Recent advances in Botrytis research
Proceedings of the IOt h International Botrytis Symposium, Heraklion, Crete, Greece 5- 10Apri l 1992
K. Verhoeff, N.E. Malathrakis & B.Williamso n (Editors)
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Printed in the Netherlands Bibliotheek Planteziektenkundig Centrum Binnenhaven 8 -Postbu s 8122 6700 ER WAGENINGEN Table of contents
Page
Preface 1
Introduction 2
Genetics 5
Genetic studieso fBotryotinia fuckeliana (Botrytis cinerea) -Faretra, F. and Grindle, M. 7 Moleculargeneti canalysi s ofpathogenesi s ofBotrytis cinerea - Van Kan, J.A.L., Bergmans, C.J.B. andVan 'tKlooster, J.W. 18 Toolsfo r moleculargeneti c analysiso fBotrytis cinerea -Bergmans, C.J.B. and VanKan, J.A.L. 22 Genetics ofCrea mmutant sof Botryotinia fuckeliana (Botrytis cinerea) withaberran tproductio n oflaccas eenzym e- Faretra, F. andMayer, A.M. 25 Pectindegradatio nb yBotrytis cinerea: amolecula rgeneti c approach - / Kusters-van Someren, M.A., Manders, B.J.G. andVisser, J. 30
Host-Pathogen interaction andphysiolog y
Botrytis cinerea: host-pathogen interactions- Kamoen, 0. 39 Activation ofhos tdefenc e mechanisms inrespons et oBotrytis cinerea - Heale, J.B. 48 Productioni nvitr oo fpolygalacturonase s byBotrytis cinerea -Johnston, D.J. andWilliamson, B. 59 Significance of polygalacturonase production byBotrytis cinerea inpathogenesi s- Leone, G. 63 Polygalacturonases ofBotryti scinere a - Van derCruyssen, G. andKamoen, O. 69 Thefunctio n oflaccas efro m Botrytis cinerea inhos tinfectio n - Viterbo, A., BarNun, N.and Mayer, AM. 76 Relationshipbetwee n greymoul d ofgrape san dlaccas eactivit y inth emus t- Roudet, J., Prudet, S.and Dubos, B. 83 Enzymaticdetoxificatio n of stilbenesb yBotrytis cinerea andinhibitio nb ygrap e berries proanthocyanidins -Pezet, R., Pont, Vand Hoang-Van, K. 87 Differing biochemical andhistologica l studieso ftw ograp ecultivar s inth evie w of theirrespectiv esusceptibilit y andresistanc et oBotryti scinere a -Pezet, R. andPont, V. 93 Somecharacteristic s ofresistanc eo fgrap eberrie st ogre ymoul dcause db y Botrytiscinerea -Prudet, S., Dubos, B.and Le Menn, R. 99 Invitr obioassay st oevaluat eth erelationshi pbetwee n grapevines andBotrytis cinerea -Bessis, R., Vannel, D.and Barbier, M. 104 Theus eo fphytoalexi n induction ando f invitr omethod s asa too lfo rscreenin g grapevines for resistancet oBotrytis cinerea -Jeandet, Ph., Sbaghi, M. and Bessis, R. 109 Ethyleneproductio n andgrowt h ofBotrytis cinerea inkiwifrui t asinfluence d by temperature andlo woxyge nstorag e- Niklis, N. D., Thanasoulopoulos, C.C. andSfakiotakis, E.M. 113 Influence ofplan tgrowt h regulators onmycelia lgrowth ,germinatio n ofconidi a andpathogenicit y ofBotrytis cinerea -Benlioglu, S.and Yümaz, D. 119 Thephysiolog yo fconidiatio n inBotrytis squamosa - Balis, F.J. andLorbeer, J.W. 123 Dectection ofBotrytis cinerea ongerber aflower s using monoclonal antibodies- Salinas, J.and Schots, A. 127 Theeffec t ofgamm airradiatio n onBotrytis cinerea andB. aclada causin g roto f pearan donio nrespectivel y -Gürer, M. andTiryaki, 0. 133 Impedance mycology,a ne wrapi d method for theevaluatio n ofmetaboli c activity ofBotrytis cinerea andB. aclada asinfluence d byenvironmenta lfactor s - Nielsen, P.V. andKampp, J. 136 Nuclearmagneti cresonanc e(NMR )microimagin g ofsof t fruits infected by Botrytis cinerea - Williamson, B.,Goodman, B.A., Chudek, J.A. and Johnston, D.J. 140
Epidemiology
Epidemiology ofgre ymould ,cause db yBotrytis cinerea invegetabl egreenhouse s- Elad, Y, Stienberg, D.,Yunis, H. andMahrer, Y. 147 Epidemiology ofBotrytis cinerea ingerber a andros egrow ni nglasshouse s- Kerssies, A. 159
Biological Control
Biologicalcontro lo fBotrytis: state-of-the-art -Dubos, B. 169 Biologicalcontro lo fBotrytis cinerea ongrapevin eb ycompos textract s andthei r microorganisms inpur ecultur e- Ketterer, N, Fisher, B.and Weltzxen, U.C. 179 Effect ofth esubstrate ,temperatur e andtim eo fapplicatio n onth eeffectivenes s of threeantagonisti c fungi againstBotrytis cinerea -Malathrakis, N.E. and Krisotaki, O. 187 Biologicalcontro lo fBotrytis lea f blighto fonions :significanc e of sporulation suppression -Kohl, J.,Molhoek, W.M.L., Van derPlas, C.H., Kessel, G.J.T. andFokkema, N.J. 192 Biological controlof Botrytis ro to f apple- Gullino, M.L., Benzi, D., Aloi,C. and Garibaldi, A. 197 Biologicalcontro l ofBotrytis cinerea oncol d storeddutc hwhit ecabbag e- Leiffert, C, Sigee, D.C. andEpton, H.A.S. 201 Droughttoleranc eo fBotrytis squamosa, B.aclada andpotentia lantagonis t- Kohl, J.,Krijger, M.C. andKessel, G.J.T. 206 Efficacy ofa grapefrui t seedextrac tBC-100 0fo r control ofBotrytis cinerea in tablegrap ei nChil e- Esterio, M.A., Auger, J.G., Vazquez, E., Reyes, M. and Scheelje.F. 211
Chemicalcontro l
Chemicalcontro lo fBotrytis spp. - Gullino, M.L. 217 Action ofstero lbiosynthesi s inhibitors againstBotrytis cinerea -De Waard, M.A. andStehmann, C. 223 Investigations onth edicarboximid e resistanceo fBotrytis cinerea. II.Incomplet e crossresistanc ei nth eRheinga u -Borge, J.F.R., Abolgassani, N. and Schlösser, E. 228 Stability ofbenzimidazol e resistancean ddiethofencar b sensitivityo fBotrytis cinerea -Ishii, H., Fukaya, M., Faretra, F., Takeda, F. andTomita, Y. 232 Effectiveness ofsom efungicide s andfungicid e combinationso nBotrytis cinerea isolates- Delen, N.and Ozbek, T. 238 Thechemica lcontro l ofBotrytis cinerea infrui t culture- Creemers, P. 242 Effect offungicid e application techniqueso nth econtro lo fBotrytis cinerea and development offunga l resistance- Besri, M.and Diatta, F. 248 Effect ongre ymoul d ofpresenc eo fBotrytis cinerea strainsshowin g reduced sensitivity todichlofluani d -Pappas, A.C. andElena, K. 252 Controlo fBotrytis cinerea incu tros eflower s bygibberelli c acid,ethylen e inhibitorsan dcalciu m -Shaul, O., Elad, Y,Krishner, B., Volpin, H. and Zieslin, N. 257 Mannansfro m Botrytis cinerea strainsresistan tt odicarboximid efungicide s - Kamoen, O., Geeraert, H.and Van der Cruyssen, G. 262 Bacillus brevis asa biocontro l agentagains tBotrytis cinerea onprotecte dchines e cabbage- Edwards, S.G. andSeddon, B. 267 Integration ofbiologica l andchemica lcontro l for greymoul d -Elad, Y and Zimand, G. 272 Controlof Botrytis spp.i ntuli pwit h reduced inputo fchemica lcro pprotectio n- Koster, A.ThJ. andVan derMeer, L.J. 277 Controlo fgre ymoul d of tomatoesi ngreenhouse s withfungicide s and antagonists- Malathrakis, N.E. andKlironomou, E.J. 282
Namesan daddresse so fParticipant san dAuthor s 287 Preface
Since 1964, plant pathologists working on diseases caused by Botrytisspp . have met regularly in the so-called European Botrytis symposia and ten of these symposia have beenheld . Atth elas fivet symposia ,specialist so nthi ssubjec tfro m othercontinent sals o participated inth ediscussions .A tth esymposium ,hel d in 1976,i twa s suggested thata book on the state-of-the-art in Botrytis research should be produced. As a result, 'The Biologyof Botrytis' waspublishe di n 1980,edite db yJ.R . Coley-Smith,K .Verhoef f and W.R.Jarvis . Since1980 ,mor ewor ko nthes efung i andth edisease sthe ycaus ehav ebee npublishe d anda nup-datin g ofth eboo kwa sneeded . In 1992, the 10th Botrytis symposium took place on Crete, Greece, and it seemed appropriate, topublis h theProceeding s of this symposium. Therefore, onespecialis to f eachsectio no fth esymposiu mwa saske dt oprepar ea revie wpaper .Thes ereview swer e followed byshorte rpresentation s anda numbe ro fposter swer epresented . Thetex to f almost allth eora lpresentation s ando f someo fth eposters ,ar epresente d inthi sbook . Wear egratefu l toth eauthor sfo r theircooperatio n inpreparin g thisbook .W e would liket oexpres sou rsincer ethank st oMrs .J. M .Mokveld ,wh odi d allth etyping . With great appreciation, we acknowledge the financial support of the Ministry of Agriculture,Natur eManagemen t andFisherie so fTh eNetherlands ,whic henable du st o publishthes eProceedings .
