Withresistanc e toman y other agricultural fungicides,knowledg e on either thebiochemica lmechanis m orth egeneti c control isincomplet e or totally lacking. Fivegene s forresistanc e to aromatic-hydrocarbon fun­ gicides (quintozene,biphenyl ,hexachlorobenzene , dicloran, chloroneb) have heenrecognise d in N. haematococca (Georgopoulos &Panopoulos , 1966); twohav ebee nrecognise d in A. nidulans (Threlfall, 1968). The same genesmus tcontro l resistance todicarboximides ,whic h are classified with the aromatic hydrocarbon group onth ebasi s ofcross-resistanc e and the ability to increase the frequency ofmitoti c segregation (Georgopou­ lose t al., 1979). Practically no conclusive evidence ongen e actioni s available.Differen tresistanc e mechanisms havebee n suggested (cf. Georgopoulos, 1977). Similarly, themechanis m ofdodin e resistance isno t known, althoughth egeneti c control inbot h N. haematococca (Kappas & Georgopoulos,1970 )an d V. inaequalis (Polach, 1973)ha sbee nclarified . Atth eothe rextreme ,w ehav e asatisfactor y knowledgeo fth ebioche ­ micalmechanis m ofresistanc e topolyoxin s in Alternaria kikuchiana, but without information onth egeneti c determinants involved.Th euptak e of polyoxin inth ewild-typ e straino fthi s fungus ishig h and isantago ­ nizedb yth edipeptid e glycylglycine (Horie t al., 1977). Incontrast ,re ­ sistantstrain s cantak eu p equally small amounts ofth e antibiotic in thepresenc e and absence ofdipeptides . Inal lcase s resistance isasso ­ ciatedwit h aver y ineffective system fordipeptid euptake . Itappear s that apeptid epermeas emus tb e coded forb y agen e forpolyoxi nresis ­ tance,bu tsuc h agen eha sno tbee n recognised. Similarly, genes forre ­ sistance topyrazopho s areno tknown ,bu t agoo dunderstandin g ofa re ­ sistancemechanis m in Pyricularia oryzae hasbee nobtaine d (deWaar d & vanNistelrooy , 1980). Thismechanis m involves alac ko f ability tocon ­ vertpyrazopho s intoth etoxi cmetabolit eP P (2-hydroxy-5-methyl-6-etho- xycarbonylpyrazolo(l,5-a)-pyrimidine).Apparently , thedeterminan tregu ­ lating thebiosynthesi s ofth e converting enzyme isresponsibl e forpyra ­ zophosresistance . Withkasugamyci n resistance in P. oryzae, involvemento fthre echro ­ mosomal lociha s clearlybee ndemonstrate d (Tagae t al., 1978). Allkasu - gamycin-resistant ascosporeprogen ywa swithou t exception resistantt o blasticidin-S.Bot hkasugamyci n andblasticidin- S actb y inhibition of

50 theprotein-synthesizin g system in P. oryzae. Although the actiono fth e genes recently recognised hasno tbee n studied atth ebiochemica llevel , iti slikel y thatthe y are involved inalteration s ofth eprotein-synthe ­ sizing system.

References

Borck,K .& H.D .Braymer ,1974 .Th egeneti canalysi so fresistanc et obenomy li n Neurospora crassa.Journa lo fGenera lMicrobiolog y85 :51-56 . Brasier,CM . &J.N . Gibbs,1975 .MB Ctoleranc e inaggressiv ean dnon-aggressiv e isolateso fCeratocysti sulmi .Annal so fApplie dBiolog y80 :231-235 . Elliott,R.W. ,1973 .Genetic so fdru gresistance .In :E .Mihi h (Ed.):Dru gresis ­ tancean dselectivity .Academi cPress ,Ne wYork ,p .41-71 . Fincham,J.R.S. ,P.R .Da y& A .Radford ,1979 .Funga lGenetics .Fourt hEdition ,Black - wellScientifi cPublications ,Oxford ,63 6p . Georgopoulos,S.G. , 1976.Th egenetic san dbiochemistr yo fresistanc et ochemical s inplan tpathogens .Proceeding so fth eAmerica nPhytopathologica lSociet y3 :53-60 . Georgopoulos,S.G. , 1977.Developmen to ffunga lresistanc et ofungicides .In :M.R . Siegel& H.D .Sisle r (Eds):Antifunga l compounds.Vol .2 ,Marce lDekker ,Inc. , NewYork ,p .439-495 . Georgopoulos,S.G .& N.J .Panopoulos ,1966 .Th erelativ emutabilit yo fth ecn bloc i inHypomyces .Canadia nJourna lo fGenetic san dCytolog y8 :347-349 . Georgopoulos,S.G. ,M .Sarri s& B.N .Ziogas ,1979 .Mitoti c instability inAspergillu s nidulanscause db yth efungicide s iprodione,procymidone ,an dvinclozolin .Pesti ­ cideScienc e 10:389-392 . Guerineau,M. ,P.P .Slonimsk i& P.R .Avner ,1974 .Yeas tepisome :oligomyci nresis ­ tanceassociate dwit ha smal lcovalentl yclose dnon-mitochondria lcircula rDNA . Biochemicalan dBiophysica lResearc hCommunication s 61:462-469 . Hori,M. ,K .Kakik i& T .Misato ,1977 .Antagonisti c effecto fdipeptide so nth e uptakeo fpolyoxi nA b yAlternari a kikuchiana.Journa lo fPesticid eScienc e 2:139-149 . Kappas,A .& S.G .Georgopoulos ,1970 .Geneti canalysi so fdodin eresistanc ei n Nectriahaematococca .Genetic s66 :617-622 . Lambert,D.H .& P.J .Wuest ,1975 .Aci dproduction ,a possibl ebasi sfo rbenomy l tolerance inVerticilliu mmalthouse iWare .Phytopatholog y65 :637-638 . Lancashire,W.E .& D.E .Griffiths ,1971 .Biocid eresistanc e inyeast :isolatio nan d generalpropertie so ftrialkylti nresistan tmutants .Federatio no fEuropea nBio ­ chemicalSocietie sLetter s 17:209-214 . Nachmias,A .& I .Barash ,1976 .Decrease dpermeabilit y asa mechanis mo fresistanc e tomethy lbenzimidazol-2-y l carbamate (MBC)i nSporobolomyce sroseus .Journa lo f GeneralMicrobiolog y 94:167-172 . Polach,F.J. ,1973 .Geneti ccontro lo fdodin etoleranc ei nVenturi ainaequalis .Phyto ­ pathology63 :1189-1190 . Ramsay,R.R. ,B.A.C .Ackrell ,C.J .Coles ,T.P .Singer ,G.A .Whit e& G.D .Thorn ,1981 . Reactionsit eo fcarboxanilide s ando fthenoyltrifluoroaceton e inComple xII . Proceedingso fth eNationa lAcadem yo fScienc eo fth eU.S.A . 78:825-828 . Shabi,E .& Y .Ben-Yephet ,1976 .Toleranc eo fVenturi apirin at obenzimidazol efun ­ gicides.Plan tDiseas eReporte r60 :451-454 . Sheir-Neiss,G. ,M.H .La i& N.R .Morris ,1978 .Identificatio no fa gen efo rß-tubuli n inAspergillu snidulans .Cel l15 :639-647 . Siegel,M.R .& S .Solel ,1981 .Effect so fimazali lo na wil d typean dfungicid e resistantstrai no fAspergillu snidulans .Pesticid eBiochemistr yan dPhysiolog y 15:222-233 . Stover,R.H. ,1977 .A non-virulen tbenomy ltoleran tCercospor a fromlea fspot scause d byMycosphaerell a fijiensisvar .diformi san dM .musicola .Transaction so fth e BritishMycologica lSociet y69 :500-502 . Taga,M. ,H .Nakagawa ,M .Tsum a& A .Ueyama ,1978 .Identificatio no fthre ediffe ­ rentloc icontrollin gkasugamyci nresistanc ei nPyriculari a oryzae.Phytopatho ­ logy69 :463-466 . Threlfall,R.J. ,1968 .Th egenetic san dbiochemistr yo fmutant so fAspergillu s nidulans resistantt ochlorinate d nitrobenzenesiJourna lo fGenera lMicrobiolog y 52: 35-44.

51 Waard,M.A .d e& J.G.M ,va nNistelrooy ,1980 .A nenergy-dependen tefflu xmechanis m forfenarimo l ina wild-typ e strainan dfenarimol-resistan tmutant so fAspergil ­ lusnidulans .Pesticid eBiochemistr y andPhysiolog y 13:255-266 . White,G.A. ,G.D .Thor n& S.G .Georgopoulos ,1978 .Oxathii ncarboxamide shighl y activeagains tcarboxin-resistan t succinicdehydrogenas e complexes fromcarboxin - selectedmutant so fUstilag omaydi san dAspergillu snidulans .Pesticid eBio ­ chemistryan dPhysiolog y9:165-182 .

52 Cross-resistance

S.G.Georgopoulo s Laboratory ofPhytopathology ,Athen s College ofAgricultura l Sciences, Votanikos,Athens, Greec e

Abstract

The scientific andpractica lvalu e ofcross-resistanc e tests isout ­ lined.Example s aregive no ftoxicant s that areuncorrelated , positively correlated andnegativel y correlated,wit hemphasi s ongroup s offungici ­ dalcompound s thatar e important inagriculture .

Keywords:positiv e correlation,negativ e correlation, collateralsensi ­ tivity,recepto r site,synergisti c action.

Introduction

When afungicid e failsbecaus e ofth edevelopmen to fresistanc eb yth e targetorganism , inpractic e iti sver y importantt okno wwhethe r theef ­ fectiveness ofothe r fungicides hasbee n affected.Th e recognition of cross-resistance relationshipsma y alsob eusefu l inothe rways . Inmod e ofactio n studies,fo rexample ,cross-resistanc e testswit h inhibitors of knownmechanis m of activityma yprovid e aver y good indicationo fth e cellularprocesse s affectedb y afungicide .Fo rexample ,th erecen tob ­ servation that fentin-resistant strains of Cercospora beticola areals o resistant tooligomyci n (Chrysayi-Tokousbalides &Giannopolitis , 1981) strongly suggests thati nthi sorganis m fentinderivative s actb y inhi­ bitiono foxidativ ephosphorylation . Ifa mutatio n affects sensitivity toon e toxicantan dno tt o another thetw o Compounds are said tob euncorrelate d interm s ofcross-resis ­ tance.A positiv e correlation existswhe nth emutan t isles s sensitiveo r lessresistan t tobot htoxicant s thanth ewild-typ e strain. Ifth emutan t ismor e resistant thanth ewil d typet oth eon echemica l andmor esensi ­ tive thanth ewil d typet o theothe ron e speaks ofnegativel y correlated cross-resistance. Inmos to fth ebacteriologica l and themedica l litera­ tureth eter m collateral sensitivity ispreferre d todescrib e negative correlation.Th eter m cross-resistance isthe nuse d only incase s ofpos -

53 itive correlation.Whateve r thepreference , the terms should notb e mis­ used.A strain, forexample ,ma yb e resistant totw odifferen t fungicides because oftw o independentmutations .The nth e fungicides areno tcorre ­ lated. Cross-resistance cannotb e assumedwithou t evidence that sensitivity tobot h chemicals iscontrolle d by the same gene.Th ebes twa y toprov e this ist ocros s aresistan twit h asensitiv e strain and showtha tal l progeny resistant toth eon e fungicide are also resistantt o theother . Ifthi s isno teas y todo ,on eca ntes tsevera l independently isolated strains resistant toth eon e toxicant forsensitivit y to theother . Oftena positiv e correlation canrathe r safelyb epredicte d onth e basis ofchemica l similaritiesbetwee ntw o compounds.Ther e are,however , somever y interesting exceptions toth erule .No tonl yma y thereb eposi ­ tivecorrelatio nbetwee n compounds thatar echemicall y unrelated,bu tal ­ socompound s chemically quite similarma yb e founduncorrelate d oreve n negatively correlated. This isno t aspeculia r asi tma y firstsound .T o remain functional,a recepto r sitecanno tchang ever y dramatically.A smallmodificatio n ofthi s sitema y reduce affinity fora toxicant ,but , depending onth emodification , iti sconceivabl e thataffinit y fora structural analoguema yno tb e affected,o rma yb eeve nincreased .

Aromatic-hydrocarbon group

This group includes some substituted aromatic hydrocarbons sucha s hexachlorobenzene, tecnazene,dicloran ,bipheny l and chloroneb. Insever ­ al fungi,mutant s obtainedb y selection onon eo fthes e fungicides are resistant toal lmember s ofth e group. (Georgopoulos & Zaracovitis,1967 ; Kuiper, 1967;Tillma n& Sisler, 1973).N o exceptions havebee n reported. Withmos to fthes ecompound s thedegre e ofresistanc e appears tob ever y high,becaus e iti spracticall y impossible to achieve aconcentratio nin ­ hibitory toth eresistan tmutants .Bu tpositivel y correlated fungicides actsynergistically , and resistantmutant s areeasil y inhibited by the concurrentus eo ftw oo rmor emember s ofth e group atlo wconcentrations . Recently, apositiv e correlationbetwee n aromatic-hydrocarbon fungi­ cides andth edicarboximide s (vinclozolin,procymidone , iprodione)ha s beenrecognise d (Lerouxe t al., 1977;Sztejnber g &Jones , 1978). Onth e basis ofthei rchemica l structures (p.102), thispositiv e correlation wouldb erathe runexpected .Howeve rther emus texis tsimilaritie s inth e mechanism ofactio nbetwee n thedicarboximide s and the aromatichydrocar ­ bonsbecause ,i nadditio nt ocross-resistance , themember s ofbot h groups have incommo n the ability to increase the frequency ofmitoti c segrega­ tion (sectoring)i ndiploi d strains of Aspergillus nidulans (Georgopoulos et al., 1979). The actiono fgene s forresistanc e toth e aromatic hydrocarbons has

54 notbee n adequately explained. Iti sno teas y therefore,t o suggesta n explanation ast owh y sensitivity tobot h the aromatic hydrocarbons and thedicarboximide s shouldb e decreasedb y the samemutation .A highl y sensitive sitewithi n the fungal nucleus appears tob e common tobot h groups of fungicides (Georgopoulos et al., 1979). However, iti sno t knownwhic h component inth enucleu s interactswit h thetoxican t and whethermutan t andwild-typ e cells differ intha tcomponent .A nalterna ­ tiveexplanatio n hasbee n suggestedb yTillma n& Sisle r (1973), whoin ­ dicated thatth emutation s may affectpartitionin g ofever y one ofth e fungicides involved atinterfaces .

Benzimidazoles

Insevera l fungi studiedb yva nTuy l (1977)a positiv e correlationbe ­ tweenth ecarbendazi m generators andthiabendazol e appears tob e the rule.However , alo wpercentag e ofmutant s of A. nidulans and A. niger, obtained onbenomy lmedium , areno tles s sensitive tothiabendazol e than thewil d type,an d somewhathighe rpercentage s ofthos e isolated onthia ­ bendazole aremor e sensitive tobenomy l (andt ocarbendazim )tha nth e wild type.Th emechanis m responsible forth eexistin g correlations is verywel l understood since sensitivity differences inviv o are reflected indifference s inth e affinity oftubuli n forth e fungicides (Davidse & Flach, 1977; 1978). With somebenzimidazole-resistan t A. nidulans mutants thatexhibi t temperature sensitivity, there isa positiv e correlationbetwee n these fungicides andp-fluorophenylalanin e (Morris& Oakley , 1979). Thiscor ­ relation appears tob e the onlypiec e of information available tou ss o faro nth emechanis m bywhic hp-fluorophenylalanin e cause non-disjunction ofchromosomes .Griseofulvin ,whic h also inhibits fungalmitosis ,wa sno t foundcorrelate d to thebenzimidazole s forcross-resistanc e (Kappas & Georgopoulos, 1974). SomeN-phenyl-carbamat eherbicide s areknow n anti­ mitotic agents.A recentstud y ofLerou x& Gred t (1979)ha s showntha t withsom emutant so f Botrytis cinerea and Pénicillium expansum theseher ­ bicides arenegativel y correlated toth ebenzimidazoles . In Cercospora beticola, however,n ocorrelatio n hasbee nobserve d (Georgopoulos,unpu ­ blished data).

Carboxamides

This group isprobabl yuniqu e among the fungicidesbecaus e ofth e greatvariet y ofcross-resistanc e relationships existing among itsmem ­ bers. In Ustilago maydis considerable resistance tocarboxi n results from mutations atth e oxr-1locu s (Georgopoulos & Ziogas,1977) . Although strains carrying amutatio n atthi s locus aremor e or lessresistan tt o

55 many carboxin analogues,includin g oxycarboxin andpyracarbolid , somein ­ teresting exceptions canver y easilyb e found (Whitee t al., 1978). The sensitivities ofth e succinic dehydrogenase complex, theenzym e system inhibitedb y thecarboxamides , aregive n forselecte d carboxamides inTabl e 1.Fro m Table 1,th emoderatel y resistant strains,carryin g the oxr-lAmutation , are 14-times less sensitive tocarboxi n thanth ewil d type.Th e samemutatio ndoe sno t affect sensitivity to4'-propyl-carboxi n (lacko fcorrelation )bu t increases sensitivity to4'-phenyl-carboxi n 25 times (negative correlation). For thehighl y resistantoxr-l Bmutants , the resistance factor is10 0wit h carboxin, almost30 0wit h3'-methylcar - boxin, closet o 3wit h3'-decyloxycarboxin , atleas t 16wit h4'-phenyl - carboxin, andbelo w 0.07 with athiophen e carboxamide, the4- n butylana- logueo f3-methylthiophene-2-carboxanilid e (White& Thorn , 1980). Similar effects ofmodification s ofth ecarboxi nmolecul e on sensitiv­ itywer e alsoobserve d for A. nidulans (White et al., 1978). With all the structural analogues available,i ti sconceivabl e tob e able toover ­ come anytyp eo fmutatio n thatdecrease s the sensitivity of afungu st o carboxin.Th e explanation thatca nb eoffere d istha tth emutation s af­ fectth ecarboxin-bindin g site,probabl y apeptide ,i nth e succinic dehy­ drogenase complex.An y suchmodificatio n ofth e site,whic hwil l decrease affinity formos tcarboxamid e structures,ma yno t affecto reve ndecreas e affinity for someothers . Itmus tb e added thati n U. maydis the ants mu­ tants,whic hhav e lowresistanc e tocarboxin , areal lhighl y sensitive to the inhibitors ofth ecytochrom e system (Georgopoulos & Sisler, 1970). Thuswhil e inth e case ofth ebenzimidazole s no compound isknow nt o sê-

Table1 .Sensitivit yo fsuccini cdehydrogenas efro mwild-typ e andcarboxin-resistan tmutan tstrain so fUstilag omaydi st o selectedcarboxamides .

Carboxamides ( M) :50 M wildtyp e oxr-lA oxr-lB

Carboxin 0.36 5.0 37.0

3'-methylcarboxin 0.075 1.7 20.0

3'-decyloxycarboxin 0.017 - 0.058

4'-propylcarboxin 0.16 0;24 15.0

4'-phenylcarboxin 4.8 0.22 >75. 0

3'-methylthiophene- 2-carboxanilide 7.2 45.0 38.0

4-nbutylanalogu eo f 3'-methylthiophene- 2-carboxanilide >100. 0 12.0 7.0

56 lectively inhibitth elarg epercentag e ofmutant s thatar eresistan tt o carbendazim andthiabendazole ,th eeffec to f allo fth ecarboxamide - resistancemutation s thathav ebee n studied canb e reservedb y theus eo f anappropriat e inhibitor.

Sterol-biosunthesis inhibitors

Fungicides ofthi s group,includin g triforine, fenarimol,nuarimol , triadimefon, imazalil,tridemorph ,bitertanol ,an dprochloraz ,ar emostl y positively correlated forcross-resistanc e (Sherald et al., 1973;Fuch s et al., 1977;Baru g& Kerkenaar , 1979). Although the group comprisesdi ­ verse chemical structures,th e similarity inth emechanis m of actioni s sufficient toexplai nwh y amutatio n should affectsensitivit y to allo f these inhibitors,i fresistanc ewa s the resulto f sensitive sitemodifi ­ cation.However ,thi s doesno t appear tob e thecase .Th e sterol patterns of sensitive and resistant strains of A. nidulans dono tdiffe rsignifi ­ cantly (Ragsdale& d eWaard , 1977), indicating thatth estero lsynthe ­ sizing system isno t affectedb y the resistancemutations .Th edat a so far available indicate thatth emechanis m ofresistanc e isrelate d tomem ­ brane transport (deWaar d& va nNistelrooy , 1980;Siege l& Solei, 1981). Whether suchdivers e chemical structures havecommo ntranspor t systems is notknown . Iti speculia r toth eergosterol-biosynthesi s inhibitors that although within thegrou p cross-resistance isno talway s reciprocal (Barug & Kerkenaar, 1979), some ofth emutant s selectedo nsuc hinhibitor s showa n altered sensitivity toquit eunrelate d toxicants.Th e ima Bmutant so f A. nidulans, selected onimazalil ,ar eresistan tt ochloramphenico l and hypersensitive tocycloheximide , acriflavin andneomyci n (vanTuyl , 1977). Someothe rmutants ,selecte d onbitertanol ,ar emor e sensitive to carboxin thanth ewil dtyp e (Georgopoulos,unpublishe d data). Thus, someo fth e mutations recognised by theus eo f ergosterol-biosynthesis-inhibiting fungicides appear tob epleiotropic .Bu t iti s far fromunderstoo d how thosemutation s cause suchpeculia r cross-resistancepatterns .

Organophosphates

Resistance of Puricularia oryzae tophosphorothiolates , e.g. 'KitazinP' ,i salway s accompaniedb y anincrease d sensitivity tophos - phoramidates,an dvic eversa .Th e twotype s of fungicides actsynergisti - callyo nwil d typebu tno to nphosphorothiolate-resistan t strains ofth e fungus (Uesugi et al., 1974). Itha sbee n showntha twild-typ e strains arecapabl e ofrapi dmetabolis m ofphosphoramidates ,bu tthi smetabolis m isinhibite db yphosphorothiolates .Mutant s selected forresistanc e to thephosphorothiolate s arecapabl e ofmuc h slowermetabolis m ofth ephos -

57 phoramidates (Uesugi& Sisler, 1978). This appears tob e a satisfactory explanation forth enegativ e correlation betweenphosphorothiolate s and phosphoramidates.

References

Barug,D .& A .Kerkenaar ,1979 .Cross-resistanc e ofUV-induce dmutant so fUstilag o maydist ovariou s fungicideswhic hinterfer ewit hergostero lbiosynthesis .Medede ­ lingenFacultei tRijksuniversitei tGen t4 4 (1): 421-427. Chrysayi-Tokousbalides,M .& C.N .Giannopolitis ,1981 .Cross-resistanc e inCercospor a beticola totriphenylti nan doligoraycin .Plan tDiseas e65 :267-268 . Davidse,L.C .& W .Flach ,1977 .Differentia lbindin go fmethy lbenzimidazole-2-y l carbamatet ofunga ltubuli na sa mechanis m ofresistand et othi santimitoti c agenti nmutan tstrain so fAspergillu snidulans .Journa lo fCel lBiolog y 72:174-193 . Davidse,L.C .& W .Flach ,1978 .Interactio no fthiabendazol ewit hfunga ltubulin . BiochimicaBiophysic aAct a543 :82-90 . Fuchs,A. ,S.P .d eRuig ,J.M .va nTuy l& F.W . deVries ,1977 .Resistanc et otrifo - rine:a nonexisten tproblem ?Netherland sJourna lo fPlan tPatholog y8 3 (Suppl.1) : 189-205. Georgopoulos,S.G. ,M .Sarri s& B.N .Ziogas ,1979 .Mitoti c instability inAspergillu s nidulans causedb yth efungicide s iprodione,procymidone ,an dvinclozolin .Pesticid e Science 10:389-392 . Georgopoulos,S.G .& H.D .Sisler ,1970 .A gen emutatio neliminatin g antimycinA toler ­ antelectro ntranspor t inUstilag omaydis .Journa lo fBacteriolog y 103:745-750 . Georgopoulos,S.G .& C .Zaracovitis ,1967 .Toleranc eo ffung it oorgani cfungicides . AnnualRevie wo fPhytopatholog y5 :109-130 . Georgopoulos,S.G .& B.N .Ziogas ,1977 .A ne wclas so fcarboxi nresistan tmutant so f Ustilagomaydis .Netherland sJourna lo fPlan tPathology ,8 3 (Suppl.1) : 235-242. Kappas,A .& S.G .Georgopoulos ,1974 .Interferenc eo fgriseofulvi nwit hth emitoti cpro ­ cessi nAspergillu snidulan sdiploids .Journa lo fBacteriolog y 199:334-335 . Kuiper,J. , 1967.Investigation so nchemica lcontro lo fcommo nbun to fwheat .Austra ­ lianJourna lo fExperimenta lAgricultur ean dAnima lHusbandr y 7:275-282 . Leroux,P. ,R .Fritz ,& M .Gredt ,1977 .Etude se nlaboratoir ed esouche sd eBotryti s cinereaPer srésistante sà l adichlozoline ,a udicloran ,a uquintozene ,à l avinchlo - zolinee ta u2601 9R P (ouglycophene) .Phytopathologisch eZeitschrif t89 :347-358 . Morris,N.R .& CE . Oakley,1979 .Evidenc e thatp-fluorophenylalanin eha sa direc tef ­ fecto ntubuli ni nAspergillu snidulans .Journa lo fGenera lMicrobiolog y114 : 449-454. Ragsdale,N.N .& M.A .d eWaard ,1977 .Stero lpattern si nfenarimol-susceptibl ean d tolerantstrain so fAspergillu snidulans .Proceeding so fth eAmerica nPhytopatho - logicalSociet y4 :21 8 (abstract). Sherald,J.L. ,N.N .Ragsdale ,& H.D .Sisler ,1973 .Similaritie sbetwee nth esystemi c fungicidestriforin ean dtriarimol .Pesticid eScienc e4 :719-727 . Siegel,M.R .& S .Solel ,1981 .Effect so fimazali lo na wil dtyp ean dfungicid eresis ­ tantstrai no fAspergillu snidulans .Pesticid eBiochemistr yan dPhysiolog y15 : 222-233. Sztejnberg,A .& A.L .Jones ,1978 .Toleranc eo fth ebrow nro tfungu sMonilini afructi - colat oiprodione ,vinchlozolin ,an dprocymidon efungicides .Phytopatholog yNew s12 : 187-188. Tillman,R.W .& H.D .Sisler ,1973 .Effec to fchlorone bo nth emetabolis man dgrowt ho f Ustilagomaydis .Phytopatholog y63 :219-225 . Tuyl,J.M .van ,1977 .Genetic so ffunga lresistanc et osystemi c fungicides.Mededelin ­ genLandbou wHogeschoo lWageninge n 77-2,13 7p . Uesugi,ï. ,M .Katagir i& 0 .Noda ,1974 .Negativel y correlated crossresistanc ean d synergismbetwee nphosphoramidate s andphosphorothiolate s inthei rfungicida lactio n onric eblas tfungi .Agricultura lan dBiologica lChemistr y38 :907-912 . Uesugi,Y .& H.D .Sisler ,1978 .Metabolis mo fa phosphoramidat eb yPyriculari aoryza e inrelatio n totoleranc ean d synergismb ya phosphorothiolat e andisoprothiolane . PesticideBiochemistr yan dPhysiolog y 9:247-254 . Waard,M.A .d ean dJ.G.M ,va nNistelrooy ,1980 .A nenergy-dependen tefflu xmechanis mfo r fenarimoli na wild-typ e strainan dfenarimol-resistan tmutan to fAspergillu snidulans . PesticideBiochemistr yan dPhysiolog y 13:255-266 .

58 White,G.A .& G.D .Thorn ,1980 .Thiophen ecarboxamid efungicides :Structure-activit y relationshipswit hsuccinat edehydrogenas e complex fromwild-typ ean dcarboxin - resistantmutan tstrain so fUstilag omaydis .Pesticid eBiochemistr yan dPhysio ­ logy 14:26-40 . White,G.A. ,G.D .Thor n& S.G . Georgopoulos,1978 .Oxathii ncarboxamide shighl y activeagains t carboxin-restistant succinicdehydrogenas e complexes fromcarboxin - selectedmutant so fUstilag omaydi san dAspergillu snidulans .Pesticid eBiochemistr y andPhysiolog y9 :165-182 .

59 Benzimidazole compounds:selectivit y and resistance

L.C.Davids e Laboratory ofPhytopathology ,Agricultura l University,Wageningen , The Netherlands

Abstract

Benzimidazole compounds inhibitmicrotubul e assembly, leading toin ­ terference with agrea tnumbe r ofprocesse s inwhic hmicrotubule s arein ­ volved, likenuclea r and cellular division,cel lmigratio n and organelle movement.Sinc emicrotubule s play arol e inmaintainin g cell shape and structure,inhibitio n ofthei r formation also affects the intracellular organization.Affinit y oftubulin , the sub-unito fmicrotubules ,t oa particular benzimidazole compound isprobabl y themos t important factor thatdetermine s the activity ina certai norganism .Modificatio n ofth e basic structurerequire d foractivit y createscompound swit h ahig hde ­ greeo f selectivity,whic h canb euse d inth e therapy of anumbe r ofdis ­ eases ofplant s and animals.Bu tth ehighl y specific interactionbetwee n tubulin andbenzimidazol e compounds isvulnerabl e toan y structuralal ­ terationo fth ebindin g site.A mutatio n that lowers thebindin g affinity oftubuli nt o abenzimidazol e compound without anyeffec to nnorma ltubu ­ lin functioning creates aresistan t strain.Thi s iswha tprobabl y hashap ­ pened alreadyman y times inth ehistor y ofbenomyl' suse . In fundamental cell research,benzimidazol e compounds arebecomin g increasingly important asvaluabl e tools inth e study ofth e structure and role ofth emicrotu - bular system,whos e originwa s one ofth ebasi cmolecula r innovations in the evolution ofprokaryote s toeukaryotes .

Keywords:benzimidazoles ,thiophanates ,benomyl ,carbendazim ,fenbendazo - le, fuberidazole,mebendazole ,nocodazole ,parbendazole ,thiabendazole , Aspergillus nidulans, Pénicillium expansum, tubulin,microtubules ,anthel ­ mintic.

Introduction

Ofth e systemic fungicides,th ebenzimidazol e compounds andthiopha ­ nates areundoubtedl y themos twidel y known,owin g tothei r excellentsys -

60 temicproperties ,thei r great efficacy incontrollin g importantplan tdi ­ seases,but ,unfortunately , also toth edisappointmen t andproblem s they evokewhe nresistanc e develops intarge t fungi.Th egenera lbiocida lprop ­ erties ofth ebenzimidazol ecompounds ,th ecurren tknowledg e ofthei r mode of action and thebiochemica l basis ofthei r selectivity andth ere ­ sistancewil lb e reviewed inthi scontribution .

Biocidal properties of benzimidazole compounds

Atpresen tvariou sbenzimidazol e compounds arebein guse d inth e the­ rapyo fa numbe r ofdisease s ofplants ,animal s andman .Th e firstcom ­ pound developed wasthiabendazole ,whic hwa s introduced in196 1a sa nan ­ tihelminthic.Late r several otherbiocida lbenzimidazol e compounds came intouse .Benomy l (Delp& Klöpping , 1968), carbendazimo rMB C (Hampel & Löcher, 1973), fuberidazole (Schumann, 1968)an d also thiabendazole (Staron& Allard , 1964)ar ewel l known assystemi c fungicides.Thiopha - nates, introduced in197 1 (Aelbers,1971 )ar eofte nplace d inthi sclas s of fungicides,sinc eunde rnatura l conditions they areconverte d tobenzi ­ midazole compounds (Sellinge t al., 1970). Fenbendazole,mebendazole ,oxi - bendazole andparbendazol e have anthelminthic properties and areuse d in veterinary medicine.Nocodazol e hasbee n described asbein g active against tumors inmammals ,includin gman .Structura l formulaeo fthes e compounds aregive n inFigur e 1.

0 0 II II jp NH-C-O-CH3 C-NH-CH2-CH2-CH2-CH3 N 0 N. R=H carbendazim Y-NH-C-OCH3 -N

R= C4H9 parbendazole benomyl

0 5 nocodazole *) N u—s 0 II R=-C mebendazole thiabendazole

= —S—v v fenbendazole CCvù fuberidazole

Figure 1. Structural formulae of biocidal benzimidazole compounds.

61 Development of resistance to these compounds is frequently encountered in crop protection, as well as in veterinary medicine. In a recent paper, Delp (1980) mentions that up to 1979 resistance to benzimidazole com­ pounds has been found in fungi belonging to 16 different genera. Accord­ ing to Webb et al. (1978, 1979), thiabendazole-resistant strains of Hae- monchus contortus, a sheep parasite, are present on over 50% of the farms in the Northern Tablelands of New South Wales, Australia.

Node of action

Optical and electron microscope studies have contributed considerably to the understanding of the mode of action of benzimidazole compounds. Benomyl and its conversion product carbendazim have been shown to inter­ fere with mitosis in fungi (Davidse, 1973; Hammerschlag & Sisler, 1973; Kunkel &Hadrich , 1977; Richmond & Phillips, 1975). Borck (1973) and Howard &Ais t (1977, 1980) described the disorganization of the fine structure of fungal hyphae induced by benomyl and carbendazim. In partic­ ular, the dislocation of the Spitzenkörper, which is presumed to function in hyphal linear elongation, was noticed in both studies. Mitosis in plants (Boyle, 1973; Marco Moll et al., 1973; Richmond & Phillips, 1975) and mammalian cells in vivo (Styles & Garner, 1974; Seiler, 1976) and in vitro (Styles & Garner, 1974; de Brabander et al., 1976b) also appears to be affected by carbendazim. In all these cases interference of carbenda­ zim with the formation or the functioning of microtubules is thought to have been responsible for the observed effects. Biochemical studies support the idea of an anti-microtubule mode of action of benzimidazole compounds. A carbendazim-binding protein was de­ monstrated in cell-free mycelial extracts of Aspergillus nidulans (Davidse & Flach, 1977) with biochemical properties characteristic for tubulin, the building block of microtubules. These properties are summarized in Table 1. Binding of colchicine, the classical inhibitor of mitosis in

Table 1. Biochemical properties of the carbendazim-binding protein in fungi. High affinity binding of carbendazim, nocodazole and thiabendazole at the same site. Binding is reversible and fast at 4 °C. Low affinity binding of colchicine at the carbendazim-binding site. Optimum pH for binding: 6.6 - 6.9. Loss of binding activity in a first order manner with t of 6.5 h. Stabilization of the binding activity by carbendazim, glycerol and sucrose. Molecular weight of 110 000. Purification by a standard procedure for mammalian brain tubulin: ammonium- sulphate fractionation (35-50 %) and DEAE -ion-exchange chromatography. a. DEAE= diethylaminoethyl.

62 animal and plant cells, to the carbendazim-binding protein unequivocally showed it to be identical with tubulin, because this compound specifical­ ly binds to tubulin and in this way prevents its assembly into microtu­ bules. A review on this subject has been written by Wilson & Bryan (1974). The carbendazim-binding protein also has a high affinity for nocodazo- le, comparable to that of mammalian tubulin (Hoebeke et al., 1976), which binds nocodazole at the colchicine-binding site. Experiments (Davidse & Flach, 1978) have shown that also thiabendazole is bound to fungal tubu­ lin at the carbendazim-binding site. A direct determination of the disso­ ciation constant of the thiabendazole-protein complex, proved to be dif­ ficult, however, because of unspecific adsorption of the labelled com­ pound to other proteins. Therefore this value was estimated by measuring the inhibition constant for the ability of thiabendazole to inhibit bin­ ding of radio-labelled carbendazim. The value obtained is the concentra­ tion of thiabendazole that would saturate half of the carbendazim-binding sites and it is therefore a reliable estimate of the dissociation con­ stant. Values obtained for thiabendazole and carbendazim in binding expe­ riments with cell-free extracts of Pénicillium expansum are given in Ta­ ble 2. Pénicillium expansum was used since it is more sensitive to thia­ bendazole than Aspergillus nidulans. Upon comparison of the dissociation constants with the ED,-0 values, it appears that the biochemical basis of the antifungal activity of thiabendazole is similar to that of carbenda­ zim. Binding studies with the anthelminthics fenbendazole, mebendazole, oxibendazole and parbendazole revealed that these drugs were bound to mammalian brain tubulin (Friedman & Platzer, 1978; Laclette et al., 1980). Binding of mebendazole and fenbendazole to nematode tubulin has recently been described by Friedman & Platzer (1980). The biochemical studies described establish that the effects of benzi- midazole compounds on cellular processes are directly due to interference with microtubule assembly.

Table 2. Sensitivity to carbendazim and thiabendazole, and the dissociation constants of the thiabendazole- and carbendazim-tubulin complex of a wild- type strain of Pénicillium expansum. ED.. (radial growth Dissociation constant on agar) (|JM) (uM) carbendazim 0.4 0.9 ± 0.21 (3) thiabendazole 7 34 ± 15.4 (4) a. The dissociation constant of the thiabendazole-tubulin complex was measured as the inhibition constant for the ability of thiabendazole to inhibit carbendazim-binding (Davidse & Flach, 1978). Values are means ± standard deviation; the number of experiments is given in parentheses.

63 Selectivity and resistance

Themicrotubula r system ispresen t inal leukaryotes . Itha sbee n showntha t ahig h degree ofconservatis m exists inth e amino-acid sequences oftubulin s fromvariou s sources (Luduena& Woodward , 1975). Nevertheless,benzimidazol e compounds arerathe r selective inthei r activ­ ityt o eukaryotes.Carbendazi m ishighl y toxic tocertai ngroup s offungi , whereas others are insensitive (Bollen& Fuchs , 1972). Sensitivity tocar ­ bendazim isno trestricte d to fungi,however , since iti s alsohighl y toxic toth eearthwor m Lumbricus terrestris (Stringer &Wright , 1976), to fresh-water organisms like the alga Chlorella pyrenoidosa, toth e fish Salmo gairdneri andth e crustacean Daphnia magna (Canton, 1976).Espe ­ cially the reproductive capacity of D. magna appears tob ever y sensitive. Theembryona l development ofmite s (Delp& Klöpping ,1968 )an dplant-par ­ asitic nematodes (Rössnere t al., 1976)i sals odisturbe d atlo wdoses . Higherplant s andmammal s areno tvisibl y affectedb y carbendazim, although cytological studieshav e revealed effects atth ecellula r level (Richmond &Phillips ,1975 ;Seiler , 1976). The curative actiono fmebendazol e inanima l andhuma n helminthiases implies selectivity forhos t andparasite .Ultrastructura l studieshav e showntha tupo ntreatmen to fth ehost ,microtubule s incell s ofth epara ­ site arecompletel y destroyed,wherea s cytoplasmic and spindlemicrotubu ­ les ofth ehos tcell sremai nundisturbed , eventhoug hbot htype s ofcell s havebee nexpose d to identical concentrations ofmebendazol e (Borgers et al., 1975a, b). Experiments inviv o onth eeffec to fnocodazol e onexperimenta l and humanneoplasm s have showntha tthi s compound specifically eliminatesmi ­ crotubules individin g andnon-dividin g neoplastic cells,wherea smicro ­ tubules ofnorma l cells ininterphas e apparently remain intact.Microtu ­ bules inmitoti c normal cells,however , areseriousl y affected (deBra - bander et al., 1975). Incompariso nwit hcarbendazim , nocodazole isles s selective init s activity for fungi and animals (Table3) . Binding studieswit h carbendazim incell-fre e extracts of fungi,plant s andmammalia nbrai nhav e ledt o abette r understanding ofth ebiochemica l

Table3 . Fungalan dmammalia ntoxicit yo fcarbendazi m andnocodazole .

Aspergillusnidulan s Mice ED.. (radialgrowt ho nagar ) LILf,(acut etoxicit y intraperitoneal) (mg/1) (mg/kg)

Carbendazim 0.9 6400* Nocodazole 0.2 135 a.Source :Seile r (1975). b. Source:Atass i& Tagno n (1975).

64 Table 4. Carbendazim-binding activity in extracts of fungal, plant and animal cells. System Binding activity (dpm 14C-carbendazim/ml extract) Aspergillus nidulans 003 (wild type) 5 640 Aspergillus nidulans 186 (extra sensitive) 12 200 Aspergillus nidulans R (resistant) 320 Pénicillium brevicompactum (sensitive) 4 140 Pénicillium brevicompactum (resistant) 220 Pénicillium corymbiferum (sensitive) 2 080 Pénicillium corymbiferum (resistant) 140 Alternaria sp. 370 Pythium irreguläre 250 pea root tips 250 porcine brain 210 a. Binding was determined with a Sephadex G-100 gel filtration assay (Davidse &Flach , 1977) at 2.8 [M 14C-carbendazim (70 000 dpm/ml) with fungal and plant extracts and at 6.1 uM 14C-carbendazim (153 000 dpm/ml) with porcine brain extract. basis of the selectivity. In extracts of carbendazim-resistant fungi no or only a low binding activity appeared to be present (Table 4) (Davidse & Flach, 1977). In strains of Aspergillus nidulans, the affinity of the binding protein, identified as tubulin, for carbendazim reflected the sensitivity of each strain (Table 5). The mutation responsible for the behaviour of the strains occured in one gene (van Tuyl, 1974, 1977), which has been identified as the structural gene for ß-tubulin, one of the monomers of the tubulin molecule (Sheir-Neiss et al., 1978). Most of the strains carrying a mutation in this gene that leads to carbendazim resistance have electrophoretically abnormal ß-tubulins. A correlation between affinity of tubulin for carbendazim and sensitivity was also found with strains of Pénicillium expansum (Table 6). It was not possible to determine the genetic background, but it can be assumed that also here a mutation in one gene is involved (van Tuyl, 1977). The correlation be­ tween degree of sensitivity and the magnitude of the affinity constant in strains of Aspergillus nidulans and Pénicillium expansum and the absence of carbendazim-binding activity in cell-free extracts of naturally resis­ tant fungi (Table 4) suggests that the selectivity of carbendazim within fungi is based on a differential affinity of fungal tubulins to this ben-

Table 5. Sensitivity to carbendazim and the dissociation constant of the carbendazim-tubulin complex in strains of Aspergillus nidulans. Strain ED_„ (radial growth on agar) (MM) (MM)

003 (wildtype ) 4.5 2.2 186(extr asensitive ) 1.5 0.6 R (resistant) 95 27 a.Dissociatio nconstant swer edetermine dwit ha charcoa lbindin gassa y (Davidse& Flach ,1977) .

65 Table6 .Sensitivit y tocarbendazi man dth edissociatio n constantso fth e carbendazim-tubulin complex instrain so fPénicilliu mexpansu m.

Strain ^=50( radialgrowt ho nagar ) Dissociationconstan t (MM) (MM) S (wildtype ) 0.4 0.9 ±0.2 1 (3) SS (extrasensitive ) 0.07 0.2 ±0.0 6 (3) R (resistant) 250 0 a.Strain shav ebee nisolate d anddescribe db yva nTuy l (1977). b.Th edissociatio n constanto fth ethiabendazole-tubuli n complexwa smeasure da s theinhibitio nconstan t forth eabilit yo fthiabendazol e toinhibi tcarben - dazim-binding (Davidse& Flach ,1978) .Value sar emean s+ standar ddeviation ; thenumbe ro fexperiment s isgive ni nparentheses . zimidazolecompound . Thatn odifference s could be foundbetwee n the Asper­ gillus nidulans strains inrate s ofuptak e andmetabolis m of carbendazim (Davidse, 1976)strengthen s this supposition.Th e existence ofothe rme ­ chanisms ofresistanc e shouldno tb e entirely excluded, however.A dif ­ ferential uptakeha sbee n suggested tob e involved inth e resistance of Botrytis cinerea, Verticillium malthousei and Sporobolomyces roseus to carbendazim (Gessler, 1976;Lamber t& Wuest , 1975;Nachmia s &Barash , 1976). Also in Aspergillus nidulans, othermechanism s ofresistanc e might operate, since in addition tomutation s inth e ß-tubulingen e othermuta ­ tions thatgiv e alo wdegre e ofcarbendazim-resistanc e havebee n found in twomor e loci (vanTuyl , 1977). Incell-fre e extracts ofpe a roottip sn o carbendazim-binding activity hasbee ndemonstrate d (Table 4). However,th epresenc e ofplan t tubulin inthes eextract s hasno tbee nproven .Th e colchicine-binding assay, whichwa suse d asa diagnosti c test,gav e anegativ e result,bu tthi s mightb edu e to alo w affinity ofplan ttubuli n forcolchicin e (Burns, 1973). Tubulinha sbee n isolated fromplan ttissu e (Rubin& Cousins , 1976)b y aprocedur e not significantly different from theon ewhic hwa s used inthes eexperiments ,s oi tmigh tb e assumed thattubuli nwa s pre­ sent. Soabsenc e ofcarbendazim-bindin g activity couldmea n thatplan t tubulinha s alo w affinity tothi sbenzimidazol ecompound .Tha t lowaf ­ finity isi nagreemen twit h the observation ofRichmon d &Phillip s (1975) thatmitosi s inplan tcell swa s only affected atrelativel y high doses of carbendazim. Sinceth eplasmalemm a ofth eplan tcel l is fullypermeabl e forcarbendazi m (Peterson& Edgington , 1976), the insensitivity ofhighe r plantsmigh tb eprimaril y duet o alo w affinity ofthei rtubulin s for this compound.Th e affinity ofmammalia nbrai n tubulin forcarbendazi m is also low,sinc en obindin g couldb e detected (Table 4). The preparation showed considerable colchicine-binding activity,whic h indicates thepre ­ sence ofnativ e tubulin.Th e selectivity ofth evariou s benzimidazole compounds isals o expressed inthei r ability to inhibitmicrotubul epoly ­ merization invitro .Nocodazole , fenbendazole,mebendazole , oxibendazole

66 andparbendazol e arever ypoten t inhibitors,wherea s carbendazim andthia ­ bendazole only slightly affectthi sproces s (Freedman& Platzer ,1978 ; Hoebeke& va nNijen , 1975;Laclett e et al., 1980) (Figure 2).However , the low affinity ofmammalia n tubulin forcarbendazim ,migh tno tb esole ­ lyresponsibl e forth enon-toxicit yo fthi s compound tomammals ,sinc e carbendazim israpidl y hydroxylated toth e 5-hydroxy derivative,whic hi s excreted from the organism asa glucuronid e ora sulphat e conjugate (Gar­ dinere t al., 1974). The low affinity ofmammalia n tubulintogethe rwit h the detoxicationmechanis m mightexplai nth erelativ e insensitivity of mammals tocarbendazim . The selectivity ofth e anthelminthic benzimidazo- lesi sprobabl y alsobase d ona differentia l binding affinity between nematode tubulin andmammalia n tubulin forthes e compounds (Friedman & Platzer, 1980). Thereha sbee nn o researchdon eo nwhethe r a differential affinity isals o involved inth e resistance ofhelminth s tothiabendazole . The selectivity ofnocodazol e with respectt o itseffec t invitr o on microtubules ininterphas emalignan tcell s andnon-malignan t cells isa n interesting phenomenon. Sincemicrotubule s inmitoti c normal cells are affected iti sdoubtfu l that adifferentia l affinity ofth e tubulins of

AA 350nm 012 control

0.09-

0.06-

0.03

l 1 20 25 time(min)

Figure2 .Effec to fnocodazol ean dthiabendazol eo nmicrotubul eassembl y invitro . Porcinebrai ntubulin ,purifie db ytw ocycle so fassembl yan ddisassembl yaccordin gt o amodifie dprocedur eo fShelansk ie tal . (1973)wa sincubate da t3 7° Cwit hnocodazol eo r thiabendazole,bot ha ta concentratio no f5 0pM .Th ecompound swer eadde d indimethyl - sulfoxide (DMSO),fina lconcentratio n 10pg/ml .Th e'contro lcontaine donl yDMSO .Assembl y wasmeasure db yth eincremen ti nradian tabsorbanc e (A)a t35 0nm .

67 the different cell types is involved. It is more likely that the selec­ tivity is based on a differential biochemical stability of the micro­ tubules to the depolymerization action of nocodazole (de Brabander et al., 1975). The difference in affinity to carbendazim between mammalian and fungal tubulin is probably due to minor differences in molecular structure, as it has been shown that tubulins from both sources can copolymerize in vi­ tro (Davidse, 1975; Sheir-Neiss et al., 1976; Water & Kleinsmith, 1976). The mutations leading to resistance to carbendazim in Aspergillus nidu- lans and Pénicillium expansum, which involve changes in affinity of tubu­ lin to carbendazim, apparently do not impair the normal functioning of the microtubular system, as the growth rate or sporulation of the strains does not change. This indicates normal functioning of the microtubular system. Evidently the ability of the altered tubulin molecules to assem­ ble has not been changed. This is an example of how a protein in spite of its conservatism can be altered when the survival of the organism is threatened.

References

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69 alsGetreidebeizmittel .Nachrichtenblat tDeutsch ePflanzenschutzdienste s20 :1-5 . Seiler,J.P. , 1975.Toxicolog y andgeneti ceffect so fbenzimidazol ecompounds . MutationResearc h32 :151-168 . Seiler,J.P. , 1976.Th emutagenitic y ofbenzimidazol e andbenzimidazol ederivatives . VI. Cytogeneticeffect so fbenzimidazol ederivative s inth ebon emarro wo fth emous e andth eChines ehamster .Mutatio nResearc h40 :339-348 . Selling,H.A. ,J.W .Von k& A .Kaar sSijpesteijn ,1970 .Transformatio no fth esystemi c fungicidemethy lthiophanat e into2-benzimidazol ecarbami cacid ,methy lester . Chemistryan dIndustry :1625 . Sheir-Neiss,G. ,B.V .Nardi ,M.A .Geal t& N.R .Morris ,1976 .Tubulin-lik eprotei nfro m Aspergillusnidulans .Biochemica l andBiophysica lResearc hCommunication s69 : 285-290. Sheir-Neiss,G. ,M.H .La i& N.R .Morris ,1978 .Identificatio no fa gen efo rß-tubuli n inAspergillu snidulans .Cel l15 :639-649 . Shelanski,M.L. ,F .Gaski n& C.R .Cantor ,1973 .Microtubul eassembl yi nth eabsenc eo f addednucleotides .Proceedin go fth eNationa lAcadem yo fSciences ,U.S.A .70 : 765-768. Staron,T .& C .Allard ,1964 .Propiéte santifongique sd u2-(4'-thiazolyl) -benzimidazol e outhiabendazole .Phytiatri ee tPhytopharmaci e13 :163-168 . Stringer,A .& M.A .Wright ,1976 .Th etoxicit yo fbenomy lan dsom erelate d 2-substituted benzimidazoles inth eearthwor mLumbricu sterrestris .Pesticid eScienc e7 :459-464 . Styles,J.A .& A .Garner ,1974 .Benzimidazolecarbamat emethy leste r- evaluatio no fit s effectsi nviv oan di nvitro .Mutatio nResearc h26 :177-187 . Tuyl,J.M .van ,1977 .Genetic so ffungicid e resistance.Mededelinge nLandbouwhogeschool , Wageningen77-2 .236p . Tuyl,J.M .van ,L.C .Davids e& J .Dekker ,1974 .Lac ko fcros sresistanc et obenomy lan d thiabendazole insom estrain so fAspergillu snidulans .Netherland sJourna lo fPlan t Pathology80 :165-168 . Water,R.D .& L.J .Kleinsmith ,1976 .Identificatio no f a- andß-tubuli n inyeast . BiochemicalBiophysica lResearc hCommunication s 70:704-708 . Webb,R.F. ,A.R.B .Jackso n& C.H .McCully ,1978 .Th eefficienc yo fvariou santhelmin ­ ticsagains tfiel dpopulation so fHaemonchu s contortus resistantt othiabendazole . AustralianVeterinar yJourna l54 :501-502 . Webb,R.F. ,C.H .McCully ,F.L .Clarke ,P .Greentre e& P .Honey ,1979 .Th eincidenc eo f thiabendazole resistance infiel dpopulation so fHaemonchu scontortu so nth eNorther n Tablelandso fNe wSout hWales .Australia nVeterinar yJourna l55 :422-426 . Wilson,L .& J .Bryan ,1974 .Biochemica lan dpharmacologica lpropertie so fmicrotubules . Advancesi nCel lan dMolecula rBiolog y4 :21-72 .

70 Resistance to ergosterol-biosynthesis inhibitors I. Chemistry and phenomenological aspects

A. Fuchs Laboratory ofPhytopathology , Agricultural University,Wageningen , TheNetherland s

M.A.d eWaar d Laboratory ofPhytopathology ,Agricultura l University,Wageningen , TheNetherland s

Abstract

The ergosterol-biosynthesis-inhibiting fungicides comprise aheteroge ­ neous group ofchemica l substances thatdespit e theirheterogeneit y have three characteristics incommon :the y allhav ea tleas ton enitrogen-con ­ taining ring;wit h someexceptions ,the ycontai n atleas ton e asymmetric carbon atom; andthe ynearl y all interferewit hergostero l synthesisb y inhibiting theC-1 4demethylatio n step.O nth ebasi s ofth echemica l structure ofthei rnitrogen-containin g ring,the yca nb e classed aspiper - azines,morpholines ,pyridines ,pyrimidines , imidazoles andtriazoles . Thoughlimited ,ther e isevidenc e tosho wtha tdifferen tstereoisomeri c formsma y differ significantly in fungitoxicity.Mos t ergosterol-biosyn- thesis-inhibiting fungicides arebroad-spectru m fungicides,toxi c to representatives ofth eAscomycetes ,Deuteromycete s andBasidiomycetes . Resistance to ergosterol-biosynthesis-inhibiting fungicides has so far notbee n observed underpractica l conditions.Howeve r under laboratory conditions resistantmutant s canb e readily obtained, thoughthe ygener ­ allyhav ereduce d fitnesswit hrespec tt o sporegermination , germ-tube elongation,mycelia l growth or sporulation.Reduce d fitness is almost invariably reflected by reducedpathogenicity , sodevelopmen t ofresis ­ tancet othi s type of fungicide underpractica l conditions isunlikely .

Keywords: azasterols,cross-resistance , diastereomers,dicarboximides , enantiomers,ergosterol-biosynthesis-inhibitin g fungicides, fitness of resistantmutants ,hypocholesteremi c agents,imidazoles , iprodione,mor ­ pholines,piperazines ,pyridines ,pyrimidines ,stereoisomerism , triazo­ les.

71 The chemistry of ergosterol-biosynthesis inhibitors

General

Among the systemic fungicides,th eergosterol-biosynthesi s inhibitors, forvariou s reasons,occup y aspecia lplace .A s distinct from virtually allothe r fungicides dealtwit h inthi sbook , the ergosterol-biosynthesis inhibitors areno tclassifie d onth ebasi s of acommo n chemical structure orstructura lmoiety ,bu t according toa commo nmechanis m ofaction .The y comprise alarg enumbe r ofchemicall y very diverse compounds that insom e way interferewit hergostero lbiosynthesis .No t allo fthe m areuse d againstplant-pathogeni c fungi.Thi smean s thatth eter m 'ergosterol-bio­ synthesis inhibitors'ha s awide r connotation thanth eter m 'ergosterol- biosynthesis-inhibiting fungicides'.Th eergosterol-biosynthesi s inhibitors arepar to fa stil l larger group ofcompounds ,th e inhibitors oflipi d synthesis (Ragsdale, 1977),whic h encompass inhibitors ofth ebiosynthesi s of sterols,gibberellins ,carotenoids ,se xhormone s and fattyacids . This contributionwil l only cover the inhibitors ofth e biosynthesis of sterols,amon gwhic h ergosterol ispredominan t in fungi.Apar t from theergosterol-synthesis-inhibitin g fungicides used inagriculture ,ther e areothe r sterol inhibitors,al lo fwhic h arewel l known for theirinter ­ ferencewit h agrea tvariet y ofbiologica l functions,an dthu s affectvir ­ tually allbiologica l systems and living organisms.Thi sma yexplai nwh y inth epas tman y ofth e so-called hypocholesteremic compounds,use d in clinical medicine to lower serum levels ofcholesterol ,hav ebee n assayed for antifungal activity.Thes e include chemicals likeAY-994 4 (trans-I,4- bis(2-chlorobenzylaminomethyl)-cyclohexanedihydrochloride )(Elliott , 1969; Matolcsy et al., 1973), SKF 525-A (ß-diethylaminoethyldiphenylpro- pyl acetatehydrochloride) ,SK F 2314 (2,2-diphenylpentanoi c acid), SKF 3301A (2,2-diphenyl-l-(ß-dimethylaminoethoxy) pentanehydrochloride) ,

SKF 7732-A3 (tris(2-dimethylaminoethyl)phosphate trihydrochloride), SKF 7997-A3 (tris(2-diethylaminoethyl)phosphatetrihydrochloride )an d SKF 16467-A (N-dimethylaminoethyl-a,a-diphenylvaleramid e hydrochloride) (Nelsone t al., 1967;Elliott , 1969); and triparanol,a compoun d thatha s some structural resemblance to ancymidol, fenarimolan dtriarimo l (Ragsdale& sisler, 1975). Thesecompound s are slightly fungitoxic to such fungi as Cochliobolus carbonum (Nelsone t al., 1967), Phytophthora cactorum, Sordaria fimicola (Elliott,1969) , Alternaria tenuis, Aspergil­ lus niger, Botrytis cinerea, Fusarium moniliforme, Helminthosporium sati­ vum, Rhizoctonia spp. (Matolcsy et al., 1973)an d Ustilago maydis (Ragsdale & Sisler, 1975). Hypocholesteremic agents are alsoknow nt o affectgrowt ho fyeas t •cells.Fo r instance,trifluperidol ,a piperidin e derivative (Figure1) , andtriparano lbot hmildl y inhibitmultiplicatio n of Saccharomyces cerevi-

72 F-f VC-(CH2)3-N

Figure 1. Structural formula of the piperidine trifluperidol.

siae cells (Aaronson, 1971;Sobu se t al., 1977). Presentknowledg esug ­ gests thatth emajo r step inth e sterolbiosyntheti c pathwayblocke db y thehypocholesteremi c agents isA 8 •»A 7 isomerization (Sobuse t al.,1977 ; cf. Schmitt& Benveniste , 1979a). Recently,anothe rgrou p ofcompounds ,th e azasteroid antifungal anti­ biotics from Geotrichum flavo-brunneum (Michel et al., 1975;Gorde e & Butler, 1975), ofwhic hA25822 B (15-aza-24-methylene-D-homocholesta-8, 14-dien-3ß-ol)i sth emos t active component,wa s reported to interfere with ergosterol biosynthesis,i nthi s caseb y inhibitiono fth e reduction ofth eA 14 bond (Woloshuk et al., 1979). Fungi affected are Aspergillus nidulans, Blastomyces dermatitidis, Cladosporium cucumerinum, Histoplasma capsulatum, Microsporum gypseum, Sporotrichum schenckii, Trichophyton mentagrophytes and Ustilago maydis; azasterols areals o active towards theyeas tspecie s Candida albicans, Cryptococcus neoformans and Saccharomyces cerevisiae (Gordee& Butler , 1975;Baile y et al.,1976 ; Woloshuk et al., 1979). Bothhypocholesteremi c agents,namel yAY-9944 ,an d azasterols,namel y A25822B,xalso interferewit h sterolbiosynthesi s inhighe rplants ,a sha s been illustratedwit hbrambl e cells.Fo rbot htype s of agents,th emai n siteso finhibitio n are identical tothos ereporte d foryeast s and fungi (Schmitt& Benveniste ,1979a ;Schmit te t al., 1980). The samehold s true forfenarimol ,whic h inbrambl e cells,a s infungi ,probabl y interferes withC-1 4demethylatio n (Schmitt& Benveniste , 1979b). Iti s noteworthy thatwit h all three sterol-biosynthesis inhibitors,stabl ebrambl e lines were obtained thatwer e able togro wpermanentl y in inhibitor-supplemen­ tedmedia .A similarphenomeno n hasbee nobserve d inlettuce ,i nwhic h resistance to triforine,anothe r ergosterol-biosynthesis-inhibiting fungicide,appear st ob edetermine db y asingl erecessiv e gene (Globerson & Eliasi,1979 ;Maxo n Smith, 1979). Thushighe rplant s appear to readily develop resistance tothes ecompounds .

Theergosterol-biosynthesis-inhibitin g fungicides used in agriculture

Although they arepotentiall y useful inagriculture ,mos to fth eergo - sterol-synthesis inhibitors dealtwit h so farar eno tuse d tocontro l plantdiseases .However ,othe rergosterol-synthesis-inhibitin g fungicides

73 are rather extensively used inagriculture .Althoug h they comprise aver y heterogeneous group of structurally unrelated chemicals,the yhav e three characteristics incommo n -b e itwit h some exceptions.First ,the y all have atleas ton enitrogen-containin g ring;the yshar ethi s characteristic with innumerable compounds,amon g them systemic fungicideswit h anen ­ tirely differentmod e of action,a swel l astrifluperido l and theaza - sterol antifungal antibiotics,which ,thoug h fungitoxic,hav eno tbee n used inagricultur e so far.Second ,wit h sevenexception s (buthiobate1, clotrimazole2, dodecyl-imidazole, EL-241, fluotrimazole1,prochloraz 1, andX E 326),the y contain at leaston e asymmetric carbon atom.Thi s too is astructura l feature occurring ina ninfinit e number ofchemicals , among them triparanol andth e azasterols.Third , all ergosterol-biosyn- thesis inhibitors,wit hth eknow nexceptio no ftridemorph , andprobabl y the other so-calledmorpholine s (Kato et al., 1980;Kerkenaar , 1980), interferewit h theergostero l synthetic pathway by inhibition ofC-1 4 demethylation. Inaddition ,othe r steps inth ebiosyntheti c pathway are blockedb y some,bu tno tall ,o fthem . Fortriarimol , for instance,a t leastthre e sites ofinhibitio n areno wknow n (Ragsdale,1975 , 1977). On thebasi s ofth e chemical nature ofth enitrogen-containin g ring present these ergosterol-biosynthesis-inhibiting fungicides canb ediv ­ ided as follows (for structural formulae seeFigure s2-7) : -Wit h asaturate d ring system .wit htw onitroge n atoms,piperazine s (triforine1) .wit h onenitroge n and one oxygen atom,morpholine s (dodemorph1, fenpropimorph1,tridemorph 1) -Wit h anunsaturate d ring system . withon enitroge nato m ina six-membere dring ,pyridine s (buthiobate1, EL-241) .wit htw onitroge n atoms ina six-membere d ring,pyrrolidine s (ancymidol3, fenarimol1,nuarimol 1, triarimol) .wit htw onitroge n atoms ina five-membere d ring, imidazoles5 (clotri­ mazole2, dodecyl-imidazole, econazole2,imazalil 1, ketonazole2'4,micona ­ zole2,phenapronil 1,prochloraz 1,X E 326)

CHO CHO I I NH NH CH-N N-CH

CC13 CCI3

Figure2 .Structura l formulao fth epiperazin etriforine ;sta rindicate sasymmetri c carbonatom .

74 CH H3C 3 yt (CH2)5\ rK CH VN O 0 N-CH2-CH-CH2—(/ V-C( 3)3 (CH2)57 ^ CH CH3 H3C 3

dodemorph fenpropimorph

CH3

C13H27-N 0

CH3

tridemorph Figure 3.Structura l formulae ofmorpholines ;sta r indicates asymmetric carbonatom .

OH N=\ ^SC4H9 N c CI <\ /r = ^SCH2-^ >-C(CH3)3

buthiobate EL-241 Figure 4.Structura l formulae ofpyridines .

OH /Cl0H F OH

£•_// \\_n0-CH- 3 ^~VA*_// V-CI

II ancymidol fenarimo nuarimol

OOHCH *

triarimol Figure 5.Structura l formulae ofpyrimidines ; star indicates asymmetric carbon atom.

75 7 // Vc-ç \) Çl2H25 Cl-/ V-CH-O-CH2—e Vci

CH2 u N—-U N— ir N- N-dodecylimidazol e econazole clotrimazole

CH2-0^f VN N-C-CH3

Cl / Vs-4 CM

CH2

ketoconazole

Cl CI CN

—^^CH-0~CH2-/j^CI ^ ^-C-C«H I 9 CH2 CH2

M U N

miconazole phenapronil

Cl

Cl—

Figure6 .Structura lformula eo fimidazoles ;sta rindicate sasymmetri ccarbo natom .

76 Cl R

V ^—V V-o-CH-CHOH-C(CH3)3

CH2 N—ü

N—u

CGA 64250 R=n-C3H7

CGA 64251 R= C2H5

O // \ 0-CH-C-C(CH3)3

fluotrimazole triadimeton Cl

Cl—^ ^>—CH -CH-CHOH-C(CH ) Cl—f V-0-CH-CHOH-C(CH3)3 2 3 3

N—u N—u triadimenol diclobutrazol

Figure 7. Structural formulae of triazoles; star indicates asymmetric carbon atom. N.B. The name diclobutrazol only applies to the 2R3R and 2S3S (threo) configuration, but not to the corresponding 2R3S and 2S3R (erythro) form.

. with three nitrogen atoms in a five-membered ring, triazoles (biterta- nol1, CGA642501'6, CGA642511'6, diclobutrazol7, fluotrimazole1, tria- dimefon1, triadimenol1).

1.Release d orpropose d foragricultura luse . 2.Release d orpropose d forclinica lus eagains thuma nfunga lpathogens . 3.Ancymidol ,primaril yknow nfo rit sgrowt hregulator yactivit y inhighe rplants ,i s slightlyfungitoxi ct oCladosporiu m cucumerinum (Sheralde tal. , 1973). 4.Th eketoconazol emolecul eals ocontain sa piperazin e ring (Thienponte tal. , 1979). 5.I naddition ,a larg enumbe ro fimidazoles ,amon gthe mbutoconazole ,isoconazole , sulconazolean dtioconazole ,ar estil li nth eexperimenta lphas e (cf.Heere s& va nde n Bossche, 1980). 6.Se eva nGeste le tal .(1980 )fo rrelate dcompounds . 7.Thi snam eapplie st oth ethre oconfiguratio nonl y (Bent& Skidmore ,1979) .

77 This listno tonl y contains agricultural fungicides,bu t also antimyco- ticsuse d inmedicine .Som eexperimenta l fungicides havebee nliste d that chemically belong toth emor ewell-know n ergosterol-synthesis-inhibiting fungicides used inagriculture ,bu twhic hma yneve rb e applied inpractice . The listshow sonc emor e that iti svirtuall y impossible todelimi tthes e fungicides unequivocally, whichmake s itdifficul t topredic t specifically the areaso fapplicatio n forprospectiv e ergosterol-biosynthesis inhibitors.