K. Verhoeff N.E. Malathrakis B. Williamson Introduction
K. Verhoeff
Thegenu sBotrytis, erected in 1729,contain s alarg enumbe ro fhost-specifi c pathogens (e.g. B.fabae onbroa dbean ,B. aclada ononion ,B. tulipae ontulip) ,an da singl ebroa d spectrum pathogen, viz.B. cinerea, which attacks a wide range of plants in temperate regions.B. cinerea attacks field-grown cropslik egrapes ,bu t isals oimportan ti nglass house-grown vegetables, flowers and fruits, during production or post-harvest during storagean dtransport .Du et oit sbroa dspectrum ,B. cinerea attractedmor eattentio ntha n anyo fth ehost-specifi c species.I ti sdifficul t toasses sth edamag ecaused ;i nman ycrops , dependingo nprevailin gweather ,economi closse so fmor etha n50 %occur .I nglasshous e crops,th elosse sals odepen d toa larg eexten to nth eclimat ei nth eglasshouse ,althoug h conduciveclimati ccondition sca nb ereduce dt osom eextent . As a pathogen, B. cinerea has received attention for more than 100year s (de Bary, 1886; Ward, 1888) studies on its biology and host-pathogen interactions were started somewhat later (Brown, 1915,1916,1917; Blackman and Welsford, 1916). Since then, manypaper shav ebee npublishe do naspect so fth ediseas ei nvariou scrops ,mos tnotabl y in grape, soft fruits, tomato and (other) glasshouse-grown crops. Although our knowledge of the fungus and the diseases caused by it has increased substantially, the work has be en hampered by one important factor: invivo, onl y asexual multinucleate conidia are found and single-spore isolates always have shown phenotypic variation when subcultured.I twa stherefor e impossiblet ocompar edirectl y results obtainedwit h B.cinerea i ngrap efro m oneregio nwit hthos eobtaine di nanothe rregion ;thi sals ohold s for othercrops . Epidemiologicalstudie swer edifficul t becausei twa sno tpossibl et oestablis hwhethe r populationsdiffere d accordingt ocrop so rgeographica lregion . Itwa sals oimpossibl et o know,whethe r apopulatio n presenti na cro pa tth ebeginnin g ofth eseaso nwa sstil lth e samea tth een do fit . With the introduction of systemic fungicides, effective control of B. cinerea was possible for a short period, but after a few years, reports were made of less effective control caused by the development of resistant isolates of the fungus (e.g. Bollen and Scholten, 1971;Jarvi s and Hargreaves, 1973;Mille r and Fletcher, 1974). This led to manypublication s whichdescribe d methodsfo r achieving adequatecontro l in thisne w situation. Thedevelopmen t of resistant strainsprovide d convenient genetic markers andle dt o therecognition ,tha tdifferen t populationso fB. cinerea d oexist .Thi sstimulate d genetic studieso fB. cinerea,bu tals owor ko nalternativ econtro lmethods ,especiall ybiologica l control(e.g .Dubos ,1992) . Breedingfo rresistanc eha sgenerall yno tbee nsuccesful , asfe wgene sassociate dwit h resistance have been identified. Defence mechanisms are however important in plants andsevera lar eelicite dupo ninfectio n byB. cinerea (e.g .Glazener , 1982;Peze tan dPon t 1992;Hoffma n andHeale , 1987),bu tthi sknowledg eha sno tbee nexploite di nbreedin g programmes. Studies on thesexua l stateo f B. cinerea wereperforme d for anumbe r of yearswit h limitedsucces ,bu trecentl y methodswer edevelope dfo r massproductio n of ascospores of B.fuckeliana (Faretr a and Antonacci, 1987). This progress, together with modern techniquesi nmolecula rgenetic sdevelope dfo rothe rfung i(e.g .Va nKa net al., 1991 )ca n nowfacilitat e researcho nB. cinerea. Iti santicipate d thatgene swhic hcod efo renzyme s essentialfo r penetrationan dsprea d ofmyceliu m withina plan twil lb eidentifie d and, in thisway ,pathogenicit y determinants willb efoun d andne wcontro lstrategie swil l focus onthis .Th esam eapproac hals oapplie st ogene sresponsibl efo rth eelicitatio no fdefenc e reactions. With such genetic information, epidemiological studies can be carried out more efficiently, and models refined topredic t outbreaks ofepidemics .Thi s information will alsosuppor tbette rstrategie s forchemica l andbiologica lcontrol . It can be expected that with the results obtained onth ebiolog y of B. cinerea, basic work on genetics of the fungus, as well as on host-pathogen interactions and epide miologywil llea dt omor eeffectiv e andmor eenvironmentall yfriendl y controlstrategies . 'RecentAdvance si nBotrytis Research ' marksth ebeginnin g ofa ne wer ai nresearc h onB. cinerea, witha spin-of f towor ko ndisease scause db yothe rBotrytis species.
References
Blackman, W.H. and Welsford, E.J., 1916. Studies in the physiology ofparasitism . II. Infection byBotrytis cinerea. Annalso fBotan y30:389-398 . Bollen, G.J. and Scholten, G., 1971.Acquire d resistance to benomyl and some other systemic fungicides in astrai n ofBotrytis cinerea in Cyclamen. Netherlands Journal ofPlan tPatholog y77:83-90 . Brown, W, 1915. Studies in the physiology of parasitism. I. The action of Botrytis cinerea. Annalso fBotan y29:313-348 . Brown,W. , 1916.Studie s in thephysiolog y of parasitism, m. On the relation between the 'infection drop' andth eunderlyin g hosttissue .Annal so fBotan y30:399-406 . Brown, W., 1917. Studies in the physiology of parasitism. IV. On the distribution of cytasei nculture s ofBotrytis cinerea. Annalso fBotan y31:489-498 . De Bary, A., 1886. Über einige Sclerotien und Sclerotienkrankheiten. Botanisches Zeitblat44:377 ,393,409,433,449,465 . Dubos,B. , 1992.Biologica l control ofBotrytis: State-of-the-art. Proceedings of theXt h Botrytissymposiu m 5-10 April 1 992,Crete ,Greec ep . 169-178. Faretra,F .an dAntonacci ,E. ,1987 .Productio no fapotheci ao fBotryotiniafuckeliana (d e Bary) Whetz. under controlled environmental conditions. Phytopathologia mediter- ranea26:29-35 . Glazener, J.A., 1982. Accumulation of phenolic compounds in cells and formation of lignin-like polymers in cell walls of young tomato fruits after inoculation with Botrytiscinerea. Physiological PlantPatholog y20:11-25 . Hoffman, R.M.an dHeale ,J.B. , 1987. Celldeath ,6-methoxymellei n accumulation,an d induced resistance to Botrytis cinerea in carrot root slices. Physiological and Molecular PlantPatholog y30:67-75 . Jarvis,W.R . and Hargreaves, A.J., 1973.Toleranc e tobenomy l inBotrytis cinerea and Pénicillium corymbiferum. PlantPatholog y 22:139-141. MillerM.W . andFletcher ,J.T. ,1974 .Benomy ltoleranc ei nBotrytis cinerea isolate sfro m glasshousecrops .Transaction s ofth eBritis hMycologica l Society62:99-103 . Pezet,R .an dPon tV , 1992. Differing biochemical andhistologica l studies oftw ograp e cultivars in the view of their respective susceptibility and resistance toBotrytis cinerea. Proceedings of theXt hBotrytis symposium 5-10 April 1992,Crete ,Greec e p.93-98 . VanKan ,J.A.L. ,Va nde nAckerveken ,G.F.J.M .an dD eWit ,P.J.G.M. , 1991.Clonin gan d characterisation of cDNA of avirulence gene avrg of the fungal pathogen Cladosporiumfulvum, causal agent of tomato leaf mold. Molecular Plant-Microbe Interactions4:52-59 . Ward,H.M. , 1888. Alil ydisease .Annal so fBotan y2:319-382 . GENETICS Genetic studieso fBotryotinia fuckeliana {Botrytis cinerea)
F. Faretraand M. Grindle
Summary
Botrytis cinereai s the pathogenic fungus responsible for 'grey mould' disease of numerous soft fruits, vegetables and ornamentals. Botryotiniafuckeliana i s the teleo- morpho fB. cinerea; i tproduce ssexua lprogen y(ascospores )b ymeiosi so fheterozygou s diploid nuclei in ascogenous hyphae derived from matings of sexually-compatible isolates.Ascospor eprogen yca nb eobtaine dreliabl yi nth elaboratory ,an ds oisolate so f B. cinerea are amenabl e togeneti c analysis by classicalMendelia n techniques. Inthi s report, we describe materials and methods for growth and genetic manipulation of B.fuckeliana i nth elaboratory ,revie winformatio n from classicalan dmolecula rgeneti c studies,an dpropos e asyste m ofnomenclatur e tob e used whencommunicatin g genetic information.
Introduction
Botrytis cinereaPers. : Fr. and Botryotinia fuckeliana (de Bary) Whetz. are asexual (anamorphic) andsexua l(teleomorphic ) stages,respectively ,i nth elif ecycl eo fth esam e filamentous fungus. The asexual stage, often referred to as the 'grey mould' fungus, consists of vegetative hyphae, sclerotia, macroconidia and microconidia. The sexual stage consists of a reproductive body, the apothecium, containing ascospores in linear asci (Fig. 1).Thus ,B. cinerea( =B. fuckeliana) isa membe ro fth elarges tclas so f fungi, theAscomycetes . Field isolates of B. cinereagrow n on synthetic media in the laboratory exhibit considerable variations in phenotype (Paul, 1929; Grindle, 1979; Coley-Smith etal., 1980). Thosedifference s whichinfluenc e thebiologica lfitnes s ofB. cinerea population s are of practical importance in agriculture; for example, field isolates which combine vigorous growth on host plants with resistance to an important fungicide used in plant protection can seriously reduce crop yields and profitability. Knowledge of thegeneti c basis of such differences in phenotype might be useful in monitoring populations of B. cinerea, assessing the role of particular genes on biological fitness, and aiding the development ofne wstrategie s tocomba tmutan tisolates . Thegenome s of B. cinerea isolatesca nb einvestigate d in thelaborator yb y classical Mendelian analysis of sexual progeny (Faretra and Antonacci, 1987; Faretra et al., 1988a,b).Isolate s ofdifferen t mating typear ecross-fertilise d toinitiat eth esexua lstag e of thelif e cycle.Analysi s ofth esexua l spores (ascopores) from apothecia has revealed thegeneti cbasi s fordif f erencesi nmatin g type,siz eo fapothecia l stalks,an dresistanc e tocertai nfungicide s (Faretraan dPollastro , 1991,1992a,b). This report discusses techniques for the maintenance and genetic analysis of Botryotiniafuckeliana, reviewsgeneti cknowledg eo f thefungus , andprovide sa frame workfo rnomenclatur e tob euse dwhe ncommunicatin g genetic information. Sourceso ffield isolate san dlaborator ymutant s
Field isolates are readily obtained from diseased plants, plant debris and soils, and by trappingaeria lspore so ndishe so fselectiv esyntheti cmedi a(Kritzma nan dNetzer ,1978 ; Kerssies, 1990).I t is advisable to obtain 'monoconidial' rather than 'mass-hyphal' or 'mass-conidial'isolate sfo rlaborator ystudies ,sinc eth efirs ttyp eo fisolat ei smor elikel y tob e genetically homogeneous (i.e .homokaryotic , rather than heterokaryotic).Mono conidialisolate s areobtaine d by spreading conidia atlo wdensit y ondishe s ofsyntheti c media andtransferrin g individual germlings totube s withfres h media. Thechance so f genetic homogeneity are increased by obtaining a succession of monoconidial isolates from monoconidialisolates ,selectin gth e 'mosttypical ' progenyo neac hoccasion . Laboratory mutants can be derived from field isolates or, preferentially, from well- documented laboratory strains of known phenotype. It is advantageous to derive most mutants from a particular wild type standard, so that the mutants are isogenic; thatis , their genomes are virtually identical, except for particular mutant genes causing a specific changei nphenotype . Spontaneous mutants can be obtained by incubating hyphae or macroconidia on selective media which discriminate between particular mutant and normal phenotypes. For example, mutants resistant to a specific antifungal chemical can be obtained by incubating spores on media containing lethal or sub-lethal amounts of that chemical. Induced mutantsca nb e obtained bydeliberatel y exposing macroconidia tochemica lo r physicalmutagens ,befor e incubation onselectiv emedia .Sinc emacroconidi a aremulti nucleate,mutant s derived from them frequently contain amixtur e of mutated and non- mutated nuclei in heterokaryotic hyphae; such mutants are phenotypically unstable, producing mutant and non-mutant progeny during subculturing. The uninucleate microconidiamigh tb ebette rsource so fmutant si fthe ycoul db einduce dt ogerminat eo n syntheticmedia . Field isolates and laboratory mutants of various phenotypes and genotypes are maintained in the culture collection of F. Faretra at the University of Bari, Italy. The collection includes standard laboratory strains ofknow nmatin g type.Culture s in tubes ofsoli dsyntheti cmediu mremai nviabl efo rsevera lmonth sa t5°C ,bu tpersisten tstorag e under these conditions can lead to accumulation of mutations, reduction of aggress iveness and loss of ability to produce sclerotia and/or apothecia. Conidia or hyphal fragments canb estore dfo r about2 year sa t5° Co nanhydrou s silicagela sdescribe db y Wilson (1986).Conidi a and ascopores remainviabl efo r atleas t 1 yeari n 10%glycero l at-70°C ;thi si sprobabl yth ebes tmetho dfo r storingstrains .