The implications of stereoisomerism

Due toth epresenc e ofon eo rmor e asymmetric carbonatoms ,mos to f these chemicals occur indifferen t stereoisomeric forms,whic hma y often differ significantly in fungitoxicity.Fo r instance,th e (-) enantiomer oftriforin e ismuc hmor e active thanit s (+)analogu e (Ost, 1976). Diclobutrazol (Bent& Skidmore, 1979), fenpropimorph (Pommer& Himmele , 1979)an d triadimenol (Buchenauer,1979b )ar ethre eothe r compounds whose activity depends onth e stereoisomer involved. Apart from the stereoisomerism due toth epresenc e ofth e asymmetric carbon atoms inth esid echains ,i nth etriforin emolecul e another form of stereoisomerism originates from thenatur e ofth epiperazin e ring.Th e chair form ofth epiperazin e ringpermit s the l-formamido-2,2,2-trichlo- roethyl sidechain s tooccu r intw odifferen t orientations ateac ho f therin gnitroge n atoms,equatoria l and axial.Accordin g toJosepovit s & Gasztonyi (1976), with one side chainequatoria l and theothe raxial , hydrogenbondin g canb e assumed tooccu rbetwee nth e formamidegrou p in onechai nan d the carbonyl group inth eother .Th e alleged higherwate r solubility ofthi s stereoisomer compared toth e stereoisomerwit hbot h side chains oriented equatorially isbelieve d tob eresponsibl e forit s greaterbiologica l activity. Thoughwidel y recognized inpharmacology , the implications of stereoisomerism, especially enantiomerism,hav ebee n almostneglecte d with respectt o agricultural fungicides. Inpharmacology , stereoselective effects ofenantiomers ,especiall y ofrepresentative s ofso-calle dconge ­ neric series ofcompounds ,ar enowaday s taken intoconsideratio nb ycon ­ trolling drug geometry indru g design (cf.Witia k et al., 1978). Direct comparisono fth e isomeric activity ratio (A/B)wit h the activity (A)o f themos tpoten tmembe r ofeac hpai r ofenantiomer s insuc h acongeneri c series (Bdenote s the activity ofth e lesser activemember )le dAriën s andco-worker s (LehmannF . etal. , 1976;Lie ne t al., 1976)t o conclude that lgA/ B isa linea r function ofl gA .Whe n the slopeo fthi s function iszero ,th e chiral centre (asymmetric carbon atom)o fth eenantiomer s is notcriticall y involved inth ebindin g ofth e enantiomers toth e target site: i.e. chirality isnon-critica l to stereoselectivity. Thismean s that insuc hcase sth e asymmetric centre doesno tpla y aselectiv e role

78 inth edifferentia l activity ofth etw omember s ofth eenantiomeri cpair . Inothe r instances (whenth e slope ofth e function isno tequa l to zero), however, stereoselectivity wholly orpartiall y depends onchirality . Two congeneric series ofergosterol-biosynthesi s inhibitors,th e imidazoles and the triazoles,see m tob e outstanding 'tools'wit hwhic h tostud y the effects of stereoselectivity inagricultura l fungicides; perhaps themorpholine s andth epyrimidine s canals ob euse d to examine thesephenomena ,whic hmigh tprovid e abette r insightint o the lacko f cross-resistance between structurally related fungicides and themecha ­ nisms ofresistanc e (p.87-100). Further,mor e knowledge aboutth eeffect s ofth e formulationuse d onth e ratio inwhic h the stereoisomers arepre ­ sentan d their fungitoxic activity, aswel l asabou tdifferentia lmetabo ­ lism of stereoisomers (cf.Miyan oe t al., 1980)seem s aprerequisit e for fullcomprehensio n ofth e significance of stereoisomerism inth emod e of actiono fergosterol-biosynthesi s inhibitors. Itma yb eessentia l also for theoptimizatio n ofbioactivit y incongeneri c series ofthes efungi ­ cides (cf.Verloo p &Tipker , 1977).

Phenomenological aspects of resistance

Introductory remarks

Ingenera lergosterol-biosynthesi s inhibitors arebroad-spectru m fun­ gicides, toxic to representatives ofAscomycetes ,Deuteromycete san d Basidiomycetes (Fuchs& Drandarevski , 1973;Buchenauer , 1979a); however Oomycetes andZygomycete s aremuc h less sensitive (Siegel et al.,1977 ; Buchenauer &Röhner , 1979;d eWaar d& Ragsdale , 1979). However,th erepor ­ ted sensitivities of fungi towards these fungicides -whic h evenamon g taxonomicallyclosel y related fungal speciesofte ndiffe r considerably -ma y depend onth epredominan t stereoisomer present inth e formulations used (cf.Ost ,1976 ;H .Buchenauer ,persona l communication, 1981). Generally, the ergosterol-biosynthesis-inhibiting fungicides used in agriculture have little effecto n sporegermination , incontras tt o germ-tubeelongation . Initial spore germinationma yb enormal ,bu t germ-tubesma ybecom e heavily distorted, resulting inabnorma lgrowth . Hyphae are frequently swollen and/or excessively branched.Nevertheless , dryweigh t increase,respirator y activity,mitosi s andnuclei c acidan d protein synthesis arehardl y orno ta tal l inhibited.However , incultu ­ res treatedwit h these fungicides,accumulatio n of free fatty acids often occurs, togetherwit h adecreas e inC- 4 desmethyl sterols (primarily ergosterol)an d an increase inmethy l and dimethyl sterols.Therefor ein ­ hibitiono fC-1 4demethylatio n inth e ergosterol biosynthetic pathway is believed tob ethei rprimar y siteo f action,althoug h additional sites of attack arepossibl e (cf.Ragsdale , 1977).

79 Fitness andvirulenc e ofresistan tmutant s

So far,practica l application of fungicides that inhibit ergosterol biosynthesis hasno t ledt o anyconfirme d caseo fresistance .Recently , however,Walmsley-Woodwar de tal . (1979a)reporte d increased levelso f resistance of Erysiphe graminis f. sp. hordei totridemorp h inbot h glass­ house and field experiments.Whethe r tridemorph exerted amutageni c effect, raising the low frequency ofresistanc e existing inwild-typ epop ­ ulations toth e levels observed, orwhethe r the reported changeswer e solely duet o selectionpressure ,i sno tknown .However , iti slikel y that once the fungicide ceases tob eused , sensitivewild-typ e strainswil l eventually predominate,becaus e ofthei rgreate r competitiveness inth e absence ofth e fungicide.A lowerdegre e of fitness inth e resistant pathogenpopulatio nma yb e thereaso nwhy , so far,ther ehav ebee nn ore ­ ports ofwidesprea d failure oftridemorph . Attempts toobtai n resistance totriadimefo n in Erysiphe graminis ff.sp . tritici and hordei, Uromyces appendiculatus and Uromyces fabae by transferring theseobligat eparasite s every fortnight onto triadimefon- treatedplant s didno tresul ti na chang e insensitivit y (Kovacs& Tüske , 1980). This agreeswit h earlier findings (Drandarevski & Schicke, 1975) thatsuccessiv e transfers ofbean-rus turedospore s tobea nplant s treated with sub-lethal concentrations oftriforin e didno tyiel d resistant strains; invariably, therewa s adeclin e ininfectio n level and sporepro ­ duction rate and after afe wpassage s nomor euredospore swer eproduced . Incontrast ,resistan tmutant s canofte nb e readily obtained invitro , by selectiono funtreate d ormutagen-treate d conidia on agarmedi awit h lethal concentrations ofan yo fth e ergosterol-biosynthesis-inhibiting fungicides.Usuall y suchresistan tmutant s arecross-resistan t tomos t otherergosterol-biosynthesi s inhibitors.A listo f fungal species for which cross-resistance invitr oha sbee nobtaine d so far isgive no n p.92 . Resistance infung i toergosterol-biosynthesis-inhibitin g fungicides isgenerall y accompanied with adecreas e in fitness orpathogenicity . This was demonstrated firstwit h triarimol- andtriforine-resistan t mutants of Cladosporium cucumerinum: thedegre e ofresistanc ewa s found tob einver ­ selyproportiona l topathogenicit y (Fuchs& Viets-Verweij , 1975;Fuch s et al., 1977) (Figure 8).Thi s relationha sbee n sinceconfirme d inexper ­ imentswit hth e same and other fungal species (Buchenauer, 1977;va nTuyl , 1977), although in Pénicillium expansum, imazalilresistanc ewa s not always coupledwit hdecrease d pathogenicity (vanTuyl , 1977). Mutants of Aspergillus nidulans with identified genes forresistanc e have displayed varying degrees ofreduce d fitnesswit h respectt o spore germination, germ-tube elongation (Figure 9),mycelia l growth (Figure 10)an dsporula ­ tion (deWaar d & Sisler,1976 ;d eWaar d& Gieskes , 1977). The rateo f

80 disease index (arbitrary units : 0 =health y to5=dead) 5-1

Ta- mutants o \ \^ r=-0.73 o OWTa-mutants \ 0 r=-0.95

"1— 0 25 50 75 resistancet o triarimol ( %uninhibite d growth)

Figure8 .Relatio no fpathogenicit y todegre eo fresistanc et otriarimo l (2.5pg/ml ) fortriarimo l (Ta)-resistantCladosporiu mcucumerinu m mutants ina cucumbe rseedlin g test,a :gree nwild-typ e strain,b :off-whit e (OW)'parent 'strai n (Source:Fuch se t al., 1977).

germ-tube elongation appeared tob enegativel y correlated withth edegre e ofresistance , and allmutan t strains resembled each other inthei rim ­ paired ability toproduc e spores.Mutan t strains of Cladosporium cucu­ merinum alsoproduce d less spores,whic hwer e oftenno tviabl e (Sherald & Sisler,1975 ;Fuch s& Drandarevski , 1976). Reduced fitnesso f resistant isolatesha s alsobee n observedwit h obligateparasites .Fo r instance,i n tridemorph-resistant strains of Erusiphe graminis f. sp. hordei pathogeni­ city andresistanc e were foundt ob enegativel y correlated (Walmsley-Wood- ward etal. ,1979b , 1980).

Practical implications of resistance

Theobservation s on fitness andvirulenc e suggesttha tresistanc e in pathogenic fungi toergosterol-biosynthesis-inhibitin gfungicide s might have significantepidemiologica l implications,sinc eth echanc e ofsuc h strains surviving inth e absence ofselectio npressur eb y the fungicide is severely reduced. Fuchs& Drandarevsk i (1976)conclude d on similar evidence thatdevelopmen t ofresistanc e tothi s typeo f fungicideunde r practical conditions israthe runlikely .Th edange ro fdevelopmen to fre ­ sistantpopulation s isals oreduce d asmos tresistan t strains canstil l be controlled withnorma l fungicide application rates (see,fo r instance Brown& Hall , 1979). 81 «5.

'' *-=•.-*

# 7 «« ^ ^

Figure 9. Spore germination and germ-tube elongation of Aspergillus nidulans after a 12 h incubation on a glucose-nitrate agar. Left: wild-type strain 003. Right: fenarimol- resistant mutant strain 193 (for details on strains see: de Waard &Gieskes , 1977).

Figure 10. Mycelial growth of some Aspergillus nidulans strains on malt agar: 003 is the wild-type strain; others are fenarimol-resistant mutant strains (for details on strains see: de Waard &Sisler , 1976).

Iprodione: a special case

One more fungicide is worth special mention: iprodione (Figure 11), a compound usually referred to as a dicarboximide fungicide, which contains a hydantoin ring that is a saturated five-membered ring with three nitro­ gen atoms. Unlike the other dicarboximides, it has been implied that

82 iprodione interfereswit hergostero lbiosynthesis ;th e accumulation of 4,4-demethylsterols indicates that itinhibit s ata sit emos tprobabl y different from thato fC-1 4demethylatio n (Pappas& Fisher , 1979). Con­ troversial data on fungal resistance tothi s fungicide (p.101-117)ma yb e due to structural rearrangement of iprodione inth e (m)ethanolicsolu ­ tionsofte nuse d todissolv e the fungicide.Unde r thesecondition sipro ­ dionerearrange s to form anisome r (Figure 11),which , like theparen t compound, isa dicarboximid e with anidentica l ring system,bu twhic hal ­ mostentirel y lacks fungitoxicity (Cooke et al., 1979). The suggestion thatth e so-called anomalousbehaviou r of Botrytis cinerea isolates on iprodione-containing media,observe d by Leroux etal . (1977), isexplai ­ nedb y this structural rearrangement seemsrathe runlikely .Bot h Leroux etal . (1977)an d Schüepp &Kün g (1978)obtaine d resistance levels (ED50s ofu p to about30 0 pg/ml, ascompare d to 0.3 pg/rolfo rth e wild-type strains)tha twer e comparablewit hthos e forth eothe r dicarboximides. Inbot h instances controlswer e included andprecaution s taken that should havepreclude d possible effects ofth e assumed isomerizations. However the resistance levels obtainedb yDenni s &Davi s (1979), who found thatthei r isolateswer e able togro w onpotato-dextros e agarcon ­ taining iprodione ata concentratio n of1 0mg/m l -3 0time shighe r than the level obtainedb y Leroux et al. and Schüepp& Kün g -migh tb eentire ­ lyo r atleas tpartiall y ascribable toth e suggestedmolecula rrear ­ rangement toth e less toxic isomer. Iti so futmos t importance toverif y the assumptions ofPappa s & Fisher (1979)tha t inhibitiono f sterolbiosynthesi s isprobabl y thepri ­ mary actiono f iprodione,an d toinvestigat e whether cross-resistance to theestablishe d ergosterol-synthesis inhibitors ispossible .Preliminar y observationswit h Aspergillus nidulans, Cladosporium cucumerinum and Pé­ nicillium italicum have revealed only low levels ofcross-resistanc e of

CL ^ CI CI ^ CI

NH ^T r C=0 L_N_c-N-CH(CH3)2 I

0 H ' N-CH(CH3)2 iprodione rearrangement product of iprodione

Figure 11.Structura l formulao fiprodion ean d itsstructura l rearrangementproduc t (cf. Cookee tal. , 1979).

83 two fenarimol-resistant Cladosporium cucumerinum isolates to iprodione andvinclozolin , andn o cross-resistance whatsoever toprocymidon e (Fuchs & deWaard ,unpublishe d data). Yetcautio n seems necessary, ifonl yt o prevent 'diseasecontro l agentswit hprove n effectiveness beingunneces ­ sarily lostt oresistance ' (Delp, 1980).

References

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Schmitt,P .& P .Benveniste ,1979a .Effec to fAY-994 4o nstero lbiosynthesi s insuspen ­ sionculture so fbrambl ecells .Phytochemistr y 18:445-450 . Schmitt,P .& P .Benveniste ,1979b .Effec to ffenarimo lo nstero lbiosynthesi s insuspen ­ sionculture so fbrambl e cells.Phytochemistr y 18:1659-1665 . Schmitt,P. ,F .Schei d& P .Benveniste ,1980 .Accumulatio no fA 8'14-sterols insuspen ­ sionculture so fbrambl ecell sculture dwit ha nazastero lantimycoti cagen t (A25822B).Phytochemistr y 19:525-530 . Schiiepp,H .& M .Kiing ,1978 .Gegenübe rDicarboximid-Fungizide n toleranteStämm evo n Botrytiscinerea .Bericht ede rSchweizerische nBotanische nGesellschaf t88 :63-71 . Sherald,J.L. ,N.N .Ragsdal e& H.D .Sisler ,1973 .Similaritie sbetwee nth esystemi c fungicidestriforin ean dtriarimol .Pesticid eScienc e4 :719-727 . Sherald,J. L &H.D .Sisler ,1975 .Antifunga lmod eo factio no ftriforine .Pesticid e Biochemistryan dPhysiolog y5 :477-488 . Siegel,M.R. ,A .Kerkenaa r& A .Kaar sSijpesteijn ,1977 .Antifunga lactivit yo fth esys ­ temicfungicid eimazalil .Netherland sJourna lo fPlan tPatholog y83S :121-134 . Sobus,M.T. ,CE .Holmlun d& N.F .Whittaker ,1977 .Effect so fth ehypocholesteremi c agenttrifluperido lo nth esterol ,stery lester ,an dfatt yaci dmetabolis mo fSaccha - romycescerevisiae .Journa lo fBacteriolog y 130:1310-1316 . Thienpont,D. ,J .va nCutsem ,F .va nGerven ,J .Heere s& P.A.J .Janssen ,1979 .Ketocona - zole- a ne wbroa d spectrumorall yactiv eantimycotic .Experienti a 35:606-607 . Tuyl,J.M .van ,1977 .Genetic so ffunga lresistanc e tosystemi c fungicides.Mededelinge n LandbouwhogeschoolWageningen ,Nederlan d 77-2:1-136 . Verloop,A .& J .Tipker ,1977 .A comparativ e studyo fne wsystemi cparameter s indru g design.In :J.A .Keverlin gBuisma n (Ed.):Biologica lActivit yan dChemica lStructure , Elsevier,Amsterdam ,p .63-81 . Waard,M.A .d e& S.A . Gieskes,1977 .Characterizatio no ffenarimol-resistan tmutant so f Aspergillusnidulans .Netherland sJourna lo fPlan tPatholog y83S :177-188 . Waard,M.A .d e& N.N .Ragsdale ,1979 .Fenarimol ,a ne wsystemi c fungicide.In :H .Ly r& C.Polte r (Eds): Systemfungizide,Akademie-Verlag ,Berlin ,p .187-194 . Waard,M.A .d e& H.D .Sisler ,1976 .Resistanc e tofenarimo li nAspergillu snidulans . Mededelingenva nd eFacultei tLandbouwwetenschappe nRijksuniversitei t Gent41 : 571-578. Walmsley-Woodward,D.J. ,F .Law s& W.J .Whittington ,1980 .Comparison sbetwee nisolate s ofErysiph egramini sf .sp .horde itoleran tan dsensitiv e toth efungicid etridemorph . Annalso fApplie dBiolog y94 :305-306 . Walmsley-Woodward,D.J. ,F.A . Laws& W.J .Whittington ,1979a .Studie so nth etoleranc e ofErysiph egramini s f.sp .horde it osystemi c fungicides.Annal so fApplie dBiolog y 92:199-209 . Walmsley-Woodward,D.J. ,F.A .Law s& W.J .Whittington .1979b .Th echaracteristic so f isolateso fErysiph egramini s f.sp .horde ivaryin g inrespons et otridemorp han d ethirimol.Annal so fApplie dBiolog y92 :211-219 . Witiak,D.T. ,H.J .Lee ,H.D .Goldma n& B.S .Zwilling ,1978 .Stereoselectiv e effectso f cis-an dtrans-cyclopropylbi s (dioxopiperazines)relate d toICRF-15 9o nmetastase s ofa hamste rlun gadenocarcinoma .Journa lo fMedicina lChemistr y21 :1194-1197 . Woloshuk,C.P. ,H.D .Sisle r& S.R .Dutky ,1979 .Mod eo factio no fth eazasteroi d anti­ biotic 15-aza-24-methylene-D-homocholesta-8,14-dien-3ß-ol inUstilag omaydis . AntimicrobialAgent san dChemotherap y 16:98-103 .

86 Resistance to ergosterol-biosynthesis inhibitors II. Genetic and physiological aspects

M.A. deWaar d Laboratory ofPhytopathology ,Agricultura l University,Wageningen , The Netherlands

A. Fuchs Laboratory ofPhytopathology , Agricultural University,Wageningen , The Netherlands

Abstract

Todate ,developmen to fresistanc e to fungicides that inhibit sterol biosynthesis hasno tbee nobserve d underpractica l conditions.Yet ,a largenumbe r ofmutant so fdifferen t fungiwit h resistance tothes efun ­ gicides havebee n isolated invitr o andcharacterized . Resistance tothes e fungicides has,a tleas ti n Aspergillus nidulans, been shownt ob ebase d ona multigeni c system. Cross-resistance between sterol-biosynthesis in­ hibitors isth erule ,eve nwhe n they arechemicall y notrelate d toeac h other.Resistanc e mayb e accompanied withpleiotropi c effects such asin ­ creased sensitivity orresistanc e tounrelate d toxicants and,possibly , reduced fitness.Th emechanis m ofresistanc e canvar y indifferen tfungi . Resistance to fenarimol in A. nidulans isprobabl ybase d ondecrease dup ­ take ofth e fungicideb yresistan tmutant s ascompare d with thewild-typ e strain.Decrease d uptake isdu et oenhance d energy-dependent efflux acti­ vity, theresul to fwhic h istha tth e fungicideprobabl y doesno t saturate the siteo faction .Th e genetic andphysiologica l data onresistanc e are discussed inrelatio n tothei r significance fordevelopmen t of resistance inpractice .

Keywords: ergosterol-biosynthesis inhibitors,imazalil , fenarimol,gene ­ ticso fimazali l resistance,mechanis m ofresistanc e to fenarimol,fungi ­ cideresistance , cross-resistance.

Introduction

Systemic fungicideswit h specific activity towards ergosterolbiosyn ­ thesis arelikel y tobecom e themos tprominen t group of chemotherapeutics inth enea r future.T o date atleas t 17chemical s ofthi styp ehav ebee n introduced foragricultura l use (Table 1).Som eo fthe mhav ebee nuse d already fora significan tperio d oftime ,other s havebee n introducedon ­ lyrecentl y andhav eno tye tbee nrelease d forpractica l application.

87 Table 1. Agricultural fungicidestha tinhibi tergostero lbiosynthesi s

Chemicalgrou p Trivialnam e Tradename(s ) Yearo fintroductio n

Piperazines triforine Funginex/Saprol 1969 Morpholines dodemorph Meltatox 1967 tridemorph Calixin 1969 fenpropimorph Corbel 1980 Pyridines buthiobate Denmert 1975 Pyrimidines fenarimol Rubigan/Rimidin 1975 nuarimol Trimidal/Triminol 1975 Imidazoles imazalil Fungaflor/Fecundal 1972 prochloraz (BTS40542 ) 1977 phenapronil Sisthane 1978 Triazoles fluotrimazole Persulon 1973 triadimefon Bayleton 1974 triadimenol Baytan 1977 bitertanol Baycor 1979 diclobutrazole Vigil 1979 propiconazole Tilt (CGA64250 ) 1979 etaconazole (CGA64251 ) 1979 a.Fo rstructura lformula ese ep.71-86 . b.Propose d trivialname .

Wide-scale use of site-specific fungicidesma y leadt odevelopmen to fre ­ sistancewithi n onet otw oyears ,a sha sbee ndemonstrate d forbenzimi - dazoles (Schroeder& Prowidenti , 1969)an d acylalanines (Davidse et al., 1981). Butprolonge d practical use of fungicides that inhibit ergosterol biosynthesis hasno t so far ledt o anyconfirme d caseo fresistanc e (p.71-86). For instance,i nth eNetherlands ,tridemorp h andtriforin e have beenuse d inglasshouse s againstresistance-pron e fungi such aspowder y mildews for atleas t fiveyear swithou t apparentdevelopmen t ofresistance . Incontrast ,resistanc e invitr o tosterol-biosynthesis-inhibitin g fungi­ cides canb e readily obtained.Thes emutan tstrain s oftendispla y reduced fitness orpathogenicit y (seep.71-86) . Probably, reduced pathogenicity and agenera l lowdegre e ofresistanc e account forresistanc e notye tbe ­ ing apparent inpractica l agriculture. This contribution describes indetai l some fundamental aspects ofre ­ sistance to fungicides that inhibitstero lbiosynthesis .Attentio n ispai d to: thegeneti cbackgroun d ofresistance ;cross-resistanc e andpleiotropi c effects associated withmutation s forresistance ; andbiochemica lmechan ­ ismso fresistance .Knowledg e ofthes ephenomen a isnecessar y tounder ­ stand thebehaviou r andcharacteristic s ofresistan tmutant sunde rprac ­ tical conditions. Genetics of resistance

Resistance to sterol-biosynthesis inhibitors may be permanent or tran­ sient. Transient, or so-called phenotypic, resistance may be obtained by subculturing fungi on fungicide-containing media. Such resistance is adapt­ ive and not due to expression of genes for resistance. Transient resis­ tance is lost upon subculturing on fungicide-free media (Fuchs & Viets- Verweij, 1975). Transient resistance may also develop in stationary phase cultures, as shown for Candida albicans to miconazole (Cope, 1980a, b; Gale et al., 1980). Permanent resistance in vitro to ergosterol-biosynthesis-inhibiting fungicides has been frequently reported (cf. Fuchs et al., 1977). General­ ly, permanent resistance is considered to be of a genetic nature, when decreased sensitivity is stable upon repeated subculturing on fungicide- free media. Determination of mutation frequencies and genetic analysis of genes for resistance have so far only been reported for imazalil resist­ ance in Aspergillus nidulans (van Tuyl, 1977). From this work, it is evi­ dent that both spontaneous and ultra-violet (UV) and N-methyl-N'-nitro-N- nitrosoguanidine (NG)-induced mutations for resistance to imazalil occur rather frequently in comparison with mutations for resistance to other fungicides, like benomyl and carboxin (Table 2). However, the degree of resistance to these two fungicides is significantly higher than to imazal­ il (Table 3). Comparable data for other fungi have been published by van Tuyl (1977). Imazalil resistance in A. nidulans is based on a multigenic system (van Tuyl, 1977). Genetic analysis of 21 imazalil-resistant mutants re­ sulted in the identification of eight loci allocated to six different linkage groups. The loci and linkage groups, in parentheses, involved are imaA (VII), imaB (V), imaC (II), imaD (VIII), imaE (II), imaF (I),imaG (III), and imaH (III). The large number of linkage groups indicates that

Table 2. Mutation frequency of resistance to various fungicides in Aspergillus nidulans•

Mutagenic Proportion (%) original Mutation frequency treatment population surviving c c c mutagenic treatment benomyl carboxin imazalil SP 100 0.02 0.25 4 UV 50 7 12 160 NG 50 22 80 250

Source: van Tuyl (1977). a. SP: spontaneous mutation; UV: ultra-violet light; NG: N-methyl-N'-nitro-N-nitrosoguanidine. b. Number of mutants per 107 surviving conidia. c. Selection medium containing benomyl, carboxin or imazalil at concentrations of 2 ug/ml, 10 pg/ml or 2 pg/ml, respectively.

89 Table3 . Highestdegre eo finduce d resistance inAspergillu snidulan st othre e fungicides.

Strain ED^ ((Jg/ml)

benomyl carboxin imazalil

Wild type 0.7 1.5 0.4 Mutant 30 200 4

Source:va nTuy l (1977). the lociconferrin g resistance areno tclustere d butdistribute d overth e fungal genome.Mutation s atth e imaA and imaB loci occurmos t frequently, since of20 2resistan t strains tested, 113wer e found tob e ofth e imaA type and 52o fth e imaB type.Th emutatio n atth e imaB locus appeared to be allelic to camD, amutatio n forresistanc e tochloramphenico l (Guna- tillekee t al., 1975), andt o amutatio n determining fenarimol resistance (deWaar d& Gieskes , 1977). Similarly, imaHwa s found tob e allelic to acte, whichconfer s resistance tocycloheximide .Tw o other genes forre ­ sistance tocycloheximide , actD and actE alsoconferre d imazalilresis ­ tance,bu ti ncontras tt oth e acte locus,the ywer e notpicke d up among mutants directly selected for imazalil resistance.Thi smean s thata t least 10differen t genes areinvolve d inresistanc e toimazalil . Allelicmutation s ina certai n locus forresistanc e to imazalil dono t always resulti nth e samedegre e ofresistance .Fo r instance,mutation s atth e imaA or imaB locusma yb e reflected by differentminimu minhibi ­ tory concentrations (MIC), which implies thatmutation s in aparticula r genema yb e dissimilar (Table 4).Mutation s inal lothe r locionl y confer a low levelo f resistance (MICo fimazali l 2.5 -5. 0 ^jg/ml), suggesting thatmutation s indifferen t genes areo fvariabl ephysiologica l consequence (vanTuyl , 1977). Studieswit hheterozygou s diploid imazalil-resistant strains of A. nidulans have showntha t imaA, imaB, imaC, and imaDmuta ­ tions are semi-dominant.Thi s implies thatth e sensitivity ofsuc hhetero ­ zygous strains isintermediat ebetwee n sensitive diploid and resistant haploid strains (vanTuyl , 1977).

Table4 . Degreeo fresistanc eo fAspergillu snidulan smutant swit halleli cima A andima Bmutation sfo rimazali lresistance .

Mutation Numbero f Numbero fstrain sfo rMIC s (pg/ml)a sindicate d strains tested 1.0-2.5 2.5-5.0 5.0-8.0 8.0-12.0

None 1 10 0 0 imaA 11 0 3 7 1 imaB 4 0 0 3 1

Source:va nTuy l (1977).

90 Table5 .Degre eo fresistanc e ofAsper ­ gillusnidulan s recombinantstrain st o imazalil.

Mutation MIC (ng/ml)

None 2 imaA 20 imaB 10 imaA,imaB ,M a 200

Source:va nTuy l (1977). a.Modifie rgen etha tfortifie sth eactio n ofth eima Agene .

Bygeneti c techniques iti spossibl e tocombin e different single-gene mutations forresistanc e to imazalil inth e same strain.Thi s hasbee n donewit h the imaA and imaB genes and amodifie r gene M(va nTuyl , 1977). The recombinant strainpossesse s arelativel y highdegre e ofresistance , which indicatespositiv e or additive interaction (Table5) .

Cross-resistance and pleiotropic effects

Cross-resistance canb e defined asresistanc e totw oo rmor e toxicants that iscause d byth e same genetic factor (Georgopoulos, 1977). This im­ plies thatmutant s selected onon etoxican t areusuall y also resistantt o toxicants that arechemicall y related orhav e asimila rmechanis m ofac ­ tion.Cross-resistanc e isofte nreciprocal ,whic hmean s thatmutant sse ­ lected inth epresenc e of acertai n toxicant are also resistant toth e other toxicantconcerned , andvic eversa .Thi s also applies tomutant s selected onmedi a containing oneo fth eergosterol-biosynthesi s inhibi­ tors (Table 6),which , although oftenunrelate d chemically,probabl y share a similarmechanis m ofactio n (cf.Ragsdale , 1977). Cross-resistance has beendemonstrate d alsot o fungitoxicantswit h asimila rmechanis m ofact ­ ion, e.g. ancymidol,clotrimazole ,EL-24 1 andmiconazol e (Sherald et al., 1973; deWaar d & Sisler,1976 ;Baru g& Kerkenaar , 1979). Data inTabl e 6indicat e thatcross-resistanc e betweenergosterol-bio ­ synthesis inhibitors isno talway s extant.Fo r instance, cross-resistance inimazalil-resistan t A. nidulans strains to fenarimolwa s almost absent ina strai ncarryin g the imaA gene (deWaar d &Gieskes , 1977). Similar resultswer e found for imazalil-resistant strains of Cladosporium cucum- erinum (Fuchse t al., 1977;va nTuyl , 1977)an d Phialophora cinerescens (vanTuyl , 1977). Also in Ustilago maudis, cross-resistance was notal ­ ways reciprocal (Barug& Kerkenaar , 1979). Perhaps cross-resistance does exist,bu t justha sno tbee n identified yetdu e to arelativel y lowmuta ­ tion frequency ofth e genes involved,o rt o differences indosag eresponse . Suchmutant sma yb emisse dwhe n alimite d number ofmutant s aretested .

91 CO r— ON en ON ON LD ^ CO ON r- •r-, ON ON ON ON ON CO o •H \o vo S« r- QJ r~-v£> \o r-*. M> ON S r- CA r-. r- o> r-. OJ w r- î-l r- I-H ON ON i-t ON -o eu p- JH u rJ ^ u u -H >1 ON +J - +J r- QJ r-. r-. r-. ON ON ON d d d i-t ta M - i-H ta • T3 r- r- -H r^ r-- ~ r- rH rH rH CU eu O) fiOJ fiQJ co m -H 4-1 0) rH rH QJ i—1 ON r~- o ON ON • ON M M ^J r-t rH r-- c/3 QJ rH - J-l (0 « *-l ON « i-l u H .M* ,*n •P ON •H « J-( o O fl) rH ta C0 U H rJ 4-> •P r- OJ QJ (U ;*S M M > +J 3 cÖ QJ OJ ta OJ r-i -T3 rH» .r-^l * .+ J rH« 3 3 3 « « X3 «*o c* 0) « w u >> OJ c0 c0 C0 3 » OJ CO >3 » 3 >3 >d d d ^ cQ QJ co «1 m u S 3 3 3 3 H Jri (Q H H w E-» O X3 >« > h > CQ M CQ CQ CQ CQ CQ CQ

+ + + + 1 + si l u Vi o w u o V-l ftu + + ++ + il o o d u fti l

u d cO (0 3 +J d en •rt il W OJ cO 1-4 3 1 d w co o rH u

+ ++ + + + + ++ + ++ +

+ +III+ + + + + + +

M) C O U *J w 3

ÏH II 4-> CO

u aj a •p W o d 3 -H ifl du i-l rH rWÛ d •p O O O ^5 O O O J2 Ä o -a *«J (U u r~i rH -H 3 -o —I -H -H -H • Ë -H E rH •H W S-llHOTJ'OOSH'a U S3 cO cQ cO cO *0 •H -O 0) n) M cO«mcOcO4H(0(0tON N N N N « cO N SH (0 "O 00 a u nj co co cO *H d to +j m o (Q c 3 o Î^S-IJ-ISHMJHJHSHUS E E S S H 0) E 3 ta t-t +J +) +J-+J +j+J +J +J *J.H

oo QJ fi d d d d d M W -H -H -H -H -H •rl ft 0 H JH U U U *i OJ QJ QJ

- o > d U (J U U U QJ CO u «E U O U O O U -i-t -H «H •ri 'H U U -H U U O T3 +J W ,>.>>>,>,>, 33333 -HCOCOW WW33tO-rt3cOfl]rO<0cOc0 S E 3 33-H-HWU.HSSSEËS IJ r-( ^ SH U M M <-\ rH U i-l O U O O f-HrHOOÄcflOOOOOOO ft ft (^ ft ft-H O0O0-H •H ft ft ft ft 00 00 00 OO 00 00 MT3 J) W C/]C/lt/)(AUI4-)(QC0O0 00 00 CA t/l O +J cQ <0 (0 CQ (Q ,n -H n o 00000>,rHrHlH iH 1-1 O O r-t >»OHrlrlHHrl ^2 M +J )H XJ'O'OTSTS JH-HTH CJJ U T3 < +J W O (oncococo+J+J-t-ift CO -ri +J CO JJ +J 4-> +J " W E/l Cfl Negatively correlated cross-resistance toergosterol-biosynthesis-in - hibiting fungicidesha sno tbee n reported. Cross-resistance in U. maydis tounrelate d fungicides like carbendazim andcarboxi nwa s absent (Leroux et al., 1976), and cross-resistance in A. nidulans topimaricin , apoly ­ ene antibioticwit hhig h affinityt oergosterol , could notb e demonstra­ ted either;som e ofth e fenarimol-resistantstrain s wereeve n slightly more sensitive tothi s antibiotic thanth ewild-typ e strain (deWaar d & Sisler, 1976). Apleiotropi c mutation isdefine d as asingle-gen e mutation affecting more thanon e characteristic. Such amutatio nma yno tonl y causeresis ­ tance to atoxican tbu tma y also affect amor e general cellpropert y (Georgopoulos, 1977), whichma y result in,fo rexample ,positively - or negatively correlated cross-resistance tounrelate d toxicants.Fo rin ­ stance,triarimol-resistan tmutant so f Botrytis cinerea have an increased sensitivity tocyclohéximid e (Lerouxe t al., 1976). Mutations forre ­ sistance toimazali l in A. nidulans alsogiv e rise topleiotropi c effects such asresistanc e or increased sensitivity tounrelate d toxicants like acriflavine,cyclohéximide ,chloramphenicol ,an dneomyci n (Table 7).Th e imaG mutationwa s alsocorrelate dwit hcol d sensitivity (vanTuyl , 1977). Inaddition ,som e imazalil-o r fenarimol-resistant mutants showedpleio ­ tropic effectswit h respectt onon-relate d fungicides,suc ha s 8-azaguani- dine,p-fluorophenylalanine ,D-serine , and thiourea (deWaar d &va nNistel - rooy, 1979), aswel l aswit h respectt o fitness (deWaar d &Gieskes , 1977). Reducedpathogenicit y ofpathogeni c fungi (seep.71-86 )migh t alsob ea pleiotropic effecto fmutation s forresistance .

Table7 . Pleiotropic effectso fimazalil-resistan tmutant so fvariou sfungi .

Fungus Mutantso r Resistance Increased sensitivity numbero fstrain s acr act cam neo acr act neo

Aspergillusnidulan s imaA +--+--- imaB -- + - + + + imaC - + + ---- imaD .___- + + imaE - + + + --- imaF ----- + + imaG - + + + --- Aspergillusnige r 1 ----++- 3 - + .- + -- Cladosporiumcucumerinu m2 ____--- Phialophora cinerescens 1 _____--

Source:va nTuy l (1977). a.Abbreviations :ac r= acriflavine ;ac t= cyclohéximide ;ca m= chloramphenicol ; neo= neomycin ;pleiotropi c effect:+ = present ;- = absent .

93 Mechanism of resistance

Resistance to fungicideswit h aspecifi cmechanis m ofactio nma yb e based ondecrease d affinity ofth e target site fora toxicant .Suc h amech ­ anism ofresistanc e hasbee ndescribe d forbenzimidazole s and oxathiins (cf. Georgopoulos, 1977). Resistance to ergosterol-biosynthesis-inhibi- ting fungicidesmigh tb ebase d ondecrease d affinity ofth e targeten - zyme(s)involve d inergostero l biosynthesis forthes e fungicides.T o date itha sno tbee n feasible to testthi shypothesi s experimentally.Alter ­ natively, resistance topolyen e antibiotics mayb ebase d on replacement ofergostero l bymor e 'primitive'sterol s (cf.Hamilton-Miller , 1974). Sucha mechanis m appearsno tt ob e involved inth eresistanc e tosterol - biosynthesis inhibitors,a smutant s of A. nidulans, C. cucumerinum and U. maydis have almostnorma l concentrations ofergostero l (Sherald & Sisler, 1975;Ragsdal e &d eWaard , 1977;Lerou x &Gredt , 1978). Factors that influence the intracellular concentration of afungicide , sucha sdecrease d uptake and increased detoxification,ma y also resulti n decreased sensitivity. Both factorsma ypla y arol e innatura l resistance totriforin e (Gasztonyi & Josepovits, 1975). Transient resistance ofC . albicans tomiconazol e instationary-phas e cultures isprobabl y also due to detoxicification. Thisma yb e theresul to funspecifi c accumulation of miconazole incel lwall s of stationary-phase cells,whic h depletes the medium andprevent smiconazol e from reaching theplasm amembrane .Th ere ­ sulting decreased sensitivity tomiconazol e iscorrelate d with decreased release ofK by thecells .Thi smechanism , rather thaninterferenc e with ergosterol biosynthesis,i sthough tt ob e theprimar ymechanis m of action ofmiconazol e (Cope,1980a ;Gal ee t al., 1980). Resistance to fenarimol in A. nidulans could notb e ascribed toincreas ­ edmetabolis m (deWaar d &Ragsdale , 1979), butrathe r to a differential uptake ofth e fungicideb ywild-typ e and resistant strains (deWaar d & vanNistelrooy , 1979). Uptake of fenarimol by thewild-typ e strainan d the three genetically defined strains J146 (imaB), M196 (imaB), andR26 4 {imaA, imaB, M) ispresente d inFigur e 1.Uptak eb yth ewild-typ e strain was characterized by arapi d initial accumulation during the first1 0min ­ utes ofincubatio n and asubsequen t gradual release;uptak eb yth emutan t strainswa snearl y always ata constan t lowlevel . Uptakeb y thewild-typ e strain appeared tob e the resultant of influx andefflu x of fenarimol. Influx couldb e inhibited byneithe r lowtempe ­ rature, anaerobiosis,starvatio n ofmyceliu m nor incubationwit h several respiratory inhibitors and istherefor e apassiv eprocess .Unde ridenti ­ cal testcondition s efflux activitywa s severely inhibited and should thus be regarded asa nenergy-dependen tprocess .Example s of sucheffect s are given inFigure s 2an d 3.Afte rprolonge d incubation (90minutes) , anequi ­ libriumbetwee n influx and effluxwa s reached, resulting ina nenergy-de -

94 uptakefenarimo l(nmol/m g dryweight ) 2.0-

60 time(min)

Figure 1. Uptake of fenarimol (30 ]M) by mycelium of wild-type strain 003 (O) and fena- rimol-resistant mutants J146 (•), M193 (•), and R264 (•) of Aspergillus nidulans in 23.4 mMK-phosphat e buffer pH 6.0 containing 0.1 mM CaCl .H.0 and 0.01 g/ml glucose (de Waard & van Nistelrooy, 1980).

pendent steady state, the efflux 'component' of which could be inhibited by addition of oligomycin or N,N'-dicyclohexylcarbodiimide, which instant­ aneously enhanced uptake (Figure 4). The establishment of an equilibrium at a low level of uptake suggests that efflux activity is inducible (de Waard &va n Nistelrooy, 1980). Uptake by the genetically defined fenarimol-resistant mutants also ap­ peared to be determined by influx and efflux. Uptake could be consider­ ably enhanced by low temperature, anaerobiosis, starvation of mycelium and incubation with respiratory inhibitors. Low uptake by these strains can be attributed to an energy-dependent efflux activity for fenarimol that rather than being inducible, like in the wild-type strain, is most probably constitutive. Upon inhibition of the efflux activity, net uptake resulted from the remaining passive influx, which in that case might be as high as in the wild-type strain. Examples of these effects are also

95 003 J 146 M 193 uptakefenarimo t (nmol/mg dry weight) 3.0-

• a

1^ ~T ~1 20 40 60 20 40 60 time (min) Figure 2. Effect of oligoraycin on uptake of fenarimol (30 uM) by mycelium of wild-type strain 003 and fenarimol-resistant mutants J146 and M193 of Aspergillus nidulans. Treatments: control (O) ; 10 (•) MM and 100 (n) |JM oligomycin (de Waard & van Nistel- rooy, 1980).

given in Figures 2, 3, and 4. The results presented in these figures suggest that wild-type and re­ sistant strains only differ in their 'efficiency' to excrete fenarimol from the mycelium. The efflux rate of resistant strains seems to be high enough to maintain a low level of uptake immediately from the moment fe­ narimol is added, so the fungicide might not be able to reach the target site at a concentration sufficient for it to exert its fungistatic action. This high efflux rate may be associated with an alteration in the regu­ lation of the imaB gene for resistance to imazalil, which all strains test­ ed have in common. The altered energy-dependent efflux in resistant mu­ tants may also account for their cross-resistance or collateral sensiti­ vity to other chemicals. The regulation of the energy-dependent efflux mechanism in the fenarimol-sensitive wild-type strain may be such that there is an initial lag before the steady-state is reached. The amount of fenarimol, that accumulates during this initial lag or is present in the steady-state may be high enough to saturate the target site(s) in the

96 003 J146 M193 uptake fenarimol (nmol/m g dry weight ) 3.0-1

2.5-

a

Figure3 .Effec to fN,N'-dicyclohexylcarbodiimid e (DCCD)o nuptak eo ffenarimol - resistant mutantsJ14 6an dM19 3o fAspergillu snidulans .Treatments :contro l (O); 20 (•) uMan d 100(D )u MDCC D (deWaar d& va nNistelrooy , 1980). fungus andconsequentl y exertit s fungistatic action (deWaar d& va n Nistelrooy, 1980).

Concluding remarks

Theergosterol-biosynthesis-inhibitin g fungicides comprise arelative ­ lyne w group of fungicides. Iti sno tsurprisin g thatdu et othei rspec ­ ificmechanis m ofaction ,resistanc e tothes e fungicidesma yreadil ydevel ­ op. However the degree ofresistanc e isrelativel y low and itofte n seems tob e correlated with adverse side-effects like decreasedpathogenicit y and fitness.Thi smigh tb e the reasonwh yu p tillno w development of resistant populations thatcanno tb e controlled bynorma l fungicide applicationha s notoccurre dunde rpractica l conditions.A crucialquestio n iswhethe r the adverse side-effects arereall ypleiotropi c and thuscouple dwit h the genes forresistanc e present,i.e . associatedwit hth emechanis m ofresist ­ ance involved.A tentative, affirmative answer issupporte d by themechan ­ ism ofresistanc e to fenarimol in A. nidulans. Therelativel y highenergy -

97 003 J146 R264 uptake fenarimol (nmol/mg dry weight) 3.0-1 ! / 0 2.5-

2.0- ^ï^f 1.5-

1.0-

0.5-

0- i i i i 1 1 ~i 1 1 1 r 20 40 60 80 100 120 20 40 60 80 100 120 20 40 60 80 100 120 time (min)

Figure4 .Effec to f10 0^ Moligomyci nan dN,N'-dicyclohexylcarbodiimid e (DCCD)o nuptak e offenarimo l (30|JM )b ymyceliu mo fwild-typ estrai n00 3an d fenarimol-resistantmutant s J146an dR26 4o fAspergillu snidulans .Treatments :contro l (O); oligomyci n (ü)an dDCC D (•),adde d6 5minute safte rfenarimo l (deWaar d& va nNistelrooy ,1980) .

dependentefflu x activity of fenarimol inresistan tmutant smigh tb econ ­ nectedwit h constitutive membrane transport activities,whic hma yb e 'fuelled'wit h energy atth ecos to fothe r cellprocesse s (e.g.germinat ­ ion, growth,an d sporulation).However ,i ti sno tknow nwhethe rth esam e mechanism ofresistanc e operates inpathogeni c fungi.Therefore , anycon ­ clusions about anegativ e correlationbetwee nresistanc e to sterol-biosyn- thesis inhibitors and fitness should notb e toodefinite .Thi svie w is supportedb yth e facttha t field isolates ofpowder y mildewswit hdecreas ­ ed sensitivity to these fungicides have alreadybee n found (Walmsley-Wood- ward et al., 1979). Atth eLaborator y ofPhytopatholog y ofth eAgricultura l University, Wageningen, resistantmutant s of Pénicillium italicum have been isolated thatd ono t show anappreciabl e loss inpathogenicit y (un­ published data). Therefore,emergenc e inpractic e ofmutan tstrain swit h normalpathogenicit y may stillb e ofconcer nsince : -multipl e application ofdifferen t fungicides inspac e and timewil l lead to increased selectionpressure ; - strainswit h arelativel y highdegre eo fresistanc ema ydevelo p duet o accumulation ofgene s forresistanc e inth e samestrain ; - resistancema yno t alwaysb e coupledwit h decreased pathogenicity; -withi n resistantpopulation s selection forth emos tcompetitiv e strain may takeplace ; - inisolate dpopulation s (glasshouses,packin g houses)mutant swit hde ­ creasedpathogenicit y may stillbuil d up resistantpopulation sbecaus e of

98 a lack of competition from the wild-type strain. These factors might lead to failure of disease control. Therefore, intro­ duction and application of these fungicides in practice should be managed carefully. This is the more significant since in the near future at least 17 ergosterol-biosynthesis inhibitors (Table 1) may be used in agricultu­ re, although not all will have the same application range. Negatively- correlated cross-resistance between these chemicals has not yet been re­ ported. This implies once more the existence of a high selection pressure by fungicides that seem to be chemically unrelated. Such a phenomenon is important when one considers the possibility of using mixtures of fungi­ cides or alternating spraying schedules to delay or prevent development of resistant populations. The energy-dependent efflux mechanism for fenarimol in A. nidulans is also an interesting subject for further research. Comparable mechanisms may occur in pathogenic fungi. If so, one might be able to manipulate fen­ arimol uptake, for instance by inhibiting efflux activity with other chem­ icals. Such chemicals might enhance fungitoxicity of fenarimol and hence could be regarded as synergists. Experiments on this aspect of research are currently being carried out at the Laboratory of Phytopathology of the Agricultural University, Wageningen.

References

Barug, D. & A. Kerkenaar, 1979. Cross resistance of UV-induced mutants of Ustilago maydis to various fungicides which interfere with ergosterol biosynthesis. Mededelin­ gen van de Faculteit der Landbouwwetenschappen, Rijksuniversiteit Gent 41: 421-427. Buchenauer, H., 1976. Hemmung der Ergosterinbiosynthese in Ustilago avenae durch Tria- dimefon und Fluotrimazol. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 83: 363-367. Buchenauer, H., 1978. Inhibition of ergosterol biosynthesis by triadimenol in Ustilago avenae. Pesticide Science 9: 507-512. Cope, J.E., 1980a. Mode of action of miconazole on Candida albicans: Effect on growth, viability and K release. Journal of General Microbiology 119: 254-251. Cope, J.E., 1980b. The interaction of [3H] miconazole with Candida albicans. Sabouraudia 18: 211-228. Davidse, L.C., D. Looijen, L.J. Turkensteen & D. van der Wal, 1981. Occurrence of metal- axyl resistant strains of Phytophthora infestans in Dutch potato fields. Netherlands Journal of Plant Pathology 87: 65-68. Fuchs, A., S.P. de Ruig, J.M. van Tuyl & F.W. de Vries, 1977. Resistance to triforine: a nonexistent problem? Netherlands Journal of Plant Pathology 83S: 189-205. Fuchs, A. & M. Viets-Verweij, 1975. Permanent and transient resistance to triarimol and triforine in some phytopathogenic fungi. Mededelingen van de Faculteit der Landbouw­ wetenschappen, Rijksuniversiteit Gent 40: 669-706. Gale, E.F., A.M. Johnson, D. Kerridge & F. Wayman, 1980. Phenotypic resistance to mico­ nazole and amphotericin B in Candida albicans. Journal of General Microbiology 117: 535-538. Gasztonyi, M. & G. Josepovits, 1975. Biochemical and chemical factors of the selective antifungal effect of triforine. Acta Phytopathologica Academiae Scientiarium Hungari- cae 10: 437-446. Georgopoulos, S.G., 1977. Development of fungal resistance to fungicides. In: M.R. Siegel & H.D. Sisler (Eds): Antifungal Compounds, Marcel Dekker, New York, Vol. II, p. 439-495. Gunatilleke, I.A.U.N., C. Scazzocchio & H.N. Arst, 1975. Cytoplasmic and nuclear muta­ tions to chloramphenicol resistance in Aspergillus nidulans. Genetic Research 26: 297-305.

99 Hamilton-Miller,J.M.T. ,1974 .Funga lsterol san dth emod eo factio no fth epolyen ean ­ tibiotics.Advance s inapplie dMicrobiolog y 17:109-134 . Leroux,P .& M .Gredt ,1976 .Comparison sde smode sd'actio n fongitoxiqued utriadimefo n (Meb 6447),d utriarimo le td el atriforine .Phytopathologisch e Zeitschrift86 : 276-279. Leroux,P .& M .Gredt ,1978 .Effet sd equelque s fongicides systémiques surl abiosynthès e del'ergostéro l chezBotryti s cinereaPers. ,Pénicilliu mexpansu m Link,e tUstilag o maydis (D.C.)Cda .Annale sd ePhytopathologi e 10:45-60 . Leroux,P. ,M .Gred t& R .Fritz ,1976 .Similitude se tdifférence sentr e lesmode sd'ac ­ tiond e1'imazalile ,d utriadimefon ,d utriarimo le td el atriforine .Phytiatrie - Phytopharmacie 25:317-334 . Ragsdale,N.N. ,1977 .Inhibitor so flipi dsynthesis .In :M.R .Siege l& H.D .Sisle r (Eds):Antifunga lCompounds ,Marce lDekker ,Ne wYork ,Vol .II ,p .333-363 . Ragsdale,N.N .& M.A .d eWaard ,1977 .Stero lpattern s infenarimol-susceptibl ean dtoler ­ antstrain so fAspergillu snidulans .Proceeding so fth eAmerica nPhytopathologica l Society4 :218 . Schroeder,W.T .& R .Prowidenti , 1969.Resistanc et obenomy li npowder ymilde wo fcu ­ curbits.Plan tDiseas eReporte r53 :271-175 . Sherald,J.L. ,N.N .Ragsdal e& H.D .Sisler ,1973 .Similaritie sbetwee nth esystemi cfun ­ gicidestriforin e andtriarimol .Pesticid eScienc e4 :719-727 . Sherald,J.L .& H.D .Sisler ,1975 .Antifunga lmod eo factio no ftriforine .Pesticid e Biochemistryan dPhysiolog y5 :477-488 . Tuyl,J.M .van ,1977 .Genetic so ffunga lresistanc et osystemi cfungicides . MededelingenLandbouwhogeschoo lWageninge n77-2 :1-136 . Waard,M.A .d e& S.A . Gieskes,1977 .Characterizatio n offenarimol-resistan tmutant s ofAspergillu snidulans .Netherland sJourna lo fPlan tPatholog y83S :177-188 . Waard,M.A .d e& J.G.M ,va nNistelrooy ,1979 .Mechanis mo fresistanc et ofenarimo li n Aspergillusnidulans .Pesticid eBiochemistr yan dPhysiolog y 10:219-229 . Waard,M.A .d e& J.G.M ,va nNistelrooy ,1980 .A nenergy-dependen tefflu xmechanis mfo r fenarimoli na wild-typ e strainan d fenarimol-resistantmutant so fAspergillu snidu ­ lans.Pesticid eBiochemistr yan dPhysiolog y 13:255-266 . Waard,M.A .d e& N.N .Ragsdale ,1979 .Fenarimol ,a ne wsystemi c fungicide.In :H .Ly r & C.Polte r (Eds):Systemfungizide .Akademie-Verlag ,Berlin ,p .187-194 . Waard,M.A .d e& H.D .Sisler ,1976 .Resistanc et ofenarimo l inAspergillu snidulans . Mededelingenva nd eFacultei tde rLandbouwwetenschappen ,Rijksuniversitei tGen t41 : 571-578. Walsmley-Woodward,D.J. ,F.A . Laws& W.J .Whittington ,1979 .Studie so nth etoleranc e ofErysiph egramini sf .sp .horde ivaryin gi nrespons et otridemorp han dethirimol . Annalso fApplie dBiolog y92 :211-219 .

100 Resistance to the dicarboximide fungicides

R.E.Beeve r * LongAshto nResearc h Station,Lon gAshton ,Bristol ,U.K .

R.J.W. Byrde LongAshto nResearc h Station,Lon gAshton ,Bristol ,U.K .

Abstract

Resistance toth e dicarboximide fungicides (dichlozoline,iprodione , procymidone,vinclozolin )i sreviewed . Fungalvariant s resistant toth e dicarboximides areusuall y cross-resistant to aromatic hydrocarbon fun­ gicides.Th emod e of actiono fth e fungicides,an d themechanis m ofresis ­ tance isunknown .Resistanc e in Neurospora crassa ismultigenic , andre ­ sistantmutant s are abnormally sensitive tomedi a ofhig h osmoticpres ­ sure.Th e genetic basis ofresistanc e in Botrytis cinerea isno tclear , butvariant s oftensho wpoore r sporulation and are lessvirulen t than theirparents .Thes e two attributesprobabl y account forth e lacko fex ­ tensive development ofresistanc e inth e field todate .Nevertheles s vari­ ants showing substantial degrees of sporulation andvirulenc e havebee n isolated and there arerecen treport s of losso fdiseas e controlb y these fungicides inth e field. Sprayingprogramme s should notrel y exclusively onthes e fungicides.

Keywords: fungicide resistance,dicarboximid e fungicides, Botrytis ci­ nerea, Neurospora crassa.

Introduction

The term dicarboximide fungicideha sbee n loosely applied toanti-fun ­ galcompound s ofth egenera l formula showni nFigur e 1.Structura l formu­ lae forth emos t important ofth edicarboximide s -vinclozoli n ('Ronilan', BASFAG , F.R.G.), iprodione ('Rovral',Rhône-Poulen c Phytosanitaire,France ; also called glycophene)an dprocymidon e ('Sumisclex', 'Sumilex', Sumitomo Chemical Company Limited,Japan ;als o called dicyclidine)ar egive ni n Figure 2.Th e firstdicarboximid e fungicide tob edeveloped , dichlozoline

* Permanentaddress :Plan tDisease sDivision ,Departmen to fScientifi can dIndustria l Research,Auckland ,Ne wZealand .

101 o II /c\

RiN R2

Figure 1.o Genera l structural formula of the dicarboximide fungicides;R has invariably been a 3,5-dichlorophenyl group,bu t R may have various structures.

('Sclex1,Sumitom o Chemical Company Limited, Japan) (Ménagere t al., 1971) waswithdraw n from themarke t soonafte r its introduction. Thepresenc e ofchlorinate d benzene residuesma yexplai nwh y fungal variants resistantt oth e dicarboximide fungicides are frequentlycross - resistant tocompound s ofth e aromatic hydrocarbon group,suc h asdiclor - anan dguintozen e (Georgopoulos &Zaracovitis , 1967). The dicarboximide fungicideshav ebee nuse dwidel y tocontro l diseases causedb yth etaxo - nomicallyrelate dpathogen s Sclerotinia, Monilinia and Botrytis; particu­ larly important aregre ymoul d ofgrape ,strawberr y andtomat o (causedb y Botrytis cinerea) andth ebrown-ro t diseases of fruit (causedb y Monili­ nia spp.). Iprodione hasbee nuse d againsta wide r spectrum ofdisease s inth e field thanhav e theothe r dicarboximides (Burgaud et al., 1975). The introduction ofth edicarboximid e fungicideswa sparticularl y welcome because ofth e onseto fresistanc e in Botrytis and Monilinia toth epre ­ viouslyver y effective carbendazim-generatingfungicides . Dichlozoline andvinclozoli n areconsidere d tob enon-systemi c (Ména­ ger et al., 1971;Eichhor n& Lorenz , 1978), butther e isgoo d evidence thatprocymidon e istranslocate d inbea n andcucumbe r (Hisadae t al., 1977), andstrawberr y (Cookee t al., 1979b). Iprodione,initiall yreport ­ ed tob enon-systemi c (Burgaud et al., 1975), istranslocate d inpotat o (Cayley& Hide , 1980).

CH ci VvCH3 Vo °\\ /CONHCH 3 >v N CH3 O-O -CH3 CI 0' CH=CH r/ iYu 2 CI 0 CH3 vinclozolin iprodione procymidone

Figure 2. Structural formulae of some common dicarboximide fungicides.

102 Because ofth e emergence ofresistan t isolates ofman ypathogen s to various typeso f fungicide,th equestio nha sbee n asked: 'Willth edicar - boximide fungicides,too ,encounte r resistance problems inth efield?' .

Field experience

Thedicarboximid e fungicides havebee nuse d extensively inth e field for severalyears ,an dman yworker shav ebee n active inmonitorin gnatu ­ ralpopulation s forresistance . Inaddition , laboratory studies havebee n used to assess theprobabilit y ofresistanc e development inth e field. Resistantvariant s havebee n recovered frombot h the field and thelabo ­ ratory (Tables 1an d 2).A lacko fdiseas e control following artificial inoculationo f strawberryplant swit h resistantvariant s of Botrytis ci­ nerea hasbee n found (Hunter et al., 1979), but relatively fewexample s of loss ofcontro l ofnaturally-occurrin g disease havebee n reported. Maraitee t al. (1981)describe d aninstanc e in Botrytis cinerea onever ­ bearing strawberries subjected torepeate d dicarboximide applications; the lacko fcontro lwa sno tmarked ,however .Mor e recently therehav e beenreport s ofa lac k ofcontro l of Botrytis cinerea on Hebe spp.i na nursery (M.J.Griffi n& C .Gay ,persona l communication, 1981), onglass ­ house tomato and cucumber (W.F.T.Hartill ,persona l communication, 1981) and onglasshous e cyclamen (A.C.Pappas ,persona l communication, 1981). Holz (1979)attribute d aloca l outbreak of Botrytis cinerea ongrap e flow­ erst oth epresenc e ofresistan tvariants .Howeve r Lorenz (1980)foun d resistantvariant swer ewidesprea d ingrap evines ,bot h inth e field and intrials ,bu tdetecte d no lacko fcontrol .Similarl y iprodione-resistant variants of Botrytis squamosa ononio nwer e controlled by iprodione in trials (Presly et al-, 1980).

Mode of action

Despite intensive investigation,th emod e ofactio no fth edicarboxi - mides remains elusive.Thei r chemical similarity, their similar effects on fungi and thecross-resistanc e patterns shownb y resistantvariant s supportth e assumptiontha tal l fungicides inth egrou p share acommo n mode ofaction .Som eresponse s thatsugges ttha tiprodion ediffer s from theother s canb e accounted forb y the finding thatiprodion e rearranges to amuc h less active isomer inethanoli c ormethanoli c solutions,whic h areofte nuse d inbiologica l testing (Cookee tal. , 1979a).

103 Table 1.Report so fresistanc e todicarboximid efungicide s invariant s isolated fromth efield .

Species Origin Reference

Botrytiscinere a strawberryfruit sfro m Davis& Dennis ,197 9 dicarboximide fungicide Dennis& Davis ,197 9 trials,197 8 (8resistan t isolates)

grapeflowers ,sever einfectio n Holz,197 9

strawberry fruitfro m Pappase tal. ,197 9 iprodionetrial ,197 8( 1 resistantisolate )

fieldsurvey ,1978-197 9 Letham& Penrose ,198 0 (14/101isolate sresistant )

fieldsurve yo fgrap evine s Lorenz,198 0 treatedwit hdicarboximin e fungicides,1978-197 9 (resistantisolate scommon , 101site sexamined )

grape-vinetria lwit h Lorenz,198 0 dicarboximidefuncides , 1979 (resistantisolate s common)

strawberry fruitsfro m Maraite et al., 1981 dicarboximide fungicide trials,1977-197 9 (3/799 isolates resistant)

everbearingstrawberrie s Maraite et al., 1981 afterintensiv edicar ­ boximide fungicidetreat ­ ment,197 9(resistan t isolatescommon )

tomato,carrot ,an dstraw ­ Davis & Dennis, 1981a berry leaflitter ,197 8

fieldsurve yo fdifferen t Griffin & Gay, personal cropstreate dwit hdicar ­ communication, 1981 boximide fungicides,198 0 (5/804isolate sresistant )

Moniliniafructicol a field screening (3isolate s Sztejnberg & Jones, 1978 resistantou to fa numbe r tested)

Morphological effects

In contrast to many protectant fungicides, the dicarboximides affect spore germination less than mycelial growth (Buchenauer, 1976; Fritz et al., 1977; Pappas & Fisher, 1979). Germ-tube elongation of Botrytis ci­ nerea in sucrose solution is stimulated by very low concentrations

104 Table2 .Report so fresistanc et odicarboximid efungicide s invariant s isolatedo n fungicide-amended medium invitro .

Species Selection Rateo fresistanc e Reference conditions (per10 7spores )

Alternaria alternata iprodione (100) McPhee,198 0

Botrytiscinere a vinclozolino r <1- 5 Lerouxe tal. ,197 7 iprodione (30) (30-140wit hU.V . mutagenesis)

vinclozolin 0-85 (0-70wit h Gullino& Garibaldi , (30) U.V.mutagenesis ) 1979

procymidone 1.7-34.7 Hisadae tal. ,197 9 (20-500)

vinclozolin <0.1-2 0 Maraitee tal. ,198 0 (8.6)

iprodione (50) 0-8 Davis& Dennis ,1981 a

vinclozolin 0.2-14.8 (50)

Botrytissquamos a iprodione Preslye tal. ,198 0 (4-2500)

Botrytistulipa e iprodione 18 Chastagner& Vassey , (50) 1979

vinclozolin 400 (50)

Monilinia functicola iprodione 26 Sztejnberg& Jones , (30) 1978

vinclozolin 18 (30)

procymidone 770 (30)

Pénicilliumexpansu m iprodione Lerouxe tal. ,197 8 (30-100)

iprodione (150) 53 Rosenbergere tal. , 1979

vinclozolin 31 (150)

Ustilagomaydi s iprodione (50-200wit hU.V . Lerouxe tal. ,197 8 (30-100) mutagenesis) a.Valu ei nbracket s isth econcentratio no fth ename d fungicide inmg/1 . 105 ofvinclozoli n (0.143 mg/1), whereas atconcentration s of 1.43 mg/1an d above, elongation isrestricted , and thegerm-tube s become swollen and distorted (Buchenauer, 1976). However,eve n ata concentratio n of 143mg/1 ,2 6% ofth econidi a germinated.Hisad a &Kawas e (1977)showe d that ifgerminatio n takesplac e ina liquid-complet e medium containing procymidone,mos tgermling seventuall y burst.A similar distortion and burstingwa s foundwit h hyphal cells after theywer e incubated inth e presence of fungicide for2 hours .Dr yweigh t increasewa sno treduce d overthi speriod , althoughhypha l cellnumbe r decreased markedly (Hisada et al., 1978). Bursting ofcell s exposed to fungicide has alsobee nre ­ portedwit hvinclozoli n and iprodione (Eichhorn &Lorenz ,1978 ;Davi s & Dennis, 1981a). With Ustilago avenae, Buchenauer (1976)foun d amuc h greater inhibition ofsporidia l multiplication thano fdry-weigh t increase inth epresenc e ofvinclozolin , and the sporidia appeared swollen and branched. Protoplastsmad e from Botrytis cinerea myceliumwer e unaffected by ahig h concentration ofprocymidon e andeve nregenerate d abnormal cell walls after2 4hour s of incubation (Hisada& Kawase , 1977). Similaref ­ fects, including conidial andhypha lburstin g (Sharpies,1961 ;Georgopou - lose t al., 1967), production of swollen,distorte d hyphae (Macris & Georgopoulos,1973 ;Threlfall , 1972), rapid inhibition ofsporidia l mul­ tiplication (Tillman& Sisler, 1973), and regeneration ofcel lwal lb y protoplasts inth epresenc e of fungicide (Macris& Georgopoulos , 1973) havebee nreporte d forth e aromatic hydrocarbonfungicides . Although theseobservation s indicate adisturbanc e ofnorma l cell-wall synthesis,on eo rmor eo fth eresponses ,includin g stunting ofapica l growth,abnorma lhypha l swelling and occasional bursting ofcell s arein ­ ducedb y diverse anti-fungal compounds,includin g theprotei n inhibitor cycloheximide (Sternlichte t al., 1973), the antitubulin carbendazim (Howard& Aist , 1977), hexose analogues such assorbos e (Moore,1981 )an d theenigmati c cytochalasins (Betina et al., 1972;Alle n et al., 1980). Thus thesemorphologica l effects,b y themselves,provid e little insight into themod e ofactio no fth edicarboximides .

Metabolic effects

Dicarboximides havevirtuall y no influence onrespiratio n (Buchenauer, 1976; Fritze t al., 1977;Hisad a et al., 1978;Pappa s& Fisher, 1979). Membrane integrity, asjudge db y efflux ofelectrolyte s (Buchenauer, 1976; Pappas &Fisher ,1979 )o r14 C-labelledmetabolite s (Hisada & Kawase, 1977), isno taffected ,no r isuptak e of C-glucos1e4 (Hisad a et al., 1978). Although someworker srepor t little orn oeffec to nsyn ­ thesis ofDNA ,RN Ao rprotei n (Buchenauer, 1976;Pappa s& Fisher, 1979), Fritze t al. (1977)an dHisad a et al. (1978)foun dreduce d rateso fin ­ corporation ofradioactively-labelle d uridine.Stero lmetabolis m is

106 little influenced by dicarboximides (Buchenauer, 1976;Frit z et al., 1977; Pappas &Fisher , 1979). Althoughmos t lipid fractions areunaffect ­ edb ythem , anincreas e inth e free fatty-acid fractionha sbee nreport ­ eda numbe r oftime s (Buchenauer, 1976;Frit ze t al, 1977;Pappa s & Fisher, 1979). Hisada etal . (1978)foun dprocymidon e ata ninhibitor y concentration stimulated cellwal l synthesis,a s judgedb ywal l dry-weightincrease , although therewa sn omarke d effecto nwal l composition.The y reported an enhancement ofth e incorporation ofradioactivel y labelled acetate,D - glucose andD-glucosamin eint oth e cellwall , incontras tt oPappa s & Fisher (1979), who found asligh treductio n inincorporatio n ofD-gluco ­ samine andN-acetyl-D-glucosamine . Thisbrie f survey indicates thatdespit e somepossibl e leads,n ometa ­ bolic stepha sye tbee n identified thatca nb eunequivocall y linkedwit h the site ofaction .A similar situation is foundwit hth e aromatichydro ­ carbons (Tillman& Sisler, 1973;Threlfall , 1968).

Genetic effects

The ability ofcertai n fungicides to stimulate somatic segregation of genetically-marked diploid strains of Aspergillus nidulans was suggested byGeorgopoulo s et al. (1976)a sa criterio n forgroupin g fungicides into those thatdo ,an d those thatd onot ,interfer ewit h thehereditar y pro­ cess.Antifunga l compounds such asth echitin-synthas e inhibitorpoly - oxinD , theprotein-synthesi s inhibitor cycloheximidean d the respiratory inhibitor carboxinha d noeffect ,wherea s theDN A intercalator actinomy- cinD , the antitubulinsgriseofulvi n andbenomy l and fungicides inth e aromatic hydrocarbon group all stimulated segregation.Th emechanis m of segregationma y differ depending onth e agentresponsible :Kappa s (1978) provided evidence thatwherea sbenomy l causesnon-disjunction , thearoma ­ tichydrocarbon s actprimaril yvi a abreakage-deletio n mechanism,al ­ thoughAzeved o et al. (1977)sugges ttha tth e aromatic hydrocarbons act mainlyvi a non-disjunction. More recently, Georgopoulos et al. (1979)foun d thatth e dicarboximide fungicides, iprodione,procymidon e andvinclozoli n also stimulate somatic segregation andthe y concluded that 'theireffec to nth echromosomes ,an d possibly onth emitoti c spindle,i sa mai n reason forthei r fungitoxici­ ty'. Thi s conclusion seemspremature ,a s sucheffect s couldb e secondary, or atleas treflec t actiona tonl yon eo f anumbe r of sites.Eve nbeno ­ myl, which isusuall y classified as afungicid e active onth enucleu s (Georgopoulos,1977 ;Lerou x & Fritz, 1978), causes arapi d reduction in hyphal linear growth rateunrelate d to itsmitoti c effect (Howard& Aist , 1977; 1980). Bothnuclea r andhypha l tipeffect s are secondary toth e primary actiono fbenomyl ,whic h ist opreven tmicrotubul e assembly,a

107 process required forbot hproductio n ofth emitoti c spindle and thecyto ­ plasmic 'cytoskeleton'.Recentl y Bellincampi et al. (1980)reporte d that the antifungalpolyene s amphotericin B andpimaricin ,th e sterol-synthe- sis inhibitor fenarimol, andmiconazole ,whic h isactiv e onth eplasm a membraneATPas e (Dufour et al., 1980), can stimulate somatic segregation. Although theseresult s cast some doubto nth e specificity ofth e somatic segregation test,the y cannotb e satisfactorily reconciled with theear ­ lier results ofGeorgopoulo s et al. (1976), who found that thepolyen e nystatin and the sterol synthesis inhibitor triarimolwer ewithou tef ­ fect.

Nodes of resistance

Themechanism s whereby fungibecom e resistant to fungicides arediver ­ se (Georgopoulos, 1977). Insom e instances themechanis m may involve the site ofaction ,givin g aninsigh tint oth emod e ofaction ,bu t inothe r instances itma yb eunrelated . Little isknow no fth e resistancemecha ­ nisms toth edicarboximides .Lerou x etal . (1978)foun d iprodione uptake by resistantvariant s of Botrytis cinerea and Pénicillium expansion tob e little different from thato f sensitive isolates,althoug huptak e ofpro - cymidonedecrease d with resistantvariants .Pappa s etal . (1979)foun dn o difference inuptak e ofthre e dicarboximides between dicarboximide-resis- tant and sensitive isolates of Botrytis cinerea. Recently Beever (unpublished data)foun d that some osmotic mutants of Neurospora crassa, incontras tt owil d types,ar e resistant to fungicides inth e dicarboximide and aromatic hydrocarbon group,i.e . these loci are in factresistanc e loci. Osmotic mutants arecharacterise d by theirab ­ normal sensitivity tohig hosmoti cpressure ,bein g inhibited, forexam ­ ple, onminima lmediu m supplemented withNaC lwit h aconcentratio n of 40g/ 1 (Mays,1969) . Some showth eremarkabl e property ofgrowin g as protoplasts inliqui dmedi a ofhig h osmoticpressur e (Hamilton& Calet , 1964). Theproduct s ofth e osmotic genes are asye tunknown ,bu ton esug ­ gestion istha tthe y are involved inth esuppl y ofprecursor s toth e cell-wall synthesizing enzymes (Mays,1969) , and, indeed, the strainsd o show somedifference s inoveral l cell-wall composition (Livingston, 1969). Their abnormal osmotic sensitivity could result from an inability toproduc e intracellular solutes,suc ha spolyol s (Jennings &Austin , 1973), ata concentratio n sufficient tocounterbalanc e the externalosmot ­ icpressure .

Genetics of resistance

Mutants resistant toth e aromatic hydrocarbons havebee n isolated ina number of fungi. In Nectria haematococca fivechromosoma l locitha tca n

108 confer resistance havebee n indentified (Georgopoulos, 1977), and in As­ pergillus nidulans twochromosoma l loci areknow n (Threlfall, 1968).Be ­ causevariant s resistant toth edicarboximide s frequently exhibitcross - resistance toth e aromatic hydrocarbons,thes e same lociwil l probably alsob e dicarboximide-resistance loci,bu tconfirmator y tests need tob e performed. Inth e discussion ofmode s ofresistanc e wenote d thatcertai n osmotic mutants of Neurospora crassa areresistan tbot h todicarboximid ean d aro­ matic hydrocarbon fungicides.Ther e are six osmotic genes in Neurospora crassa, allmappin g inlinkag e group I (Mays,1969 )and , so far,mutant s of os-1 and os-4 havebee nteste d and foundt ob e resistant (R.E.Beever , unpublished data). Inaddition , threemutant s selected asresistan t tovin - clozolinwer e foundt ob e osmoticallysensitive ,bu tw e dono tkno wwhe ­ ther allosmotically-sensitiv e strains areresistant ,o rwhethe r allre ­ sistant strainswil lprov e tob eosmoticall y sensitive.However , iti s clear thatmor e thanon e chromosomal gene canmutat e toconfe r resistance . inth e fungus.A swit h other species,spontaneou s resistant sectorsrea ­ dily ariseo n fungicide-containing media inoculated withplug s of Neu­ rospora crassa mycelium. This canb e reasonably accounted forb y acombi ­ nation ofth e fungistatic actiono fthes e fungicides,whic h allowsdeve ­ lopmento fa dens e compactcolon y fromwhic h aresistan tmutan tnucleu s cansegregat e ina rapidl y growing sector,an db y thenumbe r of loci that canmutat e togiv eresistance . Dicarboximide-resistantvariant s havebee n isolated from anumbe r of plantpathogen s (Tables 1an d 2).Althoug h little isknow no fth egeneti c basis ofresistanc e inthes e instances,som e attentionha sbee n givent o stability ofth evariants .Wit h Alternaria alternata, resistantvariant s retained their resistance following anumbe r ofsuccessiv e transfers in vitro, and single spore isolates retained the characteristics ofthei r parents (McPhee, 1980). With Botrytis cinerea, mostworker s have found thatresistan tvariant s retain their resistance after subculture ona fungicide-free medium (Leroux et al., 1977;Schüep p &Küng , 1978;Loren z & Eichhorn, 1978;Garibald i &Gullino ,1979 ;Hisad a et al., 1979).How ­ everDavi s& Denni s (1979)reporte d thatresistanc e was sometimes losti f newly isolated variantswer e notsubculture d initially on afungicide - containingmedium . Garibaldi &Gullin o (1979)foun d that less than 50% ofconidi a fromresistan t colonies growno n a fungicide-freemediu m gave resistant colonieswhe nnewl y isolated variantswer e used,bu t after five generations ofsingl e spore isolationsmos to r allproduce d resistant col­ onies.Growin g resistantvariant s on afungicide-amende d medium increased theproportio n ofresistan tconidi aproduce d (Gullino& Garibaldi ,1979 , 1980). These results arereminiscen to fthos e ofEsuruos o &Woo d (1971) for aromatic hydrocarbon fungicides.The y found thatonl y abouthal fth e sporesproduce d by resistantvariant s inth e absence of fungicide gave

109 resistantcolonies ,wherea s all sporesproduce d by thesevariant s inth e presence of fungicidevapou r gaveresistan t colonies.Furthe r testing of the sensitive coloniesproduce d inth e absence of fungicide showed that they gaveonl y sensitive colonies,wherea s second-generation resistant colonies gave amixture .Suc hinstabilit y canb e explained fromth eknow ­ ledgetha t Botrytis cinerea conidia aremultinucleat e (Epton& Richmond , 1980)an do nth ereasonable ,bu tunproven , assumptions thatresistanc e is chromosomal and dominant inheterokaryons .Newl y isolated resistantva ­ riants areprobabl y oftenheterokaryotic ; growth inth epresenc e offun ­ gicideswoul d increase theproportio n ofresistanc e nuclei inconidi a produced. Insom e instances,resultin g resistant strainsma yb ehomokary - otic forresistanc e nuclei (Webster et al., 1970). Thepersistenc e ofth eresistanc e character differswhe nmixture s of conidia of sensitive andresistan t isolates inoculated into fungicide- freemedi a are subcultured. Hisada et al. (1979)foun d arapi d reduction inth eproportio n ofresistan t conidia,bu tthe y examined onlyon ecombi ­ nation.Marait ee t al. (1980)showe d that although resistance israpidl y lost insom ecombinations ,i nothe rcombination s ahig hproportio n ofco ­ nidia retained resistance;simila r resultswer e found for subcultures transferred oncucumbe r slices.Thes e differencesma y inpar treflec t differences insporulatio n ofth edifferen t isolates,bu tthe yma y also reflectdifference s inth e extento fheterokaryo n formationbetwee nth e variouspairs . Thecross-resistanc e patterns and thedegre e ofresistanc e shownb y resistantvariant s differ.Lerou x et al. (1977)examine d arang eo f Bo­ trytis cinerea variants selected on iprodione orvinclozoli n atconcen ­ trations of3 0mg/1 , oro ndiclora n at10 0mg/1 ; they found thatth e variants couldb e classified into four groups,viz .a ,b ,c , d.Dat a for representative variants aresummarize d inTabl e 3.Late rwor kb y Leroux etal . (1978)showe d thatth ebehaviou r ofGroup s c and do fth etabl e wasno t stable;Grou p cvariant sbecam e sensitive todiclora n andquinto - zene andGrou p dvariant sbecam e resistantt o thesetw o fungicides.A distinctive feature ofsom evariant s istha tthei rgrowt h is stimulated bymoderat e concentrations of fungicide (Leroux et al., 1978;Denni s & Davis, 1979;Marait e et al., 1981).

The existence ofvariant s ofrelativel y lowresistanc e todicarboxi - mides (e.g.Grou p c)coul db e of significance inth e field (Maraite et al., 1980, 1981). By selecting forresistanc e ata lo wconcentratio n (iprodione, 2 mg/1), Beever (unpublished data)ha s recovered anumbe r of resistant variants of Botrytis cinerea with ED5Q values formycelia l growth against iprodione inth e range 2-10 mg/1.Althoug h some showed high resistance to

dicloran (ED50 > 100mg/1) , otherswer e only slightly resistant

(ED50 2-10 mg/1).

110 Table3 .Resistanc epatter no fvariou sstrain so fBotryti scinerea . T -i,_ 1/ -> ED ovalue so ffungicide s forlinea rgrowt ho fmyceliu m (mg/1) Isolate (group)5 5U ° ° 6 dichlozolin vinclozolin iprodione dicloran quintozene

CultureS 0.1 0.15 0.3 0.7 2 benomylsensitiv e

CultureB 0.2 0.25 0.4 1 1.5 benomylresistan t

SUG2 (a) >100 0 >100 0 1000 >10 0 >10 0

S0V1 (b ) >100 0 >100 0 15 >10 0 >10 0

SOD3 (c) 2 3 1.5 >100 >10 0

SOG1 (d ) >100 0 >100 0 900 1.5 1

Source:afte rLerou xe tal. , (1977,Table s2 & 6) .

1.Resistan tculture swer eselecte d invitr ofro mcultur eS ;U designate sisolate s recovered afterultra-viole t irradiationo fconidia ;0 designate sisolate sno t irradiated;G designate s selectiono nglycophen e (iprodione),V selectio no nvinclo ­ zolinan dD selectio no ndicloran .

Benomyl-resistantvariant s fromth e field areusuall y sensitive toth e dicarboximide fungicides (Buchenauer, 1976;Eichhor n& Lorenz , 1978), but variants exhibiting dual-resistance arereadil y isolated following their exposure todicarboximide s invitr o (Leroux et al., 1977).

Fitness of resistant variants

Are resistantvariant s ecologically less fittha n sensitive strains? The field results reported todat e (p.103)sugges t thatthi sma yb eso . Various attributes ofresistan tvariant s havebee nexamine d inth elabo ­ ratory inth e anticipation thatthes ema y revealpropertie s thatcoul d account forsuc hreduce d fitness.Simila r studies ofvariant s resistant to sterol inhibitors have indicatedwh yresistanc e tothes e fungicides mayneve rbecom e aproble m (Fuchse t al., 1977). There arever y fewdat a forspecie s other than Botrytis cinerea and eveninterpretatio n ofth e data forthi s species iscomplicate d by itswel l knownvariabilit y (Grindle,1979 ;Lorbeer , 1980). Manywil d strains arenaturall yhetero - karyotic (orheteroplasmons )an dmonoconidia l isolates selected from such strains oftendiffe rmarkedl y among themselves and from theirparents . Mucho fth evariatio n shownb y resistant isolatesma yb e areflectio no f thisvariability , rather thana consequenc e ofth epresenc e ofa particu ­ larresistanc egene .

111 Morphological andphysiologica l properties

Lineargrowt h rateshav eno wbee nmeasure d for aconsiderabl e number of Botrytis cinerea variants,an dvalue s range fromwel lbelo wt owel l above those ofth e respectiveparent s (Lerouxe t al., 1978;Hisad a et al., 1979;Gullin o& Garibaldi , 1979;1980 ;Marait e et al., 1980). Coni- dialproductio n on agarmedi a isusuall y less thantha to fth eparent ; values forresistan tvariants ,expresse d asa proportio n ofthei rpa ­ rents, range from 1% to3 0% (Leroux et al., 1978)an d 0% to3 4 % (Hisada et al., 1979). Gullino& Garibald i (1979)repor t amea nvalu e of 32 %fo reigh tresistan tvariants .Howeve r some laboratoryvariant s des­ cribedb yMarait e et al. (1980)showe d conidiation well inexces s oftha t ofman ywil d isolates,an dMarait e et al. (1981)not e thatconidiatio n of resistantvariant s from the field 'showedth e same range ofvariation 'a s conidiation ofsensitiv e ones. Conidialviabilit y ofresistan tvariant s was greater than 90% foral lvariant s studiedb yDavi s &Denni s (1981b). A reduction innumbe r andbranchin g ofger m tubeswa snote db yLerou x et al. (1977). Someresistan tvariant s of Botrytis cinerea produce relatively few sclerotia,bu tother sproduc e asman y asthei rparent s (Hisadae t al., 1979; Gullino& Garibaldi , 1980). Resistantvariant s are apparently much more sensitive tocoppe r oxychloride thanwil d strains,althoug h only fourvariant s havebee nteste d (Garibaldi &Gullino , 1979). Thedicarbox - imideresistanc e ofth e osmotic mutants of Neurospora crassa prompted theexaminatio n ofth eosmoti c sensitivity ofresistan tvariant s of Bo­ trytis cinerea selected inth e laboratory. Thegrowt ho fman y resistant variantswa s indeed strongly inhibited on amalt-aga rmediu m supplemented withNaC lwit h aconcentratio n of4 0g/ l (R.E.Beever ,unpublishe d data), apropert y thatcoul dwel lb eo f significance indeterminin gvirulence .

Virulence

Workers have tested thevirulenc e (relativepathogenicity )o fvariant s ina numbe r ofways .Th etest s canb e grouped intothos etha tus e aconi ­ dial inoculum, whichmimi c theestablishmen t ofprimar y diseasesites , and those thatus emycelia l plug inoculum,whic hmimi c secondary spread ofdiseas e as,fo rexample , from rotting fruit.W ewil l consider first those tests thatuse d aconidia l inoculum.Hisad a et al. (1979)measure d lesiondiamete r onseedlin g leaves ofcucumbe r and found that although many strainswer e non-virulent, ormuc h lessvirulen t thanthei rparents , themos tvirulen t strainha d avalu e of8 8% of itsparent .Rathe r simi­ lar resultswer e reportedb yGaribald i &Gullin o (1979),wh omeasure dre ­ lativenecrosi s onbroa dbea n leaves;althoug h thevirulenc e of resistant variantswa s always lesstha ntha to fthei rparents ,certai n resistant

112 variantswer emor evirulen t thansom e ofth e sensitive strains.Marait e et al. (1980)foun d that,relativ e tothei rparents ,highe rnumber s óf conidia ofresistan tvariant swer eneede d toestablis h infection oncu ­ cumberslices . Themycelial-plu gtechniqu e has alsobee nwidel y used.Measurin g the degree ofcolonizatio n of slices ofcucumbe r fruits,Lerou x etal . (1977; 1978)foun d that althoughman y resistantvariant s had lowvirulence , otherswer e atleas t asvirulen t asthei rparents .Marait e et al. (1980; 1981)reporte d thatth erat e ofcolonizatio nb y resistantvariant swa s usually reduced,bu tsom e strains,includin g those isolates fromth e field,colonize d rapidly. Similarresult swer e reported by Schiiepp& Kün g (1978)wit h apple fruits.Gullin o& Garibald i (1979)measure d weight loss from grapes and found that allvariant s incitedmuc h lessweigh tlos s thanthei r respective parents,excep t inon e instancewher e theparen t itselfwa s almostnon-virulent ;thre eo fth evariant s werenon-virulent . Itwoul db e interesting to determinewhethe r there isa correlatio nbe ­ tweenosmoti c sensitivity, asjudge db y inhibition onaga rmedia ,an dvi ­ rulence.A s grapes loseweigh tthei rconten to f soluble sugars canreac h very high concentrations.Th evariant s studiedb yDavi s& Denni s (1981a) were all able to infectan dcoloniz e strawberry fruits and tomatoleaves , from amycelia l inoculum,bu tcolonizatio n ofeithe rwhol e carrots,o r carrotdiscs ,wa sver y restricted. Inconclusion , itappear s thatwhil e resistantvariant s areusuall y lessvirulen ttha nthei r sensitiveparents ,wher e these are available for comparison,th e range ofvirulenc e shownb ybot h groups isbroa d andca n overlap.

Fieldperformanc e and thediseas e cycle

Whether resistantvariant swil l cause significant epidemics inth e field, ineithe r thepresenc e or absence ofth e selective fungicide,de ­ pends onman y factors,includin g thevariant' s ability tooverwinter , produce conidia abundantly and infectan d colonize tissue efficiently.A poorperformanc e atan yphas e ofth ediseas e cyclewil l reduce apatho ­ gen's overall effectiveness. There areman y difficulties inextrapolatin g laboratory tests toth e field,bu tonl y a fewworker s have carried out field trials.B y spraying strawberry flowers inth e fieldwit h conidial suspensions,Hunte r et al. (1979)an dDavi s& Denni s (1981b)showe d thatresistan tvariant s of Bo­ trytis cinerea cancaus ediseas e onth e fruit;treatmen twit h thedicar - boximides increased theproportio n ofrot scontainin g thedicarboximide - resistant strains.Resistan tvariant s have alsobee n shownt opersis t for manymonth s onstrawberry-lea f debris inth e field (Huntere t al.,1979 ; Maraite etal ,1981 ;Davi s& Dennis , 1981b).Tw o factors identified in

113 the laboratory tests merit consideration as major reasons for the reduced fitness of resistant variants. First is the reduced sporulation ability. Undoubtedly this can account for the poor performance of many variants, including those in the field trials of Davis &Denni s (1981b). However certain resistant variants do show high sporulation rates and data indi­ cating spread of resistance in the field have been obtained (Maraite et al, 1980, 1981). The second factor is the decreased virulence of resis­ tant variants, although a slight reduction in virulence might still give variants capable of inciting epidemics, especially in the presence of se­ lective fungicides. Such considerations, and the recent reports of breakdowns of disease control in the field, make it unwise to rely on the sole use of the di- carboximide fungicides in a spraying programme. The prudent approach must be to try to prevent resistance development by adopting programmes that alternate or mix fungicides for which fungi do not show cross-resistance (Griffin, 1980).

Acknowledgements

We are grateful to colleagues who have supplied us with unpublished information. R.E. Beever acknowledges the Department of Scientific and Industrial Research, New Zealand, the Trimble Agricultural Research Fund and the Underwood Fund for generous and financial support, which enabled him to spend a period at Long Ashton Research Station, during which this article was written.

References

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116 Pappas,A.C .& D.J .Fisher ,1979 .A compariso no fth emechanism so factio no fvinclo - zolin,procymidone ,iprodion ean dprochlora zagains tBotryti s cinerea.Pesticid e Science 10:239-246 . Pappas,A.C. ,B.K .Cook e& V.W.L .Jordan ,1979 .Insensitivit y ofBotryti scinere a toiprodione ,procymidon ean dvinclozoli nan d theiruptak eb yth efungus .Plan t Pathology28 :71-76 . Presly,A.H. ,R.B .Maude ,J.M . Miller& A .Large ,1980 .Toleranc e inBotryti ssqua ­ mosat oiprodione .Nationa lVegetabl eResearc hStatio n (U.K.)Annua lRepor t1979 : 62-63. Rosenberger,D.A. ,F.W .Meye r& C.V . Cecilia,1979 .Fungicid e strategies forcontro l ofbenomyl-toleran tPénicilliu mexpansu mi nappl estorages .Plan tDiseas eRepor ­ ter63 :1033-1037 . Schiiepp,H .& M .Kling ,1978 .Gegenübe rDicarboximi d -Fungizide ntolerant eStämm evo n Botrytiscinerea .Berich tde rSchweizerische nbotanische nGesellschaf t88 :63-71 . Sharpies,R.O. ,1961 .Th efungitoxi ceffect so fdiclora no nBotryti scinerea .Procee ­ dingso fth eBritis hInsecticid ean dFungicid e Conference 19612 .Associatio no f BritishManufacturer s ofAgricultura lChemicals ,London ,p .327-336 . Sternlicht,E. ,D .Kat z& R.F .Rosenberger ,1973 .Subapica lwal lsynthesi san dwal l thickening inducedb ycycloheximid e inhypha eo fAspergillu snidulans .Journa lo f Bacteriology 114:819-823 . Sztejnberg,A .& A.L .Jones ,1978 .Toleranc eo fth ebrow nro tfungu sMonilini a fructicolat oiprodione ,vinclozoli nan dprocymidon e fungicides.Phytopatholog y News 12:187-188 . Threlfall,R.J. , 1968.Th egenetic san dbiochemistr y ofmutant so fAspergillu snidu ­ lansresistan tt ochlorinate d nitrobenzenes.Journa lo fGenera lMicrobiology . 52:35-44 . Threlfall,R.J. , 1972.Effec to fpentachloronitrobenzen e (PCNB)an dothe rchemical s onsensitiv ean dPCNB-resistan t strainso fAspergillu snidulans .Journa lo fGene ­ ralMicrobiolog y 71:173-180 . Tillman,R.W .& H.D .Sisler ,1973 .Effec to fchlorone bo nth egrowt han dmetabolis m ofUstilag omaydis .Phytopatholog y 63:219-225 . Webster,R.K. ,J.M .Ogaw a& E .Bose ,1970 .Toleranc eo fBotryti s cinerea to2,6 - dichloro-4-nitroaniline.Phytopatholog y 60:1489-1492 .

117 Acylalanines: resistance in downy mildews, Pythiuman d Phytophthora spp.

L.C.Davids e Laboratory ofPhytopathology ,Agricultura l University,Wageningen , The Netherlands

Abstract

Inlaborator y experiments selection forresistanc e in fungit oth e acylalanines appears tob e quiteeasy . Isolateswit h ahig h level ofsta ­ ble resistance andunimpaire d virulence couldb eobtaine d from Phytoph­ thora capsici, P. megasperma f.sp . medicaginis and Pythium ultimum. Isolateswit h transient resistance orreduce dvirulence ,o rboth ,wer e frequently found.Resistanc e inviv o doesno talway s correlatewit hre ­ sistance invitro ,an d experiments with P. infestans yielded only iso­ lates thatwer e resistant invitro . Inpractice ,developmen t of resistance in P. infestans occurred quite rapidly afterexclusiv e useo fmetalaxy l under heavy diseasepressure ,an d also in Pseudoperonospora cubensis, the causal organism ofdown ymilde w ofcucumber .Th e importance of laboratory experiments toevaluat e thepotentia l oftarge t fungi todevelo presis ­ tance to fungicides isemphasized . Suchresult s canb e ofgrea tvalu e for the development ofus e strategies forne w fungicides.

Keywords: acylalanines,metalaxyl , furalaxyl,milfuram , Phytophthora cap­ sici, Phytophthora infestans, Phytophthora megasperma f.sp . medicaginis, Pythium ultimum.

Introduction

The acylalanines havebecom e ina shor ttim e increasingly important in thecontro l of several importantplan tpathogeni c fungibelongin g toth e orderPeronosporales .Damagin gpathogen s such asdown ymildew s of anum ­ ber ofcrops ,potat o lateblight ,blu emol d of tobacco and soil-borne Pythium and Phytophthora spp.ar e amongth emai n target fungi.Th e sys­ temicbehaviou r ofth e acylalanines makes them excellent forcontro l of downymildew s ofsevera l crops intropica l areas,wher e rapid crop growth andheav yprecipitatio n oftenrequire sver y frequentapplicatio n ofcon ­ ventional fungicides;wit h the acylalanines longersprayin g intervals are

118 feasible. This groupo ffungicide s includesmetalaxy lan dfuralaxyl ,whos ebio ­ logicalpropertie s havebee n studiedi ndetai l (Brücke tal. , 1980;Cohe n et al., 1979;Hicke y& Coffey ,1980 ;Stau be tal. , 1978,1980 ;Stau b& Young, 1980), togetherwit ha numbe ro fexperimenta l chemicals.Structu ­ ral formulaear egive ni nFigur e1 ;th eter m acylalanine doesno tstrict ­ lycove ral lth ecompound s since threeo fthe md ono thav ea nalanin emoie ­ ty. Sincei ti sdifficul tt ofin da mor e adequate name,an dsinc eth eter m acylalaninei sestablishe d inth eliterature , thister mwil lb euse d throughout thiscontribution . Their selectivityan dsystemi cbehaviou r suggesttha tthes e compounds acti na ver y specific way. Interference withRN Asynthesi s isa propose d mechanism ofactio n (Fisher& Hayes ,1979 ; Davidse,1981b ;Kerkenaar , 1981).

°^, 0 CH \ CH3 CH3 CH 3 CH3 Ç' " CH3 \ 3 CH_CH3 CH CH3 W CH-CH3 // \\_N( // i_N( " C-CH2 . C-CH2-0-CH3 CH3JJ VH3 fli

'Galben' metalaxyl furalaxyl

CH3 C\ CH3 /—( CH-CH3 0-N< ^={ C-CH2-O-CH3 ^^ C-CH2-CI CH II CH3 II 3 0

CGA 29212 RE 26745

C-CH2-Cl CH2 cH3 X CH3 II J 0 HH3 r0 "°-

milfuram RE 26940 Figure1 .Structura lformula eo facylalanine-typ efungicides .

119 Therefore, ifth eproblem sme twit h other specifically-acting fungicides arean yexample ,developmen t ofresistanc e toth e acylalanines is apotent ­ ial threatt othei r long-lastingusefulness . Inthi scontributio n theus e andpredictiv e value of laboratory exper­ iments toevaluat e thepotentia l oftarge t fungidevelopin g resistance to acylalanines aredescribed .Tw o examples ofdevelopmen t ofresistanc e in practice areals opresente d and aspects ofresistanc e inviv o and invitr o discussed.

Development of resistance in laboratory experiments

Selection forresistanc e inacylalanine s ina numbe r of fungiha sbee n attempted insevera l laboratories toestimat e the risko fdevelopmen t of resistance underpractica l conditionsbefor e these compoundswer eintro ­ duced. Several approaches havebee nused . Staub etal . (1979)culture d isolates of Phytophthora infestans onmedi a atsubletha l concentrations of furalaxyl.Sporangi a and zoospores from isolates showing reduced sens­ itivity wereharveste d andplate d outo nmedi a athig h concentrations. In thiswa y several isolateswer e obtainedwhic hwere ,wit hrespec tt o ED5Q values,betwee n two andmor e than 100time s less sensitive to furalaxyl thanth e original isolates.Th e isolateswer e cross-resistant toCG A 29212 andmetalaxyl ,an d theymaintaine d theirresistanc e fora tleas t2 year s with repeated subculturing onmedi awithou tth e fungicides.Bu tthes e strainsha d either lostthei rvirulenc e orthe ywer e still fully sensitive totreatment swit h acylalanine fungicides ontomat o orpotat oplants . Hence invitr o resistance isno tnecessaril y related to inviv oresistance . Ingrowt h room experiments inwhic h afiel dpopulatio n of P. infestans was exposed to sublethal dosages ofmetalaxy l onpotat oplant s for1 1in ­ fectioncycles ,n o isolateswit h adecrease d sensitivity were obtained. Based onthi s study Staub etal .stresse d the importance of including inviv o tests instudie s dealingwit hresistanc e to acylalanines.Thei r results gave the impression -i twa s never stated -tha tresistanc e to acylalanines in P. infestans wasunlikel y tooccur . Bruin (1980)studie d the developmento fresistanc e tometalaxy l insev ­ eral fungi.Usin gmor eo r less similarprocedure s asStau b et al.(1979), heobtaine d after 6-12 transfers isolateso f Phytophthora capsici andse ­ veral Pythium spp.tha t showed interm s ofED 5Q values 100 -t o3 600 - fold decreased sensitivity tometalaxyl .U V irradiation ofzoospore s of P. capsici andmyceliu m ofbot h P. capsici and Pythium ultimum alsogav e highly resistantstrains .Som eo fth emetalaxyl-resistan t strainsre ­ tained theirhig h level ofresistanc e during 8month s ofsubculturin g in the absence ofth e fungicide;othe r strains slowly reverted tointerme ­ diateresistanc e levels or lostthei r resistance completely.Al l strains were usually (someexception s occurred)cross-resistan t tofuralaxyl ,

120 milfuram,R E 26745,R E2694 0 and 'Galben'.Th emetalaxyl-resistan tstrain s of P. capsici and P. ultimum appeared tob e stillpathogeni c onpepper s andpeas , respectively, and insensitive totreatment s withmetalaxy l at dosages thatcompletel y controlled disease developmentcause db y the ori­ ginalstrains . Like Staub etal . (1979), Bruin (1980)wa s not able toobtai nmeta ­ laxyl-resistant isolates of P. infestans after several cycles ofmas sse ­ lectionwit hsporangi ao nmetalaxyl-treate dpotato-tube r tissue.A simi­ larprocedur e with Peronospora parasitica spores,eithe runtreate d or treated withU V light,an d cabbage seedlings alsodi dno tresul ti nlos s ofsensitivity . Davidse (1980, 1981a)obtaine dmetalaxyl-resistan t iso­ lateso f P. megasperma f. sp. medicaginis, the causal organism ofroo tro t ofalfalfa , insevera lways .Adaptatio n andmas s selection from zoospores yielded 19 strainswit h arelativel y lowdegre e ofresistance , allo f whichwer e lessvirulen ttha nth eorigina l isolate ina n alfalfa-seedling assay.Onl y oneo fthos e strains showed resistance invivo .Mutageni c treatmento fth e zoosporeswit hN-methyl-N'-nitro-N-nitrosoguanidin e (NG) resulted inman y highly resistant strains,however .Hal f ofthos ewer ea s virulent asth e original isolate and aconsiderabl e number oftha thal f were alsohighl y resistant invivo . Metalaxyl ateve n1 0time s thedosag eneede d tocontro l rootro ti n mature alfalfaplant s caused by asensitiv e isolate failed toprotec t theseplant s fromth ediseas ewhe nthe ywer e inoculatedwit h resistant isolates.Th e resistance tometalaxy l appeared tob ehighl y stable,sinc e virulentresistan t strains didno tlos ethei rresistanc e after 12infec ­ tioncycle s ina seedlin g assay inth e absence ofth e fungicide;neithe r didmixe dpopulation s ofa resistan t and asensitiv e isolate under simi­ larconditions . Table 1summarize s theresult s obtained insevera l laboratories.Th e dataobtaine d inth elaborator y unequivocally showtha ta numbe ro ftar ­ get fungi ofacylalanin e fungicides haveth epotentia l todevelo p ahig h level ofresistanc e invitr o aswel l asi nviv owithou t any implications ast o theirvirulence .Thi s justifies theconclusio n thatdevelopmen t of resistance underpractica l conditions islikel yt ooccur .

Development of resistance in practice

Pseudoperonospora cubensis

During the 1979-1980 growing season,cucumbe r growers from thesouth ­ ernGree kmainland , Crete and Israel reported losso feffectivenes s of metalaxyl incontrollin g cucumber downymilde w causedb y Pseudoperonos­ pora cubensis. Growth-room experiments (Georgopoulus &Grigoriu ,1981 ; Katan& Bashi ,1981 ;Malathrakis ,1980 ;Pappas ,1980 ;Reuven i et al.,

121 Table1 .Developmen to fresistanc et oacylalanine si nlaborator yexperiments .

Organism Method Resistance Resistance factor inviv o invitr o

P.infestan s adaptationan dmas sselectio n <140 selectioni nviv o(1 2cycle so n foliage)

P.infestan s selectioni nviv o(1 2cycle so n tubers)

P.parasitic a selectioni nviv o(1 2cycle so n seedlingswit han dwithou t UVirradiatio no fspores )

P.capsic i adaptation 500 yes massselectio nfro mUV-irradiate d spores 600 yes

Pythiumspp . adaptation 14-360 0

P.ultimu m adaptationfro mUV-irradiate d mycelium 5000 0 yes

P.nicotinana e adaptation >100 massselectio nfro mNG-treate d zoospores >100

P.megasperm a adaptation >500 yes f.sp .glycine a massselectio nfro mNG-treate d zoospores >500 yes

P.megasperm a adaptation <1 0 no f.sp .medicagini s massselectio nfro mNG-treate d zoospores >500 yes a.Th eresistanc e factori sth erati oo fth eE D valuefo rth eresistan tstrai n totha tfo rth ewild-typ estrain . b. Source:Stau be tal .(1979) . c.Source :Brui n (1980). d.Source :Dekke re tal .(unpublishe d data). e.Source :Davids e& Krus e (1981). f.Source :Davids e (1980, 1981a).

1980)showe d thatth e fungicidewa s almostcompletel y inactive against isolates from these areas,eve nwhe n theplant s were sprayed atdosage s several times higher than recommended. Inoculation ofdetache d leaves floating on fungicide solutions demonstrated that aconsiderabl e differ­ ence existed inminima l inhibiting concentrations forsensitiv e andre ­ sistant isolates (Table 2).Resistanc e appeared tob ever y stable.Eve n maintaining theorganis m for2 0generation s over aperio d of7 month s on untreated plants didno tresul t inan y losso fresistance .

122 Table2 .Activit yo fmetalaxy lagains tsensitiv e (S)an dresistan t (R)isolate s ofPseudoperonospor acubensi s ina detache d leafassa y

Isolate AvAveragi ediseas eratin g atvariou smetalaxy lconcentration s (ug/ml)

0 10 50 100 250 500 i 00 9 10 6 1 1 0 2 (R) 10 10 5 2 0 0 5 (R) 10 9 5 4 0 0 6 (R) 9 10 10 7 1 0 9 (S) 10 0 0 0 0 0 Source:Pappa s (1980). a.Detache d leaveswer eplace d inpetr idishe swit hth elowe rsurfac ei ncontac t withaqueou sfungicid esolution s containingbenzimidazol e aspreservativ e (50^ig/ml) .Afte r2 4h eac hlea fwa sinverte dan dwhe nth esurfac ewa sdry , inoculatedwit h1 0droplet so fa sporangia lsuspension . b.Averag eratin go na 0-1 0 scale:0 = none ;1 0= maximu m infection.Ratin g madefo rthre ereplicate s fiveday safte rinoculation .

Phytophthora infestans

In the summer of 1980 Dutch potato farmers complained about the bad performance of metalaxyl in controlling potato late blight. Although the weather was very favourable for disease development only inadequate dis­ ease control was reported in fields sprayed with metalaxyl, and later on also from fields sprayed with a mixture of metalaxyl and mancozeb. Fail­ ure did not occur in fields treated with conventional fungicides only. To determine whether resistance had developed the sensitivities of several isolates were compared in a leaf-disc assay (Table 3) (Davidse et al., 1981). An up to 1 000-fold difference in sensitivity was noticed between the isolates. Isolates originating from fields where metalaxyl failed were usually highly resistant. Sensitive isolates were found in fields in other parts of the Netherlands where the disease incidence was low due to still adequate control by metalaxyl or conventional fungicides. Resistant strains probably arose independently in several areas, be­ cause resistant isolates belonged to at least two different races of the pathogen. The failure of the mixture to control the disease adequately was probably a result of the presence of an almost entirely metalaxyl-re- sistant population. At the levels applied, mancozeb obviously was not able to withstand the high disease pressure. The isolates maintained their high level of resistance for at least four months during weekly transfers on untreated potato tuber tissue. Re­ sistant isolates were also resistant to furalaxyl, milfuram and 'Galben'. Their sensitivity to cymoxanil, fosetyl-Al and propamocarb did not change.

Discussion

Rapid development of resistance to acylalanines can be observed in the

123 Table3 .Activit yo fmetalaxy lagains tsensitiv e (S)an dresistan t (R)isolate s ofPhytophthor ainfestan s ina detache d leafassa y

Isolate Averagediseas eratin g atvariou smetalaxy lconcentration s(^g/ml )

0 0.01 0.1 1 10 100

19 (S) 3.4 3.6 0 0 0 0 24 (S) 3.2 3.0 0 0 0 0 29 (R) 4.0 4.0 4.0 4.0 4.0 1.2 52 (R) 4.0 4.0 4.0 4.0 4.0 0 84 (R) 4.0 4.0 4.0 4.0 4.0 4.0

Source:Davids e (1981a). a.Potat olea fdisc swer eplace dupsid edow no nfungicid esolution s inpetr i dishesan d inoculatedwit ha drople to fa sporangia lsuspension . b.Averag e ratingo na 0- 4 scale:0 = none ;1 ,2 ,3 an d4 = 0-25 ,25-50 ,50-7 5 and75-10 0% ,respectively ,o fth esurfac eo fth edis ccovere dwit hsporangia . Ratingmad efo rfiv ereplicates .

laboratory and the field. Inth e laboratory, inadditio nt o isolates with stable resistance andnorma lvirulence , isolates withtransien t resistance and/or decreased virulence are fequently found.Resistanc e invitr o is not always correlated with resistance invivo .Thi sha s alsobee nobser ­ ved for some field isolates of P. infestans, which althoughhighl yresis ­ tant invitr o displayed normal sensitivity tometalaxy l ontube r or leaf tissue (Bruin,1980 ;Brüc ke t al., 1980). Anexplanatio n forthes ephenomen a canonl yb e speculative.Brui n (1980)foun d thatth eE D value forgrowt h inhibition ofsingl e sporeo r hyphal tip isolates originating from asingl e resistant straino f P. cap- sici variedbetwee n 0.1 and45 0 |jg/ml. Itindicate s aheterogeneou spopul ­ ationo fnucle i consisting of 'resistant' and 'sensitive'nucle i inth e parent strain.Assumin g asimila rheterogeneit y inoriginall y sensitive isolates itmigh texplai nth eeas ewit hwhic h Phytophtora and Pythium spp. adaptt ometalaxy l invitro .Adaptatio n inthi s case implies selectiono f nuclei.Whethe r orno tthes e strains revertt o sensitivity depends onth e presence or absence oforiginall y 'sensitive'nucle i inth e adaptedstrain . A similar heterogeneous distribution ofgene s forvirulenc e among thenu ­ cleimigh tresul ti na chang e invirulenc e during adaptation forsensiti ­ vity,th eoutcom e depending onth edistributio n ofthes egene s among 'sens­ itive' and 'resistant'nuclei .Variatio n invirulenc e among the asexual progeny isa frequentl y encountered phenomenon (Erwin, 1965). The 'resis­ tant'nucle iundoubtedl y originateb ymutation ,th erat e ofwhic hca nb e enhancedb ymutagens .Muc h ofthi sexplanatio n isbase d on circumstantial evidence thatstil lha s tob e confirmed experimentally.N o data areye t available aboutth ebiochemica l mechanism ofresistance ,bu tit shig hlev ­ el indicates thata decrease d affinity atth etarge tsit emigh tb e involv­ ed.Brui n (1980)an dDavids e (unpublished data)di dno t find indications ofa nenhance dmetabolis m ordecrease duptake .

124 Although now there areenoug h ofwha t appears tob e conclusive results from laboratory experiments topredic tth edevelopmen t ofresistanc eun ­ der fieldconditions ,tha tinformatio nwa sonl ypartl yo rno ta tal lavail ­ ablewhe n the acylalanineswer e first introduced. Onth econtrar yman y datawer e available from field experiments showing thegoo dperformanc e ofthes ecompound s andwithou t any signo fdevelopmen t ofresistance , evenunde rhig h infectionpressure .Th eproduce r involved, thedistribu ­ tors of acylalanines and sometimes the local adviserswer e facedwit hth e dilemma ofdecidin gho w toevaluat e the results of the laboratoryexperi ­ ments andho wmuc h importance togiv e them indevelopin g aus e strategy. For somecrop s and somedisease s therewa s almostn ochoice . InGreec e and Israel,fo rinstance ,th egrowin go fcucumber s inplasti c housesdur ­ ingth ewinte r seasoncreate s extremely favourable conditions forth e development of P. cubensis, whichwa s difficultt ocontro lbefor e the acylalanines were introduced. The continuous and exclusiveus e ofmeta - laxyl fornearl y twoyear s there ledt oth edevelopmen t ofresistan tpop ­ ulations ofthi sfungus . For anumbe r ofdisease s thatcoul d alreadyb e controlled efficiently withconventiona l fungicides,however , severalus e strategieswer eimagi ­ nable. Insuc ha cas ecommercia lmotive s oftendetermin e the strategy, and asale s strategy rathertha n aus e strategywa s sometimes followed. Soth e distributor ofmetalaxy l inth eNetherland s recommended analmos t exclusiveus eo fthi s fungicide tocontro lpotat o lateblight .Tha tre ­ commendationprove d tob e disastrous and theus eo fmetalaxy l forcontro l ofpotat o latebligh ti nth eNetherland s had tob e discontinued little moretha ntw oyear s after itwa s firstregistere dthere . Couldmixture s ofmetalaxy l and conventional fungicides have delayed orprevente d thedevelopmen to fresistanc e in P. infestans and P. cuben­ sis, provided thatothe r fungicides effective againstth e latterwer e available? Forbot hpathogen s aresistan tpopulatio n developed only after continuous and exclusiveus eo fmetalaxyl ,admittedl y under conditions very favourable fordisease . Ifth e conditions hadbee nles s favourable, or ifonl ymixture s hadbee nused ,woul d resistance stillhav e developed? No data are available toanswe r these questions.Circumstantia l evidence indicates thatmixture s affectth edevelopmen to fresistanc e in P. infes­ tans. Inth eU.K. ,wher e inth e summero f198 0 amixtur e ofmetalaxy l and mancozeb wasuse d tocontro lpotat o lateblight ,n ocase s of resistance havebee nreported .However , inth e sameyea r in Ireland,wher e metalaxyl was used exclusively, resistancewa s found. Theus eo fmixture s topreven t ordela y the development of resistance iscontroversial ,becaus e iti salmos t impossible toprov e theireffective ­ ness experimentally, andonl ymathematica lmodel s (Kable& Jeffery,1980 ; Delp, 1980;thi sbook ,p.177-186 )provid e some insight intotha teffec ­ tiveness,bu tth e facttha tth e companion fungicideha s its own retarding

125 effecto nth e development of thediseas ema y alone justify theiruse . Should resistance develop toon ecomponen t ofth emixture ,th e second mightkee p theresistan tpopulatio n low,ideall ybelo w the threshold lev­ el forcro p losses.However ,thi s approach requires astron g companion fungicide, forwhic h theremigh tb e economical or registrational restraints. Metalaxyl isno wmixe dwit h other fungicides tocontro l foliarpatho ­ genswit h ahig h reproduction rate (Staub &Schwinn , 1980). Anotherstrat ­ egyma yb e tocombin e itwit h other fungicidesbu t limit itsus et oonl y parts of acro p andus e it for limitedperiod s only.

References

Brück,R.I. ,W.E .Fr y& A.E .Apple ,1980 .Effec to fmetalaxyl ,a nacylalanin e fungicide ondevelopmenta l stageso fPhytophthor a infestans.Phytopatholog y 70:597-601 . Bruin,G.C.A. ,1980 .Resistanc e inPeronosporale s toacylalanine-typ efungicides . Ph.D .Thesis ,Universit y ofGuelph ,Ontario ,Canada .11 0p . Cohen,Y. ,M .Reuven i& H .Eyal ,1979 .Th esystemi cantifunga lactivit yo fridomi l againstPhytophthor a infestanso ntomat oplants .Phytopatholog y 69:645-649 . Davidse,L.C. ,1980 .Resistanc et oridomi l inPhytophthor amegasperm a f.sp .medi - caginis.Act aBotanic aNeerlandic a 29:21 6 (abstract). Davidse,L.C. , 1981a.Resistanc et oacylalanin e fungicides inPhytophthor amegasperm a f.sp .medicaginis .Netherland sJourna lo fPlan tPatholog y87 :11-24 . Davidse,L.C. ,1981b .Mechanis mo factio no fmetalaxy li nPhytophthor amegasperm a f.sp. medicaginis.Netherland sJourna lo fPlan tPatholog y87 :254-255 . Davidse,L.C .& H .Kruse ,1981 .Resistanc et oacylalanin e fungicides inPhytophthor a megasperma f.sp .medicagini san dP .megasperm a f.sp .glycinea .In :H .Ly r& C.Polte r (Eds):Systemisch eFungizid ean dAntifungal eVerbindungen ,Proceeding so f theV IInternationa lReinhardsbrun nSymposiu m onSystemi c fungicides,Friedrichroda , D.D.R.,1980 .Akademi eVerlag ,Berlin .I npress . Davidse,L.C ,D .Looyen ,L.J .Turkenstee n& D .va nde rWal ,1981 .Occurrenc eo fme ­ talaxylresistan tstrain so fPhytophthor a infestans inDutc hpotat o fields.Nether ­ landsJourna lo fPlan tPatholog y87 :65-68 . Delp,C , 1980.Copin gwit hresistanc et oplan tdiseas e controlagents .Plan tDiseas e 64:652-658 . Erwin,D.C. ,1966 .Varieta lreactio no falfalf a toPhytophthor amegasperm a andvari ­ ationi nvirulenc eo fth ecausa lfungus .Phytopatholog y56 :653-657 . Fisher,D.J . &A.L .Hayes ,1979 .Fungicides ,an dmod eo faction .Phycomycet efungicides . AnnualResearc hReport ,Lon gAshto nResearc hStatio n 1979.p .137 . Georgopoulos,S.G .& A.C .Grigoriu ,1981 .Metalaxy l resistantstrain so fPseudo - peronospora cubensis incucumbe rgreenhouse so fsouther nGreece .Plan t Disease65 :729-731 . Hickey,E.L .& M.0 . Coffey,1980 .Th eeffect so fRidomi lo nPeronospor apis i parasitizingPisu m sativum:a nultrastructura l investigation.Physiologica l PlantPatholog y 17:199-204 . Kable,P.F .& H .Jeffery ,1980 .Selectio nfo rtoleranc e inorganism sexpose dt o sprayso fbiocid emixtures :a theoretica lmodel .Phytopatholog y 70:8-12 . Katan,T .& E .Bashi ,1981 .Resistanc et ometalaxy l inisolate so fPseudoperonospor a cubensis,th edown ymilde wpathoge no fcucurbits .Plan tDiseas e65 :798-800 . Kerkenaar,A. ,1981 .O nth eantifunga lmod eo factio no fmetalaxyl ,a ninhibito r ofnuclei caci dsynthesi s inPythiu msplendens .Pesticid eBiochemistr y andPhysio ­ logy 16:1-13 . Malathrakis,N.E. ,1980 .Contro lo fdown ymilde wo fcucumbe rb ysystemi can dno n systemic fungicides.Proceeding so fth e5t hCongres so fth eMediterranea nPhyto - pathologicalUnion ,Patras ,Greece ,Septembe r21-27 ,1980 .p.145-146 . Pappas,A.C. ,1980 .Effectivenes s ofmetalaxy lan dphosetyl-A lagains t Pseudo­ peronospora cubensis (Berk& Curt )Rosto wisolate s fromcucumbers .Proceeding s ofth e5t hCongres so fth eMediterranea nPhytopathologica lUnion ,Patras ,Greece , September21-27 ,1980 .146-148 .

126 Reuveni,M. ,H .Eya l& Y .Cohen ,1980 .Developmen to fresistanc et ometalaxy li n Pseudoperonospora cubensis.Plan tDiseas e64 :1108-1109 . Staub,T. ,H .Dahme n& F.J . Schwinn,1978 .Biologica lcharacterizatio no fuptak e andtranslocatio no ffungicida lacylalanine s ingrap ean dtomat oplants . Zeitschrift fürPflanzenkrankheite nun dPflanzenschut z85 :162-168 . Staub,T.H. ,H .Dahme n& F.J . Schwinn,1980 .Effect so fRidomi lR o nth edevelop ­ mento fPlasmopar aviticol aan dPhytophthor a infestanso nthei rhos tplants . Zeitschrift fürPflanzenkrankheite nun dPflanzenschut z87 :83-91 . Staub,T. ,H .Dahmen ,P .Urec h& F .Schwinn ,1979 .Failur et oselec tfo ri nviv o resistance inPhytophthor a infestanst oacylalanin e fungicides.Plan tDiseas e Reporter63 :385-389 . Staub,T .& F.J . Schwinn,1980 .Fungicida lpropertie so fmetalaxy lan dit sus estrate ­ gies.Proceeding so fth e5t hCongres so fth eMediterranea nPhytopathologica lUnion , Patras,Greece ,Septembe r21-27 ,1980 :155-157 . Staub,T.H .& T.R .Young ,1980 .Fungitoxicit yo fmetalaxy lagains tPhytophthor a parasiticavar .nicotianae .Phytopatholog y 70:797-801 .

127 Can we estimate the fungicide-resistance hazard in the field from laboratory and greenhouse tests?

J.Dekke r Laboratory ofPhytopathology ,Agricultura l University,Wageningen , TheNetherland s

Abstract

The fitness ofth e fungicide-resistant strains compared totha to f sensitivewild-typ e strains iso fparamoun t importance forth ebuild-u p of aresistan tpathoge npopulation .Parameter s of fitnessma yb emeasure d inlaborator y and greenhouse tests.Wit h some fungicides,resistanc eap ­ pears tob e linked toreduce d fitness.I nsuc hcases ,th e larger the differencebetwee nresistan t and sensitive strains themor e thebuild-u p of aresistan tpathoge npopulatio nwil lb ehampered . Ifth edifferenc e is very small or ifa lin kdoe sno texist ,i twil lusuall yb edifficul to r even impossible topredic t from laboratory orgreenhous e testswhethe r resistance problemsma y arise inpractice .Th erelatio nbetwee n resistance and fitness isreviewe d forvariou s groups offungicides .

Keywords: fungicide resistance, fitness ofresistan t strains,predictio n ofresistance ,testin g for fitness,benzimidazoles ,hydroxypyrimidines , carboxamides,organophosphates , antibiotics, ergosterol-biosynthesis inhibitors, acylalanines,dicarboximides ,organi c tincompounds .

Introduction

When failure ofdiseas e control occurs after repeated application of anoriginall y quiteeffectiv e fungicide,laborator y and greenhouse tests mayrevea l thatthi s isdu et oth e developmento fresistanc e toth ecom ­ pound concerned.Howeve r iti susuall y then too late topreven tcro p losses. Itwoul db evaluabl e if,befor e the introduction of achemica l forcontro l of aparticula r disease,informatio n couldb e obtained from experiments with fungi invitro ,i.e . on anartificia lmedium , oro n plants inth e greenhouse aboutth e chances of aresistanc eproble m aris­ ing inpractice .Ther e are several invitr o testswit h andwithou tth e useo fmutagens ,t o assess thepotentia l of fungibecomin g resistant toa particular fungicide.Ar e thesetest s ablet opredic t the occurrence of problems ofresistanc e inth e field? 128 Emergence of resistance in vitro

The capability of fungi tobecom e resistant to afungicid ema yb e tes­ ted inth e laboratory in several ways.A commonmetho d isth eplatin g out ofspore s ona n agarmediu m containing anamoun to f fungicide justgrea ­ tertha nminima l inhibitory concentration (MIC). Only thosecell s that have acquired resistanceb ymutatio n (ori nanothe rway )wil l survive and form acolony .B y counting thenumbe r ofcolonie s the frequency ofmuta ­ tion forresistanc e canb e calculated. Because the frequency ofsponta - -4 -9 neousmutatio n forresistanc e islow ,usuall ybetwee n 10 and 10 , largenumber s of spores areusuall y required forthes eexperiments .Muta ­ genic chemicals orultra-viole t (UV)irradiatio n isofte nuse d toincrea ­ seth enumbe r ofmutants .Anothe rmetho d alsoofte nuse d ist o transfer mycelium toa naga rplat e containing agrowth-inhibitin g but sublethal concentration ofth e fungicide.Developmen t ofresistanc e incell s ofth e myceliumma y result inmor e rapidly growing sectors inth ecolony .Thes e canb e isolated and studied.Afte r isolation ofresistan t colonies and exposure ofth emyceliu m orth e spores to arang e ofconcentration s of the fungicide thedegre e ofresistanc e canb e assessed. Instudie swit h Aspergillus nidulans as ates t fungus andU V irradia­ tion asa mutageni c agent,va nTuy l (1977)compare d anumbe r of systemic fungicides on frequency ofmutatio n towards resistance and the level of thatresistanc e (Table 1).Result s similar tothos e inTabl e 1wer e also obtained inexperiment s withplan tpathogeni c fungi such as Cladosporium cucumerinum and Ustilago maydis. Although the frequency ofmutatio nto ­ wards resistance andth e level ofresistanc evarie s fordifferen tfungici ­ des, insuc h invitr o experiments resistance to all systemic fungicides tested couldb e induced.Thes e fungicides,an d also afe wo fth enon-sys ­ temic conventional ones, are socalled specific-site inhibitors:the y act by interferingwit hth emetabolis m ofth e fungal cell aton eparticula r

Table 1.Inductio no fresistanc eb yU Vtreatmen to fconidi ao fAspergillu s nidulans.

Numbero fresistan t ED50 (Mg/ml) mutantspe r10 7 survivors wild-type mostresistan t fungus mutant

Benomyl 5 0.7 30 Thiabendazole 9 8 125 Carboxin 2 9 200 Chloroneb 25 10 >1000 Cycloheximide 40 150 >4000 Imazalil 100 0.5 4 Pimaricin 1 1.5 6

Source:afte rva nTuy l (1977).

129 site.Geneti c changes in fungi resulting inresistanc e to suchspecific - site inhibitors seem tooccu r readily, incontras twit hmulti-sit einhibi ­ tors,whic hmos tconventiona l fungicidesare .

Build-up of resistance in the field

The emergence invitr o ofmutant s resistant to aparticula r fungicide doesno t imply a priori that theus e ofthi s compound inth e field also will leadt o failure ofdiseas econtrol .Tha twil l onlyhappe n after a' considerable proportion ofth epathoge npopulatio n hasbecom e resistant. Thebuild-u p ofresistanc ewithi na populatio ndepend s onvariou sfac ­ tors,particularl y the fitness ofth e resistantmutant s inrelatio nt o thato fth ewild-typ epathoge n -i nbot h the absence andpresenc e ofth e fungicide.Fitnes s isa comparativ e concept:on e strain ismor e orles s fittha nanothe r undercondition s invitro ,i nth egreenhous e or inth e field.Parameter s of fitness invitr o are spore germination,myceliu m growth onaga r or ina liqui dmedium , and sporulation onaga rmedium . Importantparameter s of fitness onth ehos tplan t areth echanc e ofin ­ fection,th e speedo fcolonizatio n ofth ehos ttissu e andth e degree of sporulation. Strainswit hhighe r fitness aremor e competitive. Tocompar e fitness,competitio n testsma yb ecarrie d outbetwee ntw o ormor e strains,eithe r inth epresenc e ori nth eabsenc eo fth efungi ­ cide. Inth epresenc e ofth e fungicide theresistan t strainshav e aclea r advantageove rth esensitiv e ones,whic h arestrongl y inhibitedb yth e fungicide.Experiment s inth e absence ofth e fungicidema y give anindi ­ cation ofth e chance for survival ofresistan tmutant s during sprayinter ­ vals orwhe n application ofth e fungicide isinterrupted . Insuc hexperi ­ mentshos tplant s are inoculated with amixtur e ofspore s ofth e strains tob e compared.Whe nsporulatin g lesions appear,spore s areharveste d and used for inoculation of asecon dbatc h ofplants .Thi sprocedur e isre ­ peated anumbe r oftimes .Th e spores thenharveste d areteste d forresis ­ tance, using adiagnosti c fungicide concentration.Th e ratio of resistant tosensitiv e conidia iscompare dwit htha trati o atth ebeginnin g ofth e experiment.A decrease indicates thatth eresistan t straini sles scompe ­ titiveunde r thecondition s ofth eexperiment . As anexample ,th e results of anexperimen twit hbenomyl-resistan tan d sensitivepowder ymilde w isgive ni nTabl e 2. Itappear stha tunde rth e conditions oftha texperimen t thebenomyl-resistan t pathogen is lesscom ­ petitive thanth e sensitive fungus.However , itdoe sno t follow thati n generalbenomyl-resistan t strains are less fittha nth ewild-typ ebeno - myl-sensitive fungus:ther ema yb e other resistant strains thatd ono t have reduced fitness.Eve n ifthe y occur atextremel y lowfrequencies , the fungicidewil l select forthes emutants ,resultin g ina rapi dbuild ­ up of aresistan tpathoge npopulation .Onl y ifrepeate d experimentswit h

130 Table2 .Competitio nbetwee na benomyl-resistan t andsensitiv e straino fcucumbe rpowder ymilde w inabsenc eo fth efungicide .

Inoculation Proportiono fresistan t colonies(% ) mixture aftersuccessiv e transferso finoculu m (sensitive: resistant) 1st 2nd 3rd 4th

90 :1 0 4 7 3 0 50 :5 0 32 34 17 0 10: 9 0 45 41 23 5

fungicide-resistant strains invariably show alowe r fitnessca na lin k between resistance andreduce d fitnessb e assumed.A s thedifferenc e in fitnessbetwee n the sensitive and theresistan tpathoge nbecome sver y large, thebuild-u p of aresistan tpathoge npopulatio nwil lb e seriously hampered oreve nno ttak eplace ,dependin g onth e selectionpressur eb y the fungicide.Bu t laboratory and greenhouse testsma y notprovid e all information about fitness of strains under field conditions.Therefor e fieldobservation s mayb enecessar y forth e full answer,especiall y if differences in fitness are so small thatthe y cannotb e detected inth e greenhouse.

Resistance linked to reduced fitness

There isevidenc e thatwit h some fungicides atleast ,increase dresis ­ tance is linked todecrease d fitness.Thi sha sbee n studied forpimaricin , anantifunga l antibiotic belonging toth epolyen emacrolide s (Dekker & Gielink, 1979a). This compound isuse d againstcertai n fungal pathogens inma n and topreven tmouldin g of freshlymad e cheese.Althoug hpimaricin - resistant strains ofyeast s and other fungihav ebee nobtaine d inth e laboratory,n o resistance problemshav ebee nencountere d inpractice , i.e. inmedicin e andchees epackin g houses.Th e antibiotic isals o quite active againstman yplan tpathogeni c fungi. Inlaborator y experiments,pimari - cin-resistant strains of Cladosporium cucumerinum, thecaus e of cucumber scab, and Fusarium oxysporum f.sp. narcissi, thecaus e ofnarcissu sbul b rot,hav ebee nobtained .Al l resistant strains tested have shown reduced sporulation invitr o and strongly reduced pathogenicity. Onth ebasi s ofthes edat a and themechanis m ofactio no fpolyen e macrolide antibiotics,a lin kbetwee n resistance and reduced fitnessha s beenpostulated . Pimaricin and otherpolyen emacrolide s complexwit her - gosterol inth e fungalmembrane ,whic h causes leakage andcel ldeath . Studieswit hyeast s andnystatin , apolyen emacrolid e related topimari ­ cin, showed thatresistan t strainsha d analtere d sterolpattern ,namely , a strongly reduced amounto fergostero l inth emembran ewit h an increase insterols ,whic h arebiogeneticall y moreprimitiv e thanergostero l

131 (Lombe t al., 1975). Themajo r sterols ofresistan tculture so f Candida albicans and C. utilis arewit h increasing resistance successively more primitivebiogeni cprecursor s ofergostero l (Fryberge t al., 1976). These precursors show alowe r affinity topolyen e antibiotics thanergosterol , sotha tth e fungalmembran e ofth e resistant strains contains fewero r weakerbindin g sites toth e antibiotics,whic h results indecrease d sen­ sitivity. Inaddition , replacement ofergostero l byprecursor s appears to cripple the fungal cells,bu t iti sno tlethal . Resistance tonystati n inyeas t (Woods,1971 )an dpimarici n in Asper­ gillus nidulans (vanTuyl , 1977)ha sbee nreporte d tob e due to single genemutations . Itseem splausibl e thatsuc h amutation ,whic h leadst o analtere d sterolpattern , isresponsibl e both forresistanc e anddecrea ­ sed fitness.Thi smean s thatbuild-u p ofa resistan tpopulatio n ishampe ­ red.Ther e are indications that also forothe r groups ofsystemi cfungi ­ cides, resistance islinke d toreduce d fitness.

Type of fungicide

General

Informationo nth epossibilit y ofresistan t strains emerging invitr o isavailabl e formos tgroup s ofne w fungicides.Whethe rproblem s arise in practice depends on several factors,th ecompetitiv e ability of resistant strainsbein g an important one.Howeve r such information -obtaine d from greenhouse or fieldtest s -i s stillver y limited formos t fungicides.Som e fungicideshav ebee nuse d solon gtha tth echance s of aresistan tpopula ­ tiono fa particula rpathoge nbuildin g up areknown ,bu t forver yne w fungicides thebenefit s of suchexperienc e areno tye tavailable .Th e situation forspecific-sit e inhibitorswil lb ereviewe d here.Not e that with exception ofth e dicarboximides andth e triphenyl tincompound s most ofthes e fungicides aresystemic .

Benzimidalozes and related compounds

Development ofresistanc e tobenomy l and otherbenzimidazole s hasbee n reported fora larg e range ofplan tpathogens ,bot h inth e laboratory and the field (Dekker, 1977). Fitness ofresistan t strains appearst o varywidely . Sometimes itha sbee nreporte d toequa l thato fth esensi ­ tivepathogens ,a swa s shown forbenomy l resistance in Cercospora beti- cola inGreec e (Dovase t al., 1976). Forbenomy l theconclusio n seems justified thatresistanc e isno t absolutely linked todecrease d fitness. Moreover,resistanc e tobenzimidazol e fungicides hasbee n found stable withthi s andman y otherpathogens ,whic hwoul d aggravate theconsequen ­ ces should resistant strains that are fitoccur .

132 Hydroxypyrimidines

Soonafte r the introduction ofdimethirimo l forcontro l of cucumber powdery mildew inglasshouses ,resistanc e developed inthi spathogen . High selectionpressur eb y the fungicide, favoured by themod e ofappli ­ cation andprobabl y alsob y the isolated environmentprovide d by aglass ­ house, contributed tothi sphenomenon ; resistancewa s notreporte d from cucurbit fields inMediterranea n countries.Afte r interruption ofdime ­ thirimol application thepercentag e ofresistan t individuals inth epa ­ thogenpopulatio n gradually declined andpractica l application couldb e resumed to alimite d extentp.219-230 . Itseem s thatunde rpractica l conditions thecompetitivenes s ofresistan t strains is somewhat less thantha to fth e sensitive fungus.Thi smigh tals ob e true forethirimol , which isuse dt o controlbarle y powderymildew ,b y seed treatmento r spraying the aerialplan tparts .Althoug h resistant isolates canb e obtained fromethirimol-treate dcrops ,th e chemical continues tob e effective.Strain swit hhig hresistanc e dono tten d to increase inth e presence ofethirimo l because they are apparently less competitive (Shephard et al., 1975).

Carboxamides

Carboxin isuse d as asee d treatment against smuti nwhea t andbarley . Although resistance invitr o in Ustilago hordei was obtained easily (BenYephe te t al., 1975), therehav ebee nn o reports of disease-control failure due todevelopmen t ofresistance .Oxycarboxi n resistanceresul ­ ting incontro l failure of Puccinia horiana onchrysanthemu m hasbee n reportedb yAbik o et al. (1977); the resistant strains appeared lesscomp ­ etitive thanth e sensitivepathoge n (Uesugi, 1978). Butal lcarboxin-re - sistantmutant s of Ustilago maydis (testedb y artificial inoculation of corn seedlings inth egreenhouse )di dno t showreduce d pathogenicity (Georgopoulos et al., 1975). Georgopoulos (personal communication, 1981) has suggested thatth e lowapparen t infection ratemigh texplai nwh yn o problemswit hresistanc e tocarboxamide s inUstilaginale s have occurred inpractice .

Organophosphates

Althoughmutant s of Pyricularia oryzae resistant toedifenpho s and 'Kitazin1 were obtained easily inexperiment s invitro ,resistanc e has notbee n aproble m inth e field forabou tte nyears .Developmen t ofresist ­ ance to 'Kitazin'ha sbee nreporte d only once,i na nexperimenta l field where itwa s used intensively (Uesugi, 1978). Resistance topyrazopho s in cucumberpowder ymilde wwa s found ingreenhouse s only after several years

133 ofapplication ;whe nth eus e ofthi s fungicidewa s stopped inthos egreen ­ houses, the frequency and level ofresistanc e dropped the followingyear . The resistant isolateswer e somewhatles spathogeni c thanth e sensitive pathogen, and also lesscompetitiv e (Dekker& Gielink , 1979b).Als opyra - zophos-resistant isolates of Pyricularia oryzae, obtained inth elabora ­ toryb yd eWaar d &va nNistelroo y (1980), showed alowe r rateo f growth and sporulation onagar .Thes e observations indicate asomewha t reduced fitness of fungal isolates resistant toorgani cphosphates . Inpractice , however,resistanc e may develop under conditions ofcontinuou s severe selectionpressur eb yth efungicide .

Antibiotics

Streptomycin resistance inbacteri a hasbee nknow n for alon gtime . After the introduction of streptomycin forcontro l of aplan tdisease , namely firebligh ti npears ,cause db y Erwinia amylovora, the occurrence ofresistanc e causedproblems .Schrot h et al. (1979)showe d thatvirulenc e among resistant and sensitive strainsvarie dbu ttha tther ewa s noconsist ­ entdifferenc ebetwee n the twogroups .Th eresistan t strains appeared to be relatively stable andwer e detected evensi xyear s after termination of streptomycin application. Kasugamycin-resistantstrain s of Pyricularia oryzae havebee n easily obtained inth e laboratory. Intensive use ofthi s antibiotic againstric e blast inJapa n ledt odisease-contro l failure insom e districts thatwa s due todevelopmen to fresistance .Compariso n ofresistan t and sensitive isolates showed nodifference s inmyceliu m growth and sporulation invitro , nor inth enumbe r and length ofth e lesions. Ina mixtur e ofresistan t and sensitive isolates,th eresistan t ones still appeared lesscom ­ petitive; their incubationperio dwa s longer and their rate ofappresso - rium formation appeared tob e slower (Ito& Yamaguchi , 1979). Due to thesephenomen a andperhap s alsot odilutio no fth eresistan tpathoge n populationb y sensitive strains fromneighbourin g fieldso r areas,th e population ofth eresistan t strain decreased afterth e application ofth e antibiotic was stopped.Kasugamyci n isno wuse d again,bu t ina carefull y plannedway . Resistance topolyoxin s hasbee n reported in Alternaria kikuchiana in Japanesepea r orchards.Althoug h theresistan t strains appeared somewhat lesscompetitiv e thanth e sensitive ones, they didno tdisappea r entirely (Uesugi, 1978). The level ofresistanc e in fungit opolyen emacrolid e antibiotics, such aspimarici n andnystatin , appears tob e inversely relatedwit hth e degree ofvirulenc e (p.131-132).Therefor e lowvirulenc e greatly hampers thebuild-u p of aresistan tpathoge npopulatio n invivo .