Syntheticmedi afo r vegetativegrowt h
Most isolates grow well on various defined and undefined synthetic media. Undefined mediausuall y provide abundantorgani cnutrient sfo r luxuriantgrowth ,bu tthei rprecis e compositions areno tknown .Define d mediacontai nknow nquantitie s ofinorgani csalt s and supplements sotha t their ingredients canb ereproduce d ormodifie d precisely; this isnecessar yfo r somegeneti canalyses ,suc ha sdetectio n ofgene saffectin g biosynthetic pathways.Ou rprincipa l undefined media aremal textrac t agar (MEA;2 0g Oxoi d malt extract and2 0g agar. H water) andpotat odextros e agar(PDA ;20 0g peele d and sliced potatoes simmeredfo r 1 hi nwate r andfiltere d throughcheesecloth ; thefiltrat e volume made up to 1litre , and pH adjusted to 6.5; 20 g glucose and 20 g agar added before
8 autoclaving).Thi shome-mad ePD Ai ssuperio rt ocommercia lpreparations .Th edefine d mediause d for growth ofAspergillus nidulans (Pontecorvoet al., 1953)o rNeurospora crassa (Vogel, 1964; Davisan dd eSerres ,1970 )als osuppor tgoo dgrowt ho fB. cinerea. The composition of the media are given in Annex 1.Thes e media vary with respect to their contents, preparation and storage. The effects of the differences in media com positiono ngrowth ,conidiatio n andscleroti adevelopmen thav eno tbee nquantified . For example,i ti sno tknow n whetherth ephenotype s of strainsar esignificantl y affected by variations in traceelement s orvitamins .Althoug hB. cinerea grows reasonably wello n allthes eminima lmedia ,i tgrow sconsiderabl y bettero nth ecomplet emedia .
Sexual reproduction and classical genetics
Productiono fapotheci a
Apotheciaar eth ereproductiv ebodie swhic hemerg efro m fertilised sclerotia(Figur e1) . The procedure for obtaining apothecia has been described by Faretra and Antonacci (1987) and Faretra etal. (1988a,b):isolate st ob ecrosse d musthav eopposit ematin g ty pes; they areinoculate d ont oseparat edishe s ofME Aan dgrow n in thedar kfo r 3day s at21+ 1°C ,followe d by4 week sa t 15+1° Cand ,finally ,4 week sa t0° Ct oobtai nscleroti a which arecapabl e of carpogenesis; the sclerotia are transferred to modified test tubes containing sterile water, and fertilised with microconidia from cultures of the opposite mating type;fertilise d (spermatised) sclerotia areincubate d at 11+1°Cwit hexposur et o incandescent and fluorescent lights on a 12h photoperiod . Successful crosses produce apothecia 3-13week s after spermatisation. Isolatesca nusuall yfunctio n asth e 'male' andth e 'female' parenti nreciproca lsexua l crosses.Fo rexample , sclerotia ofisolat eA ca nb efertilise d byconidi ao fisolat eB (i.e . thecros sisA $+ xB<3)o rscleroti ao fB canb efertilise d byconidi ao fA ( B$ + x A < ?) . Isolates can occasionally produce apothecia without cross-fertilisation; self-fertile isolates are probably heterokaryons carrying the two mating type alleles in different nuclei (Faretraan dPollastro ,1992a) .
Isolationo f ascospores
Ascospores are the sexual spores which eventually develop from the nuclei in spermatised sclerotia.Fusio n ofhaploi d nuclei from thetw oparent s ofopposit ematin g typeproduce s heterozygous diploid nuclei,eac h of which undergoes meiosis andgive s riset o eight haploid ascopores contained within an ascus (Fig. 1).A n ascus comprises four pairs of ascospores, each pair having genetically identical nuclei, in a linear sequence;th e spores probably develop in the same way as theordere d spores inlinea r asci of Neurospora crassa (Raju, 1980). Each apothecium usually contains several hundredasci . Sets of eight ascospores for studies of ordered tetrads (Faretra et al., 1992a) are obtained asfollows : anapotheciu m isplace d ina dro po fsteril edistille dwate ri na Petr i dish,th ehymenia llaye ri sbroke nwit ha dissectin g needle,an dth easc iar etease dapart ; individual asciar eseparated , andth eascospore s ineac h ascus aredissecte d outi nthei r correct sequence with a micromanipulator; the dissected spores are transferred to fresh media,wher ethe ygerminat eafte r 12-16h a t21+TC . Fig. 1 Sexualreproduction ofBotryotiniafuckeliana. a)apothecia produced by sclerotia in modified test tubesexposed to incandescent andfluorescent light at U±FC b) a compl ete ascus containing eight ascospores.c) portion of an ascus,showing multinucleate ascospores s
10 Sampleso f random ascospores areobtaine d by placing several apothecia in avia lo f steriledistille d wateran d squeezing them withforcep s ordissectin g needlest oobtai na suspension of spores. Alternatively, a single apothecium can be treated in 200-300(x l water in an Eppendorf tube; this is the recommended method, since transmission of geneticmarker sma yno tb eidentica li ndifferen t apothecia.Spor esuspension sar esprea d on dishes of water agar or growth medium and incubated at 21±1°C to obtain monoascosporecolonies .
Cytology
Thehaploi d numbero fchromosome s invegetativ e hyphaeo fB. cinerea (Shiraneet al., 1988,1989)an di ndevelopin g ascio fB.fuckeliana (Faretr aan dContesini ,unpublished ) issixteen .
Molecular genetics
Wehav ecommence dmolecula rstudie si nou rlaboratorie st oexplor ean dmanipulat eth e genome of B.fuckeliana usin g techniques such as those described by Leong (1988). Differences between field isolatesan dascospor eprogen yhav ebee nidentifie d byRFL P techniques (Grindle, unpublished). Electrophoretic karyotyping has revealed consider able variability among field isolates and laboratory strains (Faretra and Grindle, unpublished). Protoplasts can be obtained by using various enzymes and osmotic stabilisers(Faretr aet al., 1992a) ;the yhav ebee ntransforme d withDN Afro m aplasmi d carrying the hygromycin B phosphotransferase gene as a selectable marker (Chabani etal., 1990) , buttransformatio n efficiency isver ylow .
Genetic nomenclature
There are various,conflictin g systems of nomenclature for describing thegenotype so f fungi.W epropos etha tth efollowin g nomenclature(base do nth esuggestion so fYode re t al.(1986 )b efollowe d whencommunicatin g genetic information onB.fuckeliana. Theter m 'strain'ca nb euse dt odistinguis hcell so rcolonie so fan yparticula risolat e from those of other isolates, whether they originated in nature or in the laboratory. A straindoe sno thav et ob echaracterise d genetically.Strain sar eassume dt ob eidentica li f they are asexual progeny (e.g. derived from conidial or hyphal inocula) of the same homokaryotic parent. Afield isolat e is any strain obtained from a natural source (e.g. from infected plants in the field, or from the air in a greenhouse) and not knowingly altered in thelaboratory . Awil d type strain has the 'normal' or 'standard' phenotypic traitso ftypica lfiel d populations(o ro fmos tlaborator ystrains ,i fth e'normal ' phenotype of field isolates is not obvious).A mutan t strain differs phenotypically from anorma l strain due to one or more heritable changes caused by mutation(s) of genes or chromosomes;th emutation sca nb espontaneou so rinduced .Mutant swhic hcanno tgro w on basal (minimal) medium unless it is supplemented with one or more specific chemicals such asa namin oacid ,ar ereferre d toa snutritiona l orauxotrophi cmutants . Mutant strains which canb egrow n on basal medium, such as those which differ from normalstrain si nthei rpigmentatio no rrespons et ofungicides , areprototrophi cmutants . Whenther ei sn oobviou s 'wild type' bywhic h a 'mutant' canb ejudged , itmigh tb e better to describe phenotypically different strains as variants. For example, natural
11 variationsi nsiz eo fscleroti aamon gfiel d isolatesar eprobabl ycause db yman yallele so f thegene sresponsibl efo rthi sphenotype .Sinc eeac hallel ewoul db ea typica lconstituen t ofth etota lgen epoo lo fa population ,a varian twoul db ea typica lindividua lcarryin gon e ofthos e alleles.Thi sphenomeno n isknow n asgeneti cpolymorphism .I tca nb eargue d thatal l naturally-occurring strains with different phenotypes caused bydifferen t alleles shouldb ereferre d toa svariants .However ,som erar eallele sma ybecom eprevalen tdu e to man-made selection pressure, and strains carrying these alleles (e. g. fungicide- resistant strains)coul d perhapsb edescribe d asmutant s sincethe y canb e distinguished from typicalstrain si nnorma lpopulations . Phenotypes are referred to by the same symbols used to designate genotypes (see below), except that the symbols are not italicised. For example, a biotin-requiring auxotrophicmutan tmigh thav eth egenotyp eBiol- andth ephenotyp eBiol-. Thegenotyp eo fa strai nca nb eidentifie d byclassica lo rmolecula r methods.Havin g identified a particular genewhic h affects a particular phenotypic character, thegen ei s givena unique ,three-lette rsymbo lwhic hrelate st otha tcharacter .Fo rexample ,mutant s requiring adeninefo rgrowt h canb edesignate d Ade,an dmutant sresistan tt o fungicides containingth eactiv eingredi en tmethy lbenzimidazole-2yl-carbamat e canb edesignate d Mbc.Appropriat esymbol sca nb efoun d infunga l geneticstext san drevie warticle s(e.g . Fincham etai, 1979;Perkin s etai, 1982). Thegen esymbo li sitalicised ,wit h acapita l first letter. Different genes which modify the same phenotype are numbered, the number following thegen esymbo lwithou ta spac eo rhyphen ;fo rexample ,thre egene s affecting theabilit yt osynthesis etryptopha ncoul db eTrpl, Trp2 andTrp3. Geneswhic hresid ei n the mitochondrial genome are enclosed in squarebracket s [] todistinguis h them from nucleargenes . Normal(wil dtype )an dmutan tallele sar edistinguishe db y+ an d- signs,respectively , if thenorma l allelei sobvious .I nth especia lcas eo f alleles of genesaffectin g astrain' s responset oantifunga l chemicals,sensitiv ean dresistanc eallele sar edistinguishe db yth e letters S and R, respectively. Alleles conferring different levels of resistance can be disinguishedb yth eletter sL R(lo wresistance) ,H R(hig hresistance) ,an ds oon . The signs and letters for different classes of alleles follow thegen e symbol, andar e not superscripts. For example, normal and mutant alleles of a gene affecting melanin biosynthesis could be Mell+ and Meli-; sensitive and resistant alleles of a gene responsible for susceptibility to the antibiotic oligomycin could be OlilS, OlilLR and OU1HR. Dominant andrecessiv e alleleso f agen ear edistinguishe d byuppe rcas ean d lower caseletters ,respectively , for allletter s of thegen esymbol .Fo rexample ,th edominant , normal andrecessive ,mutan t alleles of agen eaffectin g histidinebiosynthesi s could be HIS1+ and hisl- respectively. Tests for dominance/recessivity of alleles are often problematical, and so many allel es may notb e distinguished in this manner (Grindle, 1987,1992). Specific alleles of a gene are distinguished by numbers and/or letters in brackets, immediately after theallel edesignatio n .I ti softe n useful tohav ea descriptiv enotatio n to indicate the source of the allele. For example, the symbols His2(UBF5)an d //is2(USG8)coul ddistinguis h twoindependen t mutantscarryin g different alleles ofth e samegen eaffectin g histidinebiosynthesis ,th eforme r isolated atth eUniversit y ofBar i byFaretra ,an dth elatte ra tth eUniversit y of Sheffield byGrindle .