134 Inhibitors ofergostero l biosynthesis

Iti sknow n thatvariou s groups of fungicides actspecificall y on biosynthesis of ergosterol inth e fungal cell,namely , triazoles,imida ­ zoles,pyrimidines ,morpholines , triforine andbuthiobate .Althoug htri - forine-resistant colonies havebee nobtaine d inth e laboratory, nopro ­ blemswit h triforine resistance have arisendurin g anumbe r ofyear s of use inpractica l agriculture.Al l resistant strains have alowe r fitness invitr o aswel l as invivo ,whic h suggests thatthi smigh tb e linked to developmento f resistance.Ther e are indications thatthi smigh t alsob e true forman y ofth e other fungicides,tha t acto n sterolbiosynthesis . For amor e detailed treatmento fstero lbiosynthesi s seep.71-100 .

Acylalanines

Metalaxyl and furalaxyl,exclusivel y active against fungibelongin g to theOomycetes ,hav ebee n introduced only recently. Inlaborator ystudies , strains of Phytophthora nicotianae (Dekker,unpublishe d data)an d Phytoph- thora megasperma f.sp. glycinea (Davidse,1980 )hav ebee nobtaine dwit h decreased sensitivity tometalaxyl .Studie swit h the latter strainso n alfalfa ingrowt h room experiments did notrevea l areduce d fitness.Soo n after introduction inpractice ,resistanc e to 'Ridomil',a commercia l product containingmetalaxy l asa nactiv e ingredient, appeared indown y mildew oncucumbe r in Israel andGreec e (Georgopoulos &Gregoriu , 1981), and in Phytophthora infestans onpotatoe s inth eNetherland s (Davidse et al., 1981), causing considerable croplosses .

Dicarboximides

Vinclozolin, iprodione and dicyclidine areparticularl y active against Botrytis, Monilinia and Sclerotinia. Fungicide-resistant strains ofthes e fungihav ebee nreadil y obtained inlaborator y experiments (Leroux et al., 1977;Hisad a et al., 1979). Recently,variou s caseso f development ofresistanc e inth e fieldhav ebee nreported , e.g. in Botry­ tis on strawberries (Davis& Dennis ,1979 )an d Botrytis ongrape s (Speng­ lere t al., 1979). Some authors report from laboratory studies that fit­ ness ofth eresistan t strainswa s less thantha to fwil d strains (Speng­ lere t al., 1979;Hisad a et al., 1979), butother s found also strains with equal fitness (Sztejnberg & Jones,1978 ;Pappa s et al., 1979).There ­ fore itcanno tb e concluded thatresistanc e islinke d to adecrease d pathogenicity. Judicious application isnecessar y to avoid ordela y resistance problems inpractice .Resistanc e todicarboximide s istreate d inmor e detail onp.101-117 .

135 Organic tincompound s

Resistance toth eorgani c tincompound s hasbee nreporte d inGreec e in Cercospora beticola onbeets .Th e resistant strains testedwer e asviru ­ lenta sth e sensitive strainsbu t appeared tob e less competitive (Gianno- politis& Chrysayi-Tokousbalides , 1980).

Conclusions

From laboratory experiments circumstantial evidence hasbee n obtained that fungi are ablet odevelo p resistance toal l fungicides -whethe r sys­ temic orno t -tha tac ta s specific-site inhibitors.Th e frequency of mutation forresistanc ema yvary ,bu tthi s doesno tsee m tob e ofgrea t importance giventh e largenumbe r ofpropagule s produced by fungi.Lac k ofemergenc e ofresistanc emigh ttheoreticall y bepossibl e ifmutatio n towards resistance is lethal forth emutatin g cells.Thi s doesno tsee m tohav eoccurre d forth e fungicides tested so far,althoug h adecreas e of fitness ofresistan t strainsha s oftenbee n observed. Emergence ofresistanc e invitr o doesno tnecessaril y implytha ta resistantpathoge npopulatio nwil lbuil du p inth e field.Thi swil l depend on anumbe r of factors,amon g them the type ofpathogen ,th e nature ofth e disease,th emod e of fungicide use and the fitness of resistant strains compared to sensitive strains.Strain s that areresis ­ tant invitr oma yb e sensitive invivo ,e.g . whenth e fungicide iscon ­ verted to another fungitoxic compound orinduce s resistance inth eplan t tissue toth epathogen .Wha tthe n isth eus e andpredictiv e value of laboratory experiments? First,whe n itappear s impossible toobtai n resistance invitro ,eve n usingmutagens ,developmen t ofresistanc e inth e field isunlikely .Sec ­ ond, laboratory andgreenhous e experiments mayprovid e information about atleas t anumbe r ofparameter s of fitness,e.g . spore germination and growth ofmyceliu m invitro ,infectio n rate and speedo fcolonizatio n and sporulation onth ehos tplant . Ifdifference s infitnes s aredifficul tt o detect,competitio n experiments inth e greenhouse,usin g inoculationmix ­ tures ofspore s from resistant and sensitive strains,ma yprovid evalu ­ able information.A s fitness andpathogenicit y mayvar ywidel y amongre ­ sistanta swel l assensitiv e strains,onl y strains thatsho woptima l fit­ ness shouldb e tested.Result s from experiments withonl y afe w resistant isolates,picke d atrandom , should notb egeneralize dwithou tgrea tcare . Inspit eo f allthes eefforts ,i twil lusuall y notb epossibl e topre ­ dict, onth ebasi s oflaborator y and greenhouse experiments,tha tresis ­ tance to aparticula r fungicidewil l never occur inth e field.First ,eve n competitive experiments inth e greenhousema yno tprovid e all information that isimportan t in field situations.Second , even ifal l resistant

136 strains tested were less fit than the sensitive pathogen, often it will be not justified to exclude the possibility that rare resistant strains with high fitness will emerge. Third, resistance problems may only arise after many years of use in practical agriculture; resistant strains that in the beginning do not cause problems, due to a low level of resistance may under constant selection pressure of the fungicide evolve with higher resistance. Fourth, the age of resistance may play a role. Wild-type strains have been subjected to selection for fitness for a very long time, but resistant strains are very recent and, hence, many of them have not been substantially selected for fitness.

References

Abiko, K., K. Kishi & A. Yoshioka, 1977. Occurrence of oxycarboxin-tolerant isolates of Puccinia horania P. Hennings in Japan. Annals of the Phytopathological Society Japan 43: 145-149. Ben Yephet, Y., Y. Henis & A. Dinoor, 1975. Inheritance of tolerance to carboxin and benomyl in Ustilago hordei. Phytopathology 65 : 563-567. Davidse, L.C., 1981. Resistance to acylalanine fungicides in Phytophtora megasperma f. sp. medicaginis. Netherlands Journal of Plant Pathology 87: 11-24. Davidse, L.C., D. Looijen, L.J. Turkensteen & D. van der Wal, 1981. Occurrence of meta- laxyl-resistant of Phytopthora infestans in Dutch potato fields. Netherlands Journal of Plant Pathology 87: 65-68. Davis, R.P. & C. Dennis, 1979. Use of dicarboximide fungicides on strawberries and po­ tential problems of resistance in Botrytis cinerea. Proceedings of the 1979 of the British Crop Protection Conference, Brighton, England. British Crop Protection Council, London, p.193-201. Dekker, J., 1977. Resistance. In: R.W. Marsh (Ed.): Systemic Fungicdes. Longman, London and New York, p. 176-197. Dekker, J. & A.J. Gielink, 1979a. Acquired resistance to pimaricin in Cladosporium cu- cumerinum and Fusarium oxysporum f.sp. narcissi associated with decreased virulence. Netherlands Journal of Plant Pathology 85: 67-73. Dekker, J. & A.J. Gielink, 1979b. Decreased sensitivity to pyrazophos of cucumber and gherkin powdery mildew. Netherlands Journal of Plant Pathology 85: 137-142. De Waard, M.A. & J.G.M, van Nistelrooy, 1980. Mechanism of resistance to pyrazophos in Piricularia oryzae. Netherlands Journal of Plant Pathology 86: 251-253. Dovas, C, G. Skylakakis & S.G. Georgopoulos, 1976. The adaptability of the benomyl resistant population of Cercospora beticola in Northern Greece. Phytopathology 66: 1452-1456. Fryberg, M., A.C. Oehlschlager & A.M. Unrau, 1976. Sterol biosynthesis in antibiotic sensitive and resistant Candida. Archives of Biochemistry and Biophysics 173: 171-177. Georgopoulos, S.G., 1977. Development of fungal resistance to fungicides. In: M.R. Sie­ gel & H.D. Sisler (Eds): Antifungal Compounds, Vol. 2. Marcel Dekker Inc., New York and Basel, p. 439-495. Georgopoulos, S.G. & A.C. Grigoriu, 1981. Metalaxyl resistant strains of Pseudoperonos- pora cubensis in cucumber greenhouses of Southern Greece. Plant Disease 65: 729-731. Giannopolitis, C.N. & M. Chrysayi-Tokousbalides, 1980. Biology of triphenyl-resistant strains of Cercospora beticola from sugar beet. Plant Disease 64: 940-943. Hisada, Y., H. Takaki, Y. Kawase & T. Ozaki, 1979. Difference in the potential of Botry­ tis cinerea to develop resistance to procymidone in vitro and in the field. Annals of the Phytopathological Society of Japan 45: 283-290. Ito, I. & T. Yamaguchi, 1979. Competition between sensitive and resistant strains of Piricularia oryzae Cav. against kasugamycin. Annals of the Phytopathological Society of Japan 45: 40-46. Leroux, P., R. Fritz & M. Gredt, 1977. Etudes en laboratoire de souches de Botrytis ci­ nerea résistantes à la dichlozoline, au dicloran, au quintozene, à la vinchlozoline et au 26019 RP (ou glycophene). Phytopathologisch Zeitschrift 89: 347-358.

137 Lomb,M. ,M .Fryberg ,A.C .Oehlschalge r& A.M .Unrau ,1975 .Stero lan dfatt yaci dcompo ­ sitiono fpolyen emacrolid eantibioti c resistantTorulopsi sglabrata .Canadia nJourna l ofBiochemistr y53 :1309-1315 . Pappas,A.C. ,B.K .Cook e& V.W.L .Jordan ,1979 .Insensitivit yo fBotryti scinere at o iprodione,procymidon ean dvinclozoli nan dthei ruptak eb yth efungus .Plan tPatho ­ logy28 :71-76 . Scroth,M.N. ,S.V . Thomson& W.J .Moller ,1979 .Streptomyci n resistance inErwini a amylovora.Phytopatholog y6 9 :565-56 8 Shephard,M.C. ,K.J .Bent ,M .Woolne r& A.M .Cole ,1975 .Sensitivit yt oethirimo lo f powderymilde wfro mU Kbarle ycrops .Proceeding so fth e8t hBritis hInsecticid ean d FungicideConference .Britis hCro pProtectio nCouncil ,London ,p .59-65 . Spengler,G. ,M .Schere r& E.H .Pommer ,1979 .Untersuchunge nübe rda sResistenzenver ­ haltenvo nBotryti s cinerea gegenübervinclozolin .Mitteilunge nau sde rBiologische n Bundesanstalt fürLand -un dForstwirtschaft ,Berlin-Dahle mHef t 191:236 . Uesugi,Y. ,1978 .Resistanc eo fphytopathogeni c fungit ofungicides .Japa nPesticid e Information35 :5-9 . VanTuyl ,J.M. , 1977.Genetic so ffunga lresistanc et osystemi c fungicides.Mededelinge n Landbouwhogeschool,Wageningen ,77-2 .13 7p . Woods,R.A. , 1971.Nystati nresistan tmutant so fyeast :alteration s instero lcontent . Journalo fBacteriolog y 108:69-73 .

138 Dynamics of the pathogen population in relation to fungicide resistance*

M.S. Wolfe PlantBreedin g Institute,Trumpington ,Cambridge ,U.K .

Abstract

The emergence of fungicide resistance depends onth enatur e ofth e fungicide,it seffectivenes s inth eplan t andth e scaleo fit suse .De ­ tectiono fvariatio n forresistanc ebefor e afungicid e isintroduced ,o r shortly afterwards,i slimite db yth etechnica l difficulty ofobtainin g and recognizing rarephenotypes .Evaluatio n ofsuc hphenotype smus ttak e accounto f likely changes inthei rpathogenicit y following selectiono n untreated and treated hostplants .Th epathoge npopulatio nma yb esub ­ jected simultaneously to stabilizing, directional anddisruptiv e selec­ tion forincrease d resistance.A simplemode l forestimatin g the relative fitness and sizeo fsensitiv e and resistantpopulation s ofth epathoge n indicates therelativ e importance ofth edifferenc e inpathogenicit y between suchpopulation s onuntreate d crops,an do fth etota l crop area thatreceive s the fungicide.Strategie s of fungicide usetha texploi t population dynamics to limitth edevelopmen t ofresistanc e arebriefl y described. Fungicidemixture sma yb ehomogeneou s (allcomponent s applied toth e sameplants) , orheterogeneou s (allcomponent s applied todiffer ­ entplants) .Differen t fungicidesma y alsob e applied ina n alternating sequence. Itcanno tb e stated generallywhic h ofthes e strategies isth e more suitable forparticula r compounds,bu tth emos teffectiv e strategy islikel y tob e integrated control,fo rexampl eb yusin g limited,o r heterogeneously mixed, fungicide applications onmixture s ofhost-plan t varieties thatdiffe r inthei rresistance .

Keywords: fungicide resistance,populatio n dynamics,selectio nmodel , Erysiphe graminis, ethirimol,tridemorph , triadimefon, fungicidestrat ­ egies, integratedcontrol .

* Editors'note :i nthi scontributio n- an dth eres to fth eboo k- w econfor mwit hth e FAOrecommendatio nt odenot eth elac ko fo rdecreas ei nsensitivit y toa fungicid e byth eter mresistance .D rWolf eadvocate sth eus eo fth eter minsensitivity ,bu th e hasagree d toth ereplacemen to fthi ster mb ytha to fresistanc et omaintai ncon ­ sistantterminolog ythroughou tth ebook .

139 Occurrence of fungicide resistance

Iti sa truis m thatth epopulatio n ofa targe tpathoge n is sensitive to anewl y introduced and successful fungicide,althoug h therang e of sensitivity within thepopulatio n atth e time of introduction isusuall y unknown. Iti spossibl e thatth eorganis m mayb eunabl e tocop ewit h the fungicide:th e system required tod o so isbeyon d itsmetaboli ccapabi ­ lity.O n theothe rhand , therema yb emutant s that areresistan t toa lesser orgreate r degree;th epotentia l ceiling forresistanc ema yb e moderate orhigh . Although iti s alsotru etha tth eoccurrenc e andpotentia l range of resistance ofth epathoge nwil lb e determined by thenatur e ofth efun ­ gicide, iti susuall y impossible todistinguis h asingl e character ofth e fungicide,suc h as site specificity, asth emos t important inthi s res­ pect.Bot hth enatur e and theeffectivenes s ofth e fungicide areimpor ­ tant indeterminin g thepathogen' s response to it.Fo r example,resis ­ tancet o asite-specifi c compoundma y involve one ormor epathoge ngenes , whose easeo fmodificatio n isunpredictable . Site-specific compounds are often systemic andpersistent ,an d iti sthes echaracter s thatmak ethe m highly effective,bu ti tals oensure s ahig h degree ofselectio n for resistanceb ymaximizin g exposure ofth e fungicide toth epathogen .Re ­ sistance tomulti-sit e compoundsma y also involve oneo rmor e pathogen genes; again,th eeas eo fmodificatio n ofeac h gene isunpredictable . Multi-site compounds areofte nnon-systemi c and lackpersistence , sotha t they have loweffectiveness ,whic h doesno tgiv e suchintens e selection forresistance . Initially, resistant formsar eprobabl y ata selectiv e disadvantage and survive atonl y low frequencies inth epopulation ;the yhav e alo w fitnessprio r toth e introduction ofth e fungicide. Iti stherefor e necessary tomonito r thepopulatio n forthi scharacte r from the earliest possible stage.Unfortunately , monitoring suffers fromtw odefects . First, themajorit y ofmethod suse d arecapabl e ofdetectin g onlyrela ­ tively commonmutants ;the y areofte n lesseffectiv e thantria lplot s in detecting themor e important,bu t lesscommon , forms.Thi sproble m is illustrated inTabl e 1,whic h showsth esampl e sizes required to obtain with 95% probabilit y atleas ton eo fthos emutant s thatoccu r atlo w frequencies.Th e figureswer e obtained fromth ebinomia l expression sample size= lg (l-P)/lg (1-y) where P isth erequire dprobabilit y and y isth e frequency. Table 1als o indicates theefficienc y ofcerea l fields intrappin g mutantspore s ofpowder ymilde w (Erysiphe graminis) (derived fromsugges ­ tions ofWolf e& Schwarzbach, 1978), givena spor econcentratio n inth e atmosphere ofonl y 1pe r 100 0m 3 and aspor e depositionvelocit y of 1.5 cm/s.Onl y asmal l crop area isrequire d tob ecertai n oftrappin ga

140 Table 1.Sampl esiz ean d sampling area required toobtai n with9 5 %probabilit y anunusua lmutan toccurrin g atlo w frequencies ina pathoge npopulation ;samplin gare a required referst oth eare ape rda yo fa cerea lcro p receiving sporeso fErysiph egraminis .

Mutant Samplesiz e Sampleare a frequency required required (ha/d)

10-3 3x 10 3 0.2 10-5 3x 10 5 23.1 10-6 3x 10 6 230.8 10-8 3x 10 8 23077. 0

relatively raremutant .O fcourse ,surviva l of asingl e spore after deposition isaffecte db yman y otherfactors . The secondproble m inmonitorin g concerns theconditio n ofth efirst - foundresistan tmutants .Test s oftenindicat e thatthe y dono tgro wwel l ontreate dhos tplants, an d thatthe y are less fito nuntreate d plants thanth ecommo n sensitive strains.Unfortunately , once observed, thesecha ­ racteristics are oftenregarde d asstatic .The yma yremai n so,bu t ifse ­ lection forresistanc e ismaintaine d or increased, thengenotype s with improvedperformanc e onbot h treated anduntreate d hostplant sma y emerge. Because ofth e difficulties of adequatemonitoring , the developmento f suchchange sma y remainundetecte d fora considerabl etime . Because ofth eproblem s of scale anddynamis m inmonitoring ,man ylab ­ oratory studies onth e occurrence andperformac e ofresistan t strains of pathogens are inadequate andmisleading .Typically , ageneticall y limited sample ofth epathoge n ischallenge d with afungicid e following theus e of artificial mutagens.Th e results obtained areope nt odoubt :lac ko f response isunlikel y toindicat e the fullpotentia l ofth epathogen .Bu t eveni f arespons e does occur,i tma yno t involve the strains thatoccu r most commonly inth e field (p.207-218). Measurement ofth epathogenicit y of suchartificiall y induced mutants onuntreate d media orhos tmateria l inth e laboratoryma y alsob e mis­ leadingbecause : - itdoe sno treflec tth e total dynamicpotentia l ofth e field population - itma yb e affectedb ymutation s occurring atloc ino t involved inre ­ sistance - itmake sn o allowance forth edynamic s of fungicideus e and thedyna ­ mics ofth e sensitive and resistantpathoge npopulations .

Laboratory studies onpathoge n dynamics aregenerall y ofgreates tvalu e intestin g thecomparativ e performance ofpathoge n isolates from the field.

141 Dynamics of selection towards resistance

Giventha t the fungicide-resistant mutants dooccu r inpathoge npopu ­ lationsno tye tsubjecte d to fungicide treatments,w enee d to understand theprocesse s thatcaus e anincreas e infrequenc y ofthes emutant s when the fungicide isused .Mathe r (1953)define d three classes of selection acting onpopulation s oforganisms ,namely , stabilizing, directional and disruptive. Stabilizing selection isth eproces s thatmaintain s ahig h frequency of optimalphenotypes , i.e. those thatbes t fitth e individual to theusua l range ofcircumstance s thatth epopulatio n encounters.Becaus ether e are environmental variations,an dbecaus e similarphenotype sma yb eproduce d bydifferen t genotypes,ther ewil lb e geneticvariabilit y eveni na popu ­ lation subjectt o strong stabilizing selection. Directional selection occurs ifth e averagephenotyp e inth epopulatio n isno tclos et oth eoptimum : then selectionwil l favourthos ephenotypes , and their associated genotypes,tha tar e so;selectio nwil lb e away from the existingmeans ,toward s thene woptimum . Stabilizing selection occurs simultaneously, to stabilize thepopulatio n around thene woptimum .Di ­ rectional selection isa positiv e response tochange s inth eenvironment . Iti so fspecia l importance inth e contexto f fungicideresistance . Disruptive selection occurswhe ntw odifferen t environments thateac hfa ­ vour distinctoptima l characteristics arecreated . Selection forth etw o distinctoptim ama y cause abrea k inth econtinuit y ofpopulatio nvaria ­ tion. Stabilizing selectionwil l againoccur ,t omaintai n the selected phenotypes and theirrespectiv eoptima . All three types ofselectio nma y occur simultaneously and tovaryin g degrees inrespons e toth eenvironmenta l change causedb y introduction of a fungicide tocontro l thepathogen .Th e relative importance ofeac hwil l depend onth e degree ofus eo fth e fungicide and theexten tt owhic hdif ­ ferentmechanism s ofresistanc e are available toth eorganism . Onecriti ­ cal factor isth erelatio nbetwee n the lengtho fth ereproductiv e cycle ofth e organism andth e timedurin gwhic h the fungicide is active. Ifa n organism has alon gcycl e relative toth e duration of fungicidal activi­ ty,the nth epopulation swil l change slowly, if atall .Conversely , if thepathoge nha s ashor tcycl e relative toth eduratio no f fungicideac ­ tivity, therewil l tend tob e amor e rapidpopulatio n response,an dth e populationwil l tend to follow anychange s in fungicideuse .

The sequence ofevent s

Prior toth e introduction ofa fungicide,th e sensitivepathoge npopu ­ lationca nb e regarded asbein g inequilibriu m and subjectt o stabilizing selection forth echaracte r fungicide resistance. InFigur e 1th eoptima l

142 phenotype forresistanc ebefor e introduction ofth e fungicide isa tX j Stabilizing selection operates inth edirectio n ofth e arrows,i.e .to ­ wards Xj; genotypeswit h somedegre e ofresistanc e exist,bu t their fre­ quency islow . Introduction ofth e fungicide,an d its increasing use,re ­ present achang e inth e environment, and theoptima lphenotyp ema y then

be atx 2 orx ,dependin g onth erelationshi p between the fungicide and themetabolis m ofth epathogen , andth ewa y inwhic hth e fungicide is used. Ifth e fungicidal activity canb eeasil ymatche db y thepathogen ,

andth e fungicide isno twidel yused ,the na shif t fromx t tox 2 bydi ­ rectional selection ineac htreate d cropma yb e adequate forth epathoge n to attainoptima l fitness.Disruptiv e selectionwil l also acto nth epo ­ pulation asa whol e during thegrowin g season,sinc euntreate d andtreat ­ edcro p areaswil lb e differentniche s forrandoml y dispersedspores . Ifth e fungicidepresent s amor e severemetaboli c problem forth epa ­ thogen,o r ifi ti sver ywidel y used,th ene woptima lphenotyp ema yb e at x .I fi ti smetabolicall y possible,x mayb e attainedb yeithe r oftw o

routes. First,rar emutant sma yoccu r inth epopulatio n represented byx t that areabl et ogro w ato rclos et oth e optimum,x .I fthe y infectth e treated crop,the yma yb e ablet oincreas eeve ni fthei r fitness islo w

(Wi, Figure 1). Second, theproces sma yb e stepwise, fromX j tox 2, from x2 tox 3, and soo nt ox .Dependin g onth e appropriate fitnessvalues ,a discontinuitybetwee n the sensitive and resistant fractions ofth epopu -

frequency fitness maximum-i maximum P=0

maximum

fungicide resistance Figure 1.Stabilizing ,directiona lan ddisruptiv eselectio nfo rth echaracte rfungicid e resistance.x 1( x2 andx representdifferen toptima lphenotype seac hdependin go nth e typeo ffungicid ean dho wi ti sused .

143 lationma y develop andth e intermediate stagesma yb emor eo rles s rapidly lost. Selection forresistanc ema y alsoprocee d simultaneously alongbot h routes,bu ti ti simpossibl et opredic twhic hma y leadmos trapidl yt o x .I fo fresistan tgenotype sar efoun di tma yb edifficul tt odetermin e whether they occurreda sth e resulto fa singl emajo r change,o rserie s ofsteps .

Amode lo fdisruptiv e selection

Iftw o separatepopulation s areproduce da sa resul to fdisruptiv ese ­ lection,thei rprogres s relativet oeac hothe ri sdependen tprincipall y onthei r abilityt oreproduc eo nth epar to fth e cropt owhic hthe yar e adapted,bu tt oa lesse rdegre e alsoo nth epar tt owhic hthe y areno t adapted.Thi s canb econsidere d formallyusin ga derivatio no fth e dis­ crete generationmode l forpathoge n reproduction devisedb yBarret t (1978, 1980). Thecontinuou s generationmode lproduce s similarresults . The simplestmode l canb erepresente db ya two-wa y table,give ni n

Table 2. Nsu0 represents thenumbe ro fsensitiv e genotypeso nare a go f theuntreate d cropa tth e starto fth e epidemic, andws urepresent sth e fitnesso fth e sensitive fractiono fth epathoge npopulatio no nth eun ­ treated crop.Le tth ebasi cmultiplicatio n rateo fth epathogen , m =1+ a,wher e1 represent s theparen tcolony , and a thenumbe ro fdaughte rco ­ loniesproduced .Afte r n generations therewil lbe : -o nth euntreate d crop

Nsusu =- NsuNsu0 0.g(wsM.m).g(ws\i.m) sensitive genotypes. Buti fws ui sse t= 1 then ffsuffsu == Wsutfsuu.g(m.g(n) sensitive genotypes. n 0 0 * Similarly there are

Wru = Nru0 .gr(wru.m) resistant genotypes. - on the treated crop iVst = Nst0.h{wst.m) sensitive genotypes. But if wst = 0 then iVst n= 0.

Table 2. Reference symbols for sensitive and resistant fractions of the pathogen population onuntreate dan d treated crop areas.

Untreated crop (u) Treated crop (t) Sensitive pathogens (s) su st Resistant pathogens (r) ru rt Proportion of total g h crop area a. g+ h = 1.

144 n iVrt = Nrt0.h(wrt.m) resistantgenotypes . Therefore I Ns = Nsu + iVst = Nsun .g(m) , n n n n n

and 1 Nrn = Nrun + Nrtn = m {Nru0.g(wru) + Nrt0.h(wrt) }. Therefore n n ^Vrn/7Vsn= {iVru0.g(wru) + Nxt0 .h(wxt) }/Nsu0 .g. Note: to followth echange s inpopulation s size (andtherefor e epidemic size) mmus tb e retained.

Ifth e ratioiV r :Ns exceeds NrQ:Ns0 thenth eresistan tgenotype swil l becomeprevalent ; ifth erevers e istrue ,th e sensitive genotypes willre ­ mainprevalent . Stableequilibri a areunlikel y because thecondition sre ­ quired forstabilit y arever y restrictive (Barrett, 1978). Thismode l showstha tth eoutcom e isdependen t onth e initial frequen­ cies ofth etw o kinds ofgenotype ,thei r fitness onth euntreate d and treated fractions ofth ehost ,th erelativ e sizeo fthos etw ofractions , and thenumbe r ofpathoge n generations. Ifh is large andws t =0 ,not e that lowvalue s ofwr u andwr tma y lead toprevalenc e ofth e resistant fraction;thi sprevalenc e wouldb e accelerated ifther ewer e any increase inth evalue s ofwr u orwrt .Conversely , if h islow ,th e sensitive frac­ tionma yremai nprevalen t even ifwr u and/orwr tincreas e relative tows u andwst . Anexample :th eus eo fethirimo l inth eU K

The declining effectiveness ofethirimo l seed-dressing in controlling powderymilde wo fbarle y (Erysiphe graminis f.sp . hordei) inth eU K (Wolfe& Dinoor , 1973), indicates thatsom e adjustment ofth ebasi c model mayb enecessar ywhe nconsiderin g aparticula rproble m inpractice : -Th e relative crop areatreate dwit hethirimol , h, thoughneve rlarge , varied from seasont oseason . - Sinceth e fungicidewa s usually applied toth e seed only, itwa sef ­ fective early inth eepidemi c butgraduall ybecam e ineffective later in the seasonbecaus e of leaching andkatabolism . Thus h decreased, although theoriginall y treated crop arearemaine d constant,s otha tth e fitness ofsensitiv e relative toresistan tpathoge n genotypes increased during theseason . -Durin g the1970 sethirimo lwa s usedonl y onsprin gbarley , sotha ti n the overwintering phase ofth epathoge n onwinte rbarle y h - 0. -Durin g theperio d ofuse ,th e fungicidewa s applied todifferen tvari ­ eties covering awid e spectrum ofhos tresistanc e genes.Differen tcombi ­ nations of fungicide andhos tresistanc ema ywel lhav edelaye d theemer ­ gence ofpathogeni c recombinants,i.e . depressing wru andwr trelativ et o wsu. An exceptionwa sth epathogenicit y for-'Sultan 1 and related vari­ eties,whic hwa spositivel y associated with ethirimol resistance (Wolfe &

145 Dinoor, 1973;Wolf e& Slater, 1980). These four features together suggest thatresistanc e toethirimo lwa s noto foverridin g importance forth e survival ofth epathogen . Indeed, Bent (1978)showe d that althoughhighl y sensitive andhighl y resistant formso fth epathoge n occurred inth eU K following the introduction of ethirimol fordiseas e control ofpowder ymilde w ofbarley , the genotypes thatbecam emos tcommonl y established inth e late 1970sha d only anin ­ termediate level ofresistance , suggesting thatdirectiona l selectionha d beenprominent .A s aconsequence , theeffectivenes s ofth e fungicidede ­ clined,bu tno tdramatically ; under the rightconditions ,i tstil lha s somevalu e inpractica l agriculture. The circumstances described above inth e first,thir d and fourthpoint s also applied toth eus e oftridemorp h forcontro l ofbarle y powdery mil­ dew during the sameperiod .However ,thi s fungicidewa s onlyuse d asa foliar spray, fortreatin g established populations ofth epathogen ,s o thati tprobabl y had lesseffec ttha nethirimo l inchangin g the direction ofpopulatio n development.Consequently , althoughWalmsley-Woodwar d et al. (1979)wer e ablet odetec tresistanc e totridemorp h inth e field,th eef ­ fectiveness ofthi s fungicide appearsno t tohav e changed during thepe ­ riodo fuse .However , iti seviden ttha t adifferen t -mor e intensive- history ofus e ofethirimo l and tridemorph involving alarg e increase in h could have led toa mor e rapid and obvious decrease inth eeffective ­ ness ofbot h fungicides.Fo r thisreaso nth ecurren t largevalu e of h for theus eo fergosterol-biosynthesi s inhibitors oncereals ,an dth e finding ofresistan t formso fth ebarley-milde w pathogen inth e field in198 0an d 1981 givesconsiderabl e cause forconcern .

Strategies for fungicide use

Themajo rproble m in fungicideus e ist ominimiz e the exposure ofa particular fungicide toth epathoge npopulatio nwhil emaintainin g anac ­ ceptable levelo fdiseas econtrol .Th esimples tstrateg y ist olimi tun ­ necessary use ofth e fungicideb yno tcontrollin g disease that isno tda ­ maging. Improvements indiseas emonitorin g and forecasting,bette rus eo f disease-resistantvarieties ,an d improvedmethod s of fungicide applica­ tion, areimportan t inthi srespect .Othe r strategies involvediversifica ­ tion, eitherbetwee n fungicides,o rbetwee n fungicides and resistantva ­ rieties.

Homogeneous andheterogeneou s mixtures

Theprincipl e behind this strategy (Wolfe,1981 )i ssimpl y thata mul ­ tipleproble m interm so fdifferen t fungicides ismor e difficult fora pathogen toovercom e thana singl eproblem . Field experience now suggests

146 thatmixin g fungicides does tend todecreas e the rate ofemergenc e ofre ­ sistance (p.195-206).However ,mos texperienc e hasbee n gainedwit hhomo ­ geneousmixtures ,i.e . fungicides that are firstmixed , and then applied asa homogeneou s mixture to thecrop .Thi sma yhav eth edisadvantag e that ifon e component ismarkedl ymor epersisten t thanth eothers ,i twil lb e exposeduniforml y toth epathoge nwhe nth e dose levels ofth e lessper ­ sistentcomponent s havedeclined . Amor e effective strategymigh tb e tous eheterogeneou s mixtures,i n which thecomponent s are applied simultaneously toth ecrop ,bu tt osepa ­ rateparts . Inthi s system heterogeneity ispreserve d longer sinceth e plants treatedwit hth emos tpersisten tcomponen twil lb emixe dwith ,ef ­ fectively,untreate d plants after thedos e levels ofth eothe r components havedeclined .An y resistant isolateswil l then face competitionwit h sensitiveisolates . Inpractice ,heterogeneou s mixtures areobtaine db ymixin g seed lots treatedwit hdifferen tmaterials .Simila rmixture s of foliar spraysre ­ quireth e development ofappropriat e machinery, themos tpromisin g of which isth eelectrody n sprayer,whic hcoul d easilyb e adapted forthi s purpose.

Alternating fungicides

Theprincipl e behind this strategy ist ous e onecompoun d to reduce the absolutepopulatio n sizeo f formso fth epathoge n resistantt oth e alternate compound.Th e alternative compound canthe nb e safelyintro ­ duced tominimiz e thepopulatio n sizeo fpathoge n formsresistan t toth e firstcompound , and soon . Iti sno tpossibl e tostat e generally thatal ­ ternating of fungicides ismor e or lesseffectiv e thanmixin g fordelay ­ ingth edevelopmen to fresistance .However ,wit h alternating fungicide use, ifon e ofth ecomponent s ismuc h lesseffectiv e thanth e others,th e costo fdiseas e controlwhil e iti sbein guse dma yb ehigh ,becaus e of themor e frequentapplication s necessary.

Integrated control

Ina n analogous developmento fstrategie s fordelayin g increasedpa ­ thogenicity towards resistanthost-plan tvarieties ,Wolf e& Barret t (1980)propose d theus e ofmixture s ofresistan tplan tvarieties .The y also suggested that fungicide use shouldb e integrated withvarieta l mixtures, andmethod s fors odoin ghav ebee n described byWolf e (1981). Themos tobviou smetho d ist otrea tth e seedo fon eo rmor ecomponent s of thevarieta l mixturewit hdifferen t fungicidesbefor emixin g theseed . Alternatively, asmal lproportio n ofth e alreadymixe d seedca nb etrea ­ tedwit h fungicide and thenmixe d eitherwit huntreate d seed,o rwit h

147 seed treatedwit hothe r fungicides.Th ewithin-cro pdiversit y for disease control thatresult s isconsiderable .A pathoge n isunlikel y tomatc h such asyste m for aconsiderabl e time,particularl y ifth emixtur ecompo ­ sition ofhos t and fungicide isregularl y changed. Ifreasonabl e estimates ofth e appropriate parameters areavailable , themode l ofdisruptiv e selection described canb e extended tocompar e thebasi c strategies suggested.Howeve r for amor e realistic analysis it isnecessar y to includeparameter s ofspor edispersa l (Barrett, 1980). The simplemode l described assumes redistribution of all sporesproduce d over allhos tcomponents .Thi s isobviousl y unrealistic. Incerea lproductio nmuc hrelianc e iscurrentl yplace do nergosterol - biosynthesis inhibitors,bu tinsensitiv e forms of E. graminis f. sp. hor- dei haveno wbee ndetecte d inth e field inEnglan d in198 0 (J.T.Fletcher , personal communication)an d 1981,an d inWes tGermany , also in198 0an d 1981 (E.Limpert ,persona l communication).Ther e isn o indicationye ttha t there isa practica l problem ofreduce d effectiveness.However ,rathe r thanwai tt ose e ifa proble m doesdevelop ,i twoul db emuc hmor e rational totr y to implement some suitable strategies along the linesdescribe d in this contribution toreduc e atleas tth e likelihood of aproble m develop­ ing.

Acknowledgement

Iwis h to acknowledge thehel p ofDr .J.A .Barrett ,Genetic sDepart ­ ment, CambridgeUniversity .

References

Barrett,J.A. ,1978 .A mode lo fepidemi cdevelopmen ti nvariet ymixtures .In : P.R. Scott& A .Bainbridge . (Eds):Plan t DiseaseEpidemiology .Blackwel lScientifi c Publications,Oxford ,32 9p . Barrett,J.A. ,1980 .Pathoge nevolutio ni nmultiline san dvariet ymixtures .Zeitschrif t fürPflanzenkrankheite nun dPflanzenschut z87 :383-396 . Bent,K.J. , 1978.Chemica lcontro lo fplan tdiseases :som erelationship s topathoge n ecology.In :P.R . Scott& A .Bainbridg e (Eds):Plan tDiseas eEpidemiology .Black ­ wellScientifi cPublications ,Oxford ,32 9p . Mather,K. , 1953.Th egenetica lstructur eo fpopulations .Symposi ao fth eSociet y forExperimenta lBiolog y7 .Cambridg eUniversit yPress ,p .66-95 . Walmsley-Woodward,D.J. ,F.A .Law s& W.J .Whittington ,1979 .Th echaracteristic s ofisolate so fErysiph egramini s f.sp .horde ivaryin g inrespons et otridemorp h andethirimol .Annal so fApplie dBiolog y92 :211-219 . Wolfe,M.S. ,1981 .Integrate d useo ffungicide san dhos tresistanc e forstabl edi ­ seasecontrol .Proceeding so fth eRoya lSociet y (inpress) . Wolfe,M.S .& J.A .Barrett ,1980 .Ca nw elea dth epathoge nastray ?Plan tDiseas e64 : 148-155. Wolfe,M.S .& A .Dinoor ,1973 .Th eproblem so ffungicid etoleranc ei nth efield .Pro ­ ceedingso fth e7t hBritis hInsecticid ean dFungicid eConference .Britis hCro p ProtectionCouncil ,London ,p .11-19 . Wolfe,M.S .& E .Schwarzbach ,1978 .Pattern so frac echange si npowder ymildews .Annua l Reviewo fPhytopatholog y 16:159-180 . Wolfe,M.S .& S.E .Slater ,1980 .Annua lRepor to fth eU KCerea lPathoge nVirulenc e Survey for1979 .

148 Can we use models describing the population dynamicso f fungicide-resistant strains?

J.C. Zadoks Laboratory ofPhytopathology ,Agricultura l University,Wageningen , The Netherlands

Abstract

Vanderplank'sequivalenc e theorem leads toa nepidemiologi c evaluation of fungicides.Wit h theconcept s of 'fitness' and 'competition' frompopu ­ lationgenetic s and thoseo f 'carryingcapacity ' and 'intrinsic growth rate' frompopulatio n dynamics,i tbecome spossibl e todesig n apopulatio n biology of fungicide-resistant strains ofphytopathogeni c fungi.A first step towards aquantitativ e biology ofplan tdisease s duet ofungicide - resistant strains isventure d and afe wexample s aregiven .

Keywords: fungicides,competition , fitness,epidemiology , resistance.

The equivalence theorem

Epidemiologists cherish the disease triangle,whic h isa symbo l ofth e interactionsbetwee n environment,hos tan dpathoge n (Figure 1). The sides ofth e triangle representmutua l effectsbetwee npair s of factors;nothin g is said aboutth enatur e ofth e effects.A neconomicall y desirable effect isth ereductio no fdisease .Thi s reductionma yb ebrough t aboutb yredu ­ cingth evirulenc e ofth epathogen ,o rb y increasing the resistance of thehost ,o rb ychangin g theenvironment .Th e change ofenvironment ,i n itsturn ,ca nb e causedb y achang e incultivatio nmethods ,a chang e in crop climate,and/o r applicationo ffungicides . Epidemiologists measure therat eo fprogres s of adisease .Fo rthi s

Environment

Host Pathogen Figure 1.Th ediseas etriangle .

149 purpose they apply the logistic equation

dx/dt= r .x.( l -x ) (1) inwhic hx isth e fraction disease (0 <_x £ 1), ti stime ,an d ri sth e logistic infection rate.Th evalu e r isth e 'speedometer'o fth eepidemic . Reductiono fdiseas e canb e obtainedb y reducing the initial inoculum, x0, and/or the speed ofth eepidemic , i.e.reducin g r. Epidemiologically, resistance ofth ehost ,chang e ofmicroclimat e and application offungi ­ cides canal l lowerr .Al l thesever y different actions haveth e sameepi ­ demiological effect, i.e. they areepidemiologicall y equivalent.Thi s is the 'equivalencetheorem ' ofVanderplan k (1963), which isillustrate db y Figures 2an d 3.Not e thati nthes e figuresth evalue splotte d alongth e ordinates areno tx ,bu t logitx ,wher e

logitx =I n [x.(l-x)"1] (2) inorde rt oobtai n approximately straight lines,i.e . the logitlines . Theequivalenc e canb e carried one step further.Amon g fungicides,pro ­ tectants such asheav ymeta l compounds,sulphur , and someo fth eearl y carbamates,hav e aneffec tloosel y equivalent tohorizonta l resistance, which isa hos tresistanc e approximately equal foral l strains ofth efun -

n 26 46 66 86 time( da yo fyea r) Figure2 .Developmen to fepidemic so fCercospor aapi io nceler yi nFlorida . 0= diseaseprogres s inunspraye d celery,wil dtype ;# = diseaseprogres s inceler y receivingweekl yspray so fbenomyl ,resistan tstrain . (Source:afte rBerger ,1973) . Thebenomyl-resistan t strainstart sa ta lowe rleve ltha nth ewil dtyp ebu tovertake s it;th edifferenc ebetwee nth eslope so fth etw ologi tline si sstatisticall ysignifi ­ cant.(Source :Zadok s& Schein ,1979) .Horizonta laxis :t ,tim ea sda yo fth eyea r fromNovembe rt oMarch .Vertica laxis :logi tx ;x i sdiseas eseverity .

150 Eigenheimer x

July 1 Aug.1 Sept. 1 date Figure 3. The progress of late blight in 117 fields in potatoes in sand areas of the Netherlands. Logit lines with different slopes represent different varietal resistances; x is the proportion of diseased foliage. (Source: Anonymous, 1954; Vanderplank, 1963).

gus. By analogy, these fungicides may be called 'horizontal' fungicides. They often affect many metabolic pathways without typical site-specific action. At the opposite end of the range of variation are the 'vertical' fungicides. These are the fungicides with a very specific metabolic tar­ get. They kill all strains of the fungus except the odd one that acquires resistance to the fungicide. Many of the systemic fungicides induce resis­ tance. Their effect, then, is equivalent to 'vertical' or race-specific resistance. Resistant strains of a fungus are said to be equivalent to the classical physiologic races that have a gene-to-gene relation with their host plants.

Fitness

Thewor d 'fitness' isborrowe d frompopulatio n genetics.Th e term is easily understood ina genera lway ,bu tth econcep to f fitness isno ts o easy tohandl e ina scientifi c way. Iti sa comparativ e concept.On egeno ­ type ismor e or less fittha nanothe r genotypeunde r givenconditions ; more specifically, onegenotyp e canreproduc emore ,o r less, successfully than anothergenotype .

151 Theusua l numerical approacht ofitnes s ist ochoos eon egenotyp e asa reference andt oassig ni ta fitnes svalu eo f1 .Th eothe rgenotype ,t o be compared toth ereference ,ha sth efitnes svalu e of1 - s .Th esymbo l s stands forth edifferenc e infitnes sbetwee nth etw ogenotypes ,wit h 0 £s £ 1.Fo rou rpresen tpurpos e thenon-resistan t straino fth efungus , here indicated asa wil d strain,wil lb eth ereferenc ewit h fitness 1.Th e resistantstrai nwil lb eindicate d asmutan t strainwit h fitness 1- s ; forth etim ebein gw ewil l assume itt ob eles s fittha nth ewil d strain. Epidemiologists canb esomewha tmor eprecis e thanpopulatio ngeneti ­ cists.The yhav e techniques tomeasur evariou s components thatcontribut e to fitness,suc ha slaten tperiod ,p ,infectiou s period, i,dail ymulti ­ plication rate, R ,an ds oon .Epidemiologist s have alsodevelope d methods tocompar egenotype sb ycomponent s andcombination so fcomponents .On e method isth e 'componentsanalysis' ,whic hca nb eextende dt o 'lifeta ­ bles'an dthe n 'survivaltables' . Combinations ofcomponent s canb euse d alsot ocalculat e relative growth rateso fth esam enatur e asth elogis ­ tic infection rate,r ,mentione d before (Zadoks& Schein, 1979). Thecircl e isno wcomplete ,beginnin g atth efitnes s reductions tha t canb edetermine d experimentally,passin go nt oa detaile d analysis that leadst ovariou s rvalues ,and ,finally , comingt oth edifference s between r values,whic hca nb econsidere d interm so fdifference s infitness . Table 1give sa nexampl eo fho wa 1 0% reductio ni non ecomponen t leadst o a changei nrelativ e growth rate.Not e thata 1 0% reduction invirulenc e bywa yo fth ebasi c infection rate, R, implies adecreas e in R, buttha t a 10% reductiono fvirulenc eb ywa yo flaten tperiod ,p ,implie s anin ­ crease inp .Th eeffec to fa 1 0% reductio no fvirulenc evarie s according toth ecomponen tan dth eorigina l levelo fvirulence .Tabl e 1contain s twowarnings :i ti sdifficul tt opredic tth echang ei nr fro ma know n change inon ecomponent ;an di ti snex tt oimpossibl e tomak e conclusions aboutchange s incomponent s froma know nchang ei n r value. The logico fpopulatio n geneticswoul dno wcompel lu st oth efollowin g argument.Unde r conditionso fintensiv ean dlarge-scal e treatmentwit ha

Table1 . Theeffec to nr o fa 10 %reductio no ffitness .

Moderate r Highr

Strain wild mutant mutant wild mutant mutant P 10 11 10 10 11 10 R 1.48 1.48 1.33 21.84 21.84 19.66 pR 14.8 16.28 13.33 218.4 240.24 196.6 r 0.200 0.188 0.193 0.400 0.371 0.391 Reductiono fri n - 6 4 - 7 2 Source:Zadok s& Schei n(1979) .

152 vertical fungicideth eselectio npressur efo rresistanc ei sgreat .A mu ­ tant strainwil l appear,probabl y througha 'los smutation ' thati smor e successful thanth ewil d typea slon ga sth eselectio npressur econtinues , but lesss oa ssoo na sth eselectio npressur e ceasest oexist .Th e impli­ cationi stha tth efrequenc yo fth eresistan t straindecrease sa ssoo na s the fungicidei sn olonge rused .Thoug h the reasoningi sperfectl yplau ­ sible, real lifei sno ta ssimpl ea sthat .Ther ear e noteworthyexceptions . Figure2 show stw o epidemicso f Cercospora apii oncelery .Th ebenomyl - resistantmutan t strainha sa highe rr valu e thanth ewil d type.I twa s reported thati nsom ecase sth eresistance ,th emutan tcharacter ,persis ­ ted evenwhe nth e selectionpressur e ceasedt oexist .Suc h anomaliesca n beexplaine di ndifferen tways .Eithe rth e general conceptso fpopulatio n geneticsd ono tappl yt oresistanc eo ffungicide si nfungi ,o rth eactua l situationi smor e complex thanth etheor ypresented . Therei ssom e evi­ dence forth e lattervie w (Zadoks& Schein , 1979).

Competition

Population geneticists tendt othin ki nterm so fselectio nfo rstrain s withhig h fitness undergive nconditions .I nth e simplestcas eth efunga l populationi scompose do ftw o fractions,a fractio np ,representin gth e weaker strain,an da fractio n q, thestronge r strain.Th esu mo fth etw o fractionsmus tequa lunity :

p + q = 1. (3)

After onegeneratio n the fraction p0 ofth eweake r strainha sbecom e px:

Pj= ( 1- s).p 0/ correction factor (4) and

<ï\ -

whereth ecorrection factor =( 1- s) . p0 + q0• (6)

MultiplyEquatio n4 b y q0 andsubstitut eEquatio n6 i nEquatio n4 ,

piqo =[( 1- s) .p o .<7o]/[( l- s) . po+ q0]

= (1- s) . p0.{ q0 /[(l- s) . p0 + q0]} = (1- s) . po .q! - (7)

DivideEquatio n7 b yp 0 qo•

Pi/Po =( 1- s).(q1/q0) (8)

153 Remember thats i sth erelativ e fitness reduction and (1- s )i sth e fit­ ness ofth eweake r strainrelativ e toth e strongerone . For the second generation theprocedur e canb e repeated:

P2/P0 = (1- s)2.(<7 2/g0) (9)

Similarly after n generations:

n Pn/Po = (1- s) .(

Equation 10ca nb ewritte na s

ln(l -s )= (1/n).(logitp n -logi tp 0) (11)

Equation 11ca nb ecompare d toth e integrated form ofEquatio n 1,th elo ­ gisticequation :

_1 r = (tn-t0) .(logitx n -logi tx 0) (12)

Inthes e lasttw o equations,x andp represent fractions (0 <_x <_ 1, 0

154 In ]-x

time( d)

Figure4 .Th eprogres so fbligh ti n2 9unspraye dan d3 1spraye dfield so fth esuscepti ­ blepotat ocv .Logi tline swit hdifferen t slopesrepresen tdifferen ttreatments ;x i s theproportio no fdisease d foliage.Dat afro mth eNetherland s (Source:Anonymous ,1954 ; Vanderplank, 1963).

Figure5 .Competitio nbetwee ntw ostrain so fcucumbe rmilde w (Sphaerotheca fuliginea) underfungicide-fre e conditionswhe nmixe d in3 differen tproportion sa tn = 0 .Th ewil d benomyl-sensitive straini sth efittest .Curve ssho wth edecreas ei nth eproportion , j>> ofth emutan tfungicide-resistan t strain.Excep tfo rth elowes tlin ean dth efirs tre ­ productioncycle ,th elogi tline sar ereasonabl ystraigh tan dparallel .Fo rth euppe r line 1- s = 0.42 . (Source:dat ab ycourtes yo fD rJ .Dekker ,Laborator yo fPhytopatho ­ logy,Agricultura lUniversity ,Wageningen ,unpublished ;Zadok s& Schein ,1979) .Horizon ­ talaxis :n ,tim ei ngenerations .Vertica laxis :£ ,proportio no ffungicide-resistan t straino nlogi tscale .

155 cycle repeated, and soon .Th emutan tstrai n is less fittha nth ewil d typewhe n the selectionpressur e isremoved ; thatmean s thats isposi ­ tive (0< _ s £ 1). Calculation showstha ts = 0.5 8 and (1- s )= 0.42 .Th e relative rate ofdisappearanc e isr ,= -0.87. Ofcourse ,r ,i snegativ e asth eweake r straindisappear s gradually; the logitlin e representing theweake r strainrun s downward. Figure 5actuall y showsthre e lineso r curves, representing threemixin gproportion s at n =0 ,p bein g 0.99, 0.50, and 0.01, respectively. The line forp =0.0 1 isa n anomaly. Thecompetitio nbetwee n fungicide-resistant and fungicide-sensitive strains isno tunlik e thatbetwee n differentvertica l races ofa rust . Figure 6show sth ecompetitio nbetwee ntw oraces ,15B- 1an d56 ,o fblac k stem rust (Puccinia graminis) attw o differenttemperatures .Figur e 7 showsth e samecurve s after logittransformation .A tth e start,th etw o races arepresen t inequa lproportions .Rac e 56 isth e 'loser' ata lo w temperature.A t ahig h temperature,however , iti sth e 'winner'.Th e example illustrates three importantpoints : - arelativ e fitnessvalu e ismeaningfu l onlyunde r clearly specified conditions andpertain s exclusively tothos econditions . - the equivalence theorem isvali d also forsituation s ofcompetition . - itseem stha tth egenetic so f fitness ismor ecomple xtha n justsingl e gene inheritance.

P 1.0-

0.8-

0.6-

0.4-

0.2-

0 1—'—I—'—I—'—I—'—I—'—I 0 2 U 6 8 10 generations Figure6 .Competitio nbetwee nisolate so fblac kste mrus t (Pucciniagraminis )o nseedling s ofwhea tcv .Littl eClub .A tgeneratio n0 a 1 : 1 mixtur eo fspore so fon eisolat eo f Race 15B- lan don eisolat eo fRac e5 6wa sapplie dt oLittl eClub .Th espore sproduce d werecollected ,on epar tapplie dt ofres hplant so fLittl eClu b (nextgeneration) ,an d onepar tuse dfo rfrequenc ydetermination .Th e0 generatio nyield sapproximatel yequa l amountso fbot hraces .A thig htemperature ,th eisolat eo fRac e5 6outyield sRac e 15B-l ; atlo wtemperature ,th erevers ei strue .(Source :afte rKatsuy a& Green ,1967 ;Zadok s& Schein,1979) .Horizonta laxis :t ,tim ei nuredia lgenerations .Vertica laxis :j> ,rela ­ tivefrequenc y (orproportion )o fRac e5 6isolates .

156 logit p ./• 20 °C 4-

2- »s*

0- • K__»_^ 9 15 °C

1 1 1 2- i | i i i 0 2 4 6 8 10 uredial generations Figure 7. Competition between isolates of black stem rust (Puccinia graminis) on seed­ lings of wheat cv. Little Club. Data as used in Figure 6 are presented after logit transformation; the line for 20 °C is nearly the same as the line for 25 °C. (Source: Zadoks &Schein , 1979). Horizontal axis: t, time in uredial generations. Vertical axis: £, proportion of Race 56 isolates.

Recent studies on barley mildew (Erysiphe graminis) have shown that mutation for virulence is a prerequisite for establishment of the mildew on a new and vertically resistant cultivar, but that it is not enough (Wolfe &Barratt , 1979). Other genetic adaptations are needed for success­ ful establishment. The same may be true for fungicide-resistant mutant strains.

Carrying capacity versus intrinsic growth rate

A desk study shows what can happen in the field. Imagine a disease of which the severity, x, can be expressed as the fraction of foliage infec­

ted. Assume that the initial inoculum x0 of the wild strain is x0 (wild) = 0.001 and that x0 of the mutant strain is 10 000 times smal­ ler. Assume that the logistic growth rate r(wild) = 0.20 and that r(mutant) =0.15. In Figure 8A the heavy line represents the wild and the thin line the mutant strain. The vertical axis has a decimal logarithmic scale; leveling-off of curves represents crowding. At t = 0, the dif­

ference between the two strains is 4 l°g10 units, i.e. a 10 000-fold dif­ ference. At t = 20, it has become 5 log,« units, i.e. a 100 000-fold dif­ ference . Without treatment the mutant strain is a 'loser'. With treatment, the mutant strain follows curve c (little crowding). The wild type follows curve a if the fungicide reduces the number of sites available to the fungus, or curve b if the fungicide reduces the logistic growth rate r by a factor of 2. The experiment is designed so that in both cases the dif-

157 time ( arbitary units) Figure 8. A desk study with a mixture of two fungal strains: a wild fungicide-sensitive strain (heavy lines) and a mutant fungicide-resistant strain (thin lines). Untreated controls (solid lines); fungicide application (broken lines). Note how themutan t strain increases in frequency relative to the wild type after fungicide application. In Figure 8B, theapparen t infection rates aretw o times higher than those in Figure 8A, to represent severe epidemic conditions. ForCurve s a, thefungicida l effect is a reduction of thecarryin g capacity K; for Curves b, thefungicida l effect is a re­ duction of theinfectio n rate r. Note how Curves c, representing the mutant strain when fungicides have been applied, approach Curves a and b, slower in Figure 8A and faster in Figure 8B. (Source: Zadoks &Schein , 1979). Horizontal axis: t, time in ar­ bitrary units. Vertical axis: n, disease severity onlo gscale , to be read asx = 10°.

ference between the twostrain s at t = 20 is 3 log,n units, i.e. a thousand-fold difference. In other words, the mutant strain wins. Figure 8B shows what happens when the logistic growth rate of the wild and the mutant strains are doubled, the other things remaining unchanged. The replacement and competition process goes much faster, and the epidem­ iologic mode of action of the fungicide makes a difference. If the fun­ gicide reduces the logistic growth rate by 2 (Curve b), the difference between the strains at t = 20 is one log10 unit, a 10-fold difference, but when the fungicide reduces the number of available sites by 10, the two strains end up on a par with each other. This desk study illustrates a rule of population genetics: 'In an un- crowded environment the success of a population is determined mainly by its intrinsic rate of increase r; in a crowded environment the carrying capacity Kma y be more important' (Crow & Kimura, 1970). At what level of x does crowding begin? We assume this level to be x = 0.05 (In x = 1.30). When the fungicide is applied, crowding is not so relevant in the situa-

158 tiondepicte db y Curve b; relative growth rates determine theoutcom eo f theprocess .Fo rth esituatio ndepicte db y ath ecarryin g capacity for themutan t strain is K =x _„= 1 ,bu tth e carrying capacity ofth ewil d m iticix straini s K =0.1 ,an dth emutan trapidl y gains anadvantag e overth e wild strain. Practical findingsma ycorroborat e the foregoing reasoning. Inarea s with regular epidemics of Cercospora beticola on sugar-beet,benomyl-re - sistantmutan tstrain s aremor eprominen t (Dovase t al., 1976).Resis ­ tance in Venturia inaeanialis tododin e onapple s appeared after aparti ­ cularlywe tseaso n followingyear so fgoo dcontro l (Szkolnik& Gilpatrick , 1969). Thecommo ndenominato r inbot hcase s is anincrease , locallyo r temporarily, ofth e logistic growtho fth e epidemic,r .Th e reasoning can be extrapolated to ahighe r levelo fgenerality : resistance to fungicides ist ob e expected especially after seasons conducive tosever e epidemics i.e. epidemicswit h relatively highr values) , and inlocalitie sspecifi ­ callyconduciv e toa particula rdisease . Simplemodel s forstrategie s ofpesticid e usehav e alreadybee ndevel ­ oped.Muc h depends onth enatur e ofinheritanc e ofresistance . Isther e a genedosag e effect? Isther ea regula r sexual reproduction? Whatar e the dominance relations ofgene s fortolerance ?Tw o general conclusions seem toemerg e: -Th eus e of amixtur e of fungicideswit hdifferen tmechanism s ofaction , each applied atreduce d dosages,i sno trecommende d (Conway& Comins , 1979). -Regula r alternation of fungicideswit h differentmode s of actions applied atnorma l dosagesha s little advantage aboveth e sequential use ofthes e fungicides atnorma l dosages (i.e.usin g the firstunti l itha s become ineffective duet otoleranc e inth e fungus,the nus eth esecond , etc., etc.)(B.R .Trenbath ,persona l communication, 1979). However, forth etim ebein g conclusionsbase d onmodel smus tb ehandle d with caution,becaus e thesemodel s are stillprimitive ,incomplet e and poorly corroborated by experimental evidence.

Conclusion

Thetitl eo fthi s contribution asks 'Canw eus emodel s describing the population dynamicso f fungicide-resistant strains?'.Th e answeris ,defi ­ nitely, yes.Bu tthi sye s ispronounce d under the followingrestrictions : - Themodel smus tb e timedependan t (theduratio no fth epersistanc e of thechemica l ando fth e latentperio dp mus tb e taken into account (see p.139-148)). -Th emodel smus tcombin e elements frompopulatio n dynamics andpopula ­ tiongenetics . -Th emodel smus tcontai n stochastic elements.

159 - The models must be constructed specifically for the crop, pathogen and chemicals concerned. - Such specific models must be validated by field and laboratory observa­ tions. Such model studies could be part of the universities' contribution to the cooperative efforts toward a more sophisticated use of fungicides, in ac­ cordance with the plea by Schwinn elsewhere in this book (p.22-23).Many useful experiments can be performed with simple techniques that do not require sophisticated and expensive equipment, although they do require a considerable amount of time and labour. Such experiments will be useful for developing, first, the theory and, second, the strategy of control, with as the final objective finding a way of living with resistance to fungicides.

Acknowledgements

Thanks are due to Dr J.A. Barrett and Mr J.A.G. van Leur for their critical and constructive comments.

References

Berger, R.D., 1973. Disease progress of Cercospora apii resistant to benomyl. Plant Disease Reporter 57: 837-840. Conway, G.R. & H.N. Comins, 1979. Resistance to pesticides. 2. Lessons in strategy from mathematical models. Span 21. Crow, J.F. & M. Kimura, 1970. An introduction to population genetics theory. Harper & Row, New York, 591 p. Dovas, C, G. Skylakakis & S.G. Georgopoulos, 1976. The adaptability of the benomyl- resistant population in Cercospora beticola in Northern Greece. Phytopathology 66: 1452-1456. Katsuya, K. & G.J. Green, 1967. Reproductive potentials of races 15B and 56 of wheat stem rust. Canadian Journal of Botany 45: 1077-1091. Szkolnik, M. & J.D. Gilpatrick, 1969. Apparent resistance of Venturia inaequalis to dodine in New York apple orchards. Plant Disease Reporter 53: 861-864. Vanderplank, J.E., 1963. Epidemics and control. Academic Press, New York, 349 p. Wolfe, M.S. & J.A. Barrett, 1977. Population genetics of powdery mildew epidemics. Annals of the New York Academy Sciences 287: 151-163. Zadoks, J.C. & R.D. Schein, 1979. Epidemiology and plant disease management. Oxford University Press, New York, 427 p.

160 Fungicide resistance and microbial balance

G.J. Bollen Laboratory ofPhytopathology , Agricultural University,Wageningen , The Netherlands

Abstract

Plantpathogen s area minorit ywithi n themicroflor a ofth eplan ten ­ vironment. Innorma l situations,a well-balance d ecosystem exists through numerous interactionsbetwee n itscomponents .Fung i are amajo rcompo ­ nent, soth eus e of fungicides canaffec tth emicrobia l balance.Th e res­ ponses ofpathogen s to ashif t inthi sbalanc e are discussed. Stimulationo fth emicrobia l antagonism to apathoge n leadst o integrated orindirec tcontrol ;inhibitio no f itca nresul t ina chang e ofth e domi­ nantpathoge n orcaus e a 'boomerang'effect .Specia l attention ispai dt o responses ofpathogen s thatar eresistan t to fungicides and toth erol e ofresistan t antagonists.Whe n indirecteffect s areinvolved , closelyre ­ latedpathogen s oftendiffe r inthei rresponse s to afungicide .Th e same isals otru e forth e samepathoge n indifferen t soils. Iti s suggested thatthes edifference s arecause db y the specificity ofmicrobia l inter­ actions.

Keywords: synergistic interactions,antagonisti c interactions,competi ­ tion fornutrients ,amensalism , antibiosis,mycoparasitism , integrated control,chang e ofdominan tpathogen ,boomeran g effect, iatrogenic disease,benzimidazoles,chlorothalonil , dicarboximides,quintozene , Botrytis, Fusarium, Microdochium, Pénicillium, Pythium, Phytophthora, Rhizoctonia, Sclerotium, Trichoderma.

Introduction

Inth evas tmicrobia l world onth eplan t and inth e soil, theplan t pathogens arever ymuc h inth eminority . Compared tothei rcohabitant s of leaves androots ,mos tpathogen s arewea k competitors forth enutrient s available.However ,the y escape from this competitionb y infectinghos t plants;th e livingtissu e and itsdirec t surroundings isthei r ecological niche.Nevertheless ,durin g certain stages,o fthei r life cycle,th epatho -

161 gens are susceptible to interactions with the saprophytic microflora. These stages include sporegermination , infection ofth ehos t and,espe ­ cially, survival inth e absence ofth ecrop . Thevariou s components ofth emicroflor a inth eplan tenvironmen tar e kepti na stat eo fdynami c equilibrium throughnumerou s interactions.A s fungi constitute amajo r component,th eus eo f fungicidesma y affectth e microbialbalance .No tonl yth epopulation s ofsensitiv e specieswil lb e affected,bu t alsothos e ofresistan tmicro-organism s interactingwit h thesensitiv e ones. Inth enatura lenvironment , suppression of sensitive specieswil l soonresul t ina n increase ofresistan t species competing forth e same substrate.Beside s competition fornutrients ,othe rmech ­ anisms underlying themicrobia l balance, likeproductio n of antibiotics andmycoparasitism ,ma y alsob e affectedb yfungicides . Inrecen treview s oneffect s of fungicides onnon-targe torganisms , Hislop (1976)discusse d the effects onth emicroflor a ofaeria l plant surfaces,Wainwrigh t (1977)an dBolle n (1979)thos eo nth e soilmicro ­ flora andPapaviza s &Lewi s (1979)thos e onsoil-born epathogens .Thi scon ­ tributiondeal swit hresponse s ofpathogen s to fungicidesbrough t about by the effects ofthos e fungicides onth e interactionsbetwee npathogen s andmicroflora . Special attention ispai dt oth erol eo fthos efungi , that arenaturall y resistant to fungicides orhav ebecom e resistantunde r selectionpressur eb y afungicide .Th eexten tt owhic hthes e fungibene ­ fit fromthei r resistance incompetitio nwit h themicroflor a isdiscus ­ sed.

Types of microbial interactions

Interactions between species orpopulation sma yb e synergistic oran ­ tagonistic (Table 1); iti shar d toconceiv e aneutra l relationbetwee n organismspresen t inon emicrohabitat .Th eeffect s ofthes e interactions onth epopulation s involvedma yb epositiv e ornegativ e (Table 2). Insyn ­ ergism, growth orreproductio n ofon eo rbot h ofth e associates isstim ­ ulatedb y theother .Th eter mprobiosi swa s coinedb y Sussman (1965)t o describe anorganism' sproductio n of stimulatory substances inducing sporegerminatio n andothe r specificprocesse s inothe rorganisms .Th e greater the dependence ofon e organism onth eother ,th egreate r the effectwhe non e ofth e associates issuppresse d by afungicide .Syner ­ gistic interactions that are involved inth e etiology ofplan t diseases areknow n forassociation s ofvariou s organisms,especiall y betweenne ­ matodes and fungi.Whe n afungu spredispose s aplan tt o infectionb y an associated pathogen, application of afungicid ema y decrease disease incidence,eve nwhe n thepathoge n itself isresistan tt oth efungicide . Witheffect s ofpesticide s onantagonisti c interactions iti softe n difficultt oindicat e themechanis m involved. Iti sespeciall y difficult

162 Table 1. Microbialinteractions .

Synergistic relations Antagonistic relations

Provisiono fnutrient san d Competitionfo rnutrients ,oxyge n growthfactor sb ycross - andpossibl yspac e feedingo rcommensalis m

Productiono fprobioti c Amensalismb yproductio no fanti - substances bioticsan d simplemetabolites ,e.g . NH ,nitrite ,C H

Inactivation of substances Parasitism and prédation including pesticides toxic to the associates Additionally for plant pathogens Predisposition of the host Protection of the host plant plant to infection by from infection associated pathogens

to distinguish between effects on nutrient competition and those on amen­ salism. In amensalism, one species occupies the substrate by its ability to produce substances that are inhibitory to other organisms. In experi­ ments where the occupation of the substrate is used as a parameter, it is impossible to indicate whether this has been achieved by reduced competi­ tion for nutrients or by keep other organisms from the substrate. There-

Table 2. Effects of microbial interactions on the populations involved. Interaction Population A Population B

Positive mutualism,e.g . cross-feeding + + commensalism + 0 probiosis + 0 detoxification + 0 predispositiono fa hos tplan t byB t oinfectio nb yA + 0 mutual + +

Negative competition amensalism - 0 parasitism - + prédation - + protectiono fa hos tplan t byB fro minfectio nb yA - 0

Source:modifie d afterOdu m (1971).