12 Genesca nb eassigne dt olinkag egroup swhic har edistinguishe db yRoma nnumeral s (LGI, LGII, etc.). These are groups of genes which are probably on the same chromosome, since they tend to remain linked together during meiosis. Each gene occupiesa particula r site(locus )o na linkag egroup ,whic hca nb emappe di nrelatio nt o otherlinke dgenes .Th eterm sgen ean dlocu sar eofte n usedinterchangeably ,bu tgen ei s morespecific . Alocu scoul d encompass twoo rthre eadjacen t geneswhic har e difficult todistinguis h orseparat eb yconventiona lMendelia ntechniques . When describing the genotype of a strain carrying several genes which have been identified and assigned to linkage groups,linke d genes are separated by a single space andunlinke dgene s(o ndifferen t linkagegroups )b ya semi-colon .Fo rexample ,a strai n havingth egenotyp ehisl ; mell arg3; argl carrie srecessiv eallele so ffou rgene s(o nthre e linkagegroups) ;mell islinke dt oarg3, bu tth etw oarg gene sar eunlinked .Whe nlinkag e information isno tavailable ,al lgene s arese parate db ysingl espaces ;fo r example,tw o strainsuse di na sexua lcros smigh thav eth egenotype sadel- MbcSMATl-1 an dADE1+ MbcHRMAT1-2, respectively .
Determiningwhethe rmutan tgene sar edominant ,recessive ,o ralleli c
Testing for dominance orrecessivit y canb edon ea sfollows . Twostrains ,on e carrying thewil dtyp eallel ean dth eothe rth emutan tallel eo fa particula rgene ,ar egrow ntogethe r so that their hyphae can anastomose to form heterokaryotic mycelium (Grindle, 1987, 1992).Heterokaryo n formation in many species of fungi is restricted bynuclea r genes controllingheterokaryo ncompatibilit y(Grindle , 1992),bu tth epresenc eo fsuc hgene si n B. cinereaha sno tbee nevaluated .I tseem stha tth ematin g typegen edoe sno t influence vegetative compatibility, since pairs of strains with identical or different MAT1 alleles can form heterokaryons (Chabani and Grindle, unpublished). It might be possible to overcome heterokaryon incompatibility problemsb y fusion of protoplasts.Th e mutant allelei scompletel y(o rpartially )dominan ti fth eheterokaryo ni sphenotypicall yidentica l (orsimilar )t oth emutan tparent ;i ti scompletel y(partially )recessiv ei fth eheterokaryo n isphenotypicall y identical (similar)t oth enorma lparent . Testing for allelism couldb edon ei nth efollowin g way.Heterokaryoti c mycelium is derived from twodifferen t mutantstrain swit hth esam eo rrelate d phenotypes(Grindle , 1987, 1992).Fo r example,bot h mutants might require arginine for growth and areph e notypicallyArg- .Th emutant sar ealleli c(i.e .the ycarr ymutation si nth esam eAr ggene ) if the heterokaryon is phenotypically mutant (i.e.i t still requires arginine for growth); theyar eno talleli ci fth eheterokaryo ni sphenotypicall ynorma l(i.e .i tgrow so nminima l medium). The two mutant strains (e.g. two different Arg auxotrophs) are crossed to obtain sexualprogeny .I fther ear ever yfe wprog en ywhic har ephenotypicall ynorma l(e.g . less than0.5 %ar eprototrophi cArg +recombinants )i ti sver ylikel yma tth etw omutan tgene s occupyth esam echromosoma l locusan ds oth emutant sar eprobabl yallelic .
Testingfo rmatin gtype s
Thesexua l processi nB. fuckeliana iscontrolle d by asingl ematin g typegen ewit htw o alleles.Th eallele ssho wco-dominanc ei nheterokaryon s (Faretraet ai, 1988b),an dar e designatedMAT1-1 andMAT1-2. Thus,B. fuckeliana isa heterothalli c fungus withonl y twomatin gtypes ,an dsexua lprogenie sar eobtaine db ycross-fertilisatio n ofMAT1-1 and
13 MATl-2 isolates. Self-fertile isolates are designated MAT-1/2; most of these havebee n shownt ocarr yth eMAT- 1 andMAT- 2allele si nseparat enucle ii nheterokaryoti chyphae , andar etherefor e pseudohomothalli cisolates .Th edifferen t matingtype sca nb e referred toinformall y asMAT1 ,MAT 2an dMA T1/ 2strains . Theparen to funidentifie d matingtyp e(U )i suse di nreciproca lcrosse swit hreferenc e strains of known mating type (Rl being MAT-1an d R2bein g MAT1-2) , and it is also self-fertilized. Thus, the crosses are U$ x U Taxonomy By studying sexual reproduction of numerous field isolates of B. cinerea, Faretra etal. (1988b, 1992a) and Faretra and Pollastro (1992a,b) have shown that B.fuckeliana i s undoubtedly the sexual stage in the life cycle of this fungus. Most taxonomists recommendtha tth escientifi c nameo fth esexua lstag eshoul db epreferre d totha to fth e asexual stage. For example, Cochliobolus heterostrophus has replacedHelmintho- sporiummaydis as the scientific name for the fungus causing leaf blight of corn. Therefore, Botryotiniafuckeliana shoul d replaceBotrytis cinerea asth eLati n binomial for the fungus causing the disease 'grey mould'. However, there are precedents for a more'practical ' solution:th enam efo rth easexua lstag eo fth elif ecycl ema yb eretaine d becauseo fit sfamiliarit y andwidesprea dus eove rman yyears .Fo rexample ,Aspergillus nidulans isth eaccepte d namefo r thepopula r fungus studied byman ygeneticists ,eve n though this refers to the asexual stagei n thelif e cycle of Emericella nidulans (Eidam) Vuillemin.. Using this criterion, the familiar binomial recognised worldwide by numerous mycologists andplan tpathologist s- Botrytis cinerea- shouldb eretained . Until taxonomists agree on themos t appropriate Latin name for the fungus causing 'grey mould', both Botrytis cinerea and Botryotiniafuckeliana shoul d be included in publicationsconcernin g itsgenetics . Acknowledgement The genetic studies of F. Faretra were supported in part by a grant from the Italian National Research Council for the Bari-Sheffield bilateral project 'Classical and moleculargenetic s ofBotryotinia fuckeliana!. References Chabani, A.W., Grindle, M. and Turner, G., 1990. Genetic transformation of Botrytis cinerea. FourthInternationa l MycologicalC ongress ,Regensbur g p.177 . Coley-Smith, J.R., Verhoeff K. and Jarvis, W.R., 1980.Eds . The Biology of Botrytis, London:Academi cPress ,pp .318 . Davis,R.H . and de Serres,F.J. , 1970.Geneti c and microbiological research techniques 14 forNeurospora crassa. Methodsi nEnzymolog y27:79-143 . Faretra,F .an dAntonacci ,E. ,1987 .Productio no fapotheci ao fBotryotiniafuckeliana (d e Bary) Whetz. under controlled environmental conditions. Phytopathologia medi- terranea26:29-35 . Faretra F. and Pollastro, S., 1991. Genetic basis of resistance to benzimidazole and dicarboximide fungicides in Botryotiniafuckeliana (Botrytis cinerea). Mycological Research 95:943-951. Faretra F. and Pollastro, S., 1992a. Genetics of sexual compatibility and resistance to benzimidazole and dicarboximide fungicides in isolates of Botryotinia fuckeliana (Botrytis cinerea) from ninecountries .Plan tPathology ,i npress . FaretraF . andPollastro ,S. , 1992b. Geneticsof Botryotinia fuckeliana (Botrytis cinerea): amajo rgen ecausin g shortapothecia lstalks .Phytopathologi amediterrane a31:28-31 . FaretraF. ,Antonacci ,E .an dPollastro ,S. , 1988a.Improvemen to fth etechniqu euse dfo r obtaining apothecia of Botryotinia fuckeliana (Botrytiscinerea) unde r controlled conditions.Annal id iMicrobiologi a edEnzimologi a38:29-40 . FaretraF. ,Antonacci ,E . and Pollastro, S., 1988b.Sexua lbehaviou r and mating system ofBotryotinia fuckeliana, teleomorph ofBotrytis cinerea. Journal ofGenera lMicro biology 134:2543-2550. Faretra, F., Pollastro, S. and Santomauro, A., 1992a. Tetrad analysis in the phyto- pathogenic fungus Botryotiniafuckeliana (Botrytis cinerea). (Submitted for publi cation). Faretra, F.,Pollastro , S. and Contesini, A., 1992b. Protoplasts of the phytopathogenic fungus Botryotiniafuckeliana (Botrytis cinerea). (Submitted for publication). Fincham, J.R.S., Day, P.R. and Radford, A., 1979. Fungal Genetics, Third Edition. Oxford: Blackwell Scientific Publications. Grindle, M., 1979. Phenotypic differences between natural and induced variants of Botrytiscinerea. Journalo fGenera lMicrobiolog y 111:109-120. Grindle, M., 1987.Geneti c basis of fungicide resistance. In 'Combating Resistance to Xenobiotics'.Eds .Ford ,M.G. ,Hollomon ,D.H. ,Khambay ,B.P.A . andSawicki ,R.M. . Chichester: EllisHorwoo dLtd. ,p .75-93 . GrindleM. , 1992.Forma l Genetics.In 'Fungi: ANe w Synthesis'. Eds.Long , P.E.an d Burnett,J.H. .Oxford : Blackwell Scientific Publications,i npress . Kerssies, A., 1990.A selectiv e medium for Botrytis cinerea tob e used in aspore-trap . NetherlandsJourna lo fPlan tPatholog y96:247-250 . Kritzman,G . andNetzer , D., 1978.A selectiv e medium for isolation and identification ofBotrytis spp.fro m soilan donio nseed .Phytoparasitic a 6:3-7. Leong,S.A. , 1988. RecombinantDN Aresearc hi nphytopathogeni cfungi . In 'Advances inPlan tPathology' ,Vol . 6.Ed .Sidhu ,G.S. .London :Academi cPress ,p . 1-26. Paul,W.R.C. , 1929.A comparativ emorphologica l andphysiologica l study of strainso f Botrytis cinerea Pers.:Fr .wit h special references to their virulence.Transaction so f theBritis hMycologica l Society 14:118-135. Perkins,D.D. ,Radford , A.,Newmeyer ,D .an dBjorkman ,M. , 1982.Chromosoma l loci ofNeurospora crassa. Microbiological Reviews46:426-570 . Pontecorvo, G., Roper, J.A., Hemmons, L.M., MacDonald, K.D. and Bufton, A.W.J., 1953.Th egenetic s ofAspergillus nidulans. Advance si nGenetic s5:141-238 . Raju, N.B., 1980.Meiosi s and ascospores genesis inNeurospora. European Journalo f CellBiolog y23:208-223 . Shirane, N., Masuko, M. & Hayashi, Y., 1988. Nuclear behaviour and division in 15 germinating conidia ofBotryti scinerea .Phytop atholog y79:728-730 . Shirane,N. ,Mashuko ,M .& Hayashi ,Y. ,1989 .Ligh tmicroscopi c observation ofnucle i andmitoti c chromosomes ofBotrytis species.Phytopatholog y79:728-730 . Vogel, HJ., 1964. Distribution of lysine pathways among fungi: evolutionary implications.America nNaturalis t98:435-446 . Wilson, C, 1986. FGSC culture preservation methods. Fungal Genetics Newsletter 33:47-48. Yoder,O.C. , Valent, B. and Chumley, F., 1986.Geneti c nomenclature and practice for plantpathogeni cfungi . Phytopathology 76:383-3 85. Annex 1. Composition of media, used in studies of Botryotinia fucke liana Aspergillus medium Recipe 1 -1 (a) Minimalmediu m(MM )contain sth efollowin g (g.1 ):glucos e(10.0) ; NaN03(6.0) ; KH?P04(1.52) ;KC l(0.52) ;MgS0 4.7H20(0.152) ;trac eelement ssolutio n (5.0ml) .Th e pHi sadjuste d ifnecessar y to6.5 .Thes eingredient s (withoutglucose ) areprepare d asa 10 x concentrate (A), adjusted to pH 5.9, and stored over chloroform in 500 ml screwcapped bottles (add 5m l chloroform to each bottle) at 5°C.T omak e 1litr e solid MM,ad d 100m lA , 10g glucos e and 15g aga r todistille d water, and autoclave for 15 minatl20°Candl.lPa. (b) MMcontainin g theabov eingredient s (withoutglucos ean dmagnesiu msulphate ) isprepare da sa 1 0x concentrat e(B) :a 40 %w/ vglucos econcentrat e(C) ,an da 20 %w/ v MgS04.7H20 concentrate (D) are prepared separately. Concentrate B is stored over chloroform; concentrates Can d Dar e filter-sterilised or autoclaved for 5mi n at 120°C and 1.1.Pa ;ad d2 5m lC an d2. 5m lD t oeac hlitr eho tmedium . (c) Complete medium (CM) contains the following (g.l0 16 Recipe 2 (a) Minimalmediu m(MM )contain sth efollowin g (g.H):glucos e(20.0) ; NaN03(2.0) ; KH2P04(1.0) ;KC l(0.5) ;MgSO „.7H 20(0.5) ;FeS0 4(0.01) ;trac eelement ssolutio n(1. 0 ml). These ingredients (without glucose, trace elements and potassium phosphate) are prepared as a 100 x concentrate (A); a 100 x concentrate (B) of KH2P04 is prepared separately. Tomak e 1 litre solidMM ,ad d 10m lA , 10 mlB , 1 mltrac eelements , 20 g glucosean d2 0g aga rt owater ,an dautoclav ea sabove . (b) Complete medium (CM) contains the following (g.H) in addition to minimal medium:pepton e(10.0) ,cas ei nhydrolysat e(10.0) ;yeas textrac t(10.0) ;vitami nsolutio n (1. 0ml) .T omak e 1 litresoli dCM ,thes eingredient s(withou tvitamins )ar eadde dt oM M recipebefor e autoclaving;th evitami n solution isadde dt oautoclave d medium. The trace elements solution contains the following (g.H): ZnS04.6H20 (2.75); CuS04.5H20 (0.2); H3B03 (0.03);MnS0 4.6H20 (0.33); NaMo04(0.025) ; KI (0.006). Thesolutio n isstore d at5°C . The vitamin solution contains the following (g.H): inositol (4.0); choline chloride (2.0);nicotini c acid (2.0) ; thiamine 1.0); p-aminobenzoic acid (0.5); pyridoxine(0.5) ; riboflavin (0.5);bioti n(0.2) ;foli c acid(0.002) .Th esolutio ni sfilter-sterilised an dstore d at5°C . Neurospora medium Minimal medium (MM) contains the following (g.H): sucrose (20.0); sodium citrate, 5H20 (3.0);KH 2P04 (5.0);NH 4N03 (2.0);MgS0 47H20 (0.2);CaCL,.2H 20(0.1) ;trac e elements solution (1.0 ml); bi otin solution (0.05).I t is prepared as a 50 x concentrate (withoutsucrose) .Th e5 0x ingredient s(e.g . 150.0g sodiu mcitrate )ar eadde dseparately , in the order shown, to 750 ml water and dissolved with a magnetic stirrer at room temperature before addingth enext .Th e5. 0g calciu mchlorid ei spredissolve d in 20 ml H20 andadde d slowly tominimis e precipitation (any precipitateca n often bedissolve d byleavin g themediu m overnightan dstirrin g again).Tota lvolum ei sfinall y adjusted to 1litre ,an dth econcentrat ei sstore d overchlorofor m at5°C . Tomak e 1 litresoli dMM ,ad d2 0m lconcentrate ,2 0g sucros ean d 15 gaga rt owater , andautoclav efo r 15 min.a t 12°Can d 1.1 Pa.T omak e 1 litrecomplet emediu mCM , add 5.0g caséin ehydrolysat e and5. 0g yeas textrac tt oM Mingredient s before autoclaving. The trace elements solution contains the following (g.H): citric acid.lH20) (50.0); ZnS04.7H20 (50.0); Fe(NH4)2 (S04)2.6H20 (10.0); CuS04.5H20 (2.5); H3B03 (0.5); MgS04.7H20 (0.5);N ^ Mo04.2H20 (0.5).Eac h ingredient is added separately, in the order shown,t owate ran ddissolve d at roo mtemperatur ewit ha magneti cstirre r before addingth enext .Th econcentrat ei sadjuste d to 1 litrean dstore dove rchlorofor m at5°C ; iti susuall ymad ei nbatche so f 100ml . Thebioti nsolutio ncontain s5. 0m gbioti ndissolve di n5 0m lwater . Iti sstore d frozen, inbatche s of 5.0ml . 17 Molecular genetic analysis ofpathogenesi so f Botrytis cinerea J.A.L.van Kan,C.J.B. BergmansandJ.W. van 'tKlooster Summary To study the role of putative pathogenicity determinants of B. cinerea by molecular methods,w ear edevelopin g toolswhic henabl eu st odisrup tke ygene san dproduc esite - directed mutants.W ear eals oinitiatin ga molecula rcharacterizatio n ofgeneti cvariatio n betweendifferen t B. cinerea isolatesb yth eus eo fRFL Pan dRAPi Dmarkers . Introduction Knowledgeabou tth emolecula rgenetic so fB. cinerea i spoorl ydevelope di ncompariso n withothe rplan tpathogeni cfungi . Classicalgeneti canalyse sb yFaretr ahav eresulte di n reproducible, although time-consuming, protocols for sexual crosses (Faretra and P ollastro, 1988; Faretra etal., 1988a,b) .I norde rt operfor m moleculargeneti canalysi so f B. cinerea, several tools need to be developed. Firstly, genetic markers are required. It appearst ob edifficul t toobtai nstabl emorphologica l orauxotrophi cmarker sfo r genetic analysis(Grindle ,persona lcommunication) .Secondly ,a transformatio n procedureneed s to be established to allow over-expression, repression or disruption of specific genes. Thirdly, genetic variation can be studied by analysis of chromosome length poly morphism (CLP), restriction fragment length polymorphism (RFLP) or random amplified DNApolymorphis m (RAPiD). We aim to use all these tools in order to perform a molecular genetic analysis of B. cinerea genesinvolve d inpathogenesis .Physiologica l studieshav eindicate d rolesi n pathogenesis for hydrolytic enzymes such as cutinases, proteases and pectinases (McKeen, 1974;Va nde n Heuvel andWaterreus , 1985;Leon eet al., 1990) .B ymakin g directed mutations in, specific genes (gene disruption), we will be able to test the relevance of individual genes in pathogenesis.W ear e interested in genesencodin g the aboveextracellula rhydrolyti cenzymes ,an deventuall yals othos einvolve di nproductio n of toxins. Here we present results obtained in the development of tools to study pathogenicity andgeneti cvariatio n ofB. cinerea. Results and Discussion Toolsfo r geneticanalysi s Eight isolates of Botrytis cinerea Pers.:Fr. , obtained from theNetherland s weremate d with tworeferenc e strainswit h defined mating types,SAS5 6(containin g withMAT1- 1 allele)an dSAS40 5(containin g theMA T1- 2 allele)(Faretr aetal., 1988a). Ineac hcross , matingpartner swer euse dbot ha smal e(micro-conidia )an dfemal e (sclerotia)(Tabl e1) . Isolate Bc7, however, produced no sclerotia and could therefore only be used as male 18 partner. Self-fertilization controls aresummarize d inTabl e 1.Eac hDutc hisolat emate d with atleas t oneo f thetw oreferenc e strains.Fiv eisolate s Bc7,Bcl2 ,B e16 , Bc21an d Bc26, mated with both SAS56 and SAS40 5, suggesting that these isolates could be homothallic.Isolate sBcl 2 andals oBcl 6appeare d tob e self-fertilizing Self-fertilization g of Bc7, Bc21 andBc2 6wa sno tobserved . Table 1. Sexualcrosses of isolates ofB. cinerea. Sexualcrosse s of Botrytis cinerea in CDO msTi^CUCDOOs-lOCOOT*. enen *-T-<-ojcvjojaj Q_ < a: SAS56 =MAT1- 1 b: SAS405 =MAT1- 2 c: Bc7 producesn oscleroti a d: -, noapotheci a produced e: +,productio no fapotheci a f: USP =unspermatize dscleroti a Ineac h successful cross,man y apotheciawer eproduced .I norde r toobtai n progeny for moleculargeneti ctetra d analysis,w epropagate d subcultureso fal l eightascopore s from anascus .Fo reac hcross five, apotheci awher echosen . Fromeac hapothecium ,tw ointac t asciwer etaken ,an dgentl y hydrolysed withNovozy m for ashor tperiod . Subsequently, theorderl yrelease dascopore swer esprea dapar tove ra n agarplate ,an dlef t to germinate overnight. Germinated ascopores were transferred to individual agar plates and grown untilsporulatio n occurred.I norde rt operfor m agoo dtetra d analysis,w eusuall ycarrie d outthi sprocedur eonl ywhe na tleas tseve no fth eascopore sha dgerminated .Som eo f the crossesyielde d progenies withdistinc tmorphology ,i na 1:1 or 1:2:1 ratio.Suc hmutan t phenotypes mightb euse d forgeneti c mapping inth e future. The tenparent swil lb eanalyse db yRFL Pan dRAPi Danalysi s(William set al., 1990) todetermin epolymorphi cmolecula rmarkers .Th e segregationo findividua lmarker swil l 19 be analysed in the progeny obtained from all the crosses,thu s enabling us to establish molecular linkage maps between the different markers. By bulked segregant analysis (Michelmore et al., 1991) we might obtain markers which arelinke d to morphological characteristics, such as theinabilit y of isolate Bc7t o produce sclerotia.Th elon g term aim of theseexperiment s is theconstructio n ofa molecula r andgeneti clinkag e mapo n whichgene swit hknow n functions canb eassigne d tocertai npositions . Toolsfo rmolecula ranalysi s Toperfor m gene disruption experiments,i t will benecessar y to clone genes of interest fromB. cinerea. Fromgenomi clibrarie sorcDN Alibraries ,gene sencodin gextracellula r hydrolytic enzymes will beclone d by the use of antibodies or heterologous DNApro b es.Thes eclone swil lb emutagenize d in vitro andth emutate d genewil lb ereintroduce d into the wild type B. cinerea by transformation. By homologous recombination, the mutation can be exchanged with theresiden t chromosomal gene,resultin g in a mutant allele('gen edisruption') .Suc ha recombinatio