0 =n oeffect ;+ = positiv eeffect ;- = negativ eeffect .

163 forepreferenc e shouldb e givent o thebroa d concepto fcompetitio n of Clark (1965), inwhic h the role ofantibiotic s inth e occupation ofth e substrate andnutrien tcompetitio n are included. Reduced competition forth e substrate isprobabl y themai ncaus e for anincreas e oftoleran tpathogen s afterth eus eo fselectiv e fungistatic compounds.Example s areknow n forconventiona l and systemic fungicides: theincreas eo f Fusarium and Puthium inquintozene-treate dsoil s (Farley & Lockwood, 1969;Kata n& Lockwood, 1970), and the increase oftw o cereal pathogens, Cochliobolus sativus (Fokkemae t al., 1975)an d Rhizoctonia cerealis (Reinecke& Fehrmann ,1979 ;va nde rHoeve n& Bollen ,1980 )i n benomyl-treatedcrops ,respectively . Fungicides ofdifferen t categories,e.g .benzimidazole s anddicarbox - imidesma y affectth e synthesis of antibiotics by fungi. Stimulation as well assuppressio n ofth eproces shav ebee nreporte d (Bollen, 1979). The effect ishighl y specific and inhibitiono fgrowt hb y a fungicide does not implytha t synthesis of antibiotics isals o suppressed. Under natural conditions pathogenic fungi areofte nparasitize d by other fungi,fo rexampl eparasitis m on sclerotia ofpathogen s in soilan d onmyceliu m and spores inpustule s ofrust so nleaves . Infungicide - treated fields,thi sparasitis mwil lb e influencedwhe nth e fungushos t and itsparasit e differ inthei r sensitivity toth e fungicidesused . If themycoparasit e isles ssensitiv etha nth ehos t fungus,applicatio no fa fungicidema y result ina form of integrated control,e.g . thecoloniza ­ tiono frhizomorph so f Armillaria mellea by Trichoderma spp.i nsoi l treatedwit h CS2 andmethylbromid e (Bliss,1951 ;Oh r et al., 1973).Re ­ cently,Dave t (1979)suggeste d thatth eeffectiv e control of Sclerotinia minor invegetable sb y dicarboximides mayb e duet oth e sensitivity of thepathoge n aswel l asth eresistanc e of itsmycoparasite s tothes efun ­ gicides. Inth e reverse case -sensitiv eparasite s and toleranthos t fungi -aggravatio n ofth e diseasema yb e expected. Inspit eo fth eoc ­ currenceo fsevera lpathosystem s ofa resistan thos t fungus and asensi ­ tivemycoparasite ,dat ao n increase ofdiseas e incidence followingtreat ­ mentwit h fungicides arescarce .Th ehig hleve lo fwhite-mol d damage caus­ edb y Sclerotium rolfsii inpeanut s sprayedwit hbenomy l hasbee npartl y attributed to inhibition of Trichoderma viride (Backmane t al., 1975). Trichoderma species areeffectiv eparasite s of S. rolfsii.

Resistant pathogens and resistant antagonists

Twotype s of fungicide resistance canb e distinguished.Whe nth een ­ tirepopulatio n of aspecie s or agrou p ata highe r taxonomieleve l is resistant to afungicid e this iscalle d 'natural resistance'.Example s are theresistanc e of fusariat oquintozene , Alternaria spp.t obenzimi ­ dazoles, andoomycete s todicarboximides .Thi s shouldb e distinguished

164 from the other type ofresistance ,whic hunde r selectionpressur eb ya fungicide develops ina noriginall y sensitivepopulatio n ofa sensitive species;man y examples ofthi s kind ofresistanc e arepresente d elsewhere inthi sbook . Inbot h types resistantpathogen s will respond inth e same way to ashif ti nth emicrobia l antagonism causedb y afungicide . Emergence ofresistanc e to fungicides isno trestricte d topathogens . Saprophytes, including antagonists ofpathogens ,ma y alsobecom eresis ­ tant.Thi s hasbee n observed for Pénicillium cyclopium active asa nan ­ tagonisto fmercury-resistan t strains of Pyrenophora avenae onoa tseed s treatedwit h anorgano-mercur y fungicide (Ward& Greenaway , 1976). Another example isth e appearance ofresistan t strains of Pénicillium brevicompac- tum incyclame ncrop s treatedwit hbenzimidazoles . Ina greenhous ewher e thecro pwa s infestedwit h aresistan t straino f Botrytis cinerea, petio­ les and leaves ofsom ebenomyl-treate d plants developed agree nbloo mbe ­ causeo fabundan tsporulatio n of P. brevicompactum. Theseplant s seemed tob e less affectedb y Botrytis rottha nthos ewithou t Pénicillium spores. Antibiosis invitr ob y this fungust o B. cinerea couldb eclearl ydemon ­ strated: indua l cultures onmalt-agar ,distinc t inhibitionzone swer e observed around the colonies of P. brevicompactum. Ina greenhous eexperi ­ ment, theeffec to fth e antagonisto nth edevelopmen t ofth ediseas e ina benomyl-treated cropwa s investigated. Although thenon-inoculate d plants didno tremai n freeo f Botrytis rot,probabl ybecaus e theplant swer e kept under extremely humid conditions,i twa s showntha t inoculationwit hth e resistant straino f P. brevicompactum partly restored the antagonism to thepathogen .Th eeffec tlaste d onemont h (Figure1) . Ina stud y ofth e effecto fbenomy lo nth e fungus flora ofth ecul m base ofrye , Microdochium bolleyi (syn. Aureobasidium bolleyi) was among the species that firstdisappeare d after the treatmento fth e cropwit h the fungicide (Platenkamp& Bollen ,1973 ;Reinecke , 1977). Therefore,M. bolleyi wasuse d asa n 'indicator species'.Thi s rolewa s easilyexplai ­ ned later,whe nva nTuy l (1977)reporte d thatth e sensitivity ofmycelia l growtho f M. bolleyi wasth ehighes trecorde d foral l fungiteste d inhi s study.Nevertheless ,i nth e 1975 crop the funguswa s frequently isolated from ryeplant s inbenomyl-treate d plots ofexperimenta l fields atWage ­ ningen.Th e fungusha d lostit svalu e asa nindicato r species,becaus e parto fth epopulatio nha dbecom e less sensitive,whic hwa sconfirme d in tests invitro . Iti swell-know n that forpathogen s theemergenc e ofresistanc e occurs especially incertai n species,e.g . Botrytis cinerea, whereas thepopula ­ tionso fothe rspecie s still remainsensitiv e despite intensiveus eo f fungicides forlon gperiods .Th e sameseem s tob e true forth eantagonis ­ ticmycoflora .Amon g thenumerou s isolates of Trichoderma spp.obtaine d from roots andbulb s frombenomyl-treate d crops,th e authorneve r founda resistant strainno r could anyrecor d ofon eb e found inth eliterature .

165 affected leaves (%) 25-, BotrytisR

20-

Botrytis R+ Pénicillium R

15-

Control

10

5-

1^ 10 20 30 40 timeafte r inoculation(d )

Figure 1.Antagonis mbetwee nbenomyl-resistan tfung io ncyclame nspraye d witha suspensio no fbenomy l (1g/1) .Thre elot so f3 0densel yfoliate dplant swer e sprayedwit hbenomyl .On eda ylate rthe ywer espraye dwit hwate r (control),a spor e suspensiono fa resistan tstrai no fBotryti s cinerea,an da spor esuspensio no fth e samestrai nplu son eo fa resistan tstrai no fPénicilliu mbrevicompactum.Th eplant s werekep ta t100 %relativ ehumidity .Fo r3 2day safte rinoculatio nth emea nnumbe r ofaffecte dleave spe rplan tfo rplant sinoculate dwit hbot hresistan tstrain swa s significantly less (P< 0.05 ,Fishe rtest )tha ntha tfo rplant sinoculate dwit h B.cinere aalone .

However,resistan t isolates of Pénicillium brevicompactum were obtained frequently from thebenomyl-treate dcrops . Resistant antagonistsma y contribute todiseas e control ifthei rpop ­ ulation increases following afungicide ,treatment .The nth eultimat e disease control isth e resulto fbot hdirec tinhibitio no fth epathoge n and stimulation ofit s antagonists.Recently , successful attemptshav ebee n made to induceresistanc e ineffectiv e antagonists ofpathogens .Papaviza s (1980)obtaine d strains of Trichoderma harzianum thatwer e resistantt o captafol,chlorothalonil , iprodione andothe r fungicides.A number of thesestrain s showed anincrease d ability forbiocontro l ofwhit e roto f onioncause db y Sclerotium cepivorum.

166 Responses of resistant pathogens to fungicides due to a shift in the microbial balance

The side-effectso ffungicide so nth epopulatio no fsynergist so ran ­ tagonistso fpathogen sma yb epositiv eo rnegativ e fordiseas econtrol . Selective stimulationo fantagonis m leadst ointegrate do rindirec tcon ­ trol; selective inhibitiono fantagonis mca nresul ti na chang eo fth e dominantpathogen so rcaus e 'boomerang'effect s (Figure 2). Diseases that have increased asa resul to fthes ephenomen abelon gt oth eiatrogeni c diseases,whic hwer e definedb yHorsfal l (1979)a sthos e 'thatar einduce d orworsene db ya plan tpathologist' s prescription forth ecrop' .O fth e typeso fiatrogeni c diseases distinguishedb yGriffith s& Berri e (1978), theexample smentione di nthi s chapterbelon gt oth ecategor y inwhic h thediseas ei sinduce d through actiono fagrochemical so nth eecosystem . Invie wo fth eimportan trol eo ffung ii nmicrobia l ecosystems,on ewoul d expectmor e recordso fth eeffect so fselectiv e fungicideso nsuc heco ­ systems thanther eare . Under normal circumstances, farmerswil lno tus ea pesticid e whenthe y know thatth epathoge n infesting their cropi sresistan tt oit .Applica ­ tiono ffungicide st ocrop s infested with resistantpathogen s occursi n two situations.First ,whe nresistan t strainsar epresen t amongth epopu ­ lationo fa sensitiv epathogen . Second,whe nth ecausa l organismha sbee n wrongly diagnosed,a shappen s occasionally fordisease s thathav e similar symptomsbu tar ecause db ydifferen tpathogens .Fo rexample ,th esympto m

• Integrated control Ps Stimulated { 'Pr ^ Indirect control Antagonism to the pathogen^ Change of dominant pathogen / ( 'disease trading' ) s Suppressed { Pr. \ Boomerang effect

Ps - sensitive pathogen Pp= resistant pathogen

Figure 2.Phenomen a observed inplant-diseas e control caused bydifferentia l sensitivity to fungicides ofpathogen s andthei r antagonists.

167 damping-offca nb e caused by avariet y ofpathogens ,viz . Pythium and Phytophthora species, Rhizoctonia solani, Botrytis cinerea and Fusarium species. The effectsmentione d inFigur e 2nee dno tb ebrough t aboutonl yb y changes inth emicroflora : fungicide applicationsma y also influence the plantresistanc e mechanism or alter themicro-environmen t inth ecro p through aneffec to nplan tcanopy .

Integrated control

Disease controlbrough tabou tb y directinhibitio no fth epathoge nto ­ getherwit h anenhance dmicrobia l antagonism toth epathoge n isa for mo f integrated control.A prerequisit e forsuc hcontro l istha teithe r the population of antagonists is less affectedb y the fungicide thanth epa ­ thogen ortha tth e antagonists aremor e successful thanth epathoge n in their colonization ofplan t surfaces or soilafte r treatmento fth ecrop . Since themicroflor aplay s anessentia l role inthi s form of integrated control, theultimat e resulto f fungicide application ishighl y dependent onth ebioti c environment.A well-know n example isth e effecto fcarbo n disulphide andmethy lbromid e onth eviabilit y ofrhizomorph so f Armilla- ria mellea (Bliss,1951) . When infected roots aretreate dwit h sublethal doses ofth e fumigants andburie d innatura l soil thepathoge n dies,bu t whenburie d insteril e soil thepathoge nca nsurvive :th erhizomorph s are colonized by Trichoderma innatura l soil,bu tno t insteril e soils.Ohr & Munnecke (1974)showe d thatth etreatmen twit hmethy lbromid e inhibits the antibiotic production ofth erhizomorph s sotha tthe y cannotwar d off their antagonists,amon gwhic h Trichoderma ranks first. Inthi scontext , Baker& Coo k (1974)suggeste d anapproac h tochemica l control inth e senseo f 'rathertha nkil l thepathogen ,i tma y onlyb enecessar y towea ­ keni tan dmak e itmor evulnerabl e to antagonism ofth e associatedmicro ­ flora'. An increase ofmicrobia l antagonism asa positiv e side-effectha sbee n observed for fungicides ofvariou s categories,e.g . organo-mercury fungi­ cides, dithiocarbamates,benzimidazoles ,quintozen eand , according to Langerak (1977), alsoth e antibiotic fungicidepimaricin . Iti sno tsur ­ prising thatmos to fth e examples concern soil-bornepathogens ,sinc eth e role ofth ebioti c environment isthough tt ob emor e important forpatho ­ gens inth e soiltha no nth e aerialpart s ofplants . Inintegrate d control anumbe r ofmechanism sma yoperat esimultaneous ­ ly. Thisha sbee n shown forth eeffec to forgano-mercur y fungicides on common rootro to fwhea t (Chinn, 1971), causedb y Cochliobolus sativus, and fusarium diseases ofbulb s and rootso fdaffodil s (Langerak, 1977). Theultimat e disease controlwa s achievedb y fourdistinc teffects :a directeffec to fth e fungicide onth epathogen ; stimulation ofproductio n

168 of fungistatic substances by Pénicillium spp.;a significan t increase in thepopulation s ofantagonist s (Pénicillium spp.an d Trichoderma spp.); and a 'crossprotection 'mechanism . Cross-protection was assumed since the subcrown internodes ofwhea t seedlingswer emor e densily colonized by Pénicillium spp.i ntreate d plots. Indaffodils , Trichoderma viride and P. janthinellum becamemor e established onroot s ofbulb s treated with 2-methoxyethylmercurychlorid e ('Aretan'),thira m or aformulate d product ofpimarici ntha no n rootso funtreate dbulbs . Inmos to fth e instanceswher e themicroflor a hascontribute d toef ­ fective disease controlb y a fungicide,tha t supplementary rolewa sre ­ cognized only afterwards.Occasionally , integrated controlb y fungicides and antagonists hasbee nuse d intentionally. Carter& Pric e (1974)repor ­ tedo nth e intentional useo f integrated control for Eutypa armeniacae, anair-borne ,vascula rpathoge n of apricot.Upo n invadingwound s after pruning, thepathoge n isantagonize d by Fusarium lateritium. Benomylef ­ fectively controlled thedisease . F. lateritium was less sensitive to the fungicide thanth epathogen .Whe na conidia l suspension ofth eanta ­ gonistwa s added to the fungicide itcolonize d thewounde d tissue,resul ­ ting ina contro l supplementary totha tb y the fungicide.A n example of synergistic actionbetwee n anantagonis t and afungicid e incontrollin g soil-bornepathogen s hasbee nreporte db y Chete t al. (1979).Th e control of Sclerotium rolfsii obtainedb y supplying a Trichoderma preparation to the soilwa s significantly improvedwhe n quintozenea tsub-inhibitor ydo ­ seswa s applied togetherwit hth epreparation .

Indirect control

Examples ofeffectiv e disease control duet oeffect s onmicrobia lin ­ teractions only are scarce.Th emos t important examples of indirectcon ­ trol of fungalpathogen s inth e field are side-effects ofnematicide s on root-infecting fungi.Thi s concerns the so-called complex diseases,wher e nematodespredispos e roots to infectionb y Fusarium andothe rpathogens . An interesting laboratory example ofdiseas e control ofa toleran tpa ­ thogen istha to f Puthium debaryanum oncucumbe r seedlings treatedwit h 6-azauracil.Stankov a& Dekke r (1969)observe d thattreatmen t ofseed s with this experimental fungicide ata lo wdos erat e resulted ina signi ­ ficant increase ofth enumbe r ofbacteri a inth erhizosphere . Itwa ssug ­ gested thatth e enhancedbacteria l populationprotecte d the roots from infectionb y P. debaryanum. In fieldtrials ,reduce d incidence ofresistan tpathogen s infungicide - treated crops isoccasionall y observed. Joyner& Couc h (1976)reporte d reduced severity of Pythium blightan d red leaf spotcause db y Helmintho- sporium erythrospilum in Agrostis palustris, oneo fth egrasse s intur f treatedwit hbenzimidazol e fungicides.N oexample s ofdiseas e control have

169 been foundwher e theeffect s of fungicides onmicrobia l antagonism were highenoug h forpractica l use inth e field.

Changes ofth e dominantpathoge n

Suppression ofa targe tpathoge nb ycontro lmeasure sma yb e followed by the increase ofa pathoge ntha twa s initially ofmino r importance.Thi s phenomenonwa s firstrecognize d insoi ltreatments . Itwa s termed 'disease trading'b yKreutze r (1965), who defined ita s 'asituatio n inwhic hth e dominantpathoge n iscontrolle d by soil treatment, and amino r pathogen iselevate d tomajo r importance,thu sbecomin g thene wpathogen' .Mos t examples are associated withth eus e ofselectiv e fungicides and concern anexchang e of sensitivepathogen s forpathogen s tolerantt oth efungi ­ cides.Amon gnon-systemics , this side-effect isknow n for 'Dexon'an dquin - tozene. Suppression of Pythium by 'Dexon'ma yb e followedb y an increase in Rhizoctonia. Rhizoctonia, inturn ,ma yb e exchanged for Pythium and Fusarium in soil treatedwit h guintozene.A recentexampl e isth e shift inth epathogen s causing fruitro t instrawberr y following treatmentwit h dicarboximide fungicides.A n increase inyiel d obtainedb y thesuppres ­ siono f Botrytis cinerea waspartl y lostdu et o the incidence of Phytoph- thora cactorum (Huntere t al., 1979)o r Nucor and Rhizopus spp. (Figure3) .

Botrytis cinerea Mucoran d Rhizopus spp. infected fruits (%) 100

—i 12 20 periodo fstorag e

Figure3 .Effec to fdicarboximide so nincidenc eo fspoilag efung io nstore dstrawber ­ ries:unspraye d (O), iprodione (•),vinclozoli n (A).Th ecro pfro mwhic hth efruit s camewa sspraye ddurin gth eflowerin gperiod .Th efruit swer estore da t5 ° Can d relativehumiditie so f95-97% .Source :Davi s& Denni s (1979).

170 Sinceth eus e of systemic fungicideswit h aselectiv e antifungal spec­ trumbecam e common, farmers andplan tpathologist s havebee n confronted withne w examples.Mos treport s onth e appearance ofnon-targe tpathogen s infungicide-treate d crops concernth e incidence ofpythiaceou s fungi and otherbenzimidazole-toleran tspecie s after treatmentwit hbenzimidazol e fungicides.Nevertheless ,give nth e frequentus eo fth e fungicides,th e number ofcase so f anincrease d incidence ofresistan tpathogen s isrela ­ tively low.Onl y fewo fthes e fungi seem tobenefi tb ythei rresistance . Appearance ofnon-targe tpathogen s ina fungicide-treate d crop cannot be attributed merely to suppression ofmicrobia l antagonism by thefungi ­ cide.Anothe r causema yb e adecreas e ofhos tresistance .Swinburn e (1975) found thatbenomy lreduce d enzyme activities inbarle y seedlings.H e pointed outtha tthi smigh tresul t ininhibitio no fresistanc emechanisms . Still anothermechanis m operates through achang e ofth emicroclimat e in thecrop , for instance,brough t aboutb y adense rcanop ybecaus e ofcon ­ trol of foliarpathogens .Backma ne t al. (1975)observe d thatth e control ofpeanu t leafspo tcause db y Cercospora arachidicola and Cercosporidium personatum was associated with anenhance d damage causedb y Sclerotium rol fsii. Control oflea f spotb y sprayingbenzimidazol e fungicides and chlorothalonil promoted adens e foliage. Inth e sub-canopy environment, ahumi d atmosphere isconduciv e to infectionb y thewhit emol dfungus . Backman etal .pointe d outthat ,i nadditio nt o this side-effect,th e fungicide suppressed thepopulatio n of Trichoderma viride, anatura l antagonist of S. rolfsii. The effectwa smor epronounce d inbenomyl - thani nchlorothalonil-treate d plots,probabl ybecaus e S. rolfsii is highly resistant tobenzimidazoles .Porte r (1980)presume d change in microclimate also toaccoun t forth e increase of severity ofbligh t causedb y Sclerotinia minor inpeanut s treatedwit h captafol andchloro ­ thalonil.However ,unlik e S. rolfsii, thispathoge nwa s controlled bya highrat eo fbenomyl ,whic h ist ob eexpected , since S. minor isver y sensitive invitr ot oth e fungicide. InScierotinia-infeste dfields , peanutyiel dwa s significantly lower incaptafo l andchlorothalonil - treatedplot s than inuntreate d andbenomyl-treate d plots.Fro m thedat a givenb yPorte r itca nb eestimate d thatth e losso fyiel d andvalu edu e tothi s change ofth edominan tpathoge nwa s 13-16% . Ina few instances there issubstantia l evidence thata decrease dmi ­ crobial antagonismwa s acaus eo fenhance d incidence ofpathogens .A nex ­ ample for aerialpathogen s waswell-documente d byFokkem a et al. (1975). They showed thatbenomy l sprayings reduced themicroflor a ofry eleaves , resulting in anincrease d susceptibility to C. sativus, apathoge ntoler ­ antt oth e fungicide.Th eeffec twa s onlyobserve d ifth eleave swer e denselypopulate d with saprophytes.Anothe r example isdecrease d antago­ nism to R. cerealis, whichcause s sharpeyespot ,i ncereal s treatedwit h benzimidazoles. Sprayingwit h these fungicides have resulted ina nexchan -

171 geo feyespo tb y Pseudocercosporella herpotrichoides forshar p eyespot or, when ahig h dosewa sused , Fusarium footro t forshar p eyespot.Th e effectwa s attributed todifferentia l sensitivity ofth epathogen scompe ­ ting forhos ttissu e onth e culmbas e (Reinecke &Fehrman , 1979)an dsup ­ pression of antagonism to R. cerealis by the soilmicroflor a (vande r Hoeven& Bollen , 1980). Reduced antagonism due to fungicides can also lead to anincreas e in competitive saprophytism ofpathogen s inth e soil, asha sbee n shownfo r Pythium and Fusarium inquintozene-amende d soil (Katan& Lockwood, 1970) and Phytophthora cruptogea inbenomyl-amende d soil (Bollen, 1979).

Boomerang effect

This effect isdefine d asa naccentuatio n ofth ediseas e causedb y the targetpathoge n itself after treatment tocontro l thedisease .Th e term was used originally forth ereappearanc e ofsoil-born e pathogens indis - infested soil inquantitie s greater thanbefor e thetreatmen t (Kreutzer, 1965). During the lastdecade , farmers andplan tpathologist s havebee ncon ­ frontedwit h aspecia l category ofboomeran g effects:th e accentuation of diseases in fungicide-treated crops causedb y resistant strains appearing inth epopulatio n ofth epathogen . Ifresistanc e emerges ina treate d crop thepathoge npopulatio nma yb eeithe runaffecte d or increasedb yinhibi ­ tiono fth enatura l antagonists.Accentuatio n ofdiseas e causedb yresis ­ tant strains inbenzimidazole-treate d cropsha sbee nreporte d for Péni­ cillium roti nlil ybulb s (Rattink& Beusenberg , 1972), dollar spoti n turfgras s causedb y Sclerotinia homeocarpa Warren et al., 1974)an ddr y bubble ofcommercia lmushroo m causedb y Verticillium fungicola (Figure4) . Substantial evidence forsuppresse d microbial antagonism asa caus eo f theboomeran g effectb y resistant strains is available foronl y alimite d number ofexamples .Th epossibilit y thatresistan t strains that aremor e virulent than awil d strain incidently appear shouldno tb e overlooked. Inth eexperimen to nth e incidence ofdr ybubbl e inmushroom s (Figure 4), theyiel d ofplot s infestedwit h aresistan t strain (RO)wa s lower than ofthos e infestedwit h awil d strain (SO).Thes e results suggesttha tth e strainwa smor evirulen t thanth ewil dstrain .

Specificity of indirect effects

Records of indirecteffect s ofpesticide s onth emicroflora , including pathogens, areofte n inconsistent.Contradictor y results are sometimes obtained fordifferen tcrop so r soils,especiall ywhe n effects ofpestici ­ deswit hhig h selectivity toth emicroflor a are involved.Th e specificity ofeffect s iswel l illustrated by the study ofJoyne r& Couc h (1976), in

172 mushroom yield diseosed mushrooms LJ (kg/m2) (number per tray) * * * * 00 * -x- 0o *. *' 0o 15- 00 -300 1 00 uu oc t oo 0c M1 00 1 0c oo oo O0 oo oo oo oo oo oc oo oo oc oo oo oc 10- oo oo oo 200 oo oo 00 O0 oo 00 0o oo 0o oo # oo 0o oo * oo 0o oo *i oo DO 0o 1 O0 oo oo oo 0o oo - oo 0o 5- oo t oo 100 oo 00 oo . 0o oo 0o oo 0o oo O0 0o N oo oo oo oo 0o >o 0o oo oo oo oo •> o O0 oo oo oo oo oo 0o oo 0o oo 0o )0 oo oo 0o oo oo )o oo oo 0o Q-Q oo s? )o on oo 00 SO SB 00 R0 RB

Figure 4. Incidence of dry bubble and mushroom yield of benomyl-treated plots infested with either a sensitive or a resistant isolate of Verticillium fungicola. Treatments: no fungicide and not inoculated (00), benomyl (0.75 g/m-2) (B), inoculated with a sensitive strain (S), innoculated with a resistant strain (R). The yields and numbers of diseased mushrooms of Trial 1 (8 replicates) and Trial 2 (9 replicates) were proces­ sed by analysis of variance; * and ** are least significant differences for P = 0.05 and P = 0.01, respectively. Source: Bollen &va n Zaayen (1975).

which the effect of four benzimidazole fungicides on non-target diseases of turf grasses was estimated. Pythium blight was reduced in Agrostis pa­ lustris, but unaffected in Lolium perenne. A similar specific response was found for the incidence of leaf-spot disease, which is caused by va­ rious Helminthosporium species. In greenhouse experiments where the effects of benomyl on pythiaceous fungi were estimated, the author found that in the same soil Phytophthora cryptogea and Pythium aphanidermatum growth was enhanced, whereas growth of Pythium irreguläre was unaffected. In vitro, the pathogens were equally resistant to the fungicide. Differential effects like these can be under­ stood when it is realized that each pathogen has its specific spectrum of antagonists. It depends on the fungicide sensitivity of the dominant spe­ cies within the population of antagonists whether an effect appears or not. A similar specificity holds for the response of the antagonism to one and the same pathogen in different soils. Thé effect of benomyl on antago­ nism to R. cerealis was measured in five field soils cropped with cereals

173 (vande rHoeve n& Bollen , 1980). Twower e affected inthei r antagonistic activity, theothe rthre ewer enot .Agai nth erespons e toth e fungicide depends onth e sensitivity ofthos e species thatpredominat e inth eanta ­ gonistpopulation .Becaus e themicroflor a differs from soil to soil, the effects ofselectiv e inhibitors onmicrobia l antagonism willb e specific forth e soil.T o acertai n extentthi s isals o true for above-ground habi­ tats.

Final comments

The application offungicide s tocrop so r soilsca ncaus e ashif ti n themicrobia l population infavou ro fresistan t species,whic hma yb epa ­ thogens orthei r antagonists.Therefor e the effectsma yb eeithe rundesir ­ ableo r favourable fordiseas econtrol .Resistanc e of antagonists offers apossibilit y forintegrate d controlb y applying acombinatio n ofspore s of antagonists and lowdos e rates offungicides . Mostinstance s of accentuation ofdisease s after fungicide treatments involve selective fungicides,bot h the conventional non-systemics andth e systemics.Us eo f selective toxicants demands amor e accurate diagnosis ofth ecausa lpathoge n than forbroad-spectru m pesticides.Whe n ina nursery damping-off of seedlings iscause db y Pythium, butha serroneous ­ lybee n attributed to Rhizoctonia, theus e ofquintozen eo rbenomy lma y aggravate thedisease . During the last fewyear s thenumbe r ofreport s ofundesirabl eside - effects ofselectiv e fungicides isdeclining .Thi s isprobabl y duet oa more careful useo fth e fungicides and,especially , to theirus e incom ­ bination or alternationwit hothe r fungicides.Boomeran g effects caused bydevelopmen t ofresistanc e mayhav e declined,becaus e the increased use ofcombination s of fungicides retards thebuild-u p of aresistan tpopu ­ lation (Dekker, 1977). Changes ofdominan tpathoge n are also less likely tooccu r than afterth eus eo f asingl e fungicide,sinc e fewerpathogen s will escape thebroad-spectru m activity ofth ecombinations . Thestud y of side-effects ofpesticide s onth emicroflora , including resistantpathogens ,ha s itsow nside-effec t -a positiv e one.I nsom e cases, interactionsbetwee n organisms havebee nnotice d forth e first timewhe non e ofth e interactingpopulation s was suppressed.A meticulous study ofth e side-effects onresistan tpathogen s canprovid e information onth ekin do forganism s thatoperat e asthei r synergists orantagonists , onth eplan to r inth e soil. Such studies canprovid e the tools forinte ­ grated control.

174 References

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175 Odum,E.P. ,1971 .Fundamental so fecology .3r dEd .W.B .Saunders ,Philadelphia .57 4p . Ohr, H.D.& D.E .Munnecke ,1974 .Effect so fmethylbromid e onantibioti cproductio nb y Armillariamellea .Transaction s ofth eBritis hmycologica lSociet y62 :65-72 . Papavizas,G.C. ,1980 .Induce d toleranceo fTrichoderm aharzianu mt ofungicides .Phyto ­ pathology 70:691-692 . Papavizas,G.C .& J.A .Lewis ,1979 .Sid eeffect so fpesticide so nsoil-born eplan tpa ­ thogens.In :B .Schipper s& W .Gam s (Eds):Soil-born eplan tpathogens .Academi c Press,London :483-505 . Platenkamp,R.G.H .& G.J .Bollen ,1973 .Th eeffec to fbenomy lspray so nth eroo tmyco - florao frye .Act aBotanic aNeerlandic a 22:168-169 . Porter,D.M. , 1980.Increase d severityo fSclerotini abligh ti npeanut streate dwit h captafolan d chlorothalonil.Plan tDiseas e64 :394-395 . Rattink,H .& M .Beuzenberg ,1972 .Probleme n rondgebrui kva nBenlat e inlelieteelt . Bloembollencultuur 83:398-399 . Reinecke,P. , 1977.Untersuchunge nzu mErregerspektru m desFusskrankheitskomplexe s an Getreideunte rbesondere rBerücksichtigun gvo nRhizoctoni a solaniKühn .Thesi sUni ­ versitätGöttingen ,14 1p . Reinecke,P .& H .Fehrmann ,1979 .Rhizoctoni a cerealisva nde rHoeve na nGetreid e inde r BundesrepubliekDeutschland .Zeitschrif tfü rPflanzenkrankheite nun dPflanzenschut z 86:190-204 . Stankovâ-Opocenskâ.E .& J .Dekker ,1970 .Indirec teffec to f6-azauraci lo nPythiu m debaryanumi ncucumber .Netherland sJourna lo fPlan tPatholog y76 :152-158 . Sussman,A.S. ,1965 .Dormanc yo fsoi lmicro-organism s inrelatio nt osurvival .In : K.F.Baker ,& W.C .Snyde r (Eds):Ecolog yo fsoil-born eplan tpathogens .Universit y ofCaliforni aPress ,Berkeley ,p .99-109 . Swinburne,T.R. , 1975.Th eeffec to fbenomy lan dothe rfungicide so nphenylalanine - ammonialyaseactivit y inHordeu mvulgar ean dPhaseolu svulgaris .Physiologica lPlan t Pathology5 :81-88 . Tuyl,J.M .van ,1977 .Genetic so ffunga lresistanc et osystemi cfungicides . MededelingenLandbouwhogeschoo lWageninge n77-2 ,136p . Wainwright,M. ,1977 .Effect so ffungicide so nth emicrobiolog yan dbiochemistr yo f soils- a review .Zeitschrif tfü rPflanzenernährun g undBodenkund e 140:587-603 . Ward,S .& Y .Greenaway ,1976 .Interactio no fmercury-resistan tPénicilliu man dmercury - resistantPyrenophor aavena eo noa tseed .Transaction so fth eBritis hmycologica l Society66 :167-169 . Warren,C.G. ,P.L .Sander s& H .Cole ,1974 .Sclerotini ahomeocarp atoleranc et obenzimi - dazoleconfiguratio nfungicides .Phytopatholog y64 :1139-1142 .

176 Countermeasures for avoiding fungicide resistance

J. Dekker Laboratory ofPhytopathology ,Agricultura l University,Wageningen , The Netherlands

Abstract

Attention ispai d to factors thatma y influence thebuild-u p ofa re ­ sistantpathoge npopulation , such astyp eo f fungicide,typ eo fdiseas e and selectionpressur e exertedb y the fungicide.Measure s are discussed thatma y delay or avoid thebuild-u p ofresistanc e inpractice .

Keywords: fungicide resistance,specific-sit e inhibitor,multi-sit ein ­ hibitor, fitness,selectio npressure ,negativel y correlated cross-re­ sistance, alternating use,combine duse .

Introduction

Unexpected failure ofa fungicid e toprovid e adequatediseas e control mayb e due todevelopmen to fresistanc e inth epathoge n toth echemical . Thisma y cause serious crop lossest oth e farmer and endanger thefinan ­ cial returns forth e company thatdevelope d invariably athig hcost sth e fungicide. Iti stherefor e important fora growe r tokno wwha tt od ot o avoid ordela y thedevelopmen to f fungicide resistance inhi s fields,an d fora compan y thatdevelop sne w agricultural fungicides toobtai ninfor ­ mation aboutth eris k ofdevelopmen t ofresistanc e inth epathogen ,s o thati tca ntak e countermeasures and advise farmers aboutprope r useo f thene wproduct .Fo r suchknowledg e iti snecessar y to obtain insighti n the factors,tha t influence thedevelopmen to f fungicide resistance in the field. Inparticula r thetyp eo f fungicide,th enatur e and severity ofth e disease and the selectionpressur e exertedb y the fungicide areo f prime importance.

Type of fungicide

During acentur yo flarge-scal e useo fconventiona l fungicides,suc h ascompound sbase d oncopper , fewproblem s with fungicide resistance have

177 beenencountered . However sinceth e introduction of systemic fungicides, some 15year s ago,disease-contro l failure due to fungicide resistance is reported with increasing freguency. Itha sbee n explained (Georgopoulos, 1977;Dekker , 1977)tha tgeneti c changes ina pathoge n resulting infun ­ gicide resistance occurmuc hmor e readilywit h fungicides actingprima ­ rily aton eparticula r site inth emetabolis m ofth e fungal cell than with fungicides thatinterfer e atman y siteswit hmetaboli cprocesses . All systemic fungicides appeart ob e single-site inhibitors,whil eth e majority ofconventiona l fungicides areclassifie d asmulti-sit e inhibi­ tors. Asha sbee noutline d onp.132-136 , considerable differences also exist among single-site inhibitors ast oth echance s ofresistanc e buildingu p inpractice .Ther e are indications that,a t leastwit h some fungicides, resistance is linked todecrease d fitness (Dekker& Gielink , 1979), which will hamper thebuild-u p of aresistan tpathoge npopulation . Experience has showntha t sometype s of systemic fungicides doencounte r resistance problemsmor e readily thanother s (Table1) .

Table 1.Ris k of failure of disease control in practice due to development of resistance to specific-site fungicides; rough indication only, since risk not only depends on type of fungi­ cide, but also on type of disease and use strategy for fungi­ cide.

Site-specific fungicides Risk of failure

Antibiotics kasugamycin, streptomycin moderate to high pimaricin very low Acylalanines metalaxyl, furalaxyl high Benzimidazoles benomyl, carbendazim, thiabendazole high Carboxamides carboxin, oxycarboxin low to moderate Dicarboximides iprodione,procymidone , vinclozo lin moderate Hydroxypyrimidines ethirimol low to moderate dimethirimol high Imidazoles imazalil low Morpholines dodemorph, tridemorph, fenpropimorph very low Organic phosphorus compounds pyrazophos low edifenphos, kitazin moderate Piperazines triforine very low Pyrimidines fenarimol, nuarimol low Thiophanates thiophanate-methyl high Triazoles triadimefon, triadimenol, bitertanol low

178 Itwoul db ewrong ,however , toassum e thatresistanc eproblem s only occurwit h theus eo fsystemi c fungicides,an dno twit h conventional fun­ gicides.A fewo fthese ,althoug hno t systemic, areconsidere d tob espec ­ ific-site inhibitors,e.g . various aromatic-hydrocarbon compounds (Geor- gopoulos& Zaracovitis , 1967), dicarboximides (Pappase t al., 1979)an d organic tincompound s (Giannopolitis& Chrysayi-Tokousbalides ,1980 ; thesema y encounter resistance problems similar toth e systemic fungici­ des. Moreover, ina fe wcase s resistance has developed tosom e multi-site inhibitors, e.g. organicmercur y (Noblee t al., 1966), possibly duet oa change inpermeabilit y ofth e fungalmembran e orb y adetoxificatio nmech ­ anism.

The nature and the severity of the disease

Disease-control failurewil l onlyoccu rwhe n amajorit y of thepatho ­ genpopulatio nha sbecom e resistant.Whethe r thishappens ,an dho wrap ­ idly,depend s opth e typeo fdisease ,th enatur e ofth epathoge nan d circumstances thatpromot e orhinde r disease development.Fo rinstance , developmento fresistanc e tobenzimidazol e fungicides hasoccurre drap ­ idlywit hvariou spowder ymilde w diseases,bu tonl y after a fewyear s of usewit h apple scab andu p tono wno ta tal lwit h Pseudocercosporella footro ti ncereal s (Horsten& Fehrmann, 1980). Factors thatma ypla y arol eare : - Sporulation and spread ofpathogens . Iti seas y tounderstan d thatfun ­ gicide resistance maybuil d upmor e rapidly ina pathoge nwit h abundant sporulation onaeria lplan tpart s than ina pathoge n thatsporulate s scar­ celyo rproduce s spores that areno treadil y transported, asma yb eth e casewit h several soil-borne rooto r footdiseases . - Survival ofsensitiv e forms. Ifsom e sensitive forms survive,thes e will, duringperiod s ofreduce d selectionpressure ,compet ewit h there ­ sistant forms,s otha tth ebuild-u p of aresistan tpathoge n population willb e counteracted. Thisma yhappe nwit hpathogen s inplan tpart s that areno teasil y reachedb y fungicides applied as sprays,no teve n systemic fungicides.Example s arevascula rwil t and rootpathogens .Ther ema y also be survival ofpathogen s present inplan tpart s fromwhic h the fungicide israpidl y removedb y apoplastic transport,e.g . eyéspoto nwhea tculms . - Infection threshold.Wit hdisease s thathav e ahig h infectionthresh ­ old,th e appearance ofsingl emutan tcell swil l rarely result ininfec ­ tion, sotha tth e risko fdevelopmen t of fungicide resistance inth epo ­ pulation islow . -Multinucleat e cells.Th e rarepresenc e ofgene s forresistanc e ina multi-nucleate fungusma yunde r selectionpressur eb y the fungicide lead toa rapi d shifttoward s resistance inth epathoge npopulation .Build-u p ofresistanc e may alsooccu r throughheterokaryosi s infungi .

179 - Severity ofepidemic . Insevera l cases fungicides,despit e anumbe r of years of satisfactory performance,hav e failed to control disease insea ­ sonswit hver y severe epidemics. The absence of fungicide resistance problems inth e control ofeye - spot incereal sb ybenzimidazole smay , according toHorste n& Fehrman n (1980a, 1980b),b e attributedto : - limited sporulation and slow spread ofth epathoge n - escape of sensitive formso ncro p debris inth e soil or inth e culm, fromwhere ,afte r spraying, fungicides disappearb y apoplastic transport - high infection threshold - low selectionpressure ,becaus e sprays are applied only onceo rtwic e per season. During threeyear so f fungicide treatmentthe y foundonl yver y little increase inth e frequency ofresistan t forms (Table 2). Invie w ofthi s theyexpec ttha tth eus e ofbenzimidazol e fungicides against Pseudocercos- porella herpotrichoides atcurren trate swil l not lead to resistance pro­ blems.

Selection pressure by the fungicide.

Strains resistant to aparticula r fungicidema y arise ina numbe r of ways.Whe nth e fungicide concerned isuse d itexert s aselectiv e action uponth epathoge npopulatio n in favour ofth e cells thathav ebecom ere ­ sistant.Th e selectionpressur ema yb ehigh , intermediate or low;an d it mayb e continuous or intermittent.Whe n strains emergewit h ahig h level ofresistance , ahig h selectionpressur ewil l strongly favour thebuild ­ up of ahighl y resistantpathoge npopulatio nb y elimination ofth esensi ­ tivepathoge n and formswit h lowresistance .Th e opposite,lo w selection

Table2 .Influenc eo fon ecarbendazi mapplicatio npe rseaso no nth efrequenc yo f fungicide-resistant sporeso fPseudocercosporell aherpotrichoide s inwinte rbarley , c.v.Vogelsange r Gold.

Fungicide Numbero f Number of Numbero f Frequencyo f treatment isolates conidia (x 104) resistant resistant conidia conidia

1975 unsprayed 40 865 11 1 :7 8x 10 7 carbendazim 40 794 10 1 :7 9x 10 7

1976 unsprayed 34 161 7 23 1 :7 0x 10 7 carbendazim 46 234 6 40 1 :5 8x 10 7

1977 unsprayed 19 509 20 1 :2 5x 10 7 carbendazim 18 471 39 1 :1 2x 10 7

Source:afte rHorste n (1979).

180 pressure,wil lno tkil l formswit h low resistance,thu s saving alarge r quantity ofresistan t forms. Ifonl y strainswit h alo w level ofresis ­ tance areexpecte d toemerge ,hig h selectionpressur ema yb e favourable since itma y kill all resistant strains.Absenc e ofhighl y resistant strainsma y indeed occurwhe n alin kexist sbetwee n resistance andde ­ creased fitness,a sha sbee n suggested forpimarici n (Dekker& Gielink , 1979)an dtriforin e (Fuchs& Drandarevski , 1976). However the possibility canno tb eexclude d thatunde r constant selectionpressur eb y thefungi ­ cide, anincreas e in fitness ofthes eresistan t strainsma ytak eplace . Moreover, accumulation ofmutations ,eac h of alo w level ofresistance , may eventually lead to formswit hhighe r resistance. Thepersistenc e orcontinuit y ofth e selectionpressur ema y alsopla y a role. Interruption of selectivepressur ema y give the sensitive cells a chance tocompet ewit h resistant forms,man y ofwhic h oftenhav ea lowerpathogenicity . Thiswil l tend tocounterac t thebuild-u p ofa re ­ sistantpathoge npopulation . Ingeneral ,hig h and continuous selection pressure favoursmos tth edevelopmen t ofresistance .

Introduction of new chemicals

Before ane w fungicide isintroduce d intopractica l agriculture,know ­ ledge aboutth erisk s of fungicide resistance developing isver ydesira ­ ble ifgrower s aret ob egive nsoun d recommendations onit suse .Informa ­ tion about thepotentia l ofpathogen s tobecom e resistant to sucha fun ­ gicide iso fprim e importance. Ifa non-obligat eparasit e isconcerned , resistance canb e studied inlaborator y experiments onartificia lmedia , containing the fungicide,wit h orwithou tus eo fultra-viole t (UV)irra ­ diation ormutageni c chemicals,a sdescribe d onp.129. Itmus tb e realized,however ,tha tclassificatio n ofa pathoge n inth e lowris kcategor y doesno tguarante e thatresistanc e problemswil lneve r arise. Itcanno tb e excluded thatth epathogenicit y ofresistan t strains will increase over the course ofyears .Resistanc eproblem sma y thenoc ­ curonl y after alon gperio d ofpractica l use,a shappene d with dodine resistance in Venturia inaequalis (Gilpatrick &Blowers , 1974). Itha sbee n suggested that fungicideswit h indirectactio nmigh tb e less liable tomee tresistance .Bu tsuc h agenera l statement isno tpos ­ sible: itwil l depend onth emechanis m of actiono fth ecompoun d whether thepathoge nwil lb e ablet obrea k the artificially induced resistance of theplant .Whethe r this typeo fresistanc e canb ecompare dwit h thenat ­ urally occuring 'horizontal'o r 'vertical'resistanc e ofth ehos tplan t mayb e relevanthere . Iti simportan t thatth e search forsystemi c fungicideswit h lowris k fordevelopmen t of fungicide resistanceb e continued.

181 Fungicide management

General

Ifi ti spossibl e tochoos e different types ofchemical s forth econ ­ trol of aparticula r disease,thos ewit h the lower risk for development of fungicide resistance shouldb epreferred . This isespeciall y important whenth e typeo fdiseas e isconduciv e todevelopmen t of fungicideresis ­ tance. Iffo rcontro l ofsuc h adiseas e onlyvulnerabl e fungicides area - vailable, carefulpesticid e managementwil lb e necessary.The n special attention shouldb epai d tomeasure s thatinfluenc e selectionpressur e insuc h awa y thatth echanc e ofa build-u p of aresistan tpathoge n population arereduced ,withou t decreasing the impacto fth e fungicide. Thedose s applied, the frequency of application, themod e of application, the efficiency ofth e treatment,th eextensio n ofth e area treatedwit h one chemical and alternating orcombine d use ofdifferen t fungicides are importanthere .Earl y detection ofth ebuild-u p ofresistanc e is alsoim ­ portant, sotha ttimely , adequatemeasure s canb etaken .

Dose, frequency andmetho d of application

Ifi ti sadvisabl e toavoi d undulyhig h selectionpressure ,th e amount of fungicide sprayed onth ecro p should notexcee d theminimu m necessary foradequat e disease control.Thi s demands careful consideration ofth e doses applied and the frequencywit hwhic h they are administered. Ifth e risk ofdevelopmen t of fungicide resistance isgreat ,i tma yb ebette r torestric t application tocritica l periods inth edevelopmen to fth e disease.Fo r example,i fa vulnerabl e fungicide isuse d forpost-harves t disease control,thi so rrelate d fungicides shouldno tb e applied inth e field topreven t earlybuild-u p ofresistance . Themod e ofapplicatio nma y alsob e important.A systemic fungicide applied to the soilo r seed allows uninterrupted uptake,whic h favours continuous selectionpressur e forresistan tpathogens .Moreover , thorough treatmento fth ecro pwil lprovid e little opportunity forsensitiv epatho ­ gens to survive,s otha tresistan t formswil lmee t littlecompetition . Theexten to fth e area treatedwit h aparticula r fungicidema ycontri ­ bute toth e selectionpressur e too.Whe n alarg e area istreate dwit ha single chemical,o rwit hrelate d compounds,fe wsensitiv e formsca nen ­ terth ecro p fromoutside ,whic h limits competitionbetwee n sensitive and resistant forms during intervals oflo w selectionpressure .Growt h ofa crop ina mor e or less isolated environment, forinstanc e ina greenhouse , may alsoresul ti nlimite d competition.

182 Alternating or combined use of fungicides

To delay or prevent the development of fungicide resistance, the use - alternately or combined - of fungicides with different mechanisms of action has been advocated. With a combination of two specific-site inhib­ itors the possibility exists that the pathogen will aquire resistance to both compounds. This is much less likely if a combination of a sys­ temic fungicide and a multi-site inhibitor is used. This is now becoming a common practice. The use of different fungicides in mixtures or in an alternating strat­ egy has been studied by Kable & Jeffery (1980), who developed a mathema­ tical model for such use. The model has three variables: the efficacy of the fungicide to which resistance has developed against the sensitive and the resistant sub-population; the efficacy of the second fungicide, assumed to be equal for the two sub-populations; and the degree of spray coverage, i.e. the proportion of the fungal population not coming into contact with the fungicide. The variable that has the largest effect on the rate of selection appears to be spray coverage. With complete coverage, the use of a mixture does not slow down the build-up of a resistant pathogen population; two fungicides used alternately is then the better strategy. However complete coverage will seldom occur when the crop is sprayed: the use of mixtures appears to be increasingly effective as the coverage decreases. These relations are illustrated by the data presented in Table 3, which were calculated on the basis of assumed model values for the above mentioned parameters. Dekker & Teng (unpublished), using the same parameters as for the model of Kable & Jeffery, calculated that al­ ternating a mixture of the two fungicides with a conventional fungicide may delay the development of resistance even more efficiently (Table 4).

Table 3. The effects of combined or alternating use of two fungicides and spray coverage l on build-up of a resistant pathogen population, assuming specific model values for efficacies of the fungicides 2 and initial resistance level 3; based on data from simulation model by Kable &Jeffer y (1980).

Fungicide Number of sprays before the populations become 90 % application resistant for various values of E 0 1 5 10 30 50

S repeated 10 11 13 15 25 42 (S + C) repeated 10 13 20 29 75 157 S alternating with C 20 22 26 30 50 84 1 E (escape) = proportion of the pathogen population escaping fungicide contact S = vulnerable fungicide, e.g. systemic fungicide; efficacy against sensitive and resistant sub-populations set at 95 % and 10 %, respectively. C = conven­ tional fungicide; efficacy against both S-sensitive and S-resistant sub- populations set at 80% 3 Initial resistance frequency = 10-9

183 Table4 .Th eeffect so fcombine do ralternatin gus eo ftw ofungicide so nbuild-u p ofa resistan tpathoge npopulation ,assumin g specificmode lvalue sfo refficacie s* spraycoverag e 2 andinitia l resistance level3 .Simulatio nmode lb yJ .Dekke r& P.S.Ten g (unpublished).

Fungicide Proportiono fth e population resistant after application variousnumber so f sprayings

5 10 20 30 40

S repeated 0.000 0.826 1.000 1.000 1.000 (S+ C )repeate d 0.000 0.000 0.996 1.000 1.000 Salternatin gwit hC 0.000 0.000 0.826 1.000 1.000 (S+ C )alternatin gw i thC 0.000 0.000 0.000 0.261 0.996

1 E (escape)= proportio no fth epathoge npopulatio nescapin g fungicide contact= 5 % 2 S= vulnerabl e fungicide,e.g .systemi c fungicide;efficac yagains tsensitiv e andresistan tsub-population sse ta t9 5 %an d1 0 %, respectively.C = conven ­ tionalfungicide ;efficac yagains tbot hS-sensitiv e andS-resistan tsub - populationsse ta t8 0% 3 Initialresistanc e frequency= 10- 9

Kable& Jeffer y state thatthei rmode l isa simpl eon ean dtha t variousparameter s thatma ypla y arol ei npractic ehav eno tbee ninclu ­ ded, forexample ,th ereproductio no fth etarge torganism . They also state thatreproductio n isno tincompatibl e withth emode lprovide dth e resistantan dsensitiv e sub-populations reproduce atequa l rates.How ­ ever itappear s thatth efungicid e resistant sub-populationma yi nth e absence ofth efungicid e concerned have alowe r reproduction rate,whic h canpartl yb eascribe d toth emechanis m ofresistanc e (Dekker& Gielink , 1979; Fuchs& Drandarevski , 1976). Insuc ha case ,sequentia l fungicide usewil l favour directional selectiontoward s thesensitiv e formwhe n the applicationo fth evulnerabl e fungicide isinterrupted , thus allowing recoveryo fth esensitiv epathogen . Introductiono fparameter s forrepro ­ ductiondurin gth eselectio npressur eb yth efungicid e andrecover ydur ­ ing absenceo fselectio npressur ema ygiv ea mode l thatsimulate s reality more closely (J.Dekke r& P.S .Teng ,unpublished) .

Negatively correlated cross-resistance

Thebuild-u p offungicide-resistan t populations mightb eprevente db y usinga mixtur eo ffungicide swit hnegativel y correlated cross-resistance. Thismean s thatresistanc et ofungicid eA i slinke d tosensitivit yt o fungidiceB ,an dvic eversa .Studie so fnegativel y correlated cross-re­ sistance havebee nmad e forphosphoramidat ean dphosphorothiolat e fungi­ cides, andfo risoprothiolan e andphosphoramidat e (Uesugi, 1978),bu t no suchcombination s areye tavailabl e forpractica luse .

184 Conclusions

Among the fungicides considered to be specific site inhibitors, the probability that resistance will develop varies greatly. Therefore it is important to continue the search for new fungicides to which resistance will not develop readily, e.g. compounds where resistance is linked to decreased fitness, or a combination of compounds with negatively corre­ lated cross-resistance. When vulnerable fungicides are used, strategies to cope with resistance problems, should they arise, become necessary. Reviewing the problems of resistance in insects. Brown (1976) concluded that 'the best policy is to continue using the insecticide recommended for the job until the control effect becomes inadequate, and then re­ place it with the most insecticidal substitute'. This strategy is very costly, especially in cases of a very rapid build-up of resistance, as has occurred with some fungicides. Moreover, it seems to be possible to prolong the useful life of fungicides by strategies of fungicide treat­ ment that delay or avoid the development of resistance. The most im­ portant aspect of such strategies is to avoid a continuous high selec­ tion pressure, by restricting application to critical periods, reducing the amounts applied and limiting the area treated with one particular fungicide. Also the type of disease and the method of application should be considered. Development of resistance may be counteracted by the use of a second fungicide, preferably a multi-site inhibitor, either in com­ bination with the vulnerable fungicide or in sequence with it.

References

Brown, A.W.A., 1976. How have entomologists dealt with resistance? In: Symposium on resistance of plant pathogens to chemicals, Kansas City, July 1976. Proceedings American Phytopathological Society 3. p. 67-74. Dekker, J., 1977. Resistance. In: R.W. Marsh (Ed.): Systemic Fungicides, 2nd edition. Longman, London and New York. p. 176-197. Dekker, J. & A.J. Gielink, 1979. Acquired resistance to pimaricin in Cladosporium cucumerinum and Fusarium oxysporum f. sp. narcissi associated with decreased viru­ lence. Netherlands Journal of Plant Pathology 85: 67-73. Fuchs, A. & C.A. Drandarevski, 1976. The likelihood of development of resistance to systemic fungicides which inhibit ergosterol biosynthesis. Netherlands Journal of Plant Pathology 82: 85-87. Georgopoulos, S.G., 1977. Pathogens become resistance to chemicals. In: J.G. Horsfall & E.B. Cowling (Eds.): Plant Disease, an advanced treatise, Vol. I, Academic Press, New York. p. 327-345. Georgopoulos, S.G. & C. Zaracovitis, 1967. Tolerance of fungi to organic fungicides. Annual Review of Phytopathology 5: 109-130. Giannopolitis, C.N. & M. Chrysayi-Tokousbalides, 1980. Biology of triphenyltin- resistant strains of Cercospora beticola from sugar beet. Plant Disease 64: 940-943. Gilpatrick, J.D. & D.R. Blowers, 1974. Ascospore tolerance to dodine in relation to orchard control of apple scab. Phytopathology 64: 649-652. Horsten, J.A.H.M., 1979. Acquired resistance to systemic fungicides of Septoria nodorum and Cercosporella herpotrichoides in cereals. PhD Thesis, Agricultural University, Wageningen. Horsten, J.A.H.M. & H. Fehrmann, 1980a. Fungicide resistance of Septoria nodorum and Pseudocercosporella herpotrichoides. I. Effect of fungicide application on the fre­ quency of resistant spores in the field. Zeitschrift für Pflanzenkrankheiten und

185 Pflanzenschutz87 :439-453 . Horsten,J.A.H.M .& H .Fehrmann ,1980b .Fungicid e resistanceo fSeptori anodoru man d Pseudocercosporella herpotrichoides.II .Characterizatio n ofresistan tstrains . Zeitschrift fürPflanzenkrankheite nun dPflanzenschut z87 :513-522 . Kable,P.F .& H .Jeffery ,1980 .Selectio nfo rtoleranc e inorganism sexpose d tospray s ofbiocid emixtures :a theoretica lmodel .Phytopatholog y 70:8-12 . Noble,M. ,Q.D .Maggarvie ,A.F .Ham s& L.L .Leafe ,1966 .Resistanc et omercur yo f Pyrenophora avenaei nScottis hsee d oats.Plan tPatholog y 15:23-28 . Pappas,A.C. ,B.K .Cook e& V.W.L .Jordan ,1979 .Insensitivit yo fBotryti s cinereat o iprodione,procymidon ean dvinclozoli nan dthei ruptak eb yth efungus .Plan tPatho ­ logy28 :71-76 . Tuyl,J. Mvan ,1977 .Genetic so ffunga lresistanc et osystemi c fungicides.Mededelinge n LandbouwhogeschoolWageninge n (DissertationAgric .Univ.) . 77-2:1-136 . Uesugi,Y. ,1978 .Resistanc eo fpathogeni c fungit ofungicides .Japa nPesticid eInfor ­ mation35 :5-9 .

186 Case study 1:Cercospor a beticola of sugar-beets

S.G. Georgopoulos Laboratory ofPhytopathology ,Athen s College ofAgricultura l Sciences, Votanikos,Athens ,Greec e

Abstract

Fungicide resistance inth e sugar-beet leafspo tpathogen , Cercospora beticola, isdiscussed , drawingmainl y onexperience s inGreece .Resis ­ tancet obenzimidazole s developed after2 year s of intensive and almost exclusive use.Benzimidazole-resistan t strains arehighl y adaptable and, even inarea swher ebenzimidazole s haveno tbee nuse d for 6-8 years,the y represent aver yhig hpercentag e ofth epopulatio n ofth e fungus,renderin g the fungicides ineffective.Th e firstsign so fpoo rperformanc e offenti n derivatives were reported only after severalyear s ofuse .Resistanc e to fentindoe sno t appeart ob e equally stableunde r fieldconditions ,bu t satisfactory disease control isseldo m achieved. Some fungicidetreat ­ ments thatgiv egoo d results againstC . beticola ofsugar-beet s thatar e resistant tobot hbenzimidazole s and fentin are identified.

Keywords: sugar-beets,lea fspot ,benzimidazoles , fentin,stabilit y of resistance,selectio npressure ,maneb ,bitertanol ,nuarimol ,daconil .

Introduction

The leafspo to fsugar-beet ,du et o Cercospora beticola, isknow nt o allo fth e sugar-beet-growing countriesbu ti ti sonl y inarea swit hwar m summers thati tbecome s amajo r disease.Th epathoge nrequire shig hhumi ­ dity,bu tno t freemoisture ,fo r infection. Inirrigate d fields,humidit y ispracticall y always high inside thethic k foliage.Sprinkle r irrigation isparticularl y favourable forth edevelopmen t oflea fspo tbecaus e it providesmoistur e directly toth e foliage andwashe s off fungicidedepo ­ sits.Unde r suchcircumstances ,temperatur e isth emos tdecisiv e factor forth e devolopmento fa nepidemic ,becaus e the incubationperio dbecome s shorter asth emea ndail ytemperatur e increases. Conditions particularly favourable for Cercospora leaf-spot epidemics prevail inmos to fth e sugar-beet-growing areaso fGreece .

187 diseased foliage (% ) 100

i—rn—rn—i—i—i—i—r 10 20 30 10 20 31 10 20 31 10 20 30 June July August September date Figure 1.Si xyear sdat ao nth eprogres so fsugar-bee tlea fspo tdurin g summeri nun ­ treated sprinkler-irrigated plotsi nnorther nGreec e (Source:afte rDovas ,1975) .

Figure 1,whic h summarizes data collected byDova s (1975), shows thatth e destruction of the foliage of sprinkler-irrigated sugar-beets inGreec e iscomplet ebetwee n themiddl e ofJul y andth een d ofAugust .Th e chemical control of leaf spot is therefore indispensable, and many difficulties have been created by thedevelopmen t of fungicide resistance inC . beti- cola. Under conditions of high disease pressure mostprotectiv e fungicides fail to give satisfactory disease control.Unti l recently themos tsuc ­ cessful ofth eprotectant s were thetriphenylti n derivatives,namely ,tri - phenyltinhydroxid e (fentinhydroxide )an d triphentyltinacetat e (fentin acetate). These compounds were almostexclusivel y used against leaf spot in Greece from 1964 to 1970.Th e Hellenic Sugar Industry, S.A., which carries outmos to fth e sugar-beet disease control forth egrowers ,spraye d practically every field inmos t areaswit horganoti n fungicides,usuall y from theen d ofMa yunti l themiddl e of September, atabou t15-da yinter ­ vals. Inmos t cases the results weregoo dbu tHelleni c Sugarwa s always looking for replacements for the organotins,whic h are rather toxic to mammals and lackstron gpost-infectio n activity. Field testing of the first systemic fungicides thatbecam e available for C. beticola controlbega n inGreec e asearl y as 1967.Th e resultsob ­ tained with benzimidazole derivatives were excellent. Data from trials performed in197 0 show thatbenomy lwa spracticall y twice aseffectiv e as fentin acetate inprotectin g the foliage throughout the growing season (Table 1).O n thebasi s of fieldtest sth eus eo forganotin swa sgradual - Table 1. Comparison of benomyl and fentin acetate for the control of sugar-beet leafspo ti nGreec edurin gth e197 0growin gseason .

Fungicide Spraying Proportiono fdisease d foliage(% ) treatment interval (days) 21Jul y 2Augus t 21Augus t 4Septembe r

Benomyl30 0g/h a 12 1.5 3.6 5.9 13.8 17 4.4 4.3 8.8 25.5 22 A.5 4.6 37.5 44.0

Fentin acetate50 0g/h a 12 'A. 4 4.5 19.3 35.5 17 5.5 7.2 39.2 57.0 22 8.5 20.0 83.0 95.0

Control 100.0

Source:afte rDova s (1975).

ly reduced, and in 1972 more than300 0h awer e sprayed exclusively with benomyl.

Resistance to the benzimidazoles

The results of the commercial applications ofbenomy l againstsugar - beet leaf spot in Greece were excellent in the years 1970 and 1971.I n the summer of 1972 no signs of inadequate control were observed until the middle of July. Suddenly, the disease incidence increased very rapidly near the end of the month, andwithi n2 0 daysth eproportio n of spotted foliage increased from 5-10 % to 80-100% , in spite of regular benomyl sprayings.Th e development of the diseasewa sno t affected even by shortened spraying intervals or increasedbenomy l doses.A t firstcon ­ ditionsunusuall y favourable forth ediseas ewer ethough tt ob e responsible forth e control failures.However , indemonstratio nplot s theperformanc e ofth e organotins continued tob e satisfactory during the summer of 1972. The data inTabl e 2,collecte d only twoyear s after those ofTabl e 1, from the same area,sho wth e distinct superiority of fentinhydroxid e overbe ­ nomyl andthiophanat emethy l afterth een do fJul y1972 . Thepossibilit y ofresistanc e toth ebenzimidazole s in C. beticola was considered only towards the end ofth e 1972growin g season;specimen s were thenbrough t to the laboratory.Resistanc e tothi sgrou p of fungicides is very easy to detect, so it took only a fewday s torecognis e thatmos t conidial isolates obtained from fieldswher ebenomy l didno tcontro l leaf spotwer ehighl y resistant to that fungicide (Georgopoulos &Dovas , 1973). The dataeve n suggested acorrelatio nbetwee n thenumbe r ofbenomy l spray­ ingsdurin g 1971an d 1972an dth eproportio n ofresistan t isolates invar ­ ious fields. The same study showed that allo f 250 isolates of C. beti­ cola obtained fromgarde nbeet s inarea swher e no sugar-beets were grown

189 Table 2. Comparison of effectiveness of fungicides for control of sugarbeet leaf spoti n4 8demonstratio nplot si nGreec edurin gth e197 2growin gseason .

Fungicide Spraying Proportiono fdisease d foliage(% ) treatment interval (days) 16Jul y 30Jul y 15Augus t

Benomyl30 0g/h a 15 6.5 29.0 85.9 Thiophanatemethy l50 0g/h a 15 8.0 32.0 90.0 Fentinhydroxid e50 0g/h a 15 11.0 28.0 39.6

Control 70.0 100

Source:afte rDova s (1975).

and no benomyl had been used were sensitive to benomyl. That benomyl- resistant strains of the pathogen were responsible for the failures in thecontro l of leafspo t in197 2wa s shown ingrowth-roo m experimentscon ­ ducted duringth ewinte rmonths :resistan t and sensitive strains produced comparable amounts ofdiseas e on artificially-inoculated, untreated sugar- beet plants.Howeve r on benomyl-treated plants sensitive strains failed to produce leaf spot,bu t resistant strainsproduce d the same amounto f disease aso nuntreate d plants (Georgopoulos &Dovas , 1973). Thehig h selectionpressur e ofth ebenzimidazol e treatments onth epo ­ pulation of C. beticola was demonstrated ina larg e field experimentcon ­ ducted in the summer of 1973 (Dovas et al., 1976). Inexperimenta l plots in which only 3.5 % of leaf spotwa s causedb y resistant strains atth e end ofJune ,th eproportio n increased to 91.5% b y thebeginnin g ofAugust . Thiswa s due totw obenomy l applications,on e on3 0Jun e and oneo n 13July . As expected, inplot swit h ahig hproportio n ofbenomyl-resistan t C. beti­ cola atth ebeginnin g ofth e seasonth ebenomy l treatments hadver y little effect, but fentinhydroxid e continued togiv egoo d control.Th e reduced effectiveness of benomyl was always correlated withth e increase inth e proportion ofth e resistant conidia.Th e composition ofth epopulatio n of the pathogen with respect to sensivity to the benzimidazoles has been monitored in several areas. This monitoring has shownthat ,a sa whole , resistant strains are not less fittha nsensitiv e strains inth e absence of benzimidazoles. The proportion of conidia that areresistan t tocon ­ centrations ofbenomy l of1 ^g/m l remains constant foryears ,withou tth e useo f any related fungicide (Dovase t al., 1976). A similar stability of benzimidazole resistance has been found in other fungi, e.g. Botrytis cinerea (Miller& Jeves , 1979). In Greece, the benomyl sensitivity of C. beticola populations isde ­ termined as follows. Preferably young leaveswit h lesions are thoroughly washed with tap water to remove fungicide residues andol d conidia.The y are incubated overnight in amois t chamber. A single conidium is then picked up with a fine glass needle. Since conidia from the same lesion

190 usually behave similarly, single spore isolation isno tnecessary . Coni- dia from each lesion are transferred to apremarke dpositio n ona wate r agar plate. Of each small colonyproduced , one transfer ismad e to apo ­ tato dextrose agar slant (ifmaintainanc eo fth e isolate isdesired )an d another transfer to apremarke d position of aplat e ofcomplet emediu m containing a concentration of benomyl of 1[jg/ml . Ifexaminatio n ofth e transfers under the microscope is feasible, the distinction between resistant and sensitive isolates can be made the following day:trans ­ ferred hyphae of sensitive isolates develop swellings and other dis­ tortions, and often hyphal tips burst; normal appearance and growth of hyphae is observed forbenzimidazole-resistan t isolates (Georgopoulos & Dovas, 1973). Ifearl y determination isno trequire d theplate s areincu ­ bated for a few days and thenexamine d macroscopically.Th e distinction with the concentration of benomyl of 1M9/1* 1i sver y clear cutbecause , even after prolonged incubation, the sensitive isolates show no growth and the transferred water-agar blocks remain transparent,bu t resistant isolates formnorma lcolonies . When a large number of lesions have to be tested, it saves time to transfer conidia directly to afungicide-containin g medium (Yoder, 1979). Benomyl can be added even to thewater-aga rmediu m atth e concentration of 0.1 pg/ml and scoring can be done macroscopically four days later. Sensitive isolates do not survive on the treated medium, however, and establishment on fungicide-free medium isnecessar y ifsensitiv e aswel l asresistan t isolates need tob emaintaine d for futurestudies . For the measurement ofresistanc e dosage-response curves are obtained and compared. The effectca nb emeasure d by the inhibition ofdr y weight increase in liquid medium or, less accurately, but more easily,b y the inhibition ofradia l growth on agarplates .Whe n adjusting the concentra­ tion the water solubility of benomyl, which does not exceed 27jjg/ml , shouldb e taken intoaccount . Following the first report from Greece, resistance to benzimidazole fungicides was noticed also in other countries (D'Ambrae t al., 1974, 1980; Ruppel & Scott,1974 ;Ruppe l et al., 1980;Uesugi , 1978). Resistant strains in those countries have been found to behave similarly to the ones studied inGreec e and tob e responsible for failures indiseas econ ­ trol.