neven twil loccu rwit ha certai n frequency whichca nb escore db ygeneti c ormolecula ranalysis . To perform gene disruption, one needs to be able to introduce foreign DNA into B.cinerea b ytransformation . Atransformatio n protocoli scurrentl ybein gdeveloped .W e areattemptin g toobtai nhygromycin-resistan ttransformant s eitherb yelectroporat io no f fungal protoplastso rgermtube s(Chakrabort yet al, 1991),o rb yth estandar dPEG/CaCl 2 treatmento fprotoplast s(Mishra , 1985). Resultsobtaine dsofa r indicatetha tth emediu m on which the hygromycin selection is performed is extremely important. We have not obtainedhygromycin-resistan t coloniesyet .Clearly ,th etransformatio n procedureneed s furtherimprovemen tbefor ebein guse droutinely .T oallocat edifferen t molecularmarker s to specific chromosomes, electrophoretic karyotype analysis is being developed. Chromosomeseparatio no nagaros egel su pt osize so f 10Mb pca nb eachieve db yCHE F gel electrophoresis (Mills and McCluskey, 1990).W ehav e optimized the protocol for obtaining protoplasts of B. cinerea with high viability, but we were unable to release enoughDN Afro m theseprotoplast st ovisualis echromosom eband so nCHE Fgels .Thi s technical problem will probably be resolved in the near future. Separation of chromosomes will enable us to analyse karyotypes of different B. cinerea isolates and theirprogenies ,an dwil leventuall yallo wth emappin go fgene so rgeneti cloc it ospecifi c chromosomes. Conclusions and future prospects Many basic molecular and genetic techniques which have been applied to plant pathogenic fungi in recent years were not developed for the analysis of B.cinerea. Therefore wear eadaptin gth etechnique s tofacilitat e themolecula ran dgeneti c studyo f pathogenicity determinants in B. cinerea. Progenies from crosses have been obtained which are amenable to tetrad analysis with genetic or molecular markers. The crosses also provide means to study genetic instability unrelated to heterokaryosis. Genes encoding putative pathogenicity factors willb e isolated, mutated and reintroduced into wildtyp eB. cinerea. Transformant s willb escreene dfo rdisruptio n ofparticula rgene s of interest.Th eresultin g mutantswil lb eteste dfo r pathogenicity ontomat oan dothe rhos t plants. Such an approach will provide a better insight into the relevance of various enzymes andothe rfunga l productsfo r pathogenesis ofB. cinerea. 20 References Chakraborty, B.N., Patterson, N.A. and Kapoor, M., 1991. An electroporation-based systemfo rhigh-efficienc y transformation ofgerminate dconidi ao ffilamentou s fungi. CanadianJourna lo fMicrobiolog y37:858-863 . Faretra, F.an dPollastro ,S. , 1988.Effec t ofth esubstrat eo nth eformatio n ofscleroti ao f Botryotinia fuckeliana (Botrytis cinerea) and on their ability to germinate carpogenically.Phytopathologi aMediterrane a27:99-102 . Faretra,F. ,Antonacci ,E .an dPollastro , S., 1988a.Sexua lbehaviou r andmatin g system of Botryotinia fuckeliana, teleomorph of Botrytis cinerea. Journal of General Microbiology 143:2453-2550. Faretra,F. ,Antonacci ,E . and Pollastro, 1988b.Improvemen t of the technique used for obtaining apothecia of Botryotinia fuckeliana {Botrytis cinerea)unde r controlled conditions.Annal id iMicrobiologi ae Enzimologi a38:29-40 . Leone, G., Schoffelmeer, E.A.M. and Van den Heuvel, J., 1990. Purification and characterization of a constitutive polygalacturonase associated with the infection process of French bean leaves by Botrytiscinerea. Canadian Journal of Botany 68:1921-1930. McKeen, W.E., 1974. Mode of penetration of epidermal cell walls of Vicia faba by Botrytis cinerea. Phytopathology 64:461-467 . ' Michelmore,R.A. ,Paran ,I .an dKesseli ,R.V. , 1991.Identificatio n of markerslinke d to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings NationalAcadem y of Sciences88:9828-9832 . Mills, D. and McCluskey, K., 1990. Electrophoretic karyotypes of fungi: the new cytology.Molecula rPlan tMicrob eInteraction s 3:351 -357. Mishra,N.C. , 1985.Gen etransfe r infungi . Advancesi nGenetic s23:73-178 . Van den Heuvel, J. and Waterreus, L.P., 1985. Pectic enzymes associated with the phosphate-stimulated infection of French bean leaves by Botrytiscinerea. Nether landsJourna l ofPlan tPatholog y91:253-264 . Williams,J.G.K. ,Kubelik ,A.R. ,Livak ,K.J. ,Rafalski ,J.A . andTingey ,S.V. , 1990.DN A polymorphisms amplified byarbitrar y primersar eusefu l asgeneti cmarkers .Nuclei c AcidsResearc h 18:6531-6535. 21 Toolsfo r molecular geneticanalysi s ofBotrytis cinerea C.J.B. Bergmansand J.A.L. van Kan Summary Sexual crosses were madebetwee n eight different field isolates of B. cinerea and two strains ofknow n oppositematin g type.Progenie s of thesecrosse s areno w available for further moleculargeneti canalysis . Introduction Botrytis cinereaPers. :Fr. ,th eanamorp hBotryotinia fuckeliana (deBary )Whetzel ,i sa multinucleatefungu s anddu et oanastomosi scell sca ncontai nnuclei "o fdifferen t genetic background. This heterokaryotic nature has seriously hampered molecular genetic studieso fth efungus .T ob eabl et ostud ymolecula rgenetics ,sexua lcrosse sbetwee nte n different isolateswer eperforme d andth ehomokaryoti cprogenie scollecte di na nordere d way. Sexual crosses Sexualcompatibilit y of B. cinerea iscontrolle d by asingl e mating typegen ewit h two alleles:MA T1- 1 andMA T1-2 . Toobtai n acollectio n of homokaryotic progeny,eigh t different field isolates (prefix Be) were each crossed with two strains (prefi x SAS) of known opposite mating type (Faretrae tal. , 1988a)accordin g toa metho ddescribe db yFaretr a etal. (1988b).Strain s producing both 'male' (microconidia) and 'female' (sclerotia) structures weremate di n reciprocal crosses.Control s included unspermatised andself-fertilise d sclerotia.Tabl e1 shows allcrosse s and their success in producing apothecia. Apothecia emerged twot o fivemonth s after spermatisation and arose from any part of the sclerotial tissue.Thei r formation startedwit hth eelongatio n ofth estip ean da subsequen tdifferentiatio n ofth e apext oa head .Th eoccasiona lpresenc eo fconidiophore so nth escleroti adi dno tpreven t apothecia forming. Of the 15scleroti a used for each cross usually 25 to 100% produced apothecia of which the number varied between one to six per Sclerotium. In two cases only, one apotheciumwa sforme d onth e 15 sclerotiaused . Among the numerous apothecia formed, some weremalformed , viz.reversio n ofa n immature apothecium toth emycelia l stage,a n apothecium consisting of onestip ewit h twohead s andmalformation s ofth ehea dstructure . Crosseswhic hwer eabl et ofor m matureapothecia , aremarke dwit h a '+' inTabl e1 . IsolatesBc7 ,Bcl 2 andBcl 6 weresucces sfull y crosseda s 'male' withbot hSAS5 6an d SAS405, indicating that these B. cinerea isolates contain both mating type alleles, possibly due to their heterokaryotic behaviour. Be1 6 also produced apothecia in the reciprocal crosses and in theself-fertilisatio n control.Thi s wasno t observed for Bcl2, 22 andscleroti ao fB c1 2ar etherefor e possiblydefectiv e informatio n of apothecia.A sn os clerotialstructure sfo rBc 7ar eknow n(Salina san dSchot , 1987)reciproca lcrosse scoul d notb etested . Allothe rBc-isolate ssuccessfull y crossedeithe rwit hSAS5 6o rSAS40 5i n bothreciproca lcrosses .Th eresult ssho wthatBcl8 ,Bc21 ,Bc2 5 havematin gtypeMAT l -1an dBc2 6an dBc2 9hav ematin g typeMA T1-2 . Table 1. Sexual crosses ofBotrytis cinerea isolates. Data were compiled afew months afterthe tenth Botrytis symposium. Sexualcrosse s of Botrytis cinerea in n COO a: SAS56-MAT1-1 b: SAS405 =MAT1- 2 c: Bc7 producesn oscleroti a d: -,n oapotheci a produced e: +,productio no fapotheci a f: USP- unspermatized sclerotia For most of the successful crosses a defined collection of progenies was collected by isolatingte nasc ifro m fivedifferen t apothecia.Th ehea do fa napotheciu mwa ssquashe d in water in order to release ripe asci. A mature ascus was transferred to a droplet of Novozym (5 mg.mH in 10 mM MES buffer, pH 6.0) on a wateragar plate. After an incubation of 30 sec at 37°C the ascus was rinsed in adrople t of water. TheNovozy m treatment weakened the natural opening of the ascus and ascospores could then be released by forcing them from the ascus in the natural sequence. The spores were separated on the agar and germinated for two days. Subsequently the young colonies weretransferre d tofres h tPDAplate s(PD Acontainin g 300g crushe dtomat oleave spe r litre) and cultured until they sporulated. Conidia were collected and stored in a 10% glycerolsolutio na t-80°C . Regularlyi twa sobserve dtha tno tal leigh tspore so fon eascu s 23 wereviable .A sincomplet e sets wereno tregarde d useful for genetic studies they were discarded.Matur eapotheci awer eals ostore di n a 10%glycero lsolutio n at-80°C . Monoascosporicculture sfro m acomplet ese to fascospore so fth esam eascu susuall y showed great uniformity in morphology. Occasionally sets showed a segregation of morphological characters(1: 1o r 1:2:1) giving riset odifferen t cultures. Furtherwor k will becarrie d outusin g parental isolates and theirprogenies .Parenta l isolates will be used for karyotyping and RAPiD analysis. Segregation of polymorphic markersi nth eprogen ywil lb eanalyse dan dresult swil lb euse dfo rth econstructio no fa molecularlinkag emap . References Faretra,F. ,Antonacci ,E .an dPallastro , S., 1988a.Sexua lbehaviou r andmatin g system of Botryotiniafuckeliana, teleomorph of Botrytis cinerea. Journal of General Microbiology 134:2543-2550. Faretra, F., Antonacci, E. and Pallastro, S., 1988b. Improvement of the technique for obtaining apothecia of Botryotinia fuckeliana {Botrytis cinerea)unde r controlled conditions.Annal s ofMicrobiolog y38:29-40 . Salinas, J. and Schot CR, 1987.Morphologica l and physiological aspects of Botrytis cinerea. Mededelingen Fakulteit Landbouwwetenschappen, Rijksuniversiteit Gent 52:771-776. 