Resistance to organotins

From 1973 to 1977 thecontro l ofsugar-bee t leafspo t inGreec edepen ­ ded again almost entirely on fentinhydroxid e and fentinacetate .Durin g this period poor performance ofthes e fungicideswa s occasionally obser­ ved. Perhaps due to the increased awareness of the problem of resist­ ance -followin g theexperienc ewit hth ebenzimidazole s -thes e occurrences

191 were carefully studied. Itha sno wbee n established thatth eus eo forga - notins can also lead to selection of moderately and highly resistant strains of C. beticola and to failures indiseas e control (Giannopolitis, 1978). Itwa s first found thatconidi a collected from areaswit h andwith ­ out a history oforganoti nusag ediffe r considerably inthei r ability to germinate in solutions of fentinhydroxid e and fentinacetate .Further , the growth rate on agar media containing these fungicides differs,an d according to thatcriterio n C. beticola isolates canb e classified assensi ­ tive,moderatel y resistant, and resistantt oorganotins .Th e abundance of resistant andmoderately-resistan t strains ina give n location ispropor ­ tional toth enumbe r ofyear s triphenyltinfungicide s havebee nused . Recentstudie s (Giannopolitis &Chrysayi-Tokousbalides , 1980)hav e shown thatresistanc e of C. beticola toorganotin s isstabl e incultur e anddoe s not affectcolon ymorpholog y and growth.Ther e isn ocross-resistanc ebe ­ tweenorganotin s andbenzimidazoles ,bu tman y field isolates are resistant to both groups of fungicides, apparently because oftw o independentmu ­ tations.O nuntreate d sugar-beets,bot h fentin-acetate sensitive andresis ­ tant strains arevirulent ,bu to nplant s treated with fentin acetate (300 g/ha)onl yresistan t strains cancaus e anappreciabl e amounto flea fspot . From untreated plants inoculated with a1: 1 mixture ofsensitiv e andresis ­ tantconidia ,sensitiv e isolates areobtaine d athighe r frequency,indicat ­ ingreduce d competitive ability ofth eresistan t strains.Althoug h no extens ive monitoring for organotin resistance has been carried out,ther e are also indications thatth e resistantpopulation s inth e field tend to decline inth e absence ofth e selectiveagent . The distinction between organotin-resistant and organotin-sensitive strains of C. beticola isno t asclear-cu t aswit hbenzimidazoles .Micro ­ scopic examination of the water agar blocks the day following transfer does not reveal any distinct differences. A difference inth e amounto f growthbecome s recognizable after 2-3 days, althoughth ewate r agarbloc k is covered with mycelium, even in the case of sensitive isolates.Saf e distinction canb emad e 6day s after theblock s aretransferred : thesen ­ sitive and the moderately-resistant isolates dono tgro w outo fth e agar block into amediu m containing 0.25 |jg/mlan d 1.00 jjg/mlfenti nacetate , respectively, but colonies of resistant isolates ona mediu m containing 1.00 jjg/mlo fth e fungicide are abouthal fth e sizeo fthos e ona contro l medium (Giannopolitis &Chrysayi-Tokousbalides , 1980). Failures in C. beticola control due toresistanc e to triphenyltinfun ­ gicides have not been reported from countries other thanGreece . Inth e recent publication of D'Ambrae tal . (1980) it is stated that although resistant strains were present in an Italian population of C. beticola thatwa s studied,reduce d effectiveness oforganotin s hasno tbee nobser ­ ved in Italy.

192 Chemical control of Cercospora beticola today

There are large areas inGreec ewher e since 1978mos to fth e C. beti­ cola population hasbee nresistan t tobot hth ebenzimidazole s andth eor - ganotins, so that satisfactory control of leaf spot cannot be achieved with fungicides of either group.Giannopoliti s (1978)observe d that in areaswher e organotins perform poorly, amixtur e ofcarbendazi m andmane b was superior to fentin acetate. Itwa s first thought that partial re­ placement of organotins with such a mixture might solve the problem. However the data of Table 3 show that fora norganotin-resistan tpatho ­ gen population even maneb alone ispreferable .An d asth epopulatio nha s long been resistant to the benzimidazoles, additiono fa membe r ofthi s groupha sn oeffec t (Table3) . Extensive experimentation carried out by the Hellenic Sugar Industry S.A., in recent years hasprovide dver y useful information.Th enon-sys ­ temic bitertanol has given excellent results so far and the systemic nuarimol appears equally good. But at the doses required botho fthes e fungicides are too expensive tob e generally adopted.The yca nb erecom ­ mended for use aloneonl y once ayea r and thenonl y atth emos t critical time for disease control. Organotins are used onlyonc eo rtwic e atth e beginning of the season,whe ndiseas epressur e is low.Fo r the remaining sprayings alternation ofmixture s containingmane b (plus 'Daconil', fentin

Table 3. Comparison of fungicides and fungicide combinations for the controlo f sugar-beet leaf spot in the summer of 1980 in an area ofnorther nGreec ewher emos t of the Cercospora beticola population is resistant tobot h thebenzimidazol e andth e organotinfungicides .

a Fungicide Treatment' Proportion ofdisease d foliage(% ) (g/ha) 9Augus t 25Augus t 8Septembe r

Fentinacetat e 300 11.0 43.0 75.0 Maneb 2 000 6.0 31.0 52.0 Maneb+ carbendazi m 1 850+ 150 3.0 32.0 52.0 Maneb+ 'Daconil' 2 000+ 750 1.0 21.0 27.0 Maneb+ fenti nacetat e 2 000+ 300 3.0 22.0 35.0 Mancozeb 2 000 5.0 29.0 46.0 Propineb 1 750 9.0 38.0 69.0 Difolatan 1 500 10.0 32.0 46.0 Dithianon 750 17.0 75.0 100.0 Triadimefon 100 14.0 51.0 90.0 Bitertanol 450 1.0 17.0 23.0 Nuarimol 250 1.0 11.0 17.0 Bitertanol+ mane b 300+ 2 000 3.0 16.0 20.0 Bitertanol+ fenti nacetat e 300+ 300 2.0 25.0 34.0 Nuarimol+ mane b 55+ 2 000 5.0 20.0 30.0

Control 46.0 95.0 100.0

Source:unpublishe d datakindl yprovide db yth eHelleni cSuga rIndustr y S.A. a.Sprayin g interval 15days .

193 acetate, bitertanol or nuarimol,th e lasttw o atreduce d concentrations) havebee n adoptedwit h good results sofar .

References

D'Ambra,V. ,S..Mutt o& G .Caruba ,1974 .Sensibilit a etoleranz ed iisolat id i Cercosporabeticol a Sacc.a lbenlate .L'lndustri aSaccarifer a ItalianaN .1 :11-13 . D'Ambra,V. ,S .Mutt o& C .Caruba ,1980 .Frequenz ad iisolat id iCercospor abeticol a tolerantii lbenomy la seguit od itrattament id icamp oe sensibilit adegl iisolat i altrifenilacetat o diSn .Phytopathologisch eZeitschrif t97 :234-241 . Dovas,C , 1975.Si xyear sexperimentatio n incontrollin gCercospor a leafspo tusin g systemic fungicides.Helleni cSuga rIndustr yQuarterl yBulleti nNo .22 :355-40 1 (ingree kwit ha nenglis hsummary) . Dovas,C , G.Skylakaki s& S.G .Georgopoulos ,1976 .Th eadaptabilit yo fth ebenomyl - resistantpopulatio no fCercospor abeticol a inNorther nGreece .Phytopatholog y 66:1452-1456 . Georgopoulos,S.G .& C .Dovas ,1973 .A seriou soutbrea ko fstrain so fCercospor a beticola resistantt obenzimidazol e fungicides inNorther nGreece .Plan tDiseas e Reporter57 :321-324 . Giannopolitis,C.N. ,1978 .Occurenc eo fstrain so fCercospor abeticol aresistan t totriphenylti n fungicides inGreece .Plan tDiseas eReporte r62 :205-208 . Giannopolitis,C.N .& M .Chrysayi-Tokousbalides ,1980 .Biolog yo ftriphenyltin - resistantstrain so fCercospor abeticol a fromsuga rbeet .Plan tDiseas e64 :940-942 . Miller,M.W .& T.M .Jeves ,1979 .Th epersistenc eo fbenomy ltoleranc ei nBotryti s cinerea inglasshous etomat ocrops .Plan tPatholog y28 :119-122 . Ruppel,E.G .& P.R . Scott,1974 .Strain so fCercospor abeticol a resistantt obenomy l inth eU.S.A .Plan tDiseas eReporte r58 :434-436 . Ruppel,E.G. ,A.D .Jenkin s& L.M .Burtch ,1980 .Persistenc eo fbenomyl-toleran t strainso fCercospor abeticol a inth eabsenc eo fbenomyl .Phytopatholog y70 : 25-26. Uesugi,Y. ,1978 .Resistanc eo fphytopathogeni c fungit ofungicides .Japa nPesticid e Information35 :5-9 . Yoder,K.S. ,1979 .Method sfo rmonitorin gtoleranc et obenomy li nVenturi ainaequalis , Monilinia spp.,Cercospor a spp.,an dselecte dpowder ymilde w fungi.In :Method s forevaluatin gplan tfungicides ,nematicide san dbactericides ,America nPhytopatho - logicalSociety ,Sain tPaul ,Minnesota ,p .18-20 .

194 Case study 2:Venturi s of pome fruits and Monilinia of stone fruits

J.D.Gilpatric k NewYor k StateAgricultura l Experiment Station,Cornel l University, Geneva,Ne wYork , U.S.A.

Abstract

Dodineresistanc e inth e applesca b fungus, Venturia inaequalis, is now common ineaster n Canada andnortheaster nU.S.A . Resistantpopula ­ tions thatdevelope d after 9-10 years of intensive,an dofte nexclusive , use ofdodin e havebee ndetecte d atleas t 10year s after sprayingwit h this fungicidewa s discontinued. Resistant strains areonl y two to four times less sensitive tododin e thanwild-typ e strains.Benzimidazol ere ­ sistance in V. inaequalis isno wreporte d frommos t areaso fth eworl d where apples aregrown ,an d inth epear-sca b fungi, V. pirinia, in Israel and France,an d in V. nashicola, inJapan .Resistanc e tobenzimidazole s in Monilinia spp.,th ebrown-ro t fungi,als oha sbee nreporte dworld-wide . Benzimidazole resistance has developed after aboutthre e to four seasons of intensive use ofthes e fungicides.Th e level ofresistanc e tobenzimi ­ dazoles in Venturia and Monilinia spp.i s afacto r of atleas t five times normal sensitivity, buti softe nmuc hhigher .Preventio n ofresistanc e to fungicides onthes ecrop sma yb e achievedb y strategies thatreduc eth e selectivepressure .Thes e approaches may include theus eo fmixture s or alternative fungicides andpes tmanagemen t techniques.

Keywords:dodine ,benzimidazole ,pes tmanagement , Venturia inaequalis, Venturia pirinia, Monilinia spp.,selectiv epressure .

Introduction

Scab ofappl e andpea r andbrow n roto f stone fruits causedb y Venturia and Monilinia spp.,respectively , areeconomicall y important fungaldisea ­ seswhereve r these fruits aregrown .T opreven t severe crop losses these diseases arecontrolle d bynumerou s applications of fungicide eachgrowin g season.Unde r such intensive regimes,fungicid e resistance problems have occurredworld-wid e onpom e and stone fruits andthe yhav e severely limi­ ted theus eo fsom eo fth emos teffectiv e fungicides.

195 From experience with resistance onthes e crops there ismuc h information available thatshoul d beusefu l indealin gwit h thisproble m inth e future.

The diseases and their control with fungicides

Scab ofappl e andpea r

Apple scab caused by Venturia inaequalis (Cke.)Wint .i s agrea t problem incool ,mois t areaso fth eworld . The fungusproduce s one sexual overwintering generationpe ryea r inwhic h ascospores develop inperi - thecia in fallen leaves onth eground .Thes e spores are forcibly ejected inth e spring and causeprimar y infections on foliage and fruitunti l about fourweek s afterbloom .Man y secondary cycles ofth e asexual stage occur duringth egrowin gseaso nan deac h scab lesion iscapabl e ofpro ­ ducing thousands ofconidia .Thu s thepotentia l fordiseas e incidence in a favourable environment isextremel yhigh .T o growcommerciall y accep­ table crops inmos tappl egrowin g areas,8-2 0 fungicide applications are required eachgrowin g season,startin g atth egreen-ti p growth stage and continuing until the fruitan d leavesbecom e resistant toinfection . Several fungicides areavailabl e forth e control ofappl e scab (Table1) . Thesevar y inefficacy , controlmechanism ,biochemica l modes of action and apparentpronenes s to resistance. Inrecen ttimes ,captan ,ethylene -

Table 1. Fungicides available for apple-scab control in the U.S.A. and their properties.

Fungicides Scab Control Biochemical Resistance performance mechanism sites reported

Captan good protectant many no

Benzimidazoles good protectant one yes antisporulant curative systemic

Mancozeb good protectant many no

Dodine good protectant few yes curative antisporulant

Other carbamates moderate protectant many no

Sulfur fair protectant many no

Dichlone good curative many no

Captafol good protectant many no a. Limited to single application at green tip.

196 bisdithiocarbamates, dodine, and benzimidazoles have been used most by applegrower s againstscab ,bu tresistanc e has greatly restricted theus e of dodine and benzimidazoles. The ergosterol-synthesis-inhibiting (ESI) fungicides arepromisin gne wproduct s forapple-diseas econtrol . Pear scabs caused by V. pirina Aderh. and V. nashicola Tanaka & Yamamoto are economically important diseaseswit h life-cycles similar to V. inaequalis. Insom e areas V. pirina may overwinter inlesion s ontwig s andproduc e conidia inth e spring, aswel l as ascospores inperitheci a in fallen leaves, so that the disease pressure may be extremely intense. Control methods are similar to those for apple scab, using the same fungicides and spraying schedules, depending on the pear cultivar and geographic area. Resistance tobenzimidazole s has occurred onpears ;th e ESI fungicides areth emos tpromisin greplacements .

Brownro to f stone fruits

Brown rot is causedb ythre e species of Monilinia, including M. fruc- ticola (Wint.)Honey , M. fructigena (Aderh.& Ruhl )Honey , and M. laxa (Aderh.& Ruhl. )Honey . The disease caused by N. fructicola has twope ­ riods of major activity: thebloo m andripe-frui t stages,whic h aresep ­ arated by an interval ofsevera lweek swhe n thegree n fruiti sno thigh ­ ly susceptible unless.injured .Th ebrown-ro t fungi overwinter on infected dried fruit (mummies) or other plant parts. The following year conidia are produced in the tree or ascospores in apothecia frommummie s onth e ground.Bot h spore types infect flowers andyoun g fruit,causin g rotswit h profuse conidial sporulation, thus increasing inoculum pressure to anex ­ tremely high level byharves ttime .Extensiv e loss ofth e fruitcro p can occuro nth etre e andpost-harves t roti sextremel y destructive,especial ­ ly forstore d or shipped fruit. Control of brown rot ishighl y dependent onth e application offungi ­ cide sprays at bloom and shortly before harvest. Inrain y areas orwhe n fruit injury occurs, it also may benecessar y to spraydurin g theimma ­ ture-fruit stage. Stored or shipped fruitmus tb e treated afterharvest . Before the introduction ofth ebenzimidazoles ,brown-ro t controlwa s often unsatisfactory, requiring numerous applications of such fungicides as captan, copper,dichlone ,dichloran ,mane b and sodiumpentachlorophenate . After the benzimidazoles became available,earl y inth e1970s ,the ywer e theprimar y fungicides until resistance tothe m developed. Thebenzimida ­ zoles are now usedmostl y incombination s withbroad-spectru m fungicides such as captan. Dicarboximide andES I fungicides aregraduall ybein gap ­ proved and used for brown-rot control on stone fruits throughout the world, andthe y areexpecte d toreplac e thebenzimidazole s within thenex t fewyear sbecaus e ofproblem s ofresistance .

197 Resistance to dodine

Emergence ofresistanc e

Dodinewa s introduced asa fungicid e forapple-sca b control inth e late 1950s. Because of its low cost and excellent protectant, curative,an d antisporulantproperties ,i twa s quickly accepted and used extensively in many applegrowin g areas of theworld .Afte r about 10year s ofus e inth e western fruitgrowin g areas ofNe wYor k State,U.S.A. , widespread andun ­ precedented scab-control failures occurred in 1969. These failureswer e quickly demonstrated to be due to resistance of V. inaequalis tododin e (Szkolnik& Gilpatrick , 1969). Subsequently, workershav e documented this phenomenon in other areas of the northeasternU.S.A . and easternCanad a (Jones& Walker , 1976; McKay &Ma cNeill , 1979; Ross& Newbery, 1977). Meanwhile, the volume of dodine used onapple sha s declined from ahig h level in the 1960s to a very lowleve l today, atgrea teconomi c losst o themanufacture r ofth eproduct .I naddition ,th e losso fdodin e stimula­ ted the extensiveus eo fbenzimidazole s asreplacemen t fungicides and,n o doubt,contribute d toth e earlydevelopmen t ofresistanc e in V. inaequalis to thisne w class ofcompounds .

Detection andmeasuremen t

A disease-control failure must be carefully studiedbefor e fungicide resistance can be confirmed as the cause.Severa l factors thatca nlea d to poor disease controlwit h fungicides areliste d inTabl e 2.Afte r all other factors, such as faulty application orunusua lweather ,hav ebee n eliminated, resistance may be suspected. Unfortunately, attempts tode ­ tect resistance prior to its appearance asa contro l failure areno tof ­ ten successful with Venturia andresistanc e isusuall yprove nonl y after the failureoccurs .

Table2 .Factor sleadin gt opoo rcontro lo fplan tdiseas ewit h fungicides.

Improperfungicid eo rformulatio n Inadequateo rfault yequipmen t Improperdosage ,velocity ,volume ,o rtimin g Incompatabilitywit hothe rpesticide s Severeepidemi cconditions :hig hinoculu mlevel ,heav yrains , etc. Humanerro r Resistance

198 Proof ofresistanc e tododin e in V. inaequalis hasbee n carried outi n invitr o and inviv o tests.Fo r tests invitro ,aga rplat e or glass-slide drop-dilution methods are used.Th e sensitivity ofnon-resistan t strains toth e fungicide is firstestablishe d usingpreviousl y unexposed wildty ­ pes or standard dodine-sensitive laboratory strains. Ingerminatio ntests , spores ofdodine-sensitiv e V. inaequalis exhibitL D values of0.25-0. 5 pg/ml; resistant isolates are two to four times less sensitive (Gil- patrick& Blowers , 1974). The values aredetermine d from dosage-response curves (log10 probit) developed withinth erang eo f0.2-2. 0|jg/m lo fdo ­ dine. Inhibition of the fungus is expressed as non-germination of the spore. Because V. inaequalis grows very slowly and sporulates poorly on agar media, colony-growth tests are not as rapid as spore germination tests,bu tthe yhav ebee nuse db y someworker swit h success (Jones& Wal ­ ker, 1976;Ma cNeil l& Schooley, 1973;McKa y &Ma cNeill , 1979; Ross & Newbery, 1977). Tests in vivo may include the spraying of trees in the orchard or greenhouse with a dosage series of dodine and comparing itsperformanc e toanothe r standard,unrelate d fungicide (Szkolnik &Gilpatrick , 1969).

Build-up of aresistan tpopulatio n

Inwester n NewYor k State orchards,wher e apple scab isth e onlypri ­ mary disease requiring chemical control,dodin ewa s oftenth eonl yfungi ­ cide applied in the 9-10 years before resistance occurred. During that time, it is estimated that dodinewa s applied about8 0 times inorchard s before resistance was demonstrated. Similar use patterns were common in other areas of the U.S.A. where resistance has subsequently developed (Gilpatrick& Blowers , 1974; McKay& Ma cNeill , 1979; Ross &Newbery , 1977). To date, dodine resistance has not been reported inarea swher e the compound has had only limited use orha sbee nuse d inmixe d program­ mes with other fungicides (Gilpatrick& Blowers , 1974). Such isth e case inth eHudso nValle y ofNe wYor k State,where ,i nadditio n to scab,i ti s necessary to control cedar-apple rustan d summer diseases,agains twhic h dodine is ineffective. Carbamate fungicides are applied three or four times each year duringbloo m andearly-fruitin gperiod s tocontro lrust ; other fungicides,suc ha scaptan , areapplie d later for summer-fruit rots. Both carbamates and captan also provide adequate scab control. Thus in the HudsonValle y of NewYor k State, dodine is used forearl y scabcon ­ trolb y some growers,usuall y fortw oo rthre e sprayings, followedb ybe - nomyl or other fungicides in combination with acarbamat e duringbloom , and captan only during later sprayings.Unde r thisregime ,resistanc e in the scab fungus to dodineha sno tbee n found aftermor e than2 0year s of use. These experiences clearly indicatetha ti nth eHudso nValle yresis ­ tance has been avoided by alternating andmixin g fungicides and thati n

199 westernNe wYor k State and other areaso fth eU.S.A . exclusiveus e ofdo - dineha sresulte d inresistance . Laboratory and field studies have showntha tth e sensitivity to dodine of individuals in apopulatio n of V. inaequalis follows anorma ldistri ­ bution for concentrations of about 0.1-1.0 (jg/ml.Ma cNeil l& Schooley (1973) found a few conidia in a population of V. inaequalis that were resistant in vitro and retained this characteristic inrepeate dsubcul - turing. In the field repeated exposure of the scab fungus to dodine causes the mean sensitivity ofpopulation s tothi s fungicide todecreas e by selection of the less sensitive individuals. Eventually, a level of resistance is reached at which dodine no longer controls scabsatisfac ­ torily (Gilpatrick &Blowers , 1974; Jones& Walker , 1976;McKa y& Ma c Neill, 1979; Ross& Newbery , 1977). The change insensitivit y isusuall y only by a factor oftw ot o four and there isofte na noverlappin g ofth e distribution of dodine sensitivity between resistant and non-resistant strains.Thu s in small samplepopulation s itofte n isdifficul t toclear ­ lyseparat e thesetw o types (Gilpatrick& Blowers , 1974). The evidence therefore supports thebelie f that V. inaequalis mustb e exposed to dodine for a considerable time and that dodinemus tb euse d intensively as the principal, if not exclusive, fungicide if resistance ist odevelo p to apoin twher e apple scabn o longerca nb e controlled sat­ isfactorily withthi s fungicide.

Resistance to benzimidazoles

Venturia spp.

Resistance in V. inaequalis to benomyl was first reported inSout h Australia in 1974 (Wicks, 1974). Subsequently, resistance tobenzimida - zolesi nthi s fungusha s occurred inman y apple-growing areaso fth eworl d (Carreno& Pint o deTorres , 1979; Jones& Walker , 1976; Kiebacher & Hoffman,1976 ;Novack a et al., 1977;Olivier ,1979 ;Tat e& Samuels, 1976). Benzimidazole resistance inpea r scab causedb y V. pirina hasbee nrepor ­ ted in Israel and France and by V. nashicola in Japan (Ishii& Yama - guchi, 1977;Olivier , 1979;Shab i& Ben-Yephet , 1976). Proof of resistance to benzimidazoles in Venturia spp.i ssimila rt o that described for dodine resistance in V. inaequalis. Spore-germination tests on agar plates using either conidia or ascospores aremos tusefu l for field monitoring (Jones& Ehret ,1976 ;Umemot o& Nagai ,1979 ;Yoder , 1978). Sensitive spores germinate but exhibit marked germ tubeabnorma ­ lities at concentrations of 0.1 M

200 Resistance has occurred primarily inappl e andpea r orchardswher eben - zimidazoles have been used intensively forthre eo r fouryear s (Ishii& Yamaguchi, 1977;Jone s &Walker , 1976;Kiebache r& Hoffman , 1976;Olivier , 1979; Sawamura et al, 1976; Shabi& Ben-Yephet , 1976; Tate& Samuels , 1976; Wicks, 1974). However, in the HudsonValle y ofNe wYor k State and in certain other areas ofth enortheaster nU.S.A . where cedar-apple-rust control with other fungicides isrequired , asdescribe d under dodinere ­ sistance, no case of benzimidazole resistance in V. inaegualis has yet been recorded. However, in NorthCarolin a this is not so. In orchards surveyed in that state,benomy l was mostcommonl y used inthre eo r four pre-bloomspray s on apples,an d other fungicides unrelated tobenomy lwer e used for the remainder ofth e season (Sutton, 1978). After threet o four years, benomyl-resistant strains developed in about one-third of the orchards surveyed,bu tno t inth eothers .Th ereaso n forthi s rapidbuild ­ up of resistance in NorthCarolin a has not yet been explained (Sutton, personal communication, 1981). The rapid development ofresistanc e inth esca b fungit obenzimidazo - les may be related notonl yt oth e selection ofresistanc e throughasco - sporic and conidial inoculum by spraying during the scab season,bu tals o to a further selectivepressur e duringth eoverwinterin g period. Seasonal spraying ofbenomy l isknow nt o inhibitt o ahig hdegre eperithecia l for­ mation of sensitive strains of V. inaequalis in fallen infected apple leaves, but resistant strains are not so inhibited (Gilpatrick, unpub­ lished data). Thus, inorchard s sprayedwit hbenomyl ,developmen t ofre ­ sistance may occur very rapidly from one growing season toth enex tb y elimination of sensitive strainswithi nth eoverwinterin g funguspopula ­ tion.

Moniliniaspp .

Because ofth e superiorvalu e ofth ebenzimidazole s incontrollin g dis­ eases causedb y Monilinia spp.,thes e fungicideswer euse d extensively on stone fruits throughout the world following their introduction inabou t 1970. Within three or fouryear sman y regions reported crop failuresre ­ sulting fromresistanc e tothes e fungicides (Jones& Ehret ,1976 ;Penros e & Koffman, 1977; Szkolnik& Gilpatrick , 1977; Whan, 1976). In regions where use was less intense, resistance was delayed (Tatee t al., 1974). Detectionmethod s and sensitivities ofresistan t strains closely parallel those for Venturia spp. (Yoder, 1978). Because available alternatives toth ebenzimidazole s oftenhav egive n less than adequate control compared tobenomyl ,stone-frui t growers tend tous ebenomy l despite resistance,unti l itn o longer controlsbrow nrot . Many growershav e takent omixin gbenomy l andcapta nthemselve s totr yt o avoid severecro p losses.

201 Fitness of resistant strains

Observations in NewYor k State indicate that fungicide-resistant strains of Venturia and Monilinia are aspathogeni c as sensitive strains (Szkolnik& Gilpatrick , 1969, 1977), andtha tonc eresistanc e occurs ina fieldpopulatio n ofthes e fungi,i tcontinue s for alon g time.Dodine-re - sistant strains of Venturia have been isolated from NewYor k State or­ chards 10year s after theus e ofthi s fungicidewa s stopped, andbenomyl - resistant strains have been isolated after 3years .A similar situation exists with benomyl resistance in M. fructicola. Thus, re-use ofthes e fungicides after several years of abstinence does not seem advisable (McKay &Ma cNeill , 1979;Wicks ,1979 ;Gilpatrick ,unpublishe d data).

Strategies to avoid resistance

Apples

InNe wYor k State,grower shav ebee nprovide d with asmuc h information on fungicide resistance asi savailable .Onc e resistance is suspected or proven, the growers are advised tochang e to analternat e fungicideimme ­ diately. For scab, alis to f suitable alternatematerial s forbenzimida - zoles or dodine is available (Table 1). Apple growers have largelyre ­ frained fromusin gdodin e andbenomy l alone inarea swher e resistanceha s appeared,bu tthe y oftenus ebenomy l incombinatio nwit hmulti-sit e inhi­ bitors sucha scaptan . Resistance probably can be best prevented byusin gcertai ntreatmen t regimes from the firstus eo f ane w fungicide,rathe r thantakin gmeasu ­ res after it occurs. Based on experiences in NewYor k State, several treatment strategies may be proposed for apple-scab control (Table3) . The exclusive and intensive useo f aresistance-pron e fungicide (e.g.be ­ nomyl- »benomy l• »benomyl ) for scab control is discouraged. The use of single and multi-site fungicides in mixtures or alternations that seem successful inpreventin g resistance in V. inaequalis inth eHudso nValle y appearspromisin g (e.g.dodin e• *(benomy l +macozeb )• *captan) .Resistan ­ ce also appears tohav ebee ndelaye d inth e scab funguswhe ncaptafo l was used in the single-application technique (SAT)a tbu dburst ,followe db y benomyl later in the season (e.g.captafo l SAT• *benomyl) . Inthi scase , aboutone-hal f ofth e ascospore inoculum iseliminate d eachyea r from the selection pressure of benomylb y thecaptafol ,whic hma y account forth e delay ofresistanc e tobenomyl . Several other strategies aresuggeste d topreven t resistance in V. in­ aequalis. One ist o alternate amulti-sit e inhibitor,suc ha scaptan ,wit h a limited-site inhibitor,suc h asbenomyl ,i nth e sprayingprogramm e (e.g. captan •*benomy l •*capta n •*benomy l- »etc.) . Another ist ous ehal fdosa -

202 Table3 .Suggeste d spraying strategiest opreven t fungicideresistanc e inth eapple-sca b fungusi nNe wYor kState .

Strategy Spraying sequencedurin gseaso n Post- Probability number harvest ofresistanc e 10 foliar tobenomy l applicationo rES I

B B B B B B B B B B veryhig h

Dod Dod Dod M+Bb M+B M+B M+B C C C low 2 2 2 2

C+Bb C+B C+B C+B C+B C+B C+B C+B C+B C+B unknown 2 2 2 2 2 2 2 2 2 2

C B C B C B C B C B delay

Dif - - - B B B B B V mediumo r delay

Dif low

B+Sb B+S B+S B+S B+S B+S B+S B+S B+S B+S unknown 2 2 2 2 2 2 2 2 2 2 a.B = benomyl ;Do d= dodine ;M = mancozeb ;C = captan ;Di f= captafo lsingl eapplication , highdosage ;S = ESI . b.Hal fdosag eo ftha tnormall yrecommende d foreac hfungicide .

ges of two dissimilar fungicides at each spraying (e.g. captan + benomyl)/ 2 •+etc.) . Similarly, use of two single-site inhibitors of different bio­ chemical action in combination at reduced dosages has been suggested (e.g. (benomyl + ESIJ/2 -»etc.) . The latter three programmes involving alterna­ ting and mixing of fungicides have not yet been proven to be useful in practice. Such approaches also present certain tactical problems of opti­ mum timing for each fungicide, adequate dosages, residue dynamics, and economics. In the long run, the pest-management approach may be the best solution to prevent fungicide resistance in apple. Under Integrated Pest Manage­ ment (IPM), control tactics will be based on absolute need as determined by computerized spray-warning devices and knowledge of the dynamics and mode of action of fungicides. Furthermore, use of resistance-prone chemi­ cals can be selectively managed, so that the selection pressure on the fungus can be minimized. A scheme of scab control might involve a SAT ap­ plication of captafol at bud burst followed by two or three sprayings of a curative fungicide as needed, and a post-harvest spray of benomyl or another suitable fungicide to prevent perithecial formation during the winter e.g. captafol SAT •*ESI(curative ) •*benomy l(post-harvest) .

2(P Stone fruits

Similar strategies to prevent resistance may bepropose d forcontro l ofbrownro t in stone fruits, e.g. triforine atbloo m •*capta n incove r sprayings• *vinclozoli n (pre-harvest) •*benomyl(post-harvest) . Resistance is unlikely to develop to any ofthes e fungicides under thisregim ebe ­ cause each has a different biochemical mode ofactio n and the selection pressurewoul db e low foreac hproduct .Thi s example illustrates thenee d for avariet y of fungicides tob e available tocomba tresistance . Inad ­ dition, research isneede d todevelo p IPM strategies forston e fruits that lead to reduced dependence on or better timing of fungicide application for the control of brown rot. Such strategies may reduce the selection pressure for resistance anddela yo rpreven tresistanc ebecomin g aprac ­ ticalproblem .

Conclusions

Thehistor y ofresistanc e to fungicidesb y Venturia and Monilinia spp. onpom e fruits illustrates how resistance arises andho w thisproble m may be prevented for new fungicides. The case of dodine and V. inaequalis shows that resistance mayb e ata ver y lowleve l andrequir e alon gtim e for development. Finally, however, complete breakdown ofcontro l occurs and the use of the fungicide in disease-management strategies must be discontinued, causing the manufacturer and often the grower to suffer extensive economic losses.Thu s the detection of a low level ofresis ­ tance toa fungicide in afungu s or aslo w losso fpopulatio n sensitivity should notb e regarded as an indication that resistance will not be a problem in practice, but should be considered as awarnin g that with extended useo fth e substance its effectivenessma y decline. Inpractica l agriculture it should be possible to delay or even avoid resistance of this typeb y goodmanagemen tpractices . Benzimidazolesresistanc e onpome -an d stone-fruit diseases illustrates thatextensiv e andexlusiv eus e of aresistance-pron e fungicide canquick ­ ly lead to a high level ofresistance ,cro p losses,an dgreatl y limited use of the product. However resistance mayb e avoided ifth eproduc ti s mixed or alternated with other fungicides from its firstuse .Afte rth e discovery ofresistance ,mixture s ofbenzimidazole s andmultisit einhibi ­ torshav ebee nwidel y used,bu tth e impacto fthi s approacho nth eproble m ofresistanc e isno tye tclear .Th e future statuso fresistanc e underth e mixture regime should clarify thevalu e ofthi s strategy. New fungicides areno w available forus e onpom e and stone fruits,th e most notable of which are the ergosterol-synthesis inhibitors for both scab andbrow n rot,an d thedicarboximide s forbrow n rot.Bot h classes of compounds appear to have limited-site-inhibition characteristics. Thus,

204 "the potential for resistance in these new fungicides exists. The scab and brown-rot diseases are both multicycle diseases requiring multiple appli­ cations of fungicides each year. With dodine and the benzimidazoles this has led to resistance problems. New fungicides used on stone and pome fruits will be subjected to similar pressures and the development of resistance in the future seems probable. Thus, steps taken early in the use of new fungicides to avoid resistance would be appropriate. From past history with dodine and the benzimidazoles on these crops, it appears that strategies that reduce selective pressure on the pathogens by the fungicide offer a hopeful approach. As more and more pest management strategies are introduced that will lead to more direct control of grower practices, our ability to manage resistance should improve. Though much more research is needed before strategies are available that can be re­ lied upon to avoid fungicide resistance in Venturia and Monilinia on pome and stone fruits, these crop-disease scenarios are convenient model sys­ tems for studies of fungicide resistance in both practice and theory. The continuing economic production of these crops may depend on the out­ come of these studies.

References

Carreno, I.I. & A. Pinto de Torres, 1979. Aislamiento de razas tolerantes de Venturia inaequalis (Cke.) Wint. a fungicidas sistémicos en la zona de Curico. Agricul- tura Técnica 39: 52-58. Gilpatrick, J.D. & D.R. Blowers, 1974. Ascospore tolerance to dodine in relation to orchard control of apple scab. Phytopathology 64: 649-652. Ishii, H. & A. Yamaguchi, 1977. Tolerance of Venturia nashicola to thiophanate- methyl and benomyl in Japan. Annals of the Phytopathological Society of Japan 43: 557-561. Jones, A.L. & G.R. Ehret, 1976. Isolation and characterization of benomyl-tolerant strains of Monilinia fructicola. Plant Disease Reporter 60: 765-769. Jones, A.L. & R.J. Walker, 1976. Tolerance of Venturia inaequalis to dodine and benzimamidazole fungicides in Michigan. Plant Disease Reporter 60: 40-44. Kiebacher, H. & G.M. Hoffman, 1976. Benzimidazol-resistenz bei Venturia inaequa­ lis. Zeitschrift für Pflanzen-Krankheiten und Pflanzenschutz 83: 352-358. Mac Neill, B.H. & J. Schooley, 1973. In vitro development of dodine tolerance in Venturia inaequalis. Canadian Journal of Botany 51: 379-382. McKay, M.CR. & B.H. Mac Neill, 1979. Spectrum of sensitivity to dodine in field populations of Venturia inaequalis. Canadian Journal of Plan Pathology 1: 76-78. Novacka, H., W. Karolczak & D.F. Millikan, 1977. Tolerance of the apple scab fungus to the benzimidazole fungicides in Poland. Plant Disease Reporter 61: 346-350. Olivier, J.M., 1979. Observations sur les souches de tavelures du pommier et du poirier résistantes aux benzimidazoles. Annales de Phytopathologie 11: 135. Penrose, L.J. & W. Koffman, 1977. Tolerance of Sclerotinia fructicola to benzimi­ dazole fungicides and control of the fungus. Phytopathologische Zeitschrift 88: 153-164. Ross, R.G. & R.J. Newbery, 1977. Tolerance of Venturia inaequalis to dodine in Nova Scotia. Canadian Plant Disease Survey 57: 57-60. Sawaruma, K., T. Fujita & H. Goto, 1976. Tolerance of apple scab fungus Venturia inaequalis to thiophanate and benomyl fungicides in Japan. Bulletin of the Faculty of Agriculture, Hirosaki University 26: 1-9. Shabi, E. & Y. Ben-Yephet, 1976. Tolerance of Venturia pirina to benzimidazole fungicides. Plant Disease Reporter 60: 451-454. Sutton, T.B., 1978. Failure of combinations of benomyl with reduced rates of non- benzimidazole fungicides to control Venturia inaequalis resistant to benomyl and

205 thesprea do fresistan tstrain si nNort hCarolina .Plan tDiseas eReporte r62 : 830-834. Szkolnik,M .& J.D . Gilpatrick,1969 .Apparen tresistanc eo fVenturi ainaequali s tododin ei nNe wYor kappl eorchards .Plan tDiseas eReporte r53 :861-864 . Szkolnik,M .& J.D .Gilpatrick ,1977 .Toleranc eo fMonilini afructicol at obenomy l inwester nNe wYor kStat eorchards .Plan tDiseas eReporte r6.1 :654-657 . Tate,K.G. ,J.M .Ogawa ,B.T .Manj i& Elain eBose ,1974 .Surve y forbenomy ltoleran t isolateso fMonilini a fructicola andM .lax ai nston efrui torchard so fCalifornia . PlantDiseas eReporte r58 :663-665 . Tate,K.G .& G.J . Samuels,1976 .Benzimidazol etoleranc e inVenturi a inaequalis inNe wZealand .Plan tDiseas eReporte r60 :706-710 . Umemoto,S .& Y .Nagai ,1979 .Simpl emetho d fordeterminin gbenomy ltoleranc eo f Venturianashicola .Annal so fth ePhytopathologica l Societyo fJapa n45 :430-435 . Whan,J.H. , 1976.Toleranc eo fSclerotini a fructicola tobenomyl .Plan tDiseas e Reporter60 :200-201 . Wicks,T. ,1974 .Toleranc eo fth eappl esca bfungu st obenzimidazol efungicides . PlantDiseas eReporte r58 :886-889 . Wicks,T. ,1976 .Persistenc eo fbenomy ltoleranc ei nVenturi a inaequalis.Plan t DiseaseReporte r60 :818-819 . Yoder,K.S. ,1978 .Method sfo rmonitorin gtoleranc e tobenomy lx nVenturi ainaequalis , Monilinia spp.,Cercospor aspp. ,an dselecte dpowder ymilde wfungi .In :E.I .Zeh r (Ed.):Method so fEvaluatin gPlan tFungicides ,Nematicides ,an dBactericides . AmericanPhytopathologica l Society,p .18-20 .

206 Case study 3: Pyricularia oryzae of rice

Y.Uesug i Department ofPlan tPatholog y andEntomology ,Nationa l Institute of Argicultural Sciences,Yatabe-machi , Ibaraki,Japa n

Abstract

Fungicide-resistant strains of Pyricularia oryzae havebee n obtained invitro .Resistanc e toth e antibiotic kasugamycinan d toorganophos - phorus fungicidesha s alsobee n found inth e field.Resistanc e tokasuga ­ mycin in field isolates iscontrolle d by amajo rgene .Thre e loci for resistance were identified inmutant s obtained invitro . Inthes emutant s resistance appeared tob edu et omodificatio no fth eribosome s atth esit e ofactio no fth e antibiotic.Kasugamycin-resistan t isolates from the field are somewhat inferior tonorma l sensitive strains.Strain s resistantt o organophosphorus fungicideshav ebee nobtaine d invitr o and,onl y recently, inth e field,althoug hthes e fungicides havebee nuse d for1 5years .Th e resistantmutant sobtaine d invitr ower e cross-resistantt o isoprothiolane, whosemolecul e doesno tcontai nphosphoru s atoms,an d showed negatively- correlated cross-resistance to somephosphoramidat e compounds. Inthes e respects they clearly differ frommos tresistan t field isolates,whic h lacknegatively-correlate d cross-resistance tophosphoramidate s andhav e a lower levelo fresistanc e toorganophosphoru s fungicides.Th e resistance mechanism inth emoderatel y resistant field isolates appeared tob ein ­ creased detoxification,bu ti n laboratory-derived mutants themechanis m was different.Th e availability ofa variet y of fungicideswit h different modes of actionma yb e agrea thel p incopin gwit h the resistanceproblem .

Keywords: riceblast ,cross-resistance ,negatively-correlate d cross-resis­ tance, detoxification, kasugamycin, organophosphorus fungicides,phos ­ phoramidate,isoprothiolane .

Introduction

Riceblast ,cause db y Pyricularia oryzae, isth emos timportan tdisea ­ seo fric e inJapan . Inothe r rice-growing countries where riceblas ti s less serious,it s occurrencema y increasebecaus e ofchange s incultura l

207 methods, such asincrease d use ofnitroge n fertilizers,whic h accelerate thedevelopmen t ofth edisease . Inprinciple ,thre emethod s ofcontro l areavailable ,namely , breeding forresistance , culturalmethod s and chemical control.Th e firstmetho d doesnot ,however ,provid e lasting results,a sne wphysiologi c races of thepathoge n readily emerge.Moreover ,th emos t appreciated, tasty rice varieties areofte nsusceptibl e toth e disease.Th epossibilit y ofpre ­ venting blastb y culturalmethod s isofte nver y limited.Earl y season cultivation, forexample ,ma yhel p to avoid damage causedb ytyphoons , whichma y strike Japan justbefor e theharvest ,bu t itma y resulti nth e development ofth ediseas e ata younge r stage ofth eplant ,du et oth e lowertemperature s thenprevailing . Invie w ofthes e facts,chemica l control isindispensable , and itprobabl ywil l remain themos t important measure forcopin gwit hblast . Inth epast ,Bordeau xmixtur e hasbee nuse d to controlblast ,bu tth e effectwa sunsatisfactory . Food shortages afterth eSecon dWorl dWa rle d toth e dusting ofth eric e cropwit hphenylmercur y compounds,whic hunti l thattim eha d onlybee nuse d forsee d disinfection. From 1952t o about 1967thes e compoundswer e theprim e agents forcontro l ofric eblast . A search for antibiotics effective againstblas tstarte d in1953 . It led toth ediscover yo fblasticidin-S ,whic hwa s introduced commercially in1961 .Thi spromote d the development ofothe rnon-mercuria l fungicides, among them the antibiotic kasugamycin, organophosphorus fungicides,suc h as IBP andedifenphos ,an d other types ofcompounds ,suc h astetrachloro - phthalide,probenazol e and isoprothiolane (Figure 1).Mos to fthes enon - mercurial fungicides actspecificall y onric eblast .Howeve r someo f thesehav eencountere d problems with fungicide resistance inth e field. Fortunately, different types of fungicides areno w available,s o that control ofblas tremain spossibl eb yusin g avariet y oftype s ofchemi ­ cals.

Resistance to kasugamycin

Emergence ofresistan t strains

Mutants of P. oryzae resistantt okasugamyci n werereadil y obtainedb y platingconidi a onaga rcontainin g adiscriminatin g concentration ofth e fungicide,withou tth eus e ofmutageni c agents (Ohmori,1967 ;Uesug i et al., 1969;Katagir i &Uesugi ,1978 ;Tag ae t al., 1978). The frequency of emergence ofresistan tmutant s inthes e experimentswa s usually about 10~ ;i tvarie dwit h theparen t strains tested,but ,withi n acertai n range,no twit h theconcentratio n ofth e antibiotic.Thi s suggests that iti sunlikel y thatth e antibiotic exertsmutageni c activity (Miurae t al., 1976). Resistantmutant swer e alsoobtaine d from fast-growing sec-

208 CH3 H I CH3

n\\° "H, JiH

H2N OH| OH b!asticidin-S kasugamycin

// w /0-CH(CH3)2 S X (' ^CH2-S-PV 'l/ x C2H5OPN || 0-CH(CH3)2 \ O

IBP 'Kitazin P' edifenphos (EDDP,'Hinosan')

rS>,C-0-CH(CH3)2

^S^C-0-CH(CH3)2

isoprothiolane tetrachlorophthalide ('Rabcide')

OCH2CH=CH2

\c—N O^S

probenazole ('Oryzemate')

Figure 1,Structura lformula eo ffungicide suse dt ocomba tPyriculari aoryza e onrice .

209 tors ofmyceliu m on amediu m containing the antibiotic (Tagae t al., 1979), and fromsubculture s successively transferred tomedi a containing increasing concentrations ofth e antibiotic (Uesugi et al., 1969). Resistance of P. oryzae tokasugamyci ni nth e fieldwa s found in197 1 inth e Shönai district ofth eYamagat aPrefecture , animportan trice-grow ­ ing area inJapan ,wher e the antibiotic hadbee nuse d intensively andex ­ clusively for several years (Miurae t al., 1975). As someresistan tmu ­ tants obtained invitr o appearedvirulen t and ablet o sporulate,i ti s plausible thatth e resistant strains found inth e field also developb y mutation and that survival inth e field ispossible ,a tleas t for ali ­ mited period oftime .

Monitoring and detectiono fresistan t strains

As kasugamycin doesno t inhibitgerminatio n ofconidi a of P. oryzae, assessment ofresistanc e was carried outb y observing inhibitionb y the antibiotic ofmyceliu m growth onaga r andcalculatin g theminimu m growth- inhibiting concentration (MIC).Althoug h theseMI Cvalue s for sensitive strains appeared tovar ywidel y (Uesugi et al., 1969), resistance inth e field exceeded thisbroa d distribution ofsensitivit y (Sakurai et al., 1977; Sakurai &Naito , 1976). Agoo dmetho d tomeasur e resistance isth e agar-diffusiontechnique , inwhic h inhibition zones are formedb ydiffu ­ siono fa n antibiotic solution ina naga rmediu m uniformly seededwit h conidia ofth e strain tob e tested.Howeve r thismetho d isles sconve ­ nienttha nmeasuremen to fradia lmyceliu m growth (Katagiri& Uesugi , 1974).

Genetics

Theperfec t stage ofth e rice-blastpathoge n (Magnaporthe grisea) has recentlybee n found inculture s isolated fromric ean dvariou sothe rgra ­ minaceousplants .Strain swer e isolated from fingermille ttha tcoul db e crossedwit hothe r strains from thiscro pplan tan dwit h strains from riceplants ,s otha tgeneti c analysiswa spossible .Resistanc e tokasu ­ gamycin in field isolateswa s foundt ob e controlledb y amajo r gene (Tagae t al., 1978). Inresistan tmutant s originally isolated from finger millet, so farthre e loci forkasugamyci n resistance havebee ndetected . Ofthese ,on e appears tob e responsible also forresistanc e toblastici - din-S (Tagae t al., 1979).

Mechanism of resistance

Themod e of actiono fkasugamyci n isreporte d to involveprotei nbio ­ synthesis,b y affecting theribosome s in fungal cells.Th eresistanc eme -

210 chanism ina mutan tselecte d invitr o forresistanc e tokasugamyci n was investigated using reconstituted reaction systems ofribosome s andtRN A extracted from theresistan tmutan t and the sensitiveparen t strain.In ­ corporation oflabelle d aminoacyltRN A intoth e ribosome fractionwa s always inhibitedb y the antibiotic whenribosome s fromth e sensitive strainwer eused ,bu tno twit h ribosomes from the resistantmutant ,re ­ gardless ofth e source ofth e tRNA.Thi s indicates thatresistanc e isdu e todecrease d affinity ofth e ribosomes toth e antibiotic (Misato& Ko , 1975).

Population dynamics ofresistan t strains inth e field

Whenth eresistan t strainswer e found inth e Shönaidistric t in1971 , mosto fth e isolates from the area appeared tob e resistant.A striking decrease inth epopulatio n ofresistan t strainswa s observed afterappli ­ cation ofth e antibiotic was stopped in1972 ;th epreportio n of resistant strains inth e isolates in197 5decrease d to2-2 0 % (Miura& Takahashi , 1976). Resistant isolateswer e comparedwit h sensitive oneso nmycelia l growth, sporulation andvirulence ,bu tn o significantdifference swer e found (Miura et al., 1976). Whenric eplant swer e inoculated witha 1:1 mixture ofconidi a of sensitive andresistan t isolates,th enumbe r ofle ­ sions formedb yth e sensitive strainwas ,i nmos tcases ,fa rgreate r than forth eresistan t strain,suggestin g asuperio r competitive ability of the former overth e latter.Thi s seemed tob edu et oth ehighe r rateo f infection ofth e sensitive strain (Ito& Yamaguchi , 1979).

Resistance to organophosphorus fungicides and isoprothiolane

Emergence ofresistan t strains

Mutants of P. oryzae resistantt oorganophosphoru s fungicides,suc ha s IBP and edifenphos,ar e alsoreadil y obtainedb y selection invitr o from a largenumbe r ofconidi a (Uesugie t al., 1969). However the frequency of emergence ofresistan tmutant s seems alittl e lesstha n for kasugamycin (Katagiri& Uesugi , 1978). Note thatthes emutant s resistantt oorgano ­ phosphorus fungicides arecross-resistan t toanothe rne w fungicide, iso­ prothiolane,whic hha s nophosphoru s atom init smolecule .Mutant sselec ­ ted inth epresenc e ofisoprothiolan e areals ocross-resistan t toorgano ­ phosphorus fungicides,an d the frequency ofthei remergenc ewa s nearly thesam ea s fororganosphosphoru s fungicides (Katagiri& Uesugi ,1977 , 1978). Contrary towha ton ema y expect,som ephosphoramidat e compounds arespecificall y fungicidal to theseresistan tmutants ,bu tno tt onorma l wild-type strains (Uesugi et al, 1974). Organophosphorus fungicides havebee n inpractica l use since1965 ,

211 but fieldemergenc e ofresistan t strains of P. oryzae wasno treporte d until 1976.On eunreporte d and exceptional casewa s observed in1973 ,i n anexperimenta l greenhousewher e IBPha dbee n intensively used asa standard forevaluatin g fungicides.Th epreventiv e effecto f IBP against blastdiseas e onric e seedlingswa s remarkably diminished,bu t aphos - phoramidatewa s quite effective,suggestin g the occurrence ofresistan t strains similar to laboratory-derived resistantmutants . In1976 ,eigh t isolates resistant to IBP at0. 1 mM -a concentration towhic hnorma lwild-typ e strainsuse d tob e sensitive -wer e found among 173 isolates fromToyam a Prefecture,wher e large quantities of IBPgran ­ ules hadbee napplie d to thepadd y soil.Monitorin gwa s continued in 1977: the sensitivity ofisolate swa s comparedwit h thato f stock strains isolatedbefor e 1964,whe nn oorganophosphoru s fungicides hadbee nap ­ plied inpractica l agriculture.Th e results shown inTabl e 1indicat ede ­ velopment ofresistance ,thoug h the levelwa s ratherlow . Among the resistant field isolates,tw o levels ofresistanc ewer e found.On e isolate exhibited aphosphoramidat e sensitivity equal totha t ofresistan tmutant s obtained invitro .Mos to fth eresistan t field iso­ lates,however , showed amoderat e level ofresistance , andwer e different from resistantmutant s obtained inth e laboratory; the formerdi dno t shownegatively-correlate d cross-resistance tophosphoramidat e (Katagiri & Uesugi, 1980).

Monitoring and detectiono fresistan t strains

Themeasurement s ofMI C inTabl e 1sho w an increase inth e level ofre ­ sistance. Itwa s difficult,however ,t odistinguis h resistant strains fromoriginall y sensitive ones. Infact ,th e2 8 isolates identified in 1977,whic hha dMI Cvalue s of 0.1 mM, seemed toconsis to fresistan t and sensitivestrains . Somephosphoramidate s arespecificall y toxic to in-vitro-obtainedmu ­ tants resistant to IBP,edifenpho s and isoprothiolane,bu tno tt onorma l

Table 1.Developmen to fresistanc et oIB Po ffiel d isolateso fPyriculari a oryzae.

MIC (mM) before 1964 1974 1976 1977 ä 0.2 0 0 8 4 S 0.05 27 }165 f165 46 a.Sourc eo fisolates :stoc kstrain sisolate dbefor e 1964,whe norgano ­ phosphorusfungicide swer eno tuse di npractice ;i n197 4isolate sfro m Ibarakian dYamagat aPrefectures ;an di n197 6an d 1977isolate sfro m ToyamaPrefecture .

212 wild-type strains.Combinatio n ofth ephosphoramidate swit h IBP,edifen - phos or isoprothiolane gives,moreover , asynergisti c fungicidal effect onnorma lwild-typ e strains (Uesugi et al., 1974;Katagir i &Uesugi , 1977). The synergism, evaluated by the crossed-paper technique, isno t pronounced inlaborator y and field isolates resistantt o IBP.Th edif ­ ference inexten to f synergism makes itpossibl e todistinguis h resistant isolates from sensitive ones. Inhibition ofradia l growth ofmyceliu m on agarals omake s itpossibl e torecogniz e resistant isolates (Katagiri & Uesugi, 1980).

Genetics

Conidiao f amutan tobtaine db y selection invitr o inth epresenc e of anorganophosphoru s thiolatewer e selected againo n amediu m containing a phosphoramidate.A secondmutan twa s thus obtained. Inthi s testth e firstmutan twa s resistant to IBPan d sensitive tophosphoramidate ;th e secondmutan twa s resistant tophosphoramidat e and sensitive to IBP just liketh eorigina lwild-typ estrain .Therefor e thesecon dmutatio n seems tob e aback-mutation , and resistance to IBPwa s accompanied bysensiti ­ vity tophosphoramidat e (Uesugi &Katagiri , 1977). Genetic analyses ofresistanc e in laboratory-derived mutants of iso­ lates from fingermille tar ebein g conducted; amajo r gene for resistance to IBPan disoprothiolan e has alreadybee n identified (Tagae t al., 1980). Resistance found inth e field isdifferen t from thatobtaine d inth e laboratory,bu tgeneti c analyses ofresistan t field isolateshav eno t been reported.

Mechanism of resistance

A fieldisolat emoderatel y resistantt oorganophosphoru s fungicides detoxified IBPb ymetaboli c cleavage ofth e S-C linkage inth e IBPmole ­ cule (Uesugi et al., 1978). This detoxificationma y account forth ere ­ sistance.However , forth eresistan tmutant s obtained invitr o therat e ofdetoxificatio nwa s even lower than insensitiv ewild-typ e strains,s o thatresistanc ema yno tb e duet oincrease d detoxificationbu tt oothe r factors, such assit emodificatio n ordecrease d permeability ofth efun ­ gal cellmembran e toth e fungicide.Sinc e IBP and isoprothiolane arein ­ hibitors ofth eoxidativ emetabolis m ofphosphoramidate ,ther e isa pos ­ sibility thatth etarge t siteo fthes e fungicides isth e oxidativeen ­ zymes:modificatio n ofth eenzymes ,a ssuggeste db y decreasedphosphor ­ amidatemetabolism ,migh tb e the actual resistancemechanis m (Uesugi & Sisler, 1978). But,inhibitio no fphospholi d biosynthesis was recently proposed asth epossibl emod e ofactio no f IBP (Kodamae t al., 1979); thusbot hmod e ofactio n and resistancemechanis m haveye tt ob eex -

213 plained. Field isolatesmoderatel y resistantt oorganophosphoru sthiolat efun ­ gicides andnorma lwild-typ e strains are resistant tophosphoramidate , but laboratory-derived mutants resistant toorganophosphoru s thiolate fungicide are sensitive tophosphoramidate . Therat eo f fungal metabolism of aphosphoramidat e through hydroxylation orN demethylationwa s high in field isolateswit hmoderat e resistance to IBP and innorma l wild-type strains,bu t low inlaboratory-derive d mutants resistant to IBP.Funga l metoblism ofphosphoramidat e thuscoincide s with sensitivity tophosphor ­ amidate inal l the strains tested.

Population dynamicso fresistan t strains inth e field

Sinceemergenc e ofresistanc e inth e fieldha sbee nobserve d onlyre ­ cently, little isknow n aboutth epopulatio n dynamics ofresistan t strains.Also , itha sno tye tbee nexplained ,wh y resistant strains in the fieldonl yemerge d afterth e fungicides hadbee nuse d formor e than tenyears ,an dwh ymos to fth eresistan t strains emerging inviv ower e different from thoseobtaine d by selection invitro . Although the levelo fresistanc e inth e field isolates ismostl y only half thato fth e isolates obtained inth e laboratory, still iti s thought that itdiminishe s theeffec to fth e fungicides,especiall y whenthe y are applied asgranule s to submergedpadd y fields;the nth e fungicide is translocated within theplant . Itsconcentratio n inth eplan tremain s at amoderat e level fora lon g time,s otha tpresumabl y sensitivestrains , butno tmoderatel y resistant ones, arecontrolled . Thisma y stimulate fur­ ther selection of strainswit hmoderat eresistance .

Resistance to other fungicides

Blasticidin-S

There havebee nman y reports ofemergenc e ofmutant s of P. oruzae re­ sistantt oblasticidin- Sunde r laboratory conditions (Suzuki,1962 ;Naka - mura& Sakurai,1968 ;Uesug ie t al., 1969,Hwan g &Chung , 1977). Although the application ofthi s antibiotic israthe r limited inJapan ,an dthere ­ forether e islittl e chance ofresistanc e occurring inpractice ,cross - resistance in field isolates resistantt o kasugamycinha sbee n reported (Sakurai &Naito , 1976). However, there areals oothe rreport s ofn o cross-resistance being found (Miurae t al., 1975;It o& Yamaguchi , 1976). Maybe there are twoo rmor e types ofkasugamycin-resistan t strainswit h various cross-resistance toblasticidin-S .O nth eothe rhand , since the distribution ofMI Cvalue s ofth e antibiotic israthe rbroa d amongsensi ­ tive strains (Uesugi et al., 1969;Hwan g &Chung , 1977), itmigh tb edif -

214 ficultt odetec tresistanc e toblasticidin-S .Th emechanis m of resistance ina mutan tha sbee n investigated. Itwa s attributed todecrease dpene ­ trationb y the antibiotic into the fungal cell (Huange t al., 1964).

Benomyl

Although this fungicide isno tuse d forcontro l ofric eblas ti nJa ­ pan, iti s fairly fungicidal to P. oryzae. Inexperiment swit h alarg e number ofconidia ,th e frequency ofemergenc e ofresistan tmutant swa s _7 about 10 .Thi s is lower thanth e frequency ofresistanc e to kasugamycm and organophosphorus fungicides (Katagiri& Uesugi , 1978). The rather low frequency ofbenomy l resistance isinterestin g invie w of itsman yresis ­ tanceproblem swit hothe rplan tpathogens .

Disease-control agentshavin g littleo rn o fungicidal activity

Tetrachlorophtalide andprobenazol e have little orn o fungicidalac ­ tiono n P. oryzae, yetthe ypreven tblas tdiseas ewhe n applied toric e plants.Th equestio n remainswhethe r development ofresistanc e tothes e agents isprobabl e ornot . Ina nexperimen t of2 0 successive inoculations of fungicide-treated riceplant swit h P. oryzae, no development ofresis ­ tancewa s observedwit h tetrachlorophthalide, althoughresistanc e tocom ­ poundswit hdirec t fungicidal activityha sbee nobserve d soofte n (Aoki & Yamada, 1979).

General discussion

Under laboratory conditions P. oryzae candevelo p resistance to anti­ biotics, organophosphorus fungicides and isoprothiolane.Wit hkasugamy - cin, resistance was duet o spontaneous mutation. Selectiono nmedi acon ­ taining IBP,edifenpho s or isoprothiolane resulted inresistan t strains atnearl y the same frequency,wit h cross-resistance amongth ethre efun ­ gicides.Thi s suggests spontaneousmutatio n forresistanc e (Katagiri & Uesugi, 1978). These resistantmutants ,o r atleas tsom e ofthem , arevir ­ ulent toth ehos tplan t and seemquit e similart oth ewild-typ e parent strain inman yrespects ,excep t for fungicide resistance.Henc e they are assumed tob e able tooccu r inth e field and survive undernatura lcondi ­ tions. Nevertheless,th e following isals oconceivable .Sinc eth e resistant mutants emergeb y spontaneousmutation ,the yemerge d andexiste d alon g timebefor e the fungicideswer e developed,bu tthei rpopulation swer e lim­ ited asther ewa sno tselectio npressur e ofth e fungicide.Thu s there ­ sistantmutant smus tb e inferior andunabl e to increase their population ina natura l environment freeo fth efungicide .

215 Inlaborator y studies,kasugamycin-resistan t field isolateswer e not found tob e significantly inferior to sensitive isolates. Inth e field, however,th e fitness ofresistan t strainsmus tb e somewhatlowe rtha ntha t ofth e sensitive strains,sinc eth epopulatio n ofth e former declined after application ofth e antibiotic was stopped.A t least twotype s of IBP-resistantfiel d isolates havebee n found.On e issimila r tomutant s obtained inth e laboratory; the other isclearl y different.Th e former is rarely found inth e field,bu tth e latteroccur smor e frequently. Itseem s thatmos to fth e (moderately)resistan t strains occurring inth e field aremutant s thatemerg e far less frequentlybu tar e fitter.Mutant s that occurver y readily under laboratory conditions,however , appear tob e less fit forsurviva l under fieldconditions . The resistance mechanism inth e field isolates ispresumabl y anin ­ creased detoxification, asdistinc t from themechanis m inlaborator ymu ­ tants, forwhic h activity ofth eenzyme s involved indetoxificatio nwa s even lower. Inthi scas e changes inth ephysiolog y ofth e fungusma y cause its lower fitness inth eenvironment .Not e thatincrease ddetoxi ­ fication isth emos tcommo n resistance mechanism tomedica l drugs of clinical isolates ofbacteria ,bu tthi s isseldo m so inbacteria l mutants obtained on artificial medium inth e laboratory (Benveniste& Davis ,1973 ; Davis& Smith, 1978). Selectionpressur e under field conditions is another important factor determining the occurrence and survival of resistant strains.Tim e and frequency ofapplication , dose andpersistenc e ofth e fungicidema y all be important. Ifth e level ofresistanc e occurring inth e field islow , as is fororganophosphoru s fungicides,th e concentration of fungicide on or inth eplan tma yb e critical.A moderat e concentrationma ydiscrimi ­ natebetwee nresistan t and sensitive forms,i n favouro fth e former. Recently anove l type of fungicide thatha sn odirec t fungicidalac ­ tiono n P. oryzae, yet iseffectiv e againstth e disease,ha sbee nin ­ troduced.Th emod e of actiono fthi s typeha sno tye tbee n elucidated, but inprincipl e oneo fth e followingma yb epossible : - Itincrease s theresistanc e ofth ehos tplant . - Itdoe sno t inhibitgrowt h ofth epathogen ,bu trathe r changes itsme ­ tabolism, resulting ina decreas e ofvirulence . - Itincrease sbot hth eresistanc e ofth eplan t and decreases theviru ­ lenceo fth efungus . Strains ofpathogen s thatar e insensitive to thistyp e ofcompoun d have notye tbee nreported . Manyne w andver yeffectiv e fungicides tocomba tric eblas thav ebee n introduced duringth e lasttw odecades .Bu tther ehav e alsobee npro ­ blems, ofwhic h development ofresistanc e to some ofthes ene w fungicides isth emos t important andurgen tone .Variation ,b yusin g fungicides with differentmechanis m of action,ha shelpe d greatly tocop ewit hthes epro -

216 blems.A study ofth emod e of action and themechanis m of resistance will deepen our insightgreatly ,whic h isnecessar y ifw e aret omaintai n ade­ quatecontro lo fric eblast .

References

Aoki,K .& M .Yamada ,1979 .Rabcide ,a ne wfungicid e forcontrollin g riceblast . JapanPesticid e Information36 :32-35 . Benveniste,R .& J .Davies ,1973 .Mechanism so fantibioti c resistance inbacteria . AnnualRevie wo fBiochemistr y42 :471-506 . Davies,J .& D.I .Smith ,1978 .Plasmid-determine d resistance toantimicrobia lagents . AnnualRevie wo fMicrobiolog y 32:469-518 . Huang,K.T. ,T .Misat o& H .Asuyama ,1964 .Selectiv etoxicit yo fblasticidi nS t o Piricularia oryzaean dPelliculari a sasakii.Journa lo fAntibiotic sA 17:71-74 . Hwang,B.K .& H.S .Chung ,1977 .Acquire d tolerance toblasticidi nS i nPyriculari a oryzae.Phytopatholog y67 :421-424 . Ito,I & T .Yamaguchi ,1976 .Usag eo fkasugamyci nan doccurrenc eo fresistanc ei n blastfungus .Annal so fth ePhytopathologica lSociet yo fJapa n42 :37 2 (Abstr.). Ito,I .& T .Yamaguchi ,1979 .Competitio nbetwee nsensitiv ean d resistantstrain s ofPyriculari a oryzaeCav .agains tkasugamycin .Annal so fth ePhytopathologica l Societyo fJapa n45 :40-46 . Katagiri,M .& Y .Uesugi ,1974 .Evaluatio no fsusceptibilit yo fric eblas tfungu s tokasugamycin .Annal so fth ePhytopathologica l Societyo fJapa n40 :106-107 . Katagiri,M .& Y .Uesigi ,1977 .Similaritie sbetwee nth efungicida lactio no fiso - prothiolanean dorganophosphoru s thiolatefungicides .Phytopatholog y67 :1415-1417 . Katagiri,M .& Y .Uesugi ,1978 .I nvitr oselectio no fmutant so fPyriculari aoryza e resistantt ofungicides .Annal so fth ePhytopathologica l Societyo fJapa n44 : 218-219. Katagiri,M ., Y .Uesug i& Y .Umehara ,1980 .Developmen to fresistanc et oorganophos ­ phorusfungicide s inPyriculari a oryzaei nth efield .Journa lo fPesticid eScienc e 5:417-421 . Kodama,0. ,H .Yamad a& T .Akatsuka ,1979 .Kitazi nP ,inhibito ro fphosphatidylcholin e biosynthesis inPyriculari a oryzae.Agricultura l andBiologica lChemistr y43 : 1719-1725. Misato,T .& K .Ko ,1975 .Th edevelopmen to fresistanc e toagricultura lantibio ­ tics.In :Coulsto nan dKort e (Eds):Environmenta lQualit yan dSafety ,supplemen t 3,p .437-440 . Miura,H .& S .Takahashi ,1976 .Transitio no feffectivenes so fkasugamyci nan dpopu ­ lationshif to fresistan tstrain so fblas t fungus.Annal so fth ePhytopathologica l Societyo fJapa n42 :37 2(Abstr.) . Miura,H. ,H .It o& S .Takahashi ,1975 .Occurrenc eo fresistan tstrain so fPyricu ­ lariaoryza et okasugamyci na sa caus eo fth ediminishe d fungicidalactivit y toric eblast .Annal so fth ePhytopathologica l Societyo fJapa n41 :415-417 . Miura,H. ,M .Katagiri ,T .Yamaguchi ,Y .Uesug i& H .Ito ,1976 .Mod eo foccurrenc e ofkasugamyci n inresistan t riceblas tfungus .Annal so fth ePhytopathologica l Societyo fJapa n42 :117-123 . Nakamura,H .& H .Sakurai ,1968 .Toleranc eo fPiriculari a oryzaeCavar a toblasti ­ cidinS .Bulleti no fth eAgricultura lChemical s InspectionStatio n8 121-25 . Ohmori,K . 1967.Studie so ncharacter so fPyriculari a oryzaemad e resistantt okasu ­ gamycin.Journa lo fAntibiotic sA 20 :109-114 . Sakurai,H .& H .Naito ,1976 .A cross-resistanc e ofPyriculari a oryzaeCavar a tokasu ­ gamycinan dblasticidi nS .Journa lo fAntibiotic s29 :1341-1342 . Sakurai,H. ,M .Iwata ,N .Goh ,T .Yaoita ,K .Aoyag i& H .Naito ,1977 .Sensitivit y distributiono fphysiologi c raceso fPyriculari a oryzaeCavar a tokasugamycin . Journalo fAntibiotic s30 :607-609 . Suzuki,H. ,1962 .Fungicid eresistanc ei nblas tfungus .Annal so fth ePhytopathologi ­ calSociet yo fJapa n27 :8 5 (Abstr.). Taga,M. ,H .Nakagawa ,M .Tsud a& A .Ueyama ,1978 .Ascospor eanalysi so fkasugamyci n resistance inth eperfec t stageo fPyriculari a oryzae.Phytopatholog y 68:815-817 . Taga,M. ,H .Nakagawa ,M .Tsud a& A .Ueyama ,1979 .Identificatio no fthre edifferen t locicontrollin gkasugamyci n resistance inPyriculari a oryzae.Phytopatholog y69 : 463-466. Taga,M. ,T .Wake ,M .Tsud a& A .Ueyama ,1980 .Genetic so fIB Presistanc e inPyri -

217 culariaoryzae .Abstract so fAnnua lMeetin go fPhytopathologica l Societyo fJapa n 3-39 (Abstr.). Uesugi,Y .& H.D .Sisler ,1978 .Metabolis mo fa phosphoramidat eb yPyriculari aory ­ zaei nrelatio nt otoleranc ean dsynergis mb ya phosphorothiolat e andisoprothiolane . PesticideBiochemistr y andPhysiolog y9 :247-254 . Uesugi,Y. ,M .Katagir i& K .Fukunaga ,1969 .Resistanc e inPyriculari a oryzaet oanti ­ bioticsan dorganophosphoru s fungicides.Bulleti no fth eNationa l Instituteo f AgriculturalScience sC 23 :93-112 . Uesugi,Y. ,M .Katagir i& 0 .Noda ,1974 .Negativel y correlated cross-resistance andsynergis mbetwee nphosphoramidate s andphosphorothiolate s inthei rfungi ­ cidalaction so nric eblas t fungi.Agricultura lBiologica lChemistr y38 :907-912 . Uesugi,Y. ,0 .Kodam a& T .Akatsuka ,1978 .Inhibitio no ffunga lmetabolis mo fsom e organophosphorus compoundsb ypiperony lbutoxide .Agricultura l andBiologica l Chemistry42 :2181-2183 .

218 Case study 4: Powdery mildews of barley and cucumber

K.J. Brent LongAshto nResearc h Station,Lon gAshton ,Bristol ,U.K .

Abstract

Theoccurrenc e of acquired resistance to fungicides inpowder y mil­ dews, andth emethod s ofinvestigatin g suchphenomen a infiel dpopula ­ tions arediscusse d briefly.Response s toth e2-amino-pyrimidines , dime- thirimol and ethirimol, areconsidere d ingreate r detail.A comparison ofsurvey s ofsensitivit y ofcucumbe rpowder ymildew s todimethirimo li n glasshouses inth eNetherland s ando fbarle ypowder ymildew s to ethirimol inth eU.K . indicateswidel y divergentbehaviou rpatterns ,whic hprobabl y reflectdifferen tmethod s ofus e and agricultural situations.A wide ­ spread and rapid losso feffectivenes s ofdimethirimo l against cucumber mildewwa s experienced in1970 .Thi swa s associatedwit hth e incidence ofresistan tpopulation s ofth e fungi.Th e almostuniversa l adoptiono f dimethirimol inDutc h glasshouses and itsus e athighl y effective doses over longperiod s ofth eyea rprobabl y createdver y favourable conditions forresistance . Inth ecas eo fethirimol ,whic hha sbee n studiedmor eex ­ tensively, fieldpopulation s ofbarle ymilde wvarie d considerably in sensitivity.Diseas e controlb y ethirimol treatmentwa s associatedwit h slightly reduced sensitivity inth e survivingpopulations .However ,th e mostresistan t formsdi dno tsprea d orbecom epredominan t over afive - yearperio d ofsurvey s (1973-1977).Th econtinue d effectiveness ofethi ­ rimol duringthi sperio dprobabl y depended onlimite d use (oneapplica ­ tionpe ryear )an d the re-invasion oftreate d areasb ymor e sensitive formswhe n selectionpressure s were lowo r absent.A n interesting shift inth e spectrum ofrespons ewa sdiscerned : overth eperio d ofmonitoring , bothth emos t sensitive andth emos t resistantpopulation s decreased markedly innumber , and isolates ofintermediat e sensitivity became pre­ dominant.

Keywords: fungicide,resistance ,powder ymildew ,barley , cucumber,ethi ­ rimol,dimethirimol .

219 Introduction

Thepowder ymildew s form awell-define d group ofplan tpathogen s that cause serious damage inman y types ofcrop s throughout theworld . They arecontrolle d mainlyb y chemical treatment,an d awid e rangeo fchemi ­ cals is available forthi spurpos e (Bent, 1978). Some ofthes e fungicides (e.g. thedinitrophenol s and 2-aminopyrimidines) actonl y againstpowder y mildews.Other s (e.g.th ebenzimidazoles ,triazole s andpyrimidin ecarbi - nols)affec ta greate r range of fungal pathogens,bu t areknow nt ohav e specific mechanisms ofbiochemica l actionwithi n thecell s of susceptible fungi.Give nth emarke d selectivity ofactio n againston e group of fungi or atparticula r biochemical sites,an d giventh ewell-know n ability of powderymildew s to adaptt o 'resistant'varietie s ofcro p plants,i ti s notsurprisin g that fungicide resistance should have arisen. Indeed,th e firstreporte d caseo f acquired resistance toth ebenzimidazol e fungi­ cides -th e forerunner toman y other instances -wa s the occurrence in fieldplot s inNe wYor k State of isolates ofcucumbe rpowder y mildew (Sphaerotheca fuliginea) thatwer e resistant tobenomy l (Schroeder & Prowidenti, 1969). Subsequently, resistance tobenzimidazol e fungicides incucurbi tpowder ymildews ,couple dwit h obvious failure indiseas econ ­ trol,ha sbee n found inman y countries,bot h inglasshous e andfiel d crops (Netzer& Dishon , 1970;Kooistr a et al., 1972;Burth , 1973;Peter ­ son, 1973;Iida , 1975). Other documented instances of fungicide resistance inpowder y mildews concern therespons e ofcucumbe rpowder ymilde w todimethirimo l (Bente t al., 1971)an d triforine (Gilpatrick &Prowidenti , 1973), andbarle y powdery mildew toethirimo l (Wolfe& Dinoor , 1973;Shephard.e tal. ,1975 ; Hollomon, 1975a;Smit he t al., 1977;Walmesley-Woodwar d et al., 1979a,b). Resistance todimethirimo l and ethirimol,whic hha sbee nexamine d inex ­ tensive surveys,wil l receiveparticula r attention inthi s contribution. There arever y fewreport s of fungicide resistance amongstpowder y mil­ dews othertha nthos eo fcucurbit s andbarley , andnone ,t om yknowledge , of fungicide resistance among thepowder ymildew s ofgrapevin e (Uncinula necator), apple (Podosphaera leucotricha) orros e (Sphaerotheca pannosa). Yet itseem sver y unlikely thatthes e important andwidesprea d pathogens differ from the cucurbitan dbarle ymildew s inthei r response tofungi ­ cideuse .Th erepeate d observations ofvariatio n insensitivit y tofun ­ gicides incucurbi t andbarle ypowder ymildew sprobabl y result from the very obvious and discrete lesions causedb y thecucurbi tmildew ,whic h showu p strikingly any losso fdiseas e control,an d the extentt owhic h barleypowder ymilde w inEurop e hasbee nth e subjecto f specialinves ­ tigations designed todisclos edifference s insensitivity .Furthermore , the cucurbit andbarle ypowder ymildew s are forvariou s technical reasons easier to study inlaborator y orglasshous e experiments on leafpiece s

220 than othermajo rpowder ymildews .

Methods of assessment of fungicide resistance in powdery mildews

Most studies on fungicide resistance havebee nbase d onth edegre e of inhibitiono fgrowt h of isolates ofth e fungus on agar or inliqui dmedi a containing knownconcentration s of fungicides.However ,powder y mildews areobligat eparasites ,an dhenc eothe r techniques,whic h aremor ediffi ­ cult and lessprecise ,hav e tob eused . Ifth econdition s andprocedure s arecarefull y controlled, the conidia ofpowder y mildewswil l germinate toproduc eger m tubes and appressoriawhe nplace d on artificial substra­ tes such asglas s slides orcellulose-acetat e membranes,o rwhe n floated onwater .Assessmen to fth egerminatio no fcucumbe rpowdery-milde w spores oncellulose-acetat e membranes impregnated with dimethirimol (Bente t al., 1971)reveale drelationship sbetwee nth eresponse s ofdifferen t isolates thatwer ebroadl y similar to those revealedb y othermethods .Hollomo n (1975b)observe d germination and appressoria formationwit h sporeso f isolates ofbarle ypowder ymilde w onth e surface ofethirimol-treate dbar ­ ley leaves.Howeve r thesemicroscopi c techniques aretediou s tous ewit h a largenumbe r of samples,an d they require stringentprecaution s tomak e them accurate and reproducible.Also ,the y indicate theeffec to fth e fungicide atonl y one ortw o stages ofth e development ofth epathogen , whereas inpractic emos tpowdery-milde w fungicides exertthei r actiona t variousphase so f infection, forexampl e onhaustoria l formation,o nhy - phalgrowt h and on sporulation, and some (e.g.benomyl )d ono taffec tth e initial formationo fger m tubeso rappressori a (Schlüter& Weltzien , 1971). The amounto fvisibl e growtho fpowder ymilde w ondetache d leaveso r on leafpiece s inoculated with spores andtreate dwit h fungicide atdif ­ fering concentrations isth e commonbasi s ofth e assessmentmethod s of severalworkers .Schroede r &Provvident i (1969)use d detached cucumber leaves, someo fwhic hwer e taken fromplant s grown insoi l drenched with standard amounts ofbenomy l solution.Ben te t al. (1971)use d asimila r method, inwhic h cucumber-leaf discswer e floated ondimethirimo l solu­ tions atsi xconcentration s (0.052-5.0 jjg/ml)i npetr i dishes,inocu ­ latedwit hconidi a and incubated for7-1 0 days.Visibl emilde w oneac h discwa s assessed on asimpl e 0-4 scale according toth eproportio n of its areacovere db ymildew . Floating leafpiece s canals ob euse d toexamin e isolates ofbarle y powderymildew , althoughgrowt ho fthi smilde w is less even andharde r to assess thantha to fcucumbe rmildew ,ther e ismor evariatio n inin ­ fection frompiec e topiece ,an d curling and senescence ofth e leaf pieces cancaus e difficulties.A mor e convenient technique,adopte db y Wolfe& Dinoo r (1973), anduse d inmodifie d formb y Shephard et al. (1975), ist otrea tbarle y seedswit hdifferen t amounts ofchemica l

221 (ethirimol),gro w the seedlings under controlled environmentalcondi ­ tions, cutreplicat e leafpiece s from the seedlings,embe d the leaf pieces aton een d intap-wate r agar in apetr i dish,an d applymilde w spores. The amounto fvisibl emilde w oneac h leaf-piece isrecorde d after sixday s incubation at1 9 °C.I nth e study of Shephard et al. (1975)test s 14 with C-labelledethirimo l indicated somevariatio n inth e ethirimolcon ­ tento f replicate leafpieces ,proxima lpiece s tending tocontai n less fungicide thandista lpieces .Nevertheles s anadequat e degree ofstatist ­ ical significance between the differing results canb e obtained,particu ­ larly ifth etest s arerepeate d twoo r threetimes .Precis e standardisa­ tiono f inoculumwoul d further increase the accuracy ofth etests ,bu t this isver y difficultt o achievewit hpowder ymildews .Som edegre e of uniformity canb e achievedb y subculturing field samples on freshlea f tissueunde r controlled conditions,blowin g offol d spores and allowinga freshcro p todevelop , andthe napplyin g the spores toth e testmateria l byblowin g themwit ha standardize d airblastint o asettlin gtowe r fora givenperiod . Another approachuse db y severalworker s ist otrea twhol eplant s with fungicides atdifferen tconcentrations , and to apply conidia ofa parti ­ cular isolate toreplicate s oftreate dplants .Suc hprocedure s canprovi ­ deusefu l additional tests tocomplemen t leaf-piecemethods ,o rthe yca n beuse d asprimar y testso npathogen s forwhic h leaf-piecemethod s are notpracticabl e (e.g.appl epowder y mildew), although theyten d tob e less convenient and lessprecis e than leaf-piecemethods . Ifpre-tes t culture onplant s isrequired , tobuil du p inocula forth etests ,the n special precautions, such asth eus e ofscreenin g or individual air-flows,mus t be takent ocontai nth e individual isolates onthei r 'correct'hos tplant s andt o avoid cross-contamination. All thetechnique s described canb euse d successfully torevea lrela ­ tivedifference s insensitivit y betweenpowdery-milde w isolates.Exten ­ sivecomparison s ofth emethod shav eno tbee nmade ,bu tth e available evi­ dence indicates thatdifference s insensitivit y amongparticula r isolates shownb y thedifferen t types ofprocedure s arebroadl y similar.However , iti sver y important to avoid labelling thebehaviou r of anisolat e in absolute terms,fo rexampl e designating anisolat e as 'resistant'o r 'sensitive'o reve na s 'resistantt o adosag e of x mg/1 fungicide',with ­ outdefinin g the termsuse d carefully.On eworker' s 'resistant' formma y be anotherworker' s 'sensitive'o r 'intermediate' form, according toth e base-line adopted.Als o theconcentratio n of fungicideuse dma ybea ra greatly differentrelationshi p to fungitoxicity according toho w orwher e the fungicide is applied. Forexample , asee d treatmentwit h ethirimol at 8 000pg/ g seed givesmea nconcentration s of1.9-8. 3pg/ g freshweigh ti n leafpiece s (Shephard et al., 1975), whilstth e amounts actually reaching orpenetratin g spores atth e leafsurfac e areunknow n andvirtuall y im-

222 possible todefin e interm s ofconcentrations . Yet another difficulty isdecidin g whether tocompar e the responses ofdifferen tmilde w samples ata single ,discriminating , fungicidecon ­ centration orove r arang eo fconcentrations . Inpractica l agriculture isolates are likely toencounte r awid e rangeo f concentrations, and for this reason aswel l as for increased accuracy iti sprobabl ybes tt ous e a rangeo fconcentration s fortesting .On eca nquot e asa respons e score thehighes tconcentratio n that allowed some degree ofmilde w development, the lowestconcentratio n atwhic h complete controlwa s obtained,estima ­

ted ED-,o rED qi-values ,o r summations ofth edegree s ofcontro l givena t each concentration tested. Since eachmetho d of scoringwil l givediffer ­ entnumerica l values,i ti sadvantageou s todefin e and stick to apar ­ ticular system throughout aparticula r studyo r series ofrelate d stud­ ies.Th emerit s and limitations ofth e different scoring systemswil l vary according toth e resources,condition s and objectives ofeac hinves ­ tigation,t oth eassessmen tmetho d adopted, andt oth edegre eo fvaria ­ tionbetwee n the dose-response curves ofdifferen t isolates. Itwoul db e unwise, therefore to laydow n standardprocedure s forstudyin g fungicide resistance inpowder ymildews . Greatcar emus tb e takenwhe n interpreting the results of glasshouse or laboratory tests of fungicidal activity interm s ofpredicte d field performance. Iti sa commo n occurrence that fungicides giving complete disease controlwhe n sprayed torun-of f atconcentration s of1-1 0 ug/ml inroutin e glasshouse tests on smallplant snee d tob e sprayed atcon ­ centrations of 50-500 Mg/mlt o giveeffectiv e control ofth e samedi ­ seasei nth e field. Seedtreatment s applied ata particula r dosage appear tob emor e closely related inthei rglasshous e and fieldeffects ,bu tth e possible influence of, forexample ,soi l type,weather ,tim e of infection and fitness ofth epathoge nunde r fieldcondition s onth e relationbe ­ tweenth eeffect smus tb e carefully considered.

Methods of sampling powdery mildews for resistance testing

The selection ofsuitabl eprocedure s forobtainin g representative samples of fungi from fieldpopulation s forms adifficul tbu tver y impor­ tantpar to fth eplannin g ofal linvestigation s into fungicideresistan ­ ce. Themetho d chosenwil l depend onth eobjective s ofth eresearc h and theeffor t available. Ifth e aim ist omonito r the fungicide forconti ­ nued effectiveness over largeregions ,i ti snecessar y totak e samples fromman y siteso r trialplot swher e the fungicideha sbee nused ,to ­ getherwit h samples fromuntreate d siteso rplot s thatar eclosel ysi ­ tuated and ascomparabl e aspossible . Iti simportan t thatestimate s of thedegre e ofdiseas e control,o r atleas tth e level ofdisease ,shoul d bemad e atth etim eo fsampling , andbot hsamplin g and diseaseassess -

223 ment should berepeate d several times during the season,wheneve rpossi ­ ble. The simplestmethod , and theon e adoptedb y ICIworker s insurvey s of sensitivity toethirimo l and dimethirimol, is totak e asingl erepresen ­ tative sampleo fa larg emilde wpopulatio n ineac h field or commercial glasshouse inth e surveyb ymixin g together spores taken from anumbe r of pustules atdifferen tpoint s ineac h crop area.Suc ha sampl e shouldelic ­ ita n 'average'respons e typical ofth e site concerned. Sinceth e sample willb eheterogeneous ,i ti spossibl e thatit s composition and its res­ ponsewil l alter during incubation or sub-culturingi nth e laboratory, either inth epresenc e or absence of fungicides.Henc e any incubationo r sub-culturingbetwee n sampling andtesting ,whic h issometime s necessary toobtai n sufficientviabl e inoculum, shouldb e asbrie f aspossible .Re ­ petitive testing should give some indication of anygradua l shifts in response.However , theproblem s ofpossibl e losso fresistanc e during the pre-testperio dma yno tb e too serious,becaus eth emor etransien t the resistance and the greater itsdependenc e oncontinue d fungicide selec­ tionpressure ,th e less likely iti st obuil d up and causeproblem s in the field. Inpractice ,bul k samples ofcucumbe r andbarle ymilde w tend toretai nthei rparticula r responses topyrimidin e fungicide through several sub-cultures inth e absence ofth e fungicide;onl ymino r fluctua­ tions occurbetwee ntest s (Shephard et al., 1975). Onewa yo favoidin g any shifts in sensitivity during incubation inth e laboratory, growthroo mo rglasshouse ,an dals oo fobtainin g adirec t sample ofth e field inoculum atth e timeo f sampling, ist oplac e fungi­ cide-treated testplant s (orplan tmaterial )a tpoint swithi n ornea r the crop sotha tthe y areexpose d toth enatura l spore inoculum for acertai n lengtho ftime ,sa y4-2 4 hours,an d thent otransfe r them to thecontrol ­ led conditions ofth e growth room orglasshous e for incubation andas ­ sessment (Wolfe& Minchin , 1976). Somepossibl e drawbacks ofthi smetho d forlarge r surveys include arelativel y high labourrequirement , alogis ­ ticalproble m ofprovidin g uniform testmateria l overwid e areas,an dde ­ pendence uponth eweathe r and the inoculum level inth e crop forobtain ­ ingsatisfactor y sporedeposition s andinfection . Itca nb erewardin g totak emultipl e samples ofmildew s from oneo ra few sites,t ostud y indetai l the incidence and the spatial distribution of formswit hdifferen t degrees ofsensitivity . Mildew from singlepustu ­ leso reve nsingl e sporechain sma yb e transferred tohos tmateria l in individual containers andmaintaine d asseparat e cultivars.Th e samples obtained from onesit ema yvar y considerably inthei r responses tofungi ­ cides (Wolfe& Dinoor , 1974;Shephar d et al., 1975), and also inth esta ­ bility ofrespons e during laboratory culture (Hollomon, 1975). Indeed, uniform mildewpopulation s mayb e rareo rnon-existent .

224 Resistance of cucumber mildew to dimethirimol

Dimethirimol is asystemi c fungicidewit h ahighl y specific action againstpowder ymildews . Itwa s introduced inth eNetherland s in196 8a s a soil drench tocontro l cucumberpowder ymilde w inglasshouses . In196 9 dimethirimol treatmentwa s adoptedver ywidely ,becaus e one application provided excellentprotectio n forsevera l weeks and therewer e noef ­ fectso npredator s used forbiologica l control.Howeve rb y spring197 0a number of failures hadoccurred . Leaf-disc testso nmilde w samples taken fromman y glasshouses revealed aclos e correlation between lowersensiti ­ vity todimethirimo l and lacko fdiseas e control (Table 1).Som eresis ­ tantmilde wpopulation swer e alsodetecte d inglasshouse s inth eU.K .an d WestGermany : themor e resistant isolates appeared tob e atleas ta s vigorous ingrowt h and sporulation on leafdis c and smallcucumbe r plants assensitiv eisolates . Theproduc twa spromptl ywithdraw n fromus e inth eNetherlands .Th e incidence ofresistanc e remained high thenex tyear ,1971 ;subsequen t surveys suggest agradua l decline (J.A.W.Turne r &A.M . Cole,persona l communication), although samples stillvar y greatly, and iti sdif ­ ficultt omak evali d year-to-year comparisons because ofunavoidabl eva ­ riations insamplin g sites,experimenta l conditions andresearc hperson ­ nel. Since 1977,i nrespons e torequest s fromgrower s and advisors,a limited amounto fdimethirimo l isbein g sold again inth eNetherlands . Iti srecommende d thatonl y one applicationpe ryea r -a tth emos tne ­ cessary time -shoul db emade ,t ominimiz e theris k ofreselectio n ofre ­ sistant forms.Unrelate d fungicides areuse d atothe r times.Use d inthi s way,dimethirimo l workedwel l in197 7an d 1978 inth eNetherlands ,bu t therewer e some instances of inadequate control in1979 ,an d littleo fi t wasuse d in198 0 (B.Anema ,persona l communication).Resistanc e to

Table1 .Sensitivit yt odimethirimo lo fcucumbe rpowder ymilde w fromDutc hglasshouses .

Toleranceleve l (pg/ml) Numbero fisolate s fromsite so f fromsite so f poorcontro l goodcontro l 5.0or>5.0 8 2.0 9 0.80 10 0.32 2 1 0.13 0.05o r< 0.0 5 2 5 Source:Ben te tal.(1971) . a.Highes tdimethirimo lconcentratio ntha tallow san yvisibl e growtho fmilde wo nfloatin glea fdiscs .

225 dimethirimol inpowder ymilde w ofcucurbit s hasno tbee nreporte d during the roughly 10year s ofus e inSpai n and theMiddl e East.Glasshous e and outdoor applications havebee nmade ,b y soildrenc h andb y spraying,bu t the intensity andduratio n oftreatment s havebee nmuc h less than inth e Netherlands.

Resistance of barley mildew to ethirimol

Ethirimol ischemicall y closely related todimethirimol , andprobabl y has the samemod e of action againstpowder ymildews . Itha sbee n inwide ­ spread use since 1971 inth eU.K . asa barle y seed treatment. Inannua l trials itha s shownver y good control ofpowder ymilde w duringth e cri­ ticalphas e of infection and thisha s resulted insubstantia l yieldin ­ creases. Surveys showed little overall change insensitivit y during the period 1973-1977 (Table 2). However, ineac h surveymilde w samples from treated fieldswer e generally less sensitive than samples fromnearb yun ­ treated fields.Milde wpopulation s wereheterogeneou s inthei r response toethirimo l (single-pustule isolatesobtaine dwithi n single fieldsgav e varied responses), and the less sensitive forms survived the ethirimol treatment. Therewa sn oclear-cu t correlationbetwee n theethirimo l sensitivity ofmilde w isolates and the levels ofmilde w inthei r fields oforigi na t thetim eo f sampling.Whe n alldat a from the 1975 surveywer e amalgamated therewa s alow-significance ,positiv e correlation coefficient (P = 0.10) between sensitivity and amounto f fieldmilde w (M.Woolne r &K.J .Brent , unpublished data). This confirmed indications obtainedb y Shephard et al.

a b Table2 .Sensitivit y toethirimo lo fpowder ymilde wsample s frombarle yfield streate dan dno ttreate dwit hethirimol .

Period Scotland East Anglia

untre ated treated untreated treated

1973summe r 17.1 15.4 11.9 10.5 1974sprin g 14.8 10.5 12.3 9.3 1974summe r 13.8 10.7 14.2 10.0 1975sprin g 12.6 11.3 13.0 10.6 1975summe r 12.7 10.9 12.8 11.3 1977sprin g 13.6 12.8 14.3 12.8

Source:Bent(1978) . a.Mea nscores :0 ,insensitive ;20 ,ver ysensitive . b.Milde wsample stake nfro m2 6t o8 0field spe rregio nwer e appliedt olea fsegment sgrow nfro msee dtreate da tfiv econ ­ centrationso fethirimo l (100-8000|Jg/g )unde rcontrolle d conditions (seep.221-223) .Dat at o197 4fro mShephar de tal . (1975),i nwhic hfurthe rdetail sar egiven ,late rdat afro m M.Woolner ,D.R.E .Mill s& A.M .Skidmor e (personalcommuni ­ cation).

226 (1975)i nth e 1974surve y thatreductio n insensitivit y of fieldpopula ­ tions tendst ob e associated with the survival ofth e less sensitive ele­ ments of amixe dpopulatio n during and after aperio d ofgoo d disease control, rathertha nwit h failure ofcontrol .Ther ewa s anearl y decrease inmea n sensitivity inScotlan dbetwee n 1973 and 1974;i ti sno tknow n whether asimila r earlyeffec toccurre d inEas tAnglia ,wher e widespread treatment started ayea rearlie r than inScotland .Th emea n sensitivity ofth emilde wpopulation s ofindividua l crops inEas tAngli a and Scotland was foundt o fluctuate ineithe r direction asselectio npressure s changed. Sensitivity tended tob e lowesta tth eearlie r growth stages,whe nup ­ take ofethirimo l and disease controlwer egreatest . Table 3 shows therang eo fresponse s found inEas tAngli a andScot ­ land insuccessiv e surveys from 1973t o 1977.Initiall y isolates from EastAngli a differedwidel y inthei r responses.However ,th e rangeha s sincenarrowed . Isolates ofintermediat e sensitivity havebecom epredomi ­ nant, andbot hth emor e resistant and thehighl y sensitive isolates seem tohav e disappeared. This effecti sreminiscen t ofth e actiono fstabili ­ zing selection forpathoge nvirulence ,whic hca nlea d toth e predominance of strainswit h intermediate degrees ofvirulenc e (Vande rPlank ,1968 ; Crill, 1977). Onemigh tspeculat e thatth eextrem e forms are lesscompe ­ titive inth e field;tha tth emos tsensitiv e isolateswer e eliminated byusag e ofth e fungicide;an dtha tth emos tresistan t forms,whic hma y have increased initially, declinedwhen ,i n 1973,ethirimo l was no longer recommended forus e onwinte rbarley .Wolf e &Dinoo r (1973)als o observed differences insensitivit y toethirimo l amongstbarle ymilde wisolates , and advised thatethirimo l shouldn o longerb e applied towinte rbarley ,

Table3 .Distributio no fsensitivit yt oethirimo li npowder ymilde w samplesfro mbarle yfields .

Survey Distr:ibutio n o f sensitivity scores

<6 6-9 9-12 12-15 15-17 >17

EastAngli a 1973 5 14 41 21 12 7 1974 1 16 60 13 7 2 1975 0 3 55 37 5 0 1977 0 0 19 76 4 1

Scotland 1973 0 4 9 12 54 21 1974 0 26 21 30 12 12 1975 0 6 50 38 6 0 1977 0 0 14 80 6 0

Source:Bent(1978) . a.Value sar epercentage so fsample s (fromtreate dan duntreate d fields)i neac hcategory .Fo rexplanatio no fsensitivit yscor e andsource so fdat ase eTabl e2 .

227 to avoid selectiono fresistanc e inth eperiod sbetwee n successive spring barley crops.Thi spolic ywa s followed as far aspossible ,betwee n 1973 and 1978.Th esurve y results showa simila r reduction inScotlan d since 1973 inth erang eo fsensitivities ,particularl y ofth emos t sensitive isolates;ver y resistant formshav eneve rbee n detected inScotland , where littlewinte rbarle y isgrown . Themor e resistant forms appear togro w and sporulate normally under growth-room conditions,bu t alac ko fcompetitivenes s withmor e sensitive formsha sbee n indicated in field experiments (Hollomon, 1978). Smithe t al. (1977)reporte dtha to ftw omilde w strains differing inresistanc et o ethirimol, themor e resistant strainpersiste d inth e field longer than themor e sensitive one.However , sincecomparison s with other strainswer e notincluded , itseem spossibl e that anintermediat e formwa sbein gcompar ­ edwit h aver y sensitive one.Variatio n inth e response ofbarle ymilde w isolates totridemorp hha s alsobee n detected (Walmsley-Woodward, 1979a,b), but theresistan t forms appearno tt ocaus epractica lproblems .Geneti c studies on strains ofbarle ymilde w differing inrespons e to ethirimol havebee ndifficul tt o interpret,bu t suggest acomplex ,non-Mendelia n systemo fgeneti c control (Hollomon, 1978).A n association inbarle y mil­ dew isolatesbetwee n the frequency ofethirimo l resistance andvirulenc e tocertai nbarle yvarietie s hasbee nnote db yWolf e& Dinoo r (1973), and possible relationships between fungicide resistance and the growing of different cerealvarietie s deserve further study.

Discussion

Iti sinterestin g tocontras t therapi d onseto f failures ofdimethi - rimolt ocontro l cucumber powderymilde w inDutc hglasshouse s withth e absence ofproblem s arising from itsus e inSpai n andth eMiddl eEast , andwit h the continued goodperformanc e ofth e closely-related ethirimol onU.K .barle y crops.Wolf e (1975)pointe d outtha tvariation s inenvi ­ ronmental conditions and inth epopulatio n dynamics ofpathogen s largely determinewhethe r resistant formsbecom e dominant.Experienc e now confirms thatth e following conditions favour increase ofresistance : -Whe nmutant s ofth epathoge n arisetha tno tonl y are little affectedb y the fungicide applications,bu tals o approximate ordinary forms inthei r ability to infect andmultiply . -Whe nth e fungicide exerts apoten tselectiv e actiono n fungalpopula ­ tions. -Whe ncontac tbetwee nth e fungicide andth epathoge ni sprolonged , either by repeated application orbecaus e ofpersistence ;whe nthi scontac tex ­ tends toth ewhol epopulation ,withou t the ingress ofuntreate d populations oro fthos e treated withunrelate d fungicides. Theyear-round , almostuniversa l use ofdimethirimo l inDutc hglass -

228 houses, coupled with near-perfect levels of control in the isolated glass­ house environment, may be seen with hindsight as a recipe for misfortune. The risk of control failure with single, annual applications of ethirimol in a proportion of open barley crops is clearly lower. Note here that the resistance problems of the benzimidazole fungicides also have been ob­ served commonly in glasshouses or under other intensive horticultural practices, but rarely in cereal crops. Autumn usage of ethirimol was re-introduced in the U.K. in 1979, in response to increased farmer interest in growing winter barley and maxi­ mizing its yields. A wider range of mildew fungicides is now in use in both winter and spring barley in the U.K. Also, the survey data suggest that less sensitive forms of mildew may not persist in the spring, when the activity of the autumn-applied ethirimol has declined. Nevertheless, with applications being made in the autumn- and spring-sown crops, it is important that the performance of this fungicide be kept under review.

Acknowledgement

I am grateful to colleagues from ICI for the use of some unpublished data and for helpful discussion. Some material in this contribution was published originally in Plant Disease Epidemiology (P.R. Scott and A. Bainbridge (Eds.), 1978) and is reproduced here by kind permission of Blackwell Scientific Publications.

References

Bent, K.J., 1978. Chemical control of plant diseases: some relationships to pathogen ecology. In: P.R. Scott & A. Bainbridge (Eds.): Plant Disease Epidemiology. Blackwell, Oxford, p. 177-186. Bent, K.J., A.M. Cole, J.A.W. Turner & M. Woolner, 1971. Resistance of cucumber powdery mildew to dimethirimol. Proceedings of the 6th British Insecticide and Fungicide Conference. British Crop Protection Council, London, p. 274-282. Burth, U., 1973. Resistenz von Gurkenmehltau (Sphaerotheca fuliginea (Schlecht.) Salmon) gegenüber Systemfungiziden aus der Benzimidazol-Gruppe. Archiv für Phytopa­ thologie und Pflanzenschutz 9: 411-413. Crill, P., 1977. An assessment of stabilizing selection in crop variety development. Annual Review of Phytopathology 15: 185-202. Gilpatrick, J.D. & R. Prowidenti, 1973. Resistance to fungicides by apple scab and cucurbit powdery mildew fungi in New York. International Congress of Plant Patholo­ gy, Minneapolis. International Society for Plant Pathology, 0780 (Abstr.). Hollomon, D.W., 1975a. Behaviour of a barley powdery mildew strain tolerant to ethiri­ mol. Proceedings of the 8th British Insecticide and Fungicide Conference. British Crop Protection Council, London, p. 51-58. Hollomon, D.W., 1975b. Laboratory evaluation of ethirimol activity. In: N.R. McFarlane (Ed.): The Evaluation of Biological Activity. Academic Press, London, p. 505-515. Hollomon, D.W. 1978. Competitive ability and ethirimol sensitivity in strains of bar­ ley powdery mildew. Annals of Applied Biology 90: 195-204. Iida, W., 1975. On the tolerance of plant pathogenic fungi and bacteria to fungicides in Japan. Japanese Pesticide Information 23: 13. Kooistra, T., W.H.A. Sloot & W.H. Janssen, 1972. Resistentie van komkommermeeldauw (Sphaerotheca fuliginea) tegen Benlate, Topsin M en Bavistin. Gewasbescherming 3: 121-125. Netzer, D. & I. Dishon, 1970. Effect of mode of application of benomyl on control of

229 Sclerotinia sclerotiorumo nmusk-melons .Plan tDiseas eReporte r54 :909-912 . Peterson,R.A. ,1973 .Fiel d resistancet obenomy li ncucurbi tpowder ymildew .Austra ­ lianPlan tPatholog ySociet yNewslette r2 :27-28 . Schlüter,K .& H.C .Weltzien ,1971 .Ei nBeitra gzu rWirkungsweis e systemischerFungi ­ zideau fErysiph egraminis .Mededelinge nva nd eFakultei t Landbouwwetenschappen Rijksuniversiteit Gent36 :1159 . Schroeder,W.T .& R .Prowidenti ,1969 .Resistanc et obenomy li npowder ymilde wi n cucurbits.Plan tDiseas eReporte r53 :271-275 . Shephard,M.C. ,K.J .Bent ,M .Woolne r& A.M .Cole ,1975 .Sensitivit yt oethirimo lo f powderymilde wfro mU Kbarle y crops.Proceeding so fth e8t hBritis hInsecticid e andFungicid eConference .Britis hCro pProtectio nCouncil ,London ,p .59-66 . Smith,J.E. ,R.W .Payn e& A .Bainbridge ,1977 .Ethirimo lsensitivit y inpopulation s ofErysiph egraraini sfro mplot so fsprin gbarley .Annal so fApplie dBiolog y87 : 345-354. Vande rPlank ,J.E. ,1968 .Diseas eResistanc e inPlants .Academi cPress ,Ne wYork , p.55-107 . Walmsley-Woodward,Dianne ,J. ,F.A . Laws& W.J .Whittington ,1979a .Studie so nth etole ­ ranceo fErysiph egramini s f.sp .horde it osystemi cfungicides .Annal so fApplie d Biology92 :199-209 . Walsmley-Woodward,Dianne ,J. ,F.A .Law s& W.J .Whittington ,1979b .Th echaracteristic s ofisolate so fErysiph egramini s f.sp .horde ivaryin g inrespons et otridemorp han d ethirimol.Annal so fApplie dBiolog y92 :211-219 . Wolfe,M.S. ,1975 .Pathoge nrespons et ofungicid euse .Proceeding so fth e8t hBritis h Insecticidean dFungicid eConference .Britis hCro pProtectio nCouncil ,London , p.813-822 . Wolfe,M.S .& A .Dinoor ,1973 .Th eproble mo ffungicid e tolerance inth efield . Proceedingso fth e7t hBritis hInsecticid ean dFungicid eConference .Britis hCro p ProtectionCouncil ,London ,p .11-19 . Wolfe,M.S .& P.N .Minchin ,1976 .Quantitativ eassessmen to fvariatio ni nfiel dpopu ­ lationso fErysiph egramini s f.sp .horde iusin gmobil enurseries .Transaction s BritishMycologica lSociet y66 :332-334 .

230 Case study 5: Pénicillium decay of citrus fruits

J.W. Eckert Department ofPlan tPathology ,Universit y ofCalifornia ,Riverside , California, U.S.A.

Abstract

Sodium o-phenylphenate (SOPP), sec-butylamine,thiabendazole ,benomy l and diphenyl are applied toharveste d citrus fruits topreven t infection by Penicillim digitatum and P. italicum during storage andmarketing .Spon ­ taneousmutation s give strains of Pénicillium that areresistan tt oon e ormor e ofthes e fungicides.Thes eresistan tmutant spersis t atlo w fre­ quencies inth enatura lpopulation ,bu tunde r selectionpressur e created by thepost-harves t fungicide treatments,the yma ybecom e the dominant component ofth epopulatio n inpackin g houses.A singleconidiu m ofa re ­ sistantstrai ncoul d produce under favorable conditions aprogen y of about 108 spores inon ediseas e cycle inth e absence ofth e competing wild strain.SOPP-resistan t strains of Pénicillium areresistan t alsot o diphenyl;benzimidazole-resistan tstrain s areno tcontrolle d bythiaben ­ dazole,benomy l orthiophanate-methyl .Treatmen to f fruitwit h SOPPo r benomyl before storagema ycaus e anincreas e inth e frequency ofmutan t strains thatcanno tb e controlled bypost-storag e treatmentwit hbipheny l orthiabendazole ,respectively . Ifa strai n resistant toth ebenzimidazo - le fungicides and SOPP orsec-butylamin e ispresen t inth epopulation ,a pre-storage treatmento fSOP P orsec-butylamin e may cause an increase in benzimidazole resistance ofth e Pénicillium population during the storage period.Benomyl-resistan tstrain susuall y areles scompetitiv e thanwil d strains inuntreate d fruitstha thav ebee n inoculated with amixtur e of both strains.Thi s interactionma yexplai n the low frequencyo fbenzimi ­ dazole-resistant strains incitru s groves that areno ttreate dwit hthes e fungicides.Appropriat e strategies fordelayin g the emergence ofresis ­ tance are sanitation, fungicideprogramme s thatd ono t favor theprolifer ­ ationo fresistan t strains,an d fungicidal compounds thatth epathogen s cannotescap e readilyb ymutation .

Keywords:benomyl ;sec-butylamine ;carbendazim ;diphenyl ;gree nmould ; imazalil;lemons ;oranges ; Pénicillium digitatum; Pénicillium italicum;

231 thiabendazole; thiophanate-methyl, sodium o-phenylphenate; sodiumcarbon ­ ate; sodium tetraborate.

Disease aetiology and biology of the pathogens

Green andblu emol d ofcitru s fruits areincite db y the ubiquitous fungi, Pénicillium digitatum Sacc. and P. italicum Wehm., respectively. Greenmol d is themos t important causeo fpost-harves t decay ofcitru s fruitsproduce d inarea swit h scantrainfal l during theperio d of fruit development.Blu emol d iso f lesser over-all importance,bu ti tma ybe ­ come themajo rproble m under environmental conditions or fungicidetreat ­ ments thatselectivel y suppress the developmento fgree nmol d (Eckert, 1978). Pénicilliummold s are important also inhumi dproductio nareas , butthe yten d tob e overshadowed thereb y the stem-end rots, Diplodia na- talensis P.Evan s and Phomopsis citri Fawc. Innature , P. digitatum com­ pletes its life cycle only oncitru s fruits,wherea s P. italicum canin ­ fecta narra y ofdifferen t fruits andvegetables .Spore s (conidia)o f P. digitatum and P. italicum areproduce d onth e surface ofdisease d fruit lying onth e ground incitru s groves or inpackin g houses.Thes e spores can survive formonth s under dry conditions.The y aretransporte d by air currents tohealth y fruits,an dth e surface ofvirtuall y every citrus fruit iscontaminate d with these spores atharves t time,bu t germination and infection occurs only atinjure d sites.Potentially , asingl e spore of P. digitatum inoculated into asusceptibl e fruitma y give riset opro ­ genyo f about 108 spores inte nday sunde r optimum environmentalcondi ­ tions.Thes e spores serve as inoculum for infectiono fothe r citrus fruits andthe yma yvisibl y contaminate adjacent fruiti nth e samecon ­ tainer, giving riset oth eproble m 'soilage',whic h seriously reducesth e marketvalu e ofth efruit . Thehypha l cells of Pénicillium digitatum and P. italicum aremulti ­ nucleate,bu teac hconidiu m contains only onenucleus .Th enucle i usually arehaploid , like other species of theclas sDeuteromycotin a (Fungi Im- perfecti). No true sexual stageha sbee nreporte d foreithe r P. digitatum or P. italicum. Some isolates of P. italicum mayunderg o a 'parasexual cycle' inwhic h thehypha e oftw ocompatibl e haploid isolates anastamose to form aheterocaryon . Some ofth ehaploi d nuclei fuse to form diploid mycelium. Thediploid s revertspontaneousl y tohaploi d segregants,accom ­ paniedb y arecombinatio n ofth e chromosomes amongth eprogen y (Str0mnaes et al., 1964;Berah a& Garber , 1980). Thisparasexua l cycleha sneve r beenobserve d in P. digitatum, despite repeated attemptsusin g techniques thathav ebee n successful with other Pénicillium spp.

232 ORANGES LEMONS f harvest harvest fungicide I drench fungicide i bath ethylene I degree nin g emulsionwa x +2,4-d + .fungicidebat ho r fungicide foamwash-SOPP

emulsion A 7 store1- 4 months dry wax* flood or fungicide spray fungicide dry organic foamwash- I solvent SOPP dry i wax+ organic fungicide Ï solvent organicsolven t wax1 wax+fungicide „packi ncarton s packi ncarton s tdiphenyl withdipheny l 4 shipt omarke t 1 orstor e shipt omarke t Figure 1.Flo w diagramo fhandlin go forange san dlemon s after harvest showing fungi­ cides appliedan dothe rtreatments .

Conventional decay-control practices

Figure1 show sth esequenc eo ffungicid e treatmentso nCalifornia nci ­ trus fruitsafte rharves tt ocontro l Pénicillium decay (Dawson& Eckert , 1977).Th estructura l formulaeo fth erecommende d organic fungicides(di ­ phenyl, o-phenylphenol,thiabendazole ,benomyl , sec-butylaminean dimaza - lil)ar eshow ni nFigur e2 . Earlyan dlate-seaso norange s oftenrequir e degreeningwit h ethylene gast oattai nth eunifor m orange color thati spreferre db ymos tconsu ­ mers.Th eharveste d fruitsar ehel dfo rsevera l days (before cleaning)i n anatmospher e containing ethylenega sa tconcentration s of5-1 0n1/ 1a t 20-25 °Can d9 0% relativ ehumidity . Sincethes eenvironmenta l conditions areoptimu m forinfectio no fth efrui tb y Pénicillium spp.,th efrui tma y bedrenche dwit ha solutio no fsec-butylamin e (phosphate salt)o reithe r thiabendazoleo rbenomy lbefor e degreening.Next ,th efrui tar ecleane d

233 N„ *1 II ST-s-S diphenyl o- phenylphenol thiabendazole

C-NH-CH2-CH2-CH2-CH3 ÇH3 I 0 NL II CH3CH2C-NH2 "f^-NH-C-0CH3 —N H benomyl sec-butylamine

N=TN

I N-CH2-CH-0-CH2-CH= CH2 ~~ -Cl

Cl imazalil

Figure2 .Structure so ffungicide s thatar eeffectiv e forcontro lo fpost-harves tdeca y ofcitru sfruits . anddisinfeste dwit hon eo fth ebroad-spectru m fungicides -sodiu mcarbon ­ ate, sodium tetraborate or sodium o-phenylphenate (Eckert,1977 ,1978) . The fruitar erinse dwit h freshwate r and covered with ahydrophobi c coating ('wax')containin g a fungicide,e.g . thiabendazole (2-4 g/1),be ­ nomyl (1-2g/1 )o rsec-butylamin e (10-20g/1) .Th e 'wax'-fungicidetreat ­ mentretard swate r loss andprevent s Pénicillium decaydurin g storage and transport ofth e fruitt omarket .Finally , the fruitar eplace d ina fi - breboard box linedwit h apape r sheet impregnatedwit hth evolatil e fun- gistat,diphenyl . Severalvariation s ofthi s sequence incommercia lpractic e canhav ea major influence uponth edevelopmen t of fungicide-resistant strains ofJP . digitatum and P. italicum. Lemonsproduce d inth ecoasta l districts of Californiausuall y are stored for 1-4 months afterharves t to improve fruitqualit y and toregulat e supply and demand.Orange s and lemonsma y be stored forday so rweek s afterpackagin g forshipment ,dependin g upon

234 themarke tdeman d forcertai n fruitsize s andgrades .Sinc eth e residues of allo fthes e fungicides are stable on fruit,th epractic e of storing fungicide-treated fruitcreate s intense selectionpressur e forth efungi ­ cide-resistance genes inth epopulation s of P. digitatum and P. italicum incitru spackin ghouses . Thiabendazole,benomy lo rcarbendazim , suspended inwate r or awa x formulation, are applied tocitru s fruitsharveste d inal l production areasexcep tJapan . InJapan ,benomy l orthiophanate-methy l are applied tosatsum amandari n fruita s asingl e spraybefor eharvest ;non eo fthes e fungicides areregistere d inJapa n forpost-harves t application.Pre - harvest sprays ofbenomy l havebee nuse d effectively inFlorid a also (Brown, 1977), butthi sha sbee n discontinued since itcoul d selectfo r benzimidazole-resistant fungitha twoul d reduce theeffectivenes s ofth e post-harvestbenzimidazol etreatment ,whic h isnecessar y for successful marketing ofth ecrop . Thiabendazole isstabl e oncitru s fruits,wherea s thiophanate-methyl andbenomy l undergo transformation tocarbendazi m (Figure 3),whic hi s the stable residue onthes e fruit.Carbendazi m appears tob e theprinci ­ pal fungitoxicantpresen t inplan ttissu e and in fungi thathav ebee n treatedwit hbenomy l orthiophanate-methy l (Vonk& Kaar s Sijpesteijn, 1971; Hammerschlag & Sisler, 1973). Benomyl andcarbendazi m are roughly equally fungistatic ona nequimola rbasis ,wherea s thiabendazole issig ­ nificantly less inhibitory tomos t fungi (Eckert& Rahm , 1979). Benomyl ismor e lipophilic thanthiabendazol e orcarbendazi m and,therefore ,pe ­ netrateshydrophobi cbarrier s (waxan dcuticle )o nth eplan tsurfac emor e readily thanthiabendazol e orcarbendazi m (Eckerte t al., 1979). Forthi s reason,benomy l ismor e effective thanth e otherbenzimidazol e fungicides insituation s thatrequir epenetratio n ofth e fungicide intoth eplan t tissue, e.g. eradication oflaten tinfections ,interna lprotectiv e action and sporulation inhibition. Benzimidazole fungicides havebee nhighl y effective against Pénicil­ lium decayo fcitru s fruits,a swel l asth e stem end rots incitedb y Di-

0 II C-NH-CH2-CH2-CH2-CH3 u SO I 0 | 0 H II H II N. II -. N || ^\^N-C-N-C-OCH3 ^-NH-C-OCH3 ^f^V-NH-C-O-CHj^« f^Y -N ^ J N V\ N-C-N-C-OCH3 H II H II

benomyl carbendazim thiophanate-methyl Figure 3. Transformation of benomyl and thiophanate-methyl to carbendazim.

235 plodianatalensi s and Phomopsis citri. They are somewhatmor e effective against P. digitatum than P. italicum (Gutter, 1975), and they areinac ­ tiveagains tsou rro t (Geotrichum candidum), Alternaria rot (Alternaria citri), andbrow n rot (Phytophthora citrophthora). These diseasesma ybe ­ comemor eprominen t in fruitlot stha thav ebee n treated withbenzimida - zole fungicides andthe n stored for severalweek s (Brown, 1977).

Resistance to diphenyl and sodium o-phenylphenate

Farkas& Ama n (1940), in Israel,reporte d thatmutant swit h stable resistance todipheny l arose spontaneously inpur e cultures of P. digi­ tatum thatwer e exposed todipheny l vapors.Littaue r &Gutte r (1953)ob ­ served thatresistan tmutant s of P. italicum and Diplodia natalensis, as well as P. digitatum, appeared under similar conditions. Investigators in theUnite d Stateshav edescribe d strains of P. digitatum and P. italicum with different degrees ofresistanc e todiphenyl .Thes evariant swer eob ­ served inculture s exposed todipheny l vapor and inisolate s collected in citrus groves andpackin g houseswher eneithe r diphenyl nor related fun­ gicides had everbee nuse d (Duran& Norman , 1961;Harding , 1964;Harding , 1965; Smoot& Winston , 1967). The existence ofdiphenyl-resistan t mutants of Pénicillium spp.wa s notregarde d as apractica lproble m until the late 1960s,whe ncertai n shipments ofCalifornia n lemonspacke dwit h diphenyl-impregnatedpaper s arrived atdistan tmarket swit hhig h levels of Pénicillium decay and sporulation.Hardin g (1962, 1965)observe d thatman y ofthes e shipments originated inpackin g houseswher e the lemons hadbee ntreate d withso ­ dium o-phenylphenate (SOPP)befor e storage forsevera lmonth sprio r to shipment.Hardin g (1962)foun d that strains of P. digitatum thatwer ere ­ sistantt odipheny l were alsoresistan t toSOPP ,a swoul db e anticipated from the structural similarity ofth e two fungicides (Figure 2).Hardin g (1962)furthe rdemonstrate d that residues ofo-phenylpheno l onSOPP - treated lemonswer e sufficient to suppress the growtho fdiphenyl-sensi ­ tive strains,thereb y allowing the diphenyl-resistant mutants inth epop ­ ulation toproliferat e onth e fruit. Ina surve y ofCalifornia ncitrus - growing areas,Hardin g (1964)observe d diphenyl-resistant variants of P. digitatum ata frequenc y ofabou t 0.5 % ofth e isolates collected in citrus groves andpackin g houses thatha dneve rbee nexpose d toSOPP . Packinghouse swher e SOPP hadbee nuse d had aresistan t strain frequency of 14-18% ofth e P. digitatum population. Sixtypercen to fth e Pénicil­ lium isolateswer e resistantt o diphenyl incertai npackin g houses that haduse d SOPP continually over aperio d ofyears .Thes e observationsex ­ plained the failure ofth e diphenyl treatment to control greenmol d on fruitshippe d fromthes epackin ghouses .Diphenyl-/SOPP-resistan t isola­ tes of P. italicum were rarely recovered from the atmosphere ofCalifor -

236 nianpackin g houses,whic h explainswh y this specieswa sno t involved in thediphenyl-resistanc eproblem . Diphenyl-resistant strains of P. digita­ tum havebee nreporte d inFlorida ,bu tthes e isolates haveno tcause d a significant commercialproble mbecaus e SOPP-treated fruits areno tstore d routinely inth epackin ghouse s there (Smoot& Winston , 1967;Houck , 1977). Thus, there is little opportunity for selection and dissemination ofdiphenyl-/SOPP-resistan tmutant s inFlorida . Beraha andGarbe r (1966,1980 )investigate d thegenetic s of SOPPre ­ sistance in P. italicum and P. expansum (anapple-frui tpathogen )b y means ofth eparasexua l cycle inthes e species. In P. italicum, SOPP resistance isdu et o asingl eMendelia n gene that isdominan t inhetero ­ zygous diploids.Althoug h thepathogenicit y ofth e SOPP-resistant mutants of P. italicum was notreported , earlierwor k (Beraha et al., 1964)ha d showntha t approximately 44% ofcolo r and auxotropicmutant s of P. ita­ licum, and 67% ofth emutant s of P. digitatum, werevirulen twhe ninocu ­ lated into orange fruit.SOP P resistance in P. expansum hasbee nattribu ­ ted tothre eunlinke d genes thatar erecessiv e inheterozygou s diploids. Threemutant s resistantt oconcentration s of SOPP of 50pg/m lwer e avirulent to apple fruit,wherea s twomutant s resistantt o3 0M9/m lwer e virulent (Beraha& Garber , 1966).

Resistance to sec-butylamine

An aqueous solution ofsec-butylamin ephosphat e (pH9 )i sapplie dt o oranges and lemons afterharves t tocontro l Pénicillium molds during sto­ rage.Th etreatmen tma yb e applied toorange sbefor e degreeningwit h ethylenega san dt o lemonsbefor e storage (Dawson& Eckert ,1977 ;Eckert , 1977, 1978). Both applications create asituatio n that ishighl y favora­ ble forth eselectio n andproliferatio n ofsec-butylamine-resistan t strains of P. digitatum and P. italicum inth enatura lpopulation .There ­ foreth esever eresistanc eproble m thatdevelope dwithi nsevera lyear so f the introduction ofthi s fungicide should havebee n anticipated.

Resistance to benzimidazole fungicides

Benzimidazole fungicides havebee nuse d extensively over thepas t1 0 yearst ocontro lpost-harves t decays ofcitru s andothe r fruits (Eckert, 1977; Eckert, 1978). Thebes tknow ncompound s inthi s group of fungicides arebenomyl , thiabendazole,carbendazi m andthiophanate-methy l (Figures 2 and 3).Benzimidazole-resistanc e in Pénicillium spp.wa s observed inCali - forniancitru spackin g houses about 15month s afterthiabendazol ewa sin ­ corporated inth ewa x applied to lemonsbefor e storage (Houck, 1977). Harding (1972)reporte d thatthiabendazole-resistan t isolates ofbot h P. digitatum and P. italicum occurredwit hhig h frequency inth e atmosphere

237 oflemo npackin ghouse stha tuse d thiabendazole intensively asa pre ­ storage treatment for lemons.Thes e isolateswer eno tcontrolle db y treating inoculated fruitwit hhig h concentrations (3g/1 )o fthiabenda ­ zole. Benzimidazole-resistant isolates of P. digitatum and P. italicum havebee n isolated also inFlorid a (Smoot& Brown , 1974), Israel, Australia (Muirhead, 1974;Wil d &Rippon ,1975 )an dJapa n (Kuramoto, 1976). A survey ofth e frequency ofoccurrenc e of benzimidazole-resistant isolates of Pénicillium ondecaye d citrus fruiti nal lmajo rproductio n areasha s revealed themagnitud e ofth eproble m (Table 1).Benzimidazole - resistant strains of P. expansion havebee nreporte d onharveste d apples andpear s inOrego n (Bertrand & Saulie-Carter, 1978),Ne wYor k (Rosen- berger& Meyer , 1979;Rosenberge r et al, 1979), andAustrali a (Wicks, 1977; Koffmane t al., 1978). Mostobservation s of benzimidazole-resistant strains of Pénicillium spp.hav e followed intensiveus eo f thiabendazole orbenomy love r aperio d of somemonths, bu tKuramot o (1976)reporte d a severe resistanceproble m on satsumamandarin s inJapa n followingappli ­ cation of asingl e annual spray ofthiophanate-methy l immediately before harvest. Several investigators have recorded the isolation ofbenzimida ­ zole-resistant strains from citrus groves andpackin ghouse swher ebenzi - midazole fungicidesha dneve rbee nuse d (Harding, 1972;Kuramoto ,1976 ; Wild, 1980). Benzimidazole-resistant isolates of Pénicillium spp. found inpackin g

Table1 .Benzimidazole-resistan tstrain so fPénicilliu mdigitatu misolate d fromcitru sfruit s shippedt o Rotterdam.

Origino f Numbero f Proportiono f Proportiono f fruit isolates isolates resistant isolatesresistan t tothiabendazol e(% ) tobenomy l(% )

Algeria 27 37 30 Argentina 142 49 39 Australia 2 100 100 Brazil 86 33 19 California 388 55 50 Chile 13 15 8 Cuba 19 37 26 Cyprus 50 14 12 Egypt 22 27 14 Florida 65 28 15 Greece 57 16 9 Honduras 24 54 29 Israel 99 90 80 Italy 73 49 44 Morocco 56 57 43 SouthAfric a 115 77 32 Spain 146 31 16 Texas 57 35 26 Turkey 7 29 14 Uruguay 43 58 51 Source:McDonal de tal .(1979) .

238 houses areusuall y more sensitive tobenomy ltha n tothiabendazol e (Har­ ding, 1972;Houck , 1977;Wild , 1980)althoug hsom e isolates of P. digita­ tum and P. italicum areles s sensitive tobenomy l thant o thiabendazole (Muirhead, 1974;Wil d &Rippon , 1975). Furthermore, somethiabendazole - resistantmutant s ofP . expansum producedb ymutageni c agents invitr o weremor e sensitive tobenomy l thanth ewil d type strain (vanTuyl ,1975 ; vanTuyl , 1977). Thisphenomeno n isknow n asnegativel y correlatedcross - resistance.Mos to fth ebenzimidazole-resistan t mutants thathav ebee n isolated in fruitpackin g houses aresensitiv et oth eothe rpost-harves t fungicides, sodium o-phenylphenate, sec-butylamine, and imazalil (Smoot & Brown,1974 ;Wil d& Rippon ,1975 ;Harding , 1976;Wicks ,1977) . Less frequently, isolates of Pénicillium digitatum thatar e multiply-resistant tobenzimidazol e fungicides,SOP P andsec-butylamin e havebee n reported (Davée t al., 1980,Harding , 1976;Wild , 1980). Ingeneral ,benzimidazole-resistan t strains of Pénicillium spp.tha t havebee n isolated frompackin g houseshav ebee na svirulen t asbenzimi - dazole-sensitive isolates ofth e same species (Muirhead, 1974;Wil d & Rippon, 1975;Wicks ,1977 ;Koffma ne t al., 1978). However somebenzimi ­ dazole-resistant strainso f P. expansum collected fromrottin g apples and pearswer e lessvirulen ttha nsensitiv e strains,especiall y whenteste d oncertai nvarietie s of apples (Bertrand & Saulie-Carter, 1978;Rosenber - ger& Meyer , 1979). Beraha& Garbe r (1980)produce dmutant so f P. itali­ cumb y treatmento fconidi awit hultra-viole t lightan dnitrosoguanidine . They isolatedmutant swit hlo w (L),intermediat e (I)an dhig h (H)re ­ sistance tothiabendazol ebase dupo nth e ability ofth emutant s togro w ona mediu m amendedwit hconcentration s ofthiabendazol e of0.5 , 7.0 or 500 jjg/ml,respectively . Thiabendazole resistance inthi s fungus involved twoo rthre e linked genes.Berah a& Garbe r (1980)foun dtha t asensitiv e (wild)strai ncoul dno tb emutate d directly toa nintermediat e orhig h level ofthiabendazol e resistance.Rather , itwa s obligatory thatgen eL firstunderg omutatio nt ogiv elow-leve lmutant stha tcoul db emutate d againt ogiv e strainswit h intermediate andhig hresistance .

Levels ofbenzimidazol e resistance innatura lpopulation s ofP . digitatum

Wild (1980)investigate d thecharacteristic s ofth enatura l population ofbenzimidazole-resistan t strains of P. digitatum inCalifornia .Approxi ­ mately 130carbendazim-resistan tisolate s of P. digitatum were collected inpackin g houses and citrus groves inCaliforni a and classified accord­ ingt odosag e required toalte rcolon y appearance,ED,- 0 for inhibition ofgrowt hrate ,minimu m growth-inhibitingconcentratio n (MIC)an d dosage required toinhibi tsporulation .Th eED 5Q values (ugcarbendazim/ml )fo r therecognize d categories ofresistanc ewere :Categor y 0 (wildtype )- 0.03;Categor y I- 1.3 ;Categor y II -5.5 ;Categor y III -6.5 ; Category

239 IV- 40 .Isolate s resistantt ocarbendazi mwer e cross-resistant tothia ­ bendazole atappropriat e concentrations,bu t the relative resistance of each straint oth etw o fungicideswa s notconstant . Isolates thatbe ­ longed tocarbendazi m resistance Category IIwer e themos t resistantt o thiabendazole (ED,-n=60ug/ml) . None ofth e isolateswer enegativel ycross - resistant. Isolates inCategor y IIan d IIIcoul d notb e separated solely onth ebasi s ofED ^ ordosag erequire d to alter colony appearance.How ­ ever, thesecategorie swer e readily differentiated whenth e additional criteria ofMI C and sporulation inhibitionwer e applied toth eclassifi ­ cation.Approximatel y 5% o fth eresistan t isolates obtained frompackin g houses fell intoCategor y I,3 3% intoCategor y II,5 8% Category IIIan d 4 % inCategor y IV. Ina simila r survey ofpackin g houses inJapan ,Kura - moto (1976)foun dtha tth eMI Co fbenomy l forth egrea tmajorit y ofresist ­ antisolate swa s400-80 0 |jg/ml (whichtransform s into 263-527ug/m lcar ­ bendazim), approximately 9-18 times greater than forth emos t abundant category (Category III,MI C =3 0|jg/ml )o fresistan t strains inCalifor ­ nia. Wild (1980)als ocollecte d spores from citrus groveswher ebenomy l and thiabendazole hadneve rbee n applied, toasses s therang eo fcarben - dazim-resistance inth ewil dpopulatio n of P. digitatum. Only afe wcar - 8 9 bendazim-resistant isolates (Category III)wer e recoveredwhe n1 0-1 0 spores fromthre ecitru s groveswer eplate d onmediu m ammendedwit hcar ­ bendazim ata concentratio n of 1ug/ml . Isolatesbelongin g toresistanc e Category IVwer e less infectious than isolatesbelongin g toCategorie s 0, I, IIo r IIIwhe n spores (10/ml )fro m apur ecultur eo feac h strainwer e inoculated intountreate doranges . Although thebenzimidazole-sensitiv e strains of P. digitatum werewel l controlled by thetreatmen t of inoculated fruitwit h carbendazim (asbe ­ nomyl)concentration s of0.0 3 g/1o r thiabendazole concentrations of 0.08 g/1,non eo fth ebenzimidazole-resistan tstrains ,irrespectiv e of resistance category,wer econtrolle d by carbendazim at1- 2 g/1 (asbenomyl ) orthiabendazol e at2- 4 g/1 -concentration s used incommercia l packing houses.