24 Genetics of cream mutants ofBotryotinia juckeliana (Botrytis cinerea)wit h aberrant production of laccase enzyme F Faretra andAM. Mayer Summary Two UV-induced albino mutants of B. fuckeliana (B. cinerea), with conidia and conidiophores ofa crea mcolour ,showe daltere dproductio n oflaccas eenzyme .Geneti c defects of mutants were investigated through genetic analysis of ascospore progenyo f sexual crosses with a wild-type strain. In both mutants, single Mendelian genes were responsiblefo r theaberran tmorphology .Laborator y testso fagressivenes so ncucumbe r cotyledons and grape berries showed that ascospore progenies with cream phenotypes weren oles s aggressivetha nwild-typ eprogenies . Introduction In mutagenesis experiments aimed at obtaining genetic markers, two mutants of B. cinerea werefoun d yielding sporulating colonieswit h acrea mcolou rrathe r thanth e grey-brown colouro fth ewild-typ estrain . Fungiproduc ea variet yo fdar kpigment sknow ngenericall ya smelanins ,althoug hthei r synthetic pathways are different (Bell and Wheeler, 1986). It is known that for some fungi, sucha sColletotrichum an dMagnaporthe spp. ,melani nha sa nessentia lrol ei nth e penetration ofhos tplant s(i.e. ,Chumle yan dValent , 1990; Kuboe tal. , 1987). Studieso f several fungi suggested that laccase is involved in melanin biosynthesis (Bell and Wheeler, 1986). In B.fuckeliana, there is evidence that laccase is also involved in pathogenicity (Bar Nun and Mayer, 1989;Ba r Nun etal, 1988; Hodson et al, 1987; Marbache tal. , 1984,1985) . This paper deals with genetic studies of cream mutants and the evaluation of their laccaseproductio n andaggressiveness . Material and Methods The two mutants of Botryotiniafuckeliana (d e Bary) Whetz., teleomorph of Botrytis cinerea Pers.: Fr., BAM28 and BAM19 , were obtained from strain SAS56 after UV- irradiation. They were mated with the strain SAS405 as described (Faretra et al., 1988a, b). Random ascospores from individual apothecia were collected singly and grown onmal textrac taga r(MEA )a sdescribe db yFaretr a andGrindl e(thi svolume) . Theaggressivenes s of B.fuckelian a isolates wasteste d oncucumbe r (cv.Bet a Alfa) cotyledons floating on a thin layer of water in Petri dishes,a s well as ongrap e berries (cvs. Italiaan dRegina )kep ti na humi dchamber . Conidialsuspension s(1 0JJU Icontainin g 105spores.m Hi n0.05 %Twee n20 )o rmycelia lplug s( 1mm 3block s or3-m m diameter disks) were used as inoculum. Conidia were collected from seven day-old colonies 25 «5 'è S g f-OO^ON I U ^ 60 C S '5b ON NO ai «M O 00 p •rt 00 r~ ON c od i-i M" -^000 sa •3 3 •a a "a, e u 10 ON en NO tv H a" c *& .23 -H ON f- NO £| ON su *8 -<' Ö O -H" §3 £§. -a 1 NO «O .v> S --H t^ NO 00 NO a. 'O3 , Pu, S- d ?i?î?l l o «5 2 s u Ü I-; p >o p o - (N ^ rn ri-' | > 1 Ol X o c 0O NO O •* 3 •s .•a Ira S O §5N p iri »o TJ- U3 m' m' m' en C 8 o 60 •< u O 3 ,-< 'S f—| w#. S (D « 4H s•us a Dû O vo o° ö © O Ci B Q o ^ NO >o 00 o e CL, •s s O ON NO Table3. Aggressiveness of creammutants and wild-type reference strain^ Strain Conidi a Cucumber cotyledonsb> Grapeberries 0) Mycelial plug Conidia (wound) Mycelial plug (1 mm3) 1 mm3 3 mm3 'Italia' 'Regina' 'Italia' 'Regina' SAS56 0.7 2.0 1.3 3.0 3.0 0.7 \a SAS405 2.0 1.7 2.3 2.7 2.0 1.0 1.0 BAM1 8 2.7 0.3 1.3 3.0 2.7 0.7 0.7 BAM19 0.7 2.7 3.0 3.0 3.0 0.0 0.0 a) Figures represent classes of empirical scales ranging from 0 (healthy) to 3 (symptom spot > 11 mm) for cucumber cotyledons and from 0 (healthy) to 6 (berry rotten) for grape berries. b) Survey carried out after two,fou r and six days of incubation for conidia, 1mm 3 and3 - mm diameter plugs of mycelium, respectively. c) Survey carried out after six days of incubation. grown on potato dextrose agar and exposed to white and near UV-radiation on a 12 h photoperiod; mycelium was collected from the margin of actively growing colonies on MEA. Grape berries were wounded with a needle immediately before inoculation with conidia. In experiments with cucumber leaves conidial suspensions were prepared in solutions containing 25 g.H glucose and 50 mM KH2P04 (Akutsu et al, 1987). Symptoms were evaluated at two-day intervals by using the following infection scales: a) cucumber leaves, 0 = healthy leaf, 1 = rot spot diameter < 5 mm, 2 = 6-10 mm, 3 = > 11mm ; b) grapes: 0 = healthy berry, 1 = rot spot diameter 1-5 mm, 2 = 6-10 mm, 3 = rot on 25%berr y surface, 4 = 50%, 5 = 75%,6 = berry entirely rotten. All experiments were carried out twice with three replicates. Fortest s oflaccas eenzym eproduction , thefungu s wasgrow n in 1 Lflask s containing 130 ml medium at 20°C (static culture). The culture medium contained (per 1000 ml) 20g mal textrac t and 16g citru s pectin in0.01 M KPi-citratebuffe r pH 3.5; 1 ggalli c acid was added after autoclaving. Enzyme activity was measured by using an oxygen elec trode with 10m M quinol as substrate, pH 4.6 at 25°C. 27 Results and discussion Thetw omutant swer ecrosse d withMAT1- 1 strain SAS56an dMAT1- 2strai nSAS405 . Theywer efertil eonl ywit hS AS405 ,thu sretainin gth ematin gtyp eo fth eparenta lisolat e SAS56. Wild-typean dcrea mphenotype ssegregate di na 1: 1 ratioi nascospor eprogenie sfro m crosses of strain SAS405wit h both cream mutants(Tabl e 1).Thi sindicate s thatmutan t phenotypeswer edu et osingl echromosoma lgenes ,whic hwer ebot hprovisionall ycode d Crm. Both mutants produced dark sclerotia but were unable to produce apothecia; therefore itwa sno tpossibl et omak ecrosse sfo r testing allelism. Crm genes segregated independently during meiosis from either the Mbcl geneo f resistancet obenzimidazole s andth eDa f1 genefo rresistanc et odicarboximide s(Faretr a andPollastro ,1991,1992) .Th egene sMbc l andDafl showeda loos elinkag ei nth ecros s SAS405x BA M1 8 butwer eindependentl y reassorted inth ecros s SAS405x BA M19 . Strains SAS56 and SAS405 produced similar amounts of endo- and exo-laccase.I n comparison,BA M1 8produce dles sendo-laccas ebu tmor eexo-laccase ;an dBA M1 9wa s almost normal in endo-laccase production, but deprived of exo-laccase (Table 2). Preliminary acrylamide gel electrophoresis indicated differences in enzyme mobility among mutants. The enzyme produced by BAM1 8appeare d as one band while thato f BAM1 9 separated into two bands, one of which was very close to starting point. The control(a nINR Astrain )ha d 1-2 bandscorrespondin g toth efaste r band ofBA M19 . Evaluations of aggressiveness yielded datavaryin g with theassa y systems used.Fo r example,BA M1 8 wasth emos taggressiv eisolat ei ntest swit hconidi ao ncucumber ,bu t the least aggressive in tests with mycelium on the same host (Table 3). Thus, no conclusive evidence of correlation between production of laccase and aggressiveness couldb e obtained.Meioti c progenies ofmutant swer eals oevaluated . Inal ltests ,crea m progenies were consistently more aggressive than wild-type progenies (Table 4).Thi s suggeststha taberran tphenotype sd ono tnecessaril ycaus elos so faggressiveness . Meiotic progenies derived from themutant s areno wbein g investigated to ascertain whether, as the preliminary results indicate, the aberrant production of laccase and aberrantpigmentatio nco-segregat edurin gmeiosis ;tha ti st osay ,whethe rthe yar eeffect s ofth esam egeneti c defect. References Akutsu, K., Takutsu, Y, Sakiyama, H. and Okuyama, S., 1987. Stimulative effect of potassium phosphateo ninfectio n ofcucumbe r leavesb yconidi a ofBotrytis cinerea. Annalso fth ePhytopathologica l Societyo fJapa n 53:175-181. BarNun ,N .an dMayer , A.M., 1989. Cucurbitacins -repressor s of induction of laccase formation. Phytochemistry28:1369-1371 . Bar Nun, N., Tal Lev, A., Harel, E. and Mayer, A.M., 1988. Repression of laccase formation in Botrytis cinerea and its possible relation to phytopathogenicity. Phytochemistry 27:2505-2509. Bell, A.A. and Wheeler, M.H., 1986.Biosynthesi s and functions of fungal mélanines. Annual Review ofPhytopatholog y 24:411-451. Chumley, F.G. and Valent, B., 1990. Genetic analysis of melanin-deficient, non pathogenic mutants of Magnaporthe grisea. Molecular Plant-Microbe Interactions 3:135-143. 28 T Faretra, F. and Pollastro, S., 1991.Geneti c basis of resistance to benzimidazole and dicarboximide fungicides in Botryotiniafuckeliana (Botrytis cinerea). Mycological Research95:943-951 . Faretra, F. and Pollastro, S., 1992. Genetics of sexual competibility and resistance to benzimidazole and dicarboximide fungicides in Botryotinia fuckeliana(Botrytis cinerea). PlantPathology ,i npress . Faretra,F. ,Antonacci ,E .an dPollastro ,S. , 1988a. Improvemento fth etechniqu euse dfo r obtaining apothecia of Botryotinia fuckeliana (Botrytis cinerea) under controlled conditions.Annal id iMicrobiologi a eEnzimologi a 38:29-40 . Faretra,F. ,Antonacci ,E .an dPollastro ,S. , 1988b.Sexua lbehaviou r andmatin g system ofBotryotinia fuckeliana, teleomorph ofBotrytis cinerea. Journalo fGenera lMicro biology 134:2543-2550. Hodson,M.J. ,Bar-Nun ,N .an dMayer ,A.M. ,1987 .Cytochemica llocalizatio no flaccas e inBotrytis cinerea. Transactionso fth eBritisc hMycologica l Society89:572-575 . Kubo,Y. ,Furusawa ,I .an dShishiyama ,J. , 1987.Relatio nbetwee npigmen tintensit yan d penetrating ability inappressori ao fColle totrichu m lagenarium.Canadia nJourna lo f Microbiology33:871-873 . Marbach, I., Harel, E. and Mayer, A.M., 1984. Molecular properties of extracellular Botrytiscinerea laccase .Phytochemistr y2 3:2713-2717 . Marbach, I., Harel, E. and Mayer, A.M., 1985. Pectin, a second inducer for laccase productionb yBotrytis cinerea. Phytochemistry24:2559-2561 . 29 Pectin degradation byBotrytis cinerea'. a molecular genetic approach M.A.Kusters-van Someren, B.J.G. Manders andJ. Visser Summary Thehomokaryoti c strainB. cinerea, SAS56, wasinvestigate d byisoelectri c focusing in combination with overlay staining and Western analysis of samples of the growth medium to determine its pectinolytic activity. It was shown that this strain produced several isoforms of polygalacturonase (PG) and pectinesterase (PE),an d evidence was found thaton eo rmor epecti nlyase s(PL )wer eals oproduced .Usin g pectinolyticgene s from Aspergillus niger as probes, it was possible to isolate genes in B. cinerea by heterologoushybridisation .A gen elibrar yo fB. cinerea SAS5 6ha sbee nconstructe d for thispurpose .Ther ewer eindication so fa polygalacturonas ean dpecti nlyas egen efamil y inB. cinerea. Introduction Botrytis cinerea Pers.:Fr .produce s several extracellular enzymes which are implicated in pathogenesis.Cutinase s may play arol e inth eprimar y penetration of thehos t plant; cellulases,hemicellulase s and pectinases degradeth eplan t cellwall ,an dprotease san d phospholipases are able to degrade the cell membrane. Pectinases have received parti cular attention, since these enzymes are the first to be produced by several phyto- pathogenic fungi when grown on cell wall material.Thei r rolei n theinfectio n process has been established in bacteria (Collmer and Keen, 1986) as well as in fungi such as Verticillium albo-atrum(Durrand s and Cooper, 1988), Fusarium solani f.sp. pisi (Crawford and Kolattukudy, 1987) andB .cinere a (Leone, 1990;Movahed i and Heale, 1990).Althoug h multiple forms of polygalacturonase aredescribe d for B. cinerea, iti s not clear whether these are all encoded by different genes. Over the past 5 years, our group has isolated and characterised several pectin lyase and polygalacturonase genes from thesaprophyti c fungus Aspergillus nigeran d studied theregulatio n of thesegene s (Gysler et al, 1990; Harmsen et al, 1990; Bussink et al, 1991,1992; Kusters-van Someren et al, 1991,1992). We also have purified and characterised the encoded pectinolytic enzymes. Now we have investigated which pectinolytic enzymes are producedb yB. cinerea S AS5 6an dwhether ,usin gth eA. nigergene sa sprobes ,w emigh t be able to isolate the homologous genes from B. cinerea. Analysis of the genes and especiallyo fth eencode denzymes ,wil llea dt oa bette runderstandin g ofth ephysiolog y ofth einfectio n process. Material and Methods Strainsan dplasmid s B.cinerea homokaryoti cstrai nSAS5 6wa skindl yprovide db yDr . F.Faretr a(Bari ,Italy ) 30 and used for the construction of a gene library in the lambda vector EMBL3 from Promega. Plasmid pGW820 contains the A. nigerN40 0 pectin lyase A (pelA) gene (Harmsenet al, 1990),plasmi dpGW180 0contain sth eA . nigerN40 0polygalacturonas e II (pgaTT) gene (Bussink et al., 1990) and plasmid pIM305 contains the nigerN40 0 pectinesterase gene (pme) (Küsters-van Someren et ai, unpublished results). Esche richia coliLE39 2 (F-,hsdR5U(r k-,m k-), supEM, SK/?F58, lacYl or delta (laclZY) 6, galK2,gaYT22, meiBl, trpR55, lambda-) wasuse d asth elambd ahos tstrain . Growthcondition san danalysi so fpectinolyti cenzyme s The medium used for pectinolytic enzyme production contained per litre distilled water5 g KN0 3,2.5g KH 2P04,0.25g MgS0 4,0.4g FeCL 3wit h 1% pectin(fro m apple, degree of esterification 72.8%, Obi-pektin, Bischofszeil) and 1% glucose as carbon sources. The medium was inoculated with 106 spores.mH and incubated in a rotary shaker at 20°C for varying time periods. PG and PE zymograms were made by iso electricfocusin g ofcultur efiltrate s (10|xl) ,followe d byoverlayin gth efocusin gge lwit h a pectin-containing agarose gel,incubatin g itfo r 30mi nan d staining itwit h ruthenium red. ForWester n analysis, proteins from thegrowt h medium were separated on a10 % SDS-polyacrylamide gel. After electrophoresis, the proteins were blotted onto nitro cellulose and detected using the alkaline phosphatase assay (Biorad). Polyclonal anti bodiesraise dagains tA. nigerPL Ian dPLI Io rPGI Iwer euse dt oprob eth eWester nblots . DNAmanipulation s Standard methods were used for plasmid DNA isolation, random primed labelling, plaque screening and Southern analysis (Sambrook etal, 1989).DN A fragments were isolated from agarosegel susin gth eGeneClea nki t from BiolOl. Genelibrar y construction A medium containing per liter distilled water 2.0 g KH2P04, 2.5 g K2HP04, 1.0 g (NH4)2S04,0.5g MgS0 4.7H20,2.0g yeas textrac t(Gibco-BRL )an d 10.0g glucos ewa s inoculated with B. cinerea spores (lO^mF) and grown for 2 days at 20°C in a rotary shaker. The mycelium was collected by centrifugation (10 min, 10.000 rpm in aG SA rotor) and lyophilised. DNA was isolated as described for Aspergillus chromosomal DNA(d eGraaf f etal. , 1988),takin gcar et oshea rth eDN Aa slittl ea spossible .T oisolat e fragments of 14-20kb ,3 0 |xg of the DNA waspartiall y digested for 1 h at37° Cusin g 0.075unit sSau3A .Afte r additiono fEDT At oa fina l concentration of2 0mM ,th eDN A fragments wereseparate do na 0.4 %agaros ege li nTA E(5 0x TA Econsist so f24 2g Tri s base, 57.1 ml glacial acetic acid and 100 ml 0.5 M EDTA [pH8.0] per litre) and the fragments ofinteres twer ecu tou to fth egel .Th eDN Awa srecovere db yelectro-elution . For ligation, 125 ng B. cinerea chromosomal DNA fragments were used with 500n g EMBL3BamHI-digested ,dephosphorylate d lambdaarm s(Promega )i na 10\xl reaction mix, further containing 1uni t T4 ligase (Gibco-BRL) and ligase buffer (Gibco-BRL). After 4 h incubation at room temperature, 50 JJLI packaging mix (Packagene, Promega) was added. Packaging and titration was done as recommended by Promega (Promega Protocols and Applications Guide). The gene library was amplified as described by Sambrooke tal (1989) . 31 Hybridisationcondition s Hybridisation wasdon efo r 16 ha t60° Ci nstandar dhybridisatio n buffer containing 6x SSC( 1 xSS Cconsist so f0.1 5M NaCl ,0.01 5M sodiu mcitrate) .Th eblot so rfilter swer e then washed twice for 30mi n with 4 xSSC ,0.5 %SD S and twicefo r 30mi nwit h 2x SSC,0.5 %SD Sa t60°C . Results and Discussion Pectinolytic enzymeproductio n B. cinerea SAS56wa sgrow n onvariou s carbon sources (polygalacturonic acid,pectin / sugarbee tpulp ,pectin/glucose )fo r5 days .Eac hda ya sampl eo fth ecultur emediu mwa s takenan danalyse db yiso-electri cfocusin g andactivit ystaining .Wit hal lthre emedi aP G and PEband s were visible after several days. At least two forms of polygalacturonase were detected and four forms of pectinesterase (not shown). From these preliminary resultsw econclude dtha tth ebes tconditio nfo rproductio no fthes eenzyme swa sgrowt h for 5day s in amediu m with 1% pectin and 1% glucose ascarbo n source.Th eWester n analysisshowe dtha tpolyclona lantibodie sraise d againstA . nigerpecti nlyas ear eusefu l todetec tth ecorrespondin gB. cinereaenzyme s(Fig . 1).Th eA. nigerPGI Iantibod ywa s not very specific, and detected some other proteins, besides several A. niger poly galacturonases. 12 3 4 A B Fig.1. Western analysis ofproteins, producedby B. cinerea ina culture medium after 5days of growth. Lane1 :growt ho n 1%pectin/1 %glucose ;lan e2 :A. nigerPUl; lan e3 :growt ho n1 %pectin/1 %glucose ; lane4 : A.niger P GI . A: a-PLIan da -PLI Ipolyclona lantibody ; B:a-PGI Ipolyclona lantibody . 32 H B E S H B E S HBES i W' m Fig. 2. Southern analysis of B. cinerea SAS56 chromosomal DNA using A. niger pectinolytic genes as probes. H: Hindlll; B: BamHI; EcoRI; Sail. Probes used: A:A. niger N400 pelA 1.6 kb Clal fragment; B: A. niger N400pgal l 1.2 kbBamHI/Bgll lfragment ; C: A. nigerN40 0pm e0. 7k bSai lfragment . The hybridisationcondition swer ea s describedi nMateria lan dMethods . Southern analysis Chromosomal DNA was isolated from B. cinerea SAS56 and digested with Hindlll, BamHI, EcoRI and Sail. After Southern blotting onto nitrocellulose, DNA fragments containing the A. niger genes encoding pectin lyase A, polygalacturonase II, and pectinesterase were used as probes to detect the homologous B. cinerea genes. Fig. 2 shows thathybridisin g bands couldb edetecte d using thesegene s asprobes .Th e fact that multiple bands of different intensity are visible in Fig.2 A and 2B is indicative of a gene family for pectin lyases and endo-polygalacturonases. InA. niger, genefamilie s for these functions have also been described and cloned (Harmsen et al, 1990; Bussink et al, unpublished data).Ther e is probably only onegen e encoding pectin esterase, although a second hybridising band was seen in Southern blots using this gene as a probe (Khanh etal., 1991;Kusters-va n Someren etal., unpublishe d results). Construction of the B. cinerea gene library High molecular weight chromosomal DNA (30 |xg)fro m B. cinerea SAS5 6 was partially digested using 0.075 units Sau3A. Fragmente of 14-20 kb were isolated and used for subcloning in the lambda vector EMBL3 digested with BamHI. After ligation and 33 packagingc .2. 5x 104plaque swer efound , thuscontainin gc .3- 4x 105k bDN Ai ntotal . Assuming thatth egenom esiz eo fB. cinerea approximates thato fA .niger ( 5x 104 kb), thecomplexit yo f thegen elibrar yi s6- 8time sth egenom esize . Screeningo fth egen elibrar ywit hA. nigergene sa sprobe s Fiveplate swit h3 x 104plaque seac hwer euse dfo rth eplaqu escreening .Th esam epecti n lyase and polygalacturonase probes were used as previously in the Southern analysis