Competitionbetwee n sensitive and resistant isolates indisease d fruit

The low frequency ofcarbendazim-resistan tisolate s inth enatura lpo ­ pulation of P. digitatum incitru s grovesprompte d aninvestigatio n of the competitionbetwee n carbendazim-sensitive and -resistant isolates in infected oranges (Wild, 1980). Healthy orangeswer e inoculatedwit ha standarizedmixtur e oftw oo rmor e strains of P. digitatum, incubated at 26 °C for 5days , and arepresentativ e sample ofth e sporepopulatio nwa s collected from thesurfac e ofth edisease d fruits.A second loto fheal ­ thyorange swa s then inoculatedwit h asuspensio no fthes e spores (first generation).Th erelativ e abundance of fungicide-resistant and -sensitive

240 spores inth epopulatio n ofeac h spore generationwa s determined bypla ­ ting asampl e ofth e spores intopotato-dextros e agar amendedwit hcar - bendazim oranothe r fungicide. Insom e experiments the fruitwer e treated with a fungicide after theyha dbee n inoculated with each sporegenera ­ tion.Treatmen t oforange swit h abenomy lconcentratio n of 0.5 g/1 (which transforms into 0.33 g/1 carbendazim), after inoculationwit h amixtur e ofcarbendazim-resistan t and -sensitive spores,resulte d inalmos ttota l selection forth e resistant isolate inth e firstspor e generation (Figure 4). The addition of asecon dunrelate d fungicide to thebenomy l treatment, asrecommende d by some authorities tocontro l resistance development,ha dn omeasurabl e influenceupo nth e selection andprolife ­ rationo fth eresistan t strain.Treatmen to fth e inoculated fruitwit ha non-benzimidazole fungicide alone,o rn o fungicide atall ,usuall yresul ­ ted ina rapi d decline inth e frequency ofth e benzimidazole-resistant strain insubsequen t sporegeneration s (Figure 4). The rapid disappearan­ ceo fth eresistan t strains fromth e sporepopulatio nwa sunexpecte dbe ­ cause the resistant isolateswer e asvirulen t asth ecarbendazim-sensiti - veisolates . These investigations showtha tresistan t strains canb e rapidlyselec ­ ted from anatura lpopulatio n of P. digitatum sporesunde r the selection pressure ofa benzimidazol e fruittreatment .Furthermore ,th ewea kcompe ­ titivebehaviou r ofresistan t strains inmixtur ewit h sensitive strains ininfecte d fruit (nottreate dwit h abenzimidazol e fungicide)coul dex ­ plain the low frequency ofbenzimidazole-resistan t strains inth enatura l sporepopulation s ofcitru s groves.Benzimidazol e fungicideshav eneve r beenuse d forcontro l of field diseases ofcitru s trees inCalifornia , butenormou s numbers ofbenzimidazole-resistan t sporeshav ebee ndischar ­ ged intoth e atmosphere frompackin ghouses . Despite thecompetitiv e advantage ofbenzimidazole-sensitiv e strains overresistan t strains inuntreate d fruit,i tha sbee nobserve d thatben ­ zimidazole-resistant strains dono tdisappea r from the spore population ofpackin g houses after thebenzimidazol e fungicideshav ebee n replaced by sodium o-phenylphenate (SOPP)o rsec-butylamine .Wil d (1980)foun d thatman y carbendazim-resistant isolates collected inthes epackin g houseswer e alsoresistan t tosec-butylamin e or SOPP.Orange swer einocu ­ latedwit h amixtur e of astrai nsensitiv e tocarbendazim , sec-butylamine and SOPP and astrai ntha twa s doubly-resistant tobot h carbendazim and sec-butylamine.Treatmen t of fruitinoculate d with this sporemixtur e with eitherbenomy l orsec-butylamin e resulted in animmediat e increase inth e frequency ofth edoubly-resistan t strain inth epopulatio no fth e firstspor egeneratio n (Figure4 ,RRS) ,wherea s treatmentwit h unrelated fungicides didno tcreat e selectionpressur e foreithe ro fth e resistance genes forcarbendazi m orsec-butylamine .Treatmen to forange s inoculated with amixtur e of sensitive andcarbendazim/SOP P doubly-resistant spores

241 proportiono f carbendazim-resistan t spores(% )

100-

spore generation Figure 4. Effect of treatment of several lots of Valencia oranges with the same fun­ gicide upon the frequency of carbendazim-resistant spores of P^ digitatum in the popu­ lation after two disease cycles (spore generations). The fruit were treated with the specified fungicides after infection was established by inoculation with a spore mix­ ture containing: Upper plot, a fungicide-sensitive strain and a strain doubly-resistant to carbendazim and sec-butylamine but sensitive to SOPP (RRS); Lower plot, a fungi­ cide-sensitive strain and a strain doubly-resistant to carbendazim and SOPP but sen­ sitive to sec-butylamine (RSR) (Wild, 1980).

with either carbendazim or SOPP alone had an analogous influence on the build-up of SOPP/carbendazim doubly-resistant strains in the population (Figure 4, RSR). *

242 Factors promoting the build-up of a fungicide-resistant population

Thedevelopmen t ofa seriou s resistance problem depends uponth erapi d multiplication anddisseminatio n ofth eresistan t straint oman y other hosts treated with the same fungicide.Th enumbe r ofresistan tpropagule s (spores)o f P. digitatum can increaseb y about afacto r of 10t oth e power 8i n1 0days , and they arereadil y disseminated toothe r selective environments,sinc e all fruits aretreate d uniformlywit h the samefun ­ gicide.Th epoint s inth ehandlin g sequence forcitru s fruittha tar e most favorable to thedevelopmen t andproliferatio n offungicide-resis ­ tantstrain s of Pénicillium are shown inFigur e 1. Early-season oranges aredrenche dwit h asolutio no fthiabendazole , benomyl, orsec-butylamin ean d thenplace d ina nethylen e degreening room at22-2 5 °C.Th e fungicide residues suppress the sensitive strains that aredominan t inth e fieldpopulatio n of Pénicillium spp.,thereb ypermit ­ ting the fungicide-resistant strains tocoloniz e the fruitan d sporulate after several days.When ,afte r degreening,th e fruitar edumpe d fromth e fieldboxe sth e spores ofth e resistant strains areborn eb y the airt o thenewly-harveste d fruita sthe y areplace d inth edegreenin g room.Af ­ ter de-greening, the fruitcontaminate d withbenzimidazole-resistan t spores receive awea kdisinfectan ttreatmen t and thenpas s intoth e packing housewher e they receive asecon d treatmentwit h abenzimidazol e fungicide in 'wax',befor e shipmentt omarket . Inevitably, the firstben ­ zimidazole treatment selects forresistan t strains and,therefore ,th e second treatment failst ocontro l fruitdecay . Figure 1show stha tlemo n fruitar ewashe d ina bat ho fSOP P or sodium carbonate,coate dwit h astorag e 'wax'containin g 2,4-D andthiabendazole , benomyl orsec-butylamine , and stored at1 5 °Cfo r severalmonths .Whe n the lemons areremove d from storage,benzimidazole-resistan t strains that areselecte d during the storageperio d contaminate the sound fruit,lea ­ dingt oth e failure ofthei r secondbenzimidazol e treatment (inwax) . Sim­ ilarly,th e application ofSOP P to lemonsbefor e storagema y selectfo r SOPP-/diphenyl-resistant strains thatma ygreatl y reduce theeffective ­ ness ofth edipheny l treatmentdurin g shipment.Anothe r seriousproblem , andperhap smor e difficultt o solve,i sth econtaminatio n of lemonsente ­ ring storagewit h spores ofresistan t strains thathav ebee nselecte db y fungicide residues onth epreceedin g loto f fruitdurin gstorage . A final scenario forth edevelopmen to fa seriou s fungicide-resistance problem iswhe n lemonso r oranges arepacke d formarket ,bu tcanno tb e soldbecaus e ofwea kdemand .Thes e fruitremai n incol d storage forsev ­ eralweek sbefor e they arereturne d toth epackin ghous e forremova l of decayed fruit fromth eboxe sprio r to shipment.Fungicide-resistan t Péni­ cillium spp.,selecte d duringstorag eb y the fungicidedeposit so nth e

243 fruit, contaminate all fruitpassin g through thepackin g house,resultin g ina grea treductio n ineffectivenes s ofth e final fungicidetreatments .

Detection and evaluation of the fungicide-resistance problem

Iti scommo npractic e tomonito r the level of fungicide resistance in thepopulatio n of Pénicillium spores inpackin g houses.Petr i dishescon ­ tainingpotato-dextros e agar,amende dwit hconcentration s ofbenomy lo f 10(jg/m lo rsec-butylamin e (HClsalt )o f50 0jjg/ml ,ar eexpose d toth e atmosphere ofth epackin g house for alengt h oftim e thatdepend supo n thesiz eo fth e Pénicillium sporepopulation .Diclora n ata concentratio n of3 pg/m lma yb e added toth emediu m torestric tth eradia l growtho f the Pénicillium colonies.Th eexpose dplate s are incubated forsevera l days at2 5 °Can d thecolonie s counted.Th enumbe r ofcolonie s that develop onth e fungicide-treated medium comparedwit hth e colonies that appearo npaire d plates containingbasa lmediu m only indicates the level of fungicide-resistance inth epopulatio n inth epackin g house.Di - phenyl and sodium o-phenylphenateresistanc e canb emonitore d ina simi ­ lar fashion,excep ttha ti tma yb emor e convenient toplac e crystals of diphenyl inth e lido fth e invertedpetr i dish thani nth egrowt hmedium .

Strategies to avoid build-up of resistant strains

Since theeffectiv eus eo fth ebenzimidazol e fungicides is essential forth e success ofcurren tprocedure s inmarketin g citrus fruits,severa l strategies forth e control ofresistan t strains of Pénicillium arebein g investigated.

Sanitation

Duringperiod s ofth eyea rwhe n fruit areno tharvested , packing houses canb e disinfested withbroad-spectru m antimicrobial agents (e.g., formaldehyde,guarternar yammoniu m compounds)tha tcanno tb euse dwhe n fruit arepresent .Thi s assures aver y lowpopulatio n ofspore s inth e packing house atth ebeginnin g ofth eharves tperiod , and results inhig h efficiency ofal lpost-harves t fungicide treatments.However ,withi na month ofoperatio no fth epackin g house the level of fungicide-resistance inth e Pénicillium populationbegin s tobuild-up , accompanied by agra ­ dual deterioration ineffectivenes s ofon eo rmor e fungicidetreatments . Eventually, apoin ti sreache dwher e the decaycontro lprogramm e isn o longercost-effective .Thi s economic threshold canb edelaye db y excluding contaminated fruit from thepackin g house andb y arigorou s sanitation programme inside thepackin g house,consistin g ofa dail y disinfestation ofth e fruit-handling equipmentwit h abroad-spectru m sanitizing agent.

244 Thesepractice s dono treduc e the frequency ofresistan t strains existing inth epopulation ,no rd othe y influence intrinsically theselectio npro ­ cess.Bu t sanitation does reduce the absolutenumbe r ofresistan t spores inth epackin g house toth e same extent that itreduce s the total spore population.A reduction inth enumbe r of resistant spores should delay the developmento fa fungicide-resistance epidemic because theprobabili ­ tyo f successful infection (bya resistan t strain)depend s uponth e spore concentration (inrelatio n toth enumerica l inoculum threshold ofinfec ­ tion)a ta limite dnumbe r ofinjure d siteso nth e fruitsurface .A reduc ­ tion intota l sporepopulatio n also should delayth ebuild-u p ofresis ­ tance, since fungicide-sensitive spores can increase theprobabilit y of infectiono fbenzimidazole-treate d fruitb y abenzimidazole-resistan t strain (Wild, 1980).

Non-selective treatments

The elimination of selective fungicides and structurally-related com­ pounds fromth epost-harves t fungicideprogramm e shouldproduc e adecrea ­ sei nth e frequency ofth e resistance strains,becaus e ofth e competition ofth e sensitive strains (Figure 4). This strategywoul db ecounter-pro ­ ductive,however , since heavydeca y losseswoul d arisethroug h fruitin ­ fectionb yth euncontrolle d fungicide-sensitive strainso f Pénicillium. Furthermore,reductio n ofth e fungicide-resistant population to aleve l thatcoul db e controlled by abenzimidazol e treatmentwoul d requirese ­ veral disease cycles,an d restoration ofth ebenzimidazol e treatment could cause animmediat e increase inth e frequency ofth e resistant strains (Figure4) . Heated solutions ofbora x and sodium carbonate reduce fruit infection by Pénicillium. Moreover,the yhav eno tbee n implicated ina fungicide - resistanceproblem .Th e application ofbora x israthe r limited todaybe ­ cause ofth epossibilit y thati twil lpollut e the ground-water,bu tsodiu m carbonate (30g/1 , at4 3 °C)i sa neffectiv etreatmen t for disinfecting citrus fruitbefor e they arebrough t into thepackin ghouse .Non e ofth e otherwell-know nmulti-sit e fungicides (e.g.,captan ,maneb ,chlorothalo - nil)ar eeffectiv e against Pénicillium decay.

Mixtures of selective andnon-selectiv e fungicides

This strategyha sbee n suggestedb y several authorities todela y the build-up ofresistan t strains ofpathogen s inth e field.Unfortunately , there islittl epublishe d data tosuppor tthi scontention .Mixture s of systemic fungicides (e.g.benomyl )an dprotectiv e fungicides (e.g.cap - tan)ma ypreven tpenetratio n ofbenzimidazole-resistan tstrain s intotreat ­ ed fruits,thereb y reducing thetota l pathogenpopulation , including the

245 benzimidazole-resistant component (Koffmane tal. ,1978 ;Rosenberge r etal. ,1979) . However,th e combination ofbenzimidazol e and protective fungicides doesno t appear tob e asuitabl e strategy forcontro l ofre ­ sistantstrain s of Pénicillium oncitru s and other fruits thatma yb ein ­ fectedwit h amixtur e ofresistan t and sensitive strainsbefor e thefun ­ gicide treatment is applied.Non-systemi c fungicides cannot eradicate deep-seated infections nor inhibitsporulatio n efficiently (Brown,1977 ; Eckerte tal ,1979) . Thus, the rapidbuild-u p of benzimidazole-resistant strains following treatmento finfecte d fruitwit h amixtur e ofa benzi ­ midazole and aprotectiv e fungicide ispredictable .Th ecombinatio n of twounrelate d systemic fungicides,suc h asbenomy l and imazalil (Figure2) , eachwit h auniqu emechanis m ofaction ,ma yb e amor epromising ,bu tex ­ pensive, strategy forth e control ofresistan t strains (Laville et al., 1977; Koffmane t al., 1978;Rosenberge r et al., 1979). Thepossibilit y of combining two synergistic compounds ortw o fungicides thatexhibi tnega ­ tively correlated cross-resistance isa n intriguing,bu tunproven ,stra ­ tegy forcontrollin g fungicide-resistant plantpathogen s (Dekker,1977 ; Georgopoulos, 1977).

Alternation oftw oo rmor e unrelated fungicides

This strategy involves the application oftw oo rmor eunrelate d fun­ gicides ina sequenc e that,hopefully ,wil l suppress strains ofth epa ­ thogentha t areresistan tt oth e final fungicide inth e series.Thi s pro­ cedure is feasible forcitru s fruitsbecaus e they arealread y treated with several fungicides afterharvest .Fo rexample ,lemon sma yb e treated with SOPP and/orsec-butylamin ebefor e storage and, after storage,wit h SOPP, abenzimidazol e fungicide,an ddipheny l (Dawson& Eckert ,1977 ; Eckert, 1978). This sequence isrequire dbecaus e thebenzimidazole sar e theonl y approved fungicides thatca ncontro l sporulation of Pénicillium ondisease d fruits.Th e success ofthi s schedule forlemon sdepend s upon anefficien t sanitationprogramme ,sinc eth ebuild-u p ofa larg epopula ­ tiono f SOPP-o rsec-butylamine-resistan tstrain s during storage could result inth e selection ofmutant s thatar edoubly-resistan t toon eo f these compounds andth ebenzimidazol e fungicides (Figure 4).A pre-stora - ge sequenceo fSOP P followedb y sec-butylaminemigh tb eeffectiv e insup ­ pressing in Pénicillium population, sincedouble-resistanc e tothes etw o fungicides isrelativel y rare,whic h indicates thatsuc h doubly-resistant (SOPP/sec-butylamine)mutant sma yb epoorl y adapted as fruitpathogen s (Wild, 1980). Themultipl e siteso factio no fSOP P and sec-butylamine should further discourage thedevelopmen to fstrain s resistant tobot h of these fungicides.A tlo wconcentrations ,sec-butylamin e inhibitspyruvi c dehydrogenase in P. digitatum (Yoshikawa &Eckert ,1976 ;Yoshikaw a et al., 1976); athighe r concentrations thetranspor to famin o acids into

246 thehypha ei sinhibite d (Bartz& Eckert , 1972). SOPP increasesmitoti c abnormalities indiploi d strains of Aspergillus nidulans, which suggests thatthi s inhibitor may adversely effecthereditar y processes inothe r fungi aswel l (Georgopoulos et al., 1976; 1979). SOPP also inhibitsse ­ veral tricarboxylic acid (TCA)cycle enzymes in P. italicum (Rehm, 1969).

Fungicides thatth epathoge n cannotescap e easilyb ymutatio n

Tests conducted over thepas tsevera lyear shav edemonstrate d that imazalil (Figure2 )i shighl y effective inpreventin g infection ofcitru s fruitsb y strainso f Pénicillium spp.tha tar eresistan tt oothe rfungi ­ cides, and alsohighl y effective insuppressin g sporulation ofthes efun ­ gio ndisease d fruits (Harding,1976 ;Lavill e et al., 1977). Imazalil- resistant strains of P. digitatum wereno tobserve d following treatment ofspore swit hmutageni c agents thatproduc emutant s resistant toothe r fungicides (vanHoor n& Eckert ,unpublishe d data),va nTuy l (1977b)foun d thatth emutationa l frequency forresistanc e to imazalil in P. expansum washig h (70x 10~ )compare d totha t forbenomy l (1x 10 ~ ),bu ttha t the increase inresistanc e level accompanying asingl e genemutatio nwa s rather small.Th e increase inresistanc e level (ED5Q mutant T ED5Q wild type)fo r asingl emutatio nwa s 330 0 forbenomy l and4 for imazalil.Mu ­ tations forimazali l resistance atdifferen t lociyielde d strains that werepleiotropicall y resistant orhypersensitiv e toothe r fungicides. Imazalil inhibits ergosterol synthesis in P. italicum (Siegel &Ragsdale , 1978), P. expansum (Leroux& Gredt , 1978), and Vstilago avenae (Buchen- auer, 1977). The strikingpleiotropi c effects accompanying imazalilre ­ sistance in A. nidulans mayb e related to asit eo factio ninvolvin g the cellmembran e (vanTuyl , 1977A). Other fungicideswit h asimila rmecha ­ nism ofactio nhav eno tye tencountere d apractica l problemwit hpathoge n resistance (Dekker, 1977). A recentrepor to fimazalil-resistan t strains ofP . expansum causing decay ofpear s in Israel (Pruskye t al., 1980)make s itclea r thateve n this fungicidemus tb euse d cautiously to avoidth edevelopmen to fresis ­ tance.Nonetheless ,geneti c andbiochemica l investigations of imazalil and related inhibitors in fungiprovid ehop etha tth e development ofresis ­ tance in Pénicillium spp.wil lb e slow and involvemutant swit hdimini ­ shedvirulence .Relativel y highdosage s ofimazali l shouldb e applied to fruitt o suppress thedevelopmen t oflow-leve l resistantmutants ,which , ifallowe d topersis t inth epopulation ,migh tacquir e additionalresis ­ tancegenes ,resultin g instrain swit h ahighe r level ofresistance . All strategies forus eo f imazalil should include astric t sanitation programme tominimiz e theresistance-gen e pool available forselectio nb y imazalil.Trial s areno wunde rwa y inlemo npackin ghouse s to evaluate several fungicide sequences including imazalil andbenzimidazoles .Ta -

247 Table2 .Estimate d decaylosse s in197 9o fCalifornia n citrusfruit s treatedwit hcurrentl yuse dfungicide so rimazalil .

Citrus Decay losses (numbero f35-poun d cartons) Decreasei n fruit r— decay(% ) currentlyuse d fungicides imazalil

Lemons 532 340 0 12910 8 98 Oranges 442 400 0 165 900 0 63 Grapefruit 62700 0 23500 0 63 Tangerines 11040 0 5520 0 50

Total 1048 480 0 324 028 0

a.SOPP ,sec-butylamine ,thiabendazol ean dbenomyl . b.Imazali lwa sno tuse dextensivel y inCaliforni ai n1979 .Estimate so f decay losses,i fal lfrui tha dbee ntreate dwit himazalil ,wer emad eb y CaliforniaCitru sQualit yCounci l (Anonymous,1980) .

ble2 contain s estimates ofth epotentia l reduction indeca y anticipated if allCaliforni a citrus fruitsha dbee ntreate dwit h imazalili n1979 .A majorportio n ofth ebeneficia l effects ofimazali lwoul d result from the control of Pénicillium strains that areresistan t toothe r fungicides currently inuse .

References

Anonymous,1980 .Imazalil :a ne wweapo ni nth efrui tdeca ybattle .Citrograp h65 : 95-96. Bartz,J.A .& J.W .Eckert ,1972 .Studie so nth emechanis m ofactio no f2-aminobutane . Phytopathology 62:239-246 . Beraha,L .& E.D .Garber ,1966 .Genetic so fphytopathogeni c fungi.XV .A geneti cstud y ofresistanc e tosodiu mortho-phenylphenat ean d sodiumdehydroacetat e inPénicilliu m expansion).America nJourna lo fBotan y53 :1041-1047 . Beraha,L .& E.D .Garber ,1980 .A geneti c studyo fresistanc et othiabendazol ean d sodiumo-phenylphenat ei nPénicilliu mitalicu mb yth eparasexua l cycle.Botanica l Gazette 141:204-209 . Beraha,1. ,E.D .Garbe r& 0 .Str0mnaes ,1964 .Genetic so fphytopathogeni c fungi.X . Virulenceo fcolo ran dnutritionall y deficientmutant so fPénicilliu mitalicu man d Pénicilliumdigitatum .Canadia nJourna lo fBotan y42 :429-436 . Bertrand,P.F .& J.L .Saulie-Carter ,1978 .Th eoccurrenc eo fbenomyl-toleran tstrain s ofPénicilliu mexpansu man dBotryti s cinerea inth emid-Colbumbi aregio no fOrego n andWashington .Plan tDiseas eReporte r62 :302-305 . Brown,G.E. ,1977 .Applicatio no fbenzimidazol e fungicides forcitru sdeca ycontrol . Proceedingso fth eInternationa lSociet yCitricultur e 1:273-277 . Buchenauer,H. ,1977 .Mechanis mo factio no fth efungicid eimazali l inUstilag oavenae . ZeitschriftPflanzenkrankheite nun dPflanzenschut z84 :440-450 . Dave,B.A. ,H.J .Kapla n& J.F .Petri ,1980 .Th eisolatio no fPénicilliu mdigitatu m Sacc.strain stoleran tt o2-AB ,SOPP ,TB Zan dbenomyl .Proceeding so fth eFlorid a StateHorticultura lSociety ,93 :344-347 . Dawson,A.J .& J.W .Eckert ,1977 .Problem so fdeca ycontro li nmarketin gcitru sfruits : strategyan dsolutions ,Californi aan dArizona .Proceeding so fth eInternationa l SocietyCitricultur e 1:255-259 . Dekker,J. , 1977.Th efungicide-resistanc e problem.Netherland sJourna lPlan tPatholog y 83 (Suppl.1) : 159-167. Duran,R .& S.M .Norman ,1961 .Differentia l sensitivityt obipheny lamon gstrain so f Pénicilliumdigitatu mSacc .Plan tDiseas eReporte r45 :475-480 .

248 Eckert,J.W. ,1977 .Contro lo fpostharves tdiseases .In :M.R .Siege l& H.D .Sisle r (Eds): AntifungalCompounds ,Vol .1 .Marce lDekker ,Inc. ,Ne wYork .p .269-352 . Eckert,J.W. , 1978.Postharves tdisease so fcitru sfruit .Outloo ko nAgricultur e9 : 225-232. Eckert,J.W .& M.L .Rahm ,1979 .Th eantifunga lactivit yo falky lbenzimidazole-2-ylcar - bamatesan drelate d compounds.Pesticid eScienc e 10:473-477 . Eckert,J.W. ,M.J .Kolbezen ,M.L .Rah m& K.J .Eckard ,1979 .Influenc eo fbenomy lan d methyl2-benzimidazolecarbamat eo ndevelopmen to fPénicilliu mdigitatu mi nth eperi ­ carpo forang efruit .Phytopatholog y69 :934-939 . Farkas,A .& J .Aman ,1940 .Th eactio no fdipheny lo nPénicilliu man dDiplodi amoulds . PalestineJourna lBotan yJerusale mSerie s2 :38-45 . Georgopoulos,S.G. , 1977.Developmen to ffunga lresistanc et ofungicides .In : M.R.Siege l& H.D .Sisle r (Eds): AntifungalCompound sVol .2 .Marce lDekke rInc. , NewYork .p .439-495 . Georgopoulos,S.G. ,A .Kappa s& A.C .Hastie ,1976 .Induce dsectorin g indiploi dAsper ­ gillusnidulan sa sa criterio no ffungitoxicit yb yinterferenc ewit hhereditar ypro ­ cesses.Phytopatholog y66 :217-220 . Georgopoulos,S.G. ,M .Sarri s& B.N .Ziogas ,1979 .Mitoti cinstabilit y inAspergillu s nidulans causedb yth efungicide s iprodione,procymidon ean dvinclozolin .Pesticid e Science 10:389-392 . Gutter,Y. ,1975 .Interrelationshi p ofPénicilliu mdigitatu man dP .italicu m inthia - bendazole-treated oranges.Phytopatholog y65 :498-499 . Hammerschlag,R.S .& H.D .Sisler ,1973 .Benomy lan dmethyl-2-benzimidazole-carbamat e (MBC): biochemical,cytologica lan dchemica laspect so ftoxicit yt oUstilag omaydi s andSaccharomyce s cerevisiae.Pesticid eBiochemistr yan dPhysiolog y3 :42-45 . Harding,P.R .Jr. ,1962 .Differentia l sensitivityt osodiu morthophenylphenat eb y biphenyl-sensitive andbiphenyl-resistan t straino fPénicilliu mdigitatum .Plan t DiseaseReporte r46 :100-104 . Harding,P.R . Jr.,1964 .Assayin g forbipheny lresistanc ei nPénicilliu mdigitatu mi n California lemonpackin ghouses .Plan tDiseas eReporte r48 :43-46 . Harding,P.R .Jr. ,1965 .Th enatur eo fbiphenyl-resistan tmutant so fPénicilliu mdigi ­ tatum.Plan tDiseas eReporte r49 :965-969 . Harding,P.R .Jr. ,1972 .Differentia l sensitivityt othiabendazol eb ystrain so fPéni ­ cillium italicuman dP .digitatum .Plan tDiseas eReporte r56 :256-260 . Harding,P.R .Jr. ,1976 .R23979 ,a ne wimidazol ederivativ eeffectiv eagains tpost - harvestdeca yo fcitru sb ymold sresistan tt othiabendazole ,benomy lan d2-amino - butane.Plan tDiseas eReporte r60 :643-646 . Houck,L.G. , 1977.Problem so fresistanc et ocitru sfungicides .Proceeding so fth eIn ­ ternationalSociet yCitricultur e 1:263-269 . Koffman,W. ,L.J .Penrose ,A.R .Menzies ,K.C .Davie s& J .Kaldor ,1978 .Contro lo fben - zimidazole-tolerantPénicilliu mexpansu m inpom efruit .Scienti aHorticultura e9 : 31-39. Kuramoto,T. ,1976 .Resistanc et obenomy lan dthiophanate-methy l instrain so fPénicil ­ liumdigitatu man dP .italicu m inJapan .Plan tDiseas eReporte r60 :168-171 . Laville,E.Y. ,P.R .Harding ,Y .Dagan ,M .Rahat ,A.J .Kragh t& L.E .Rippon ,1977 . Studieso nimazali la spotentia ltreatmen t forcontro lo fcitru sfrui tdecay .Pro ­ ceedingso fth eInternationa lSociet yCitricultur e 1:269-273 . Leroux,P .& M .Gredt ,1978 .Effet sd equelque sfongicide s systémiquessu rl abiosyn ­ thèsed el'Ergostéro lche zBotryti sciner aPers. ,Pénicilliu mexpansu mLink ,e t Ustilagomaydi s (DC.)Cda .Annal sPhytopatholog y 10:45-60 . Littauer,F .& Y .Gutter ,1953 .Diphenyl-resistan t strainso fDiplodia .Palestin e JournalBotany ,Rehovo tSerie s8 :185-189 . McDonald,R.E. ,L.A .Riss e& B.M .Hillebrand ,1979 .Resistanc et othiabendazol ean d benomylo fPénicilliu mdigitatu man dP .italicu m isolated fromcitru sfrui tfro m severalcountries .Journa lAmerica nSociet yHorticultura lScienc e 104:333-335 . Muirhead,I.F. ,1974 .Resistanc et obenzimidazol efungicide si nblu emoul do fcitru si n Queensland.Australia nJourna lExperimenta lAgricultur eAnima lHusbandr y14 : 698-701. Prusky,D. ,M .Baza k& R .Bendrie ,1980 .Toleranc eo fPénicilliu mexpansu m inIsrae l topostharves tfungicid etreatments .Proceeding so fth e5t hCongres so fth e MediterraneanPhytopathologica nUnion ,Patras ,Greece ,21-2 7Septembe r1980 . HellenicPhytopathologica lSociety ,p .158-159 . Rehm,H.J. ,1969 .Inhibitin gactio no fsodiu mo-phenylphenat e (SOPP)an dbipheny lo n specific reactionso fth emetabolis mo fmicroorganisms .Proceeding so fth eFirs t InternationalCitru sSymposiu m3 :1325-1331 .

249 Rosenberger,D.A . &F.W .Meyer ,1979 .Benomyl-toleran tPénicilliu mexpansu mi nappl e packinghouses inEaster nNe wYork .Plan tDiseas eReporte r63 :37-40 . Rosenberger,D.A. , F.W.Meye r& C.V .Cecilia ,1979 .Fungicid e strategies forcontro l ofbenomyl-toleran tPénicilliu mexpansu m inappl estorages .Plan tDiseas eReporte r 63:1033-1037 . Siegel,M.R .& N.N .Ragsdale ,1978 .Antifunga lmod eo factio no fimazalil .Pesticid e Biochemicalan dPhysiolog y9 :48-56 . Smoot,J.J .& G.E .Brown ,1974 .Occurrenc eo fbenzimidazole-resistan t strainso fPéni ­ cilliumdigitatu m inFlorid a citruspackinghouses .Plan tDiseas eReporte r58 : 933-934. Smoot,J.J .& J.R .Winston ,1967 .Biphenyl-resistan t citrusgree nmol d reportedi n Florida.Plan tDiseas eReporte r51 :700 . Str0mnaes,0 ,E.D .Garbe r& L .Beraha ,1964 .Genetic so fphytopathogeni cfungi . IXHeterocaryosi s andth eparasexua lcycl ei nPénicilliu mitalicu m andP .digitatum . CanadianJourna lo fBotan y42 :423-427 . Tuyl,J.M .van ,1975 .Geneti caspect so facquire d resistancet obenomy lan dthiaben ­ dazole ina numbe ro ffungi .Mededelinge nva nd eFacultei tde rLandbouwwetenschap ­ penRijksuniversitei tGen t40 :691-697 . Tuyl,J.M .van ,1977a .Geneti caspect so fresistanc et oimazali li nAspergillu snidu - lans.Netherland sJourna lo fPlan tPatholog y8 3 (Suppl.1) : 169-176. Tuyl,J.M .van ,1977b .Genetic so ffunga lresistanc et osystemi c fungicides.Doctora l Thesis,Agricultura lUniversity ,Wageningen ,Netherlands ,13 7pp . Vonk,J.W .& A .Kaar sSijpesteijn ,1971 .Methy lbenzimidazole-2-y l carbamate,th e fungitoxicprincipl eo fthiophanate-methyl .Pesticid eScienc e2 :160-164 . Wicks,T. ,1977 .Toleranc et obenzimidazol e fungicidesi nblu emol d (Pénicilliumex ­ pansum)o npears .Plan tDiseas eReporte r61 :447-449 . Wild,B.L. ,1980 .Resistanc eo fcitru sgree nmol dPénicilliu mdigitatu m Sacc.t oben ­ zimidazole fungicides.Doctora lThesis ,Universit yo fCalifornia ,Riverside .8 9p . Wild,B.L .& L.E .Rippon ,1975 .Respons eo fPénicilliu mdigitatu m strainst obenomyl , thiabendazolean dsodiu mo-phenylphenate .Phytopatholog y65 :1176-1177 . Yoshikawa,M .& J.W .Eckert ,1976 .Th emechanis mo ffungistati cactio no fsec-butyla - mine.I .Effect so fsec-butylamin eo nth emetabolis mo fhypha eo fPénicilliu mdigi ­ tatum.Pesticid eBiochemistr yPhysiolog y 6:471-481 . Yoshikawa,M.J. ,J.W .Ecker t& N.T .Keen ,1976 .Th emechanis mo ffungistati cactio no f sec-butylamine.II .Th eeffec to fsec-butylamin eo npyruvat eoxidatio nb ymitochon ­ driao fPénicilliu mdigitatu m ando nth epyruvat edehydrogenas e complex.Pesticid e Biochemistryan dPhysiolog y 6:482-490 .

250 Participants of the 1980 and 1981 international post-graduate courses on Fungicide Resistance in Crop Protection

Md.Bahadu rMeah ,Departmen t ofPlan tPathology ,Agricultura l University, Mymensingh,Bangladesh . H.Baltruschat ,Ruh r StickstoffA.G. , Landwirtschaftliche Forschung Hanninghof,Hanningho f35 ,D-440 8Dülmen , F.R.G. D. Baumert,Scherin gA.G. ,Gollancz-Strass e 91-99,D-1000 ,Berli n28 , F.R.G. B.Berggren ,Departmen t ofPlan t and ForestProtection ,Swedis h University ofAgricultura l Sciences,P.O . Box 7044, S-7500 7Uppsal a Sweden. M.Besri , InstitutAgronomiqu e etVétérinair eHassa n II,B.P .704 , Rabat-Agdal,Morocco . G.J. Bollen,Laborator y ofPhytopathology ,Agricultura l University, Binnenhaven 9, 6709P D Wageningen,Th eNetherlands . K.J. Brent,Lon gAshto nResearc h Station,Lon gAshton ,Bristo l BS189AF , England,Unite d Kingdom. I.F.Brown ,El iLill y andCompany ,Agricultura l ResearchCenter , Greenfield, Indiana 46280, U.S.A. G.C.A.Bruin ,Departmen to fEnvironmenta l Biology,Universit y of Guelph,Guelph ,Ontari oNI G2WI ,Canada . H.Buchenauer , Instituteo fPlan tDiseases ,Nussalle e 9, 5300Bonn ,FRG . R.W.J. Byrde,Lon gAshto nResearc h Station,Lon gAshton ,Bristo l BS189AF , England,Unite d Kingdom. G.A. Carter,Lon gAshto nResearc h Station,Lon gAshton ,Bristo l BS189AF , England,Unite d Kingdom. L.C.Davidse ,Laborator y ofPhytopathology , Agricultural University, Binnenhaven 9, 6709P D Wageningen,Th eNetherlands . A.Davis ,E.I .Dupon tD eNemour s &Co. ,Experimenta l Station,Wilmington , Delaware 19898, U.S.A. A.J.Dawson , SunkistGrower s Inc.,Researc h andDevelopmen tDepartment , 760E .Sunkis t Street,Ontari o CA 91761, U.S.A. J.Dekker ,Laborator y ofPhytopathology ,Agricultura l University, Binnenhaven 9, 6709P D Wageningen,Th eNetherlands .

251 N.Delen ,Departmen t ofPhytopatholog y andAgricultura l Botany,Uni ­ versity ofEge , Izmir,Turkey . C.J. Delp,E.I .Dupon tD eNemour s & Co., Biochemical Department,Expe ­ rimental Station,Wilmington ,Delawar e 19898, U.S.A. M.Didier ,Departmen t ofPhytopathology , InstitutNationa l de laRecherch e Agronomique,Rout e de St.Clément ,4900 0Angers ,France . P.Diepenhorst ,Luxa nB.V. , Industrieweg 2,666 2P A Eist,Th eNether ­ lands. K.Dorn ,Dr .R .Maa gA.G .CH-815 7Dielsdorf , Switzerland. G.Doudoulakakis ,Helleni c Sugar Industry,Orestia s SugarFactory , P.O. Box 6,Orestias ,Greece . C.A. Drandarevski,Celamerc k Biological Station,D-6501 , Schwabenheim, F.R.G. J.W. Eckert,Departmen t ofPlan tPathology ,Universit y ofCalifornia , Riverside,Californi a 92502, U.S.A. M.J. Faulkner,Lill yResearc h Centre Ltd,Ear lWoo dManor ,Windlesham , Surrey,England ,Unite d Kingdom. F. Florelli,Procida-RU , Field TestingDepartment ,2 7ru eMauric eBer - teany, 78540Vernouillet ,France . J.F. Fortes,Embrapa ,Kepa e deCascata ,Caix aPosta l403 ,96.10 0Pelo ­ tas, R.S.Brazil . A. Fraile,Departmen t ofPlan tPathology ,Escuel a Technica de Ingenieros Agronomos, Cindad Universitaria,Madri d 3,Spain . J.R. French,Diamon d Shamrock Corporation,Agricultura l Chemicals Division, 1100 SuperiorAvenue ,Cleveland , Ohio 44114, U.S.A. A. Fuchs,Laborator y ofPhytopathology ,Agricultura l University, Binnenhaven 9, 6709P D Wageningen,Th eNetherlands . L.V. Gangawane,Soi lMicrobiolog y &Pesticid e Laboratory,Botan y Department,Marathwad a University, Aurangabad 431004,India . A.Gamier , JanssenPharmaceutic aN.V. ,Agricultura l Division, Turnhoutseweg 30,B-234 0 Beerse,Belgium . S.G. Georgopoulos,Athen s College ofAgricultura l Sciences,Votanikos , Athens 3001,Greece . J.D. Gilpatrick,Ne wYor kAgricultura l Experiment Station,Geneva , NewYor k 14456,U.S.A . M.J.Griffin ,Agricultura l Development andAdvisor y Service,Governmen t Buildings,Burghil l Road,Westbury-on-Trym , Bristol BS10 6NJ,England , United Kingdom. M.L. Gullino, Institute ofPlan tPathology ,Vi aGiuri a 15,1012 6Torino , Italy. M.J.Henry ,Departmen t ofBotany ,Universit y ofMaryland , CollegePark , Maryland 20742, U.S.A. J.Herzog , SwissFedera lResearc h Station forFrui tgrowing ,Viticultur e andHorticulture ,CH-882 0Wädenswill , Switzerland.

252 L.D.Houseworth , Ciba-Geigy Corp.,P.O . Box 11422,Greensboro ,Nort h Carolina 27409, U.S.A. Ph. Ioannidis,Laborator y ofPhytopathology ,Helleni c Sugar Industry, Sugar Factory ofLarissa ,Larissa ,Greece . F.E.O. Ikediugwu,Departmen t ofBiologica l Sciences,Universit y ofBenin , Benin City,Nigeria . A.L. Jones,Departmen to fBotan y andPlan tPathology ,Michiga n StateUni ­ versity,Eas tLansing ,Michiga n48824 ,U.S.A . A.Kaar s Sijpesteijn, Institute forOrgani c ChemistryTNO ,Croese - straat79 ,350 2J A Utrecht,Th eNetherlands . T.Katan ,Departmen t ofPlan tPathology ,Volcan i Center,P.O . Box 6, Bet-Dagan 50-200,Israel . A.Kerkenaar , Institute forOrgani c ChemistryTNO ,Croesestraa t79 , 3502 JA Utrecht,Th eNetherlands . J. Kiebacher,Hoechs tA.G. , Landwirtschaftliche Entwicklungsabteilung, Hessendam 1-3, 6234Hattersheim , F.R.G. M.Kosi mKardin ,Departmen t ofPlan tPathology ,Universit y ofMinnesota , 1519Gortne rAve. ,St .Paul ,M N 55108,U.S.A./Centra lResearc hInsti ­ tuteo fAgriculture ,Jala nMerdek a 99,Bogor , Indonesia. K.H.Kuck ,Baye rA.G. ,Agrochemical sDivision ,Biologica l Research, D-5090Leverkusen , F.R.G. LimTon gKwee ,Departmen t ofPlan tProtection ,Universit y ofAgricultur e Malaysia,Serdang , Selangor,Malaysia . G.D. Laude,Merc k Sharp& Dohme , 3Avenu eHoche ,75008 ,Paris ,France . H.M. LeBaron,Ciba-Geig y Corp.,P.O . Box 11422,Greensboro ,Nort h Carolina 27409,U.S.A . T.Locke ,Agricultura l Development andAdvisor y Service,Governmen tBuil ­ dings,Burghil l Road,Westbury-on-Trym , Bristol BS106NJ ,England , United Kingdom. D. Lorenz,Landes -Lehr -un d Forschungsanstalt,D-673 0Neustad t 1,FRG . G. Lorenz-Spengler,BASF ,Versuchsstatio nAbteilun g Fungizidforschung, 6703 Limburgerhof, F.R.G. N.E. Malathrakis,Plan tProtectio n Institute,Heraklion ,Crete ,Greece . B.Mallet ,Rhône-Poulenc , 13,Qua i Jules Guesde,9440 0Vitry/Seine ,France . H.Maraîte ,Laboratoir e dePhytopathologie ,Universit é Catholiqued e Louvain,Plac e Croix du Sud 3,B 1348Lourai nL aNeuve ,Belgium . R.T.Mercer ,Ma y& Bake rLtd ,Onga rResearc h Station,Fyfiel d road, Ongar, Essex,CM SOHW ,England ,Unite d Kingdom. Y.Miyagi ,Biologica l ResearchCenter ,Niho nNohyak u Co,4-3 1 Kawachi- Nagano,Osak a 586,Japan . T.Nakanishi ,Sanky o Co.Ltd ,Yasu-Gun ,Shiga-Ken ,Japan . P.M. Nelson,FM CCorporation , CitrusMachiner y Division, 6446 Fremont street.Riverside ,Californi a 92504,U.S.A .

253 B.J. Nielsen,Nationa l Research Centre forPlan tProtection , Institute forPesticides ,Lottenborgve j2 ,280 0Lyngby ,Denmark . H.Nowacka ,Researc h Institute ofPomolog y and Floriculture, ul.Pomolo - giczna 18,96-10 0 Skierniewice,Poland . D.M. Olivier, Stationd ePathologi e Végétale,Rout e de SaintClément , Beaucouze-49000,Angers ,France . H.Olvang ,Departmen t ofPlan tan d ForestProtection , Swedish University ofAgricultura l Sciences,P.O . Box 7044, S-7500 7Uppsala ,Sweden . A.C. Pappas,Benak iPhytopathologica l Institute,Kiphissia ,Athens , Greece. J.B.va nde rPas ,Fores tResearc h Institute,Privat eBag ,Rotorua , New Zealand. L.Pere zVicente ,Cuban aPlan tProtectio nDivision ,Havana ,Cuba . N.Petsikos-Panayotarou , Benaki Phytopathological Institute,Kiphissia , Athens,Greece . H. Rattink, Research Station forFloriculture ,Linnaeuslaa n2A , 1431J V Aalsmeer,Th eNetherlands . Abdel-RazekOsman , Faculty ofAgriculture ,Cair oUniversity , Giza,Egypt . P.Reinecke , Institute forPlan tPathology , BayerA.G. ,D-509 0Leverkusen , F.R.G. B. Sachse,Pflanzenschutsforschun g Biologie,Hoechs tA.G. , 6230 Frankfurt amMai n80 ,F.R.G . P.L. Sanders,Departmen t ofPlan tPathology ,Pennsylvani a StateUniversity , 211Buckhou tLaboratory ,Universit y Park,Pennsylvani a 16802,U.S.A . H. Scheinpflug, Institute forPlan tPathology ,Baye rA.G. ,D-509 0Lever ­ kusen, F.R.G. W.J. Scholtens,BAS FNederlan d B.V., P.O. Box 1019,680 1M C Arnhem TheNetherlands . J. Schreurs,Merc k Sharp& Dohme ,Waarderwe g 39,203 1B N Haarlem, The Netherlands. F.J. Schwinn,Ciba-Geig y Ltd, Schwarzwaldallee 215,CH-400 2Basel , Switzerland. H. Siegle,Dr .R .Maa gA.G. , CH-8157Dielsdorf , Switzerland. H.D. Sisler,Departmen t ofBotany ,Universit y ofMaryland , CollegePark , Maryland 20742, U.S.A. J.L. Smilanick,Departmen t ofPlan tPathology ,Universit y ofCalifornia , Riverside,Californi a 92502, U.S.A. J.H. Smith,Consolidate d CitrusEstate s Ltd,P.O . Box 6, Letaba 0870, Republic of SouthAfrica . D. Snieder,Colleg e ofAdvance d Agriculture KNTB,Bade nPowellstraa t 1, 5212B W 's-Hertogenbosch,Th eNetherlands . D. Sozzi,Ciba-Geig y Ltd, Schwarzwaldallee 215,400 2Base l Switzerland. Th. Staub,Ciba-Geig y Ltd, Schwarzwaldallee 215,400 2Basel ,Switzerland . A. Strömberg,Kenogar dA.B. ,P.O . Box 11033,S-10 06 1 Stockholm, Sweden.

254 T.B. Sutton,Nort hCarolin a StateUniversity , P.O. Box 5397,Raleigh , NorthCarolin a 27609,U.S.A . A. Svanold, SwedishUniversit y ofAgricultura l Sciences,P.O . Box 7044, 75007Uppsala ,Sweden . A.Tempel ,Researc h Institute forPlan tProtection ,Binnenhave n12 , 6700A A Wageningen,Th eNetherlands . J.M. vanTuyl , Institute forHorticultura l PlantBreeding ,Mansholt - laan15 ,670 0A A Wageningen,Th eNetherlands . Y.Uesugi ,Nationa l Institute ofAgricultura l Sciences,Divisio no f Agricultural Chemicals,Yatabe-machi ,Tsukuba , Ibaraki 305,Japan . M.A.d eWaard ,Laborator y ofPhytopathology ,Agricultura l University, Binnenhaven 9, 6709P D Wageningen,Th eNetherlands . M.Wade ,Shel lResearc h Ltd,Sittingbourn eResearc h Centre,Sitting - bourne,Ken tME 9 8AG,England ,Unite d Kingdom. E.J.G.L.va nWambeke ,Laborator y ofPhytopatholog y andPlan tProtection , Faculty ofAgronomy ,Kardinaa lMercierlaa n92 ,B-303 0Heverlee ,Leuven , Belgium. M.S. Wolfe,Plan tBreedin g Institute,Mari s Lane,Trumpington , Cambridge CB2 2LQ,England ,Unite d Kingdom. C.P.Woloshuk ,Departmen t ofBotany ,Universit y ofMaryland , College Park,Marylan d 20742,U.S.A . E.C.Wynn ,Nationa l Vegetable Research Station,Wellesbourne ,Warwic k CV35 9EF,England ,Unite d Kingdom. KyoYoun gKang ,Departmen to fPlan tPathology ,Universit y ofCarolina , Riverside,Californi a 92502,U.S.A . J.C.Zadoks , Laboratory ofPhytopathology ,Agricultura l University, Binnenhaven 9, 6709P D Wageningen,Th eNetherlands . M. Ziedan, Institute ofPlan tPathology ,Agricultura l ResearchCentre , Giza,Egypt . J. Ziffer,Departmen to fFoo d engineering andBiotechnology , TheTech - nion, 3200Haifa ,Israel .

255 Sponsors

The following companiesmad e afinancia l contribution to the international course onFungicid eResistanc e inCro p Protection,hel d in198 0 and 1981 inWageningen ,Th eNetherlands .

BASFAktiengesellschaft , Limburgerhof, F.R.G. BayerA.G. ,Leverkusen , F.R.G. TheBoot s Company Ltd,Nottingham ,Unite d Kingdom. CelamerckG.m.b.H. , Ingelheim, F.R.G. Chevron Chemical Company, Paris,France . Ciba-Geigy A.G.,Basel , Switzerland. Diamond Shamrock Corporation, Cleveland, Ohio,U.S.A . E.I. Dupontd eNemour s &Company ,Wilmington ,Delaware , U.S.A. Eli Lilly and Company,Elanc o Products Company,Greenfield , Indiana, U.S.A. HoechstA.G. , Frankfurt, F.R.G. Imperial Chemical Industries Ltd, PlantProtectio nDivision ,Jealott' s Hill,Unite d Kingdom. N.V. JanssenPharmaceutica ,Beerse ,Belgium . Ligtermoet ChemieB.V. , Roosendaal,Th eNetherlands . Merck& Co. ,Rahway ,Ne w Jersey, U.S.A. NihonNohyak u Company Ltd,Osaka ,Japan . PennwaltCorporation , Fresno,California , U.S.A. Charles Pfizer& Company Inc.,Brooklyn ,Ne wYork , U.S.A. Procida,Group eRousse l Uclaf,Paris ,France . ScheringA.G. ,Berlin , F.R.G. Shell Research Ltd,London ,Unite d Kingdom. Uniroyal Chemical,Bethany , Connecticut,U.S.A .

256 Index

'A25822B1,7 3 apoplastic transport,17 9 Achlya radiosa, 37 apple scab, 194,195,197 acquired resistance inpowder y applicationmethod , 185 mildews, 219,220 'Aretan',16 9 acriflavine, 57,93 Armillaria spp., 13 actinomycinD , 107 A. mellea, 164,168 acylalanines,25,88,118,120,121 , aromatic hydrocarbons,54,5 5 135 Aspergillus nidulans, 4,26,35, adenosine deaminase,3 4 48-50,54-56,62,63,65,66,68, adviser,rol eo fthe ,17,1 9 73,80,83,87,89,91,93,94,97- agrochemicalindustry , 17 99,107,109,129,132,247 'Miette', 137 A. nicer, 55,72 allelicmutation ,9 0 Aureobasidium bolleui, 165 Alternaria spp.,13,16 4 'AY-9944', 72,73 A. alternata, 105,109 8-azaguanidine, 93 A. citri, 236 15-aza-24-methylene-D-homo- A. kikuchiana, 3,25,50,134 cholesta-8,14-dien-3ß-ol,73 A. tenuis, 72 azasteroidal antifungalanti ­ alternation of fungicides,147 , biotics,7 3 246 azasterols, 73,74 amensalism,16 3 6-azauracil,16 9 2-amino-pyrimidines, 219,220 amphotericinB , 108 ancymidol, 72,74,77,91 barleypowder ymildew ,219,221 , antagonism, selectivestimu ­ 224,226,227 lationof ,16 7 base line sensitivity, 24 antagonisticmycoflora ,16 5 benomyl,35,55,60-62,107,111 , anthelmintics,61,63,6 7 132,164,165,169,171,173,174, antibiotics, 207,208 188-191,199,200-204,215,220, anti-microtubulemod e ofaction , 221,231,233-235,237-241,243, 62 244,246,247 antimycotics,7 8 benzimidazoles,19,24-26,46,48 ,

257 55,56,60-66,88,94,132,168, C. apii, 153 169,171-173,178,180,187-193, C. arachidicola, 26,171 195-198,200-202,204,205,220, C. beticola, 18,25,26,53,55,132, 229,235,238,239,241,243-247 136,159,187-190,192,193 biochemicalmode s ofaction ,19 6 Cercosporidium personatum, 171 trans-1,4-bis(2-chlorobenzylami- 'CGA49104' , 37 nomethyl)cyclohexane dihy- 'CGA64250' , 36,77 drochloride,7 2 'CGA64251' ,7 7 bitertanol, 36,57,77,187,193 chirality, 78 blasticidin-S, 34,207,210,214 chitin synthetase,3 5 Blastomyces dermatitidis, 73 chloramphenicol, 57,9 3 boomerang effect,167,17 2 chlorinated nitrobenzenes,2 7 borax,24 5 chloroneb, 26,35,50,54 Bordeauxmixture ,20 7 chlorothalonil, 34,166,171,245 Botrytis spp., 13,102,135,165 cholesterol,3 7 B. cinerea, 25,26,55,66,72,83, Chondrostereum spp.,1 3 93,101-104,106,108,109-113, chromosomal loci,50,10 8 165,168,170,190 Cladosporium cucumerinum, 2,73, B. squamosa, 103,105 77,80,81,83,91,94,129,131 B. tulipae, 105 clotrimazole,74,9 1 brown rot,195,19 7 Cochliobolus carbonum, 72 buthiobate, 36,74,135 C. sativus, 164,168,171 butoconazole, 77 colchicine,6 2 sec-butylamine,33,233,234,237 , collateral sensitivity,5 3 239,241-244,246 competition tests,13 0 congeneric series ofcompounds , 78 Candida albicans, 73,89,94,132 consumer, 17,18 C. utilis, 132 control of applescab ,19 6 captafol, 34,166,196,202,203 conventional fungicides,2 captan,34,196,197,199,201-203 , coppercompounds ,12,177,19 7 245 copper oxychloride,11 2 carbamates, 196,199 copper sensitivity, 14 carbendazim,35,48,49,55,61-68 , crop losses, 1,3 93,106,193,235,237,240-242 cropprotectio nmeasures ,1 carbendazim-binding protein,62 , cross-resistance,36,50,53,54 , 63 79,91,109,110,120,121,211, carbendazim generators,5 5 214,215,240 carboxamides,25,26,5 5 crowding, 157,158 carboxin,33,49,55,56,89,90,93 , Cryptococcus neoformans, 73

107,133 CS2, 164 carboxinbindin g site,5 6 cucumber downymildew ,12 1 Ceratocystis spp.,1 3 cucumberpowder ymildew ,219 - Cercospora spp.,1 3 221,224,225,228

258 curzate,3 7 directional selection, 142,144 cycloheximide,26,34,57,93,106 , disease control,degre e of,22 3 107 diseasecycle ,11 3 cymoxamil,12 3 diseasepressure ,11 8 cytochalasins,10 6 disease,rat eo fprogress ,14 9 disruptive selection,14 2 distortiono fhypha l cells,10 6 2,4-D,24 3 disturbance ofcel lwal l 'Daconil',187,19 3 synthesis,10 6 DDT, 17 dithianon,19 3 3'-decyloxycarboxin, 56 dithiocarbamates,11,12,15,168 , degreeningo foranges ,23 3 196-200,202-205 C-14demethylation ,7 9 dodecyl-imidazole,7 4 detoxification, 213,216 dodemorph, 74 'Dexon',33,17 0 dodine, 25,26,36,195,196,203 dicarboximides,25,26,35,50,54 , dominantpathogen ,change sof , 55,101-111,132,135,164,170, 170 179,197,204 dosage-response,2 7 dichlone, 34,196,197 downymildews ,1 3 diclozoline, 101,102 dyrene,2 5 diclobutrazol,77,7 8 dicloran,35,50,54,102,110,197 , 244 econazole,7 4 dicyclidine,13 5 economic impacto fresistance , iV,iV-dicyclohexylcarbodiimide, 95 18 ß-diethylaminoethyldiphenylpro- edifenphos,35,133,207,211-213 , pyl acetatehydrochloride ,7 2 215 dimethirimol,34,133,219-221 , •EL-241', 74,91 224-226,228 Emericella nidulans, 4 iV-dimethylaminoethyl-a,a-diphe- enantiomerism,7 8 nylvaleramid ehydrochloride , energy-dependent efflux offena - 72 rimol, 94,96,98 dimethyldithiocarbamates,3 3 ergosterol,3 6 dinitrophenols,33,22 0 ergosterol-biosynthesis inhib­ dinocap, 14,33 itors,49,72,87,135,197,203 , diphenyl,25,26,35,50,54,234 , 204 236,237,243,244,246 ergosterol synthetic pathway, 74 2,2-diphenyl-l-(ß-dimethylami- Erwinia amylovora, 134 noethoxy)pentanehydrochlori ­ Erysiphe graminis, 140,156 de, 72 E. graminis f.sp . hordei, 80, 2,2-diphenylpentanoi c acid,7 2 81,145,148 Diplodia natalensis, 235 E. graminis f.sp . tritici, 80 D. natalensis P.Evans ,23 2 ethirimol,34,133,145,147,219 - difolatan,19 3 222,224,226-229

259 ethirimol sensitivity, 226 Gauemannomyces spp.,1 3 ethylene,24 3 'Galben', 121,123 ethylenebisdithiocarbamates,15 , gene action,4 9 197 genetic control,5 0 etridiazole,3 7 Geotrichum spp.,1 3 eukaryotes, 64 G. candidum, 236 Eutypa armeniacae, 169 G. flavo-brunneum, 73 evolution,1 germination ofcucumbe r powdery mildew spores,22 1 grapes, Botrytis on,13 5 fenarimol,36,50,57,72-74,87 , griseofulvin, 55,107 91,93-98,108 fenbendazole, 61,63,66 fenpropimorph,36,74,7 8 ffe.bespp. , 103 fentin,2 6 Heliothis virescens, 17 fentinacetate , 33,188,191-193 H. zea, 17 fentinderivatives ,53,18 7 Helminthosporium spp.,13,17 3 fentinhydroxide , 188-192 H. erythrospilum, 169 fitness H. sativum, 72 conceptof ,149,15 1 heterokaryosis,17 9 parameters, 130,136 hexachlorobenzene, 35,50,54 reduced, 131 Histoplasma capsulatum, 73 ofresistan t strains,20 2 horizontal resistance,15 0 p-fluorophenylalanine, 55,93 hydroxyisoxazole,3 5 fluotrimazole,74,7 7 hydroxypyrimidines,13 3 folpet,3 4 hypocholesteremic agents,72,7 3 formaldehyde,24 4 fosetyl-Al,12 3 free fatty acid fraction,10 7 iatrogenic diseases,16 7 fuberidazole,6 1 IBP, 35,207,212-216 fungicide-resistance genes,23 5 imazalil,36,50,57,74,91,239 , fungicide resistance inpowder y 246-248 mildews,220,221,22 3 imidazoles, 71,77,79,135 furalaxyl, 34,119-121,123,135 infection threshold, 179 Fusarium spp.,13,164,168,170 , inhibition of 172 cellwal l synthesis,3 5 F. lateritium, 169 lipidbiosynthesis ,3 5 F. moniliforme, 72 nucleic acid synthesis,3 4 F. oxysporum, 25,35 protein synthesis,3 4 F. oxysporum f. sp. narcissi, inhibitors of lipidsynthesis , 131 72 inoculumpressure ,19 7 inosine, 34 integrated control,147,16 4

260 iprodione,35,54,82-84,101-103 , Microsporum gypseum, 73 106-108,110,135,166 microtubules,35,60,62-64,67 , isoconazole,7 7 107 isoprothiolane,35,184,207,211 - milfuram, 121,123 213,215 mitosis, 35,66 mitotic effect,10 7 mitotic segregation,5 4 kasugamycin,25,34,134,206,208 - mitotic spindle,10 7 211,214,215 mixtures of fungicides,147,24 6 ketonazole,7 4 Monilinia spp.,102,135,195,201 , 'KitazinP' ,57,13 8 202,204,205 M. fructicola, 197,202 M. fructigena, 197 lifetables ,15 2 M. laxa, 197 Lolium perenne, 173 monitoring, 29,140,192 lowwate r solubility of morpholines, 71,74,79,135 fungicides,2 7 Mucor spp.,17 0 M. mucedo, 37 multi-site compounds,14 0 Magnaporthe grisea, 210 multi-site fungicides,20 2 mammalian tubulin,6 7 multi-site inhibitors,13 0 managementproblems ,3 mutation frequency,89,91,129 , mancozeb, 123,125,196,202,203 136 maneb,34,187,193,197,24 5 mycoparasitism, 162 MBC, 61 Mycosphaerella fijiensis, 48 mebendazole, 61,63,66 M. musicola, 48 mebenil,3 3 mechanism ofactio no ffungici ­ des, 32,178 NADHdehydrogenation ,3 3 mercurials,1 2 national regulatory authorities, mercury, 14,179 17,20 metalaxyl,34,118-121,123-126 , naturalresistance ,9 4 135 Nectria spp.,1 3 metalaxyl, adaptationto ,12 4 N. haematococca, 4,25,26,47,50, methfuroxam,3 3 108 2-methoxyethylmercurychloride , negatively correlatedcross - 169 resistance,53,184,185,239 , methylbromide,16 4 246 3'-methylcarboxin,5 6 neomycin,57,9 3 iV-methyl-W-nitro-iV-nitrosogua - Neurospora crassa, 4,26,47,101, nidine, 121 108,109,112 miconazole, 74,89,91,108 nitrosoguanidine,23 9 microbial antagonism, 168 nocodazole, 61-64,66-68 Microdochium bolleyi, 165 non-mercurial fungicides,20 7

261 nuarimol, 57,74,187,193 phenylmercury compounds,20 7 nutrientcompetition ,16 3 o-phenylphenol,23 6 nystatin, 108,131,132,134 Phialophora cinerescens, 91 Phomopsis citri, 236 P. citri Fawc, 232 obligateparasites ,22 1 phosphatidyl choline,3 5 oligomycin,47,5 3 phospholipid biosynthesis,21 3 organic fungicides,23 3 phosphoramidates,57,184,211 - organic tincompounds ,13 6 214 organomercurials,25,165,16 8 phosphorothiolates, 57,184 organophosphorus fungicides, phthalimides,12,1 5 206,207,211,212,215,216 Phycomycetes ,3 7 organophosphorus thiolate,21 3 Phymatotrichum spp.,1 3 organotinresistance ,19 2 Phytophthora spp.,13,37,118 , organotins, 25,189,191-193 124,168 osmoticmutants ,108,109,11 2 P. cactorum, 72,170 oxathiincarboxanilides ,49,9 4 P. capsici, 118,120-122,124 oxibendazole, 61,63,66 P. citrophthora, 236 oxycarboxin,5 6 P. cryptogea, 172,173 oxydativephosphorylation ,3 3 P. cubensis, 125 P. infestans, 3,118,120-125,135 P. megasperma f. sp. glycinea, parbendazole, 61,63,67 122 pear scab,195-19 7 P. megasperma f. sp. medicaginis, Pénicillium spp.,165,169,232 , 118,125,135 236-239,243-248 P. nicotianae, 122,135 P. brevicompactum, 165 P. parasitica, 122 P. cyclopium, 165 pimaricin,93,108,129,131,132 , P. digitatum, 33,231,232,234, 134,168,169,181 236-241,243,246,247 piperazines, 71,74 P. digitatum Sacc, 232 planttubulin ,6 6 P. expansion, 55,63,65,68,80,108, plasmatoxicants, 4 237-239,247 Plasmodiophora spp.,1 3 P. italicum, 83,98,231,232,234, pleiotropic mutation,9 3 236-239,247 Podosphaera leucotricha, 220 P. italicum Wehm.,23 2 polyenemacrolides , 131 P. janthinellum, 169 polyols,10 8 Peronosporales, 34,37,118 polyoxinD , 25,35,107 Peronospora parasitica, 121 population genetics,15 3 pestmanagemen t approach,20 3 positively-correlated phenapronil,36,7 4 fungicides, 54 Ä-phenyl-carbamateherbicides , post-harvest decay ofcitru s 55 fruits,23 2 4'-phenylcarboxin,5 6 post-harvest benzimidazole

262 treatment,23 2 quarternary ammoniumcompounds , potato lateblight ,12 3 244 powdery-mildew fungicides,22 1 quintozene,35,50,102,110,164 , powderymildew , 13,219 168-170,172,174 ofapple ,22 0 ofgrape-vine ,22 0 ofrose ,22 0 'RE26745' , 121 PP, 37,50 'RE26940' , 121 probenazole,37,207,21 5 relative fitness of resistant probiosis,16 2 strains,5 prochloraz, 36,57,74 resistance procymidone,35,54,84,101,102 , buildu pof ,177,181,182,185 , 105-107 243 proneness toresistance ,19 6 conditions favouring increase propamocarb, 37,123 of,22 8 propineb,19 3 countermeasures for,17 7 4'-propylcarboxin,5 6 degreeof ,24,27,2 8 prothiocarb,3 7 extentof ,24,2 9 Pseudocercosporella spp.,1 3 toacylalanines ,12 0 P. herpotrichoides, 26,172,180 tobenomyl , 89,153,154,159 Pseudoperonospora cubensis, 118, tobenzimidazoles ,48,55,62 , 121,123 164,200,204,237,239,241,243, Puccinia graminis, 156 246 P. horiana, 133 toblasticidin-S , 50,211 pyracarbolid, 33,56 tosec-butylamine ,23 7 pyrazophos, 37,46,50,134 tocarbendazim ,48,57,66 , Pyrenophora avenae, 25,165 239-241 Pyricularia oryzae, 3,25,34,35, tocarboxin ,8 9 50,57,133,134,206,208,210- tochloramphenicol ,9 0 212,214-216 todicarboximides ,50,109 , pyridines, 71,74 110,112,113,164 pyrimidine carbinols,22 0 todiphenyl ,23 6 pyrimidines,19,71,74,79,135 , tododine ,50,159,18 1 224 toethirimol ,145,22 6 pyruvatedehydrogenation ,3 3 to fenarimol,84,90,93-95 , Pythium blight,17 3 97 Pythium spp.,12 2 to IBP,213,21 4 P. aphanidermatum, 173 to imazalil,80,89,90,93,96 , P. debaryanum, 169 247 P. irreguläre, 173 to isoprothiolane,211,213 , P. splendens, 34 215 P. ultimum, 118,120,122 tokasugamycin , 50,210,215 tometalaxyl ,120,12 1 tomiconazole ,9 4

263 tooligomycin ,4 7 site-specific compounds,14 0 toorganophosphoru sfungici ­ 'SKF525-A' ,7 2 des, 211,215 'SKF2314' , 72 tooxycarboxin ,13 3 'SKF330 1A' ,7 2

topolyoxins ,50,13 4 'SKF7732-A 3', 72

topyrazophos ,5 0 'SKF7997-A 3', 72 toquintozene ,16 4 'SKF16467-A' ,7 2 tosodium-o-phenylphenat e sodium carbonate, 234,243,245 (SOPP), 236 sodiumpentachlorophenate , 197 tothiabendazole ,57,6 2 sodium o-phenylphenate (SOPP), totriarimol ,80,9 3 234,236,237,239,241-244,246, totridemorph ,98,14 6 247 to triforine,80,9 4 sodium 2-phenylphenolate,25,2 6 socio-economic impact,1 6 sodium tetraborate,23 4 socio-economic problems,1 7 somatic segregation, 107,108 resistantvariants ,109-11 4 SOPP-resistance locus,23 9 Rhizoctonia spp.,13,7 2 Sordaria fimicola, 72 R. cerealis, 171,173 specific-site inhibitors,129 , R. solani, 168 132,136,179 Rhizopus spp.,17 0 Sphaerotheca fuliginea, 154,220 riceblast ,20 6 S. pannosa, 220 •Ridomil',13 5 Sporobolomuces roseus, 49,66 'Ronilan',10 1 Sporotrichum schenckii, 73 rootro to f alfalfa,12 1 springbarley ,22 8 •Rovral',10 1 stabilizing selection,14 2 stable resistance,12 4 Stemphylium spp.,1 3 Saccharomyces cerevisiae, 4,26, stereoisomerism, 78 49,72,73 stereoisomers,7 9 Sclerotinia spp.,102,13 5 stereoselectivity, 79 S. homoeocarpa, 25,172 sterol-biosynthesis inhibitors, S. fructicola, 25 36,57 S. minor, 164,171 strawberries, Botrytis on,13 5 Sclerotium cepivorum, 166 streptomycin, 34,134 S. rolfsii, 164,169,171 sub-population, 184 •Sclex1, 102 succinate dehydrogenation,3 3 selectionpressure ,133,134,153 , succinate-ubiquinone reductase 180-182,185,190,202,224,235 complex,3 3 selectivepressure ,195,20 5 sugar-beet, 187-190 Septoria spp.,1 3 sugar-beet leafspot,187,190 - S. nodorum, 26 193 D-serine, 93 sulconazole,7 7 single-genemutation ,9 1 sulphur, 14,196 single-site inhibitors,17 8 'Sumilex',10 2

264 'Sumisclex',10 2 tubulin,bindin g affinity of,6 0 survivorship tables,15 2 systemic fungicides, 2,11 UDP-tf-acetylglucosamine,3 5 Uncinula necator, 220 tecnazene,5 4 Uromyces appendiculatus, 80 tetrachlorophthalide, 207,215 U. fabae, 80 thiabendazole,35,55,61,63,67 , Ustilago avenae, 106,247 233-235,237-240 V. hordei, 4,133 thiabendazole loci,23 9 V. maydis, 4,26,47,49,55,56,72, thiophanatemethyl ,35,189,235 , 73,91,93,94,129,133 237 thiophanates,6 0 thiourea,9 3 vector-borne disease,1 7 thiram, 33,169 vector resistance,1 7 tioconazole,7 7 Venturia spp.,13,195,200-202 , transientresistance ,94,118 , 204,205 124,224 V. inaequalis, 3,4,18,26,47,50, triadimefon, 36,57,77,80,193 159,181,195-202,204 triadimenol,36,77,7 8 V. nachicola, 195,197,200 trialkyltinderivatives ,4 9 V. pirina, 195,197,200 triarimol, 36,72,74,80,108 Verticillium spp.,1 3 triazoles,71,77,79,135,22 0 V. fungicola, 172 Trichoderma spp.,164,165,168 , V. malthousei, 49,66 169 vinclozolin,35,54,84,101,102 , T. harzianum, 166 106,107,110,135,204 T. viride, 164,169,171 virulence, 112-114 Trichophyton mentagrophytes, 73 tricyclazole,3 7 tridemorph,36,57,74,80,81,88 , winterbarley , 227 146,228 world foodproduction ,1 trifluperidol,72,7 4 worldpesticid emarket ,8 triforine,36,57,73,74,78,80 , 88,135,181,204,220 triparanol, 72,74 •XE326' , 74 triphenyltinfungicides ,33,49, 132,192 tris (2-diethylaminoethyl) zineb, 14,34 phosphate trihydrochloride, 72 tris (2-dimethylaminoethyl) phosphate trihydrochloride, 72 (ß-)tubulin,48,60,63,65-6 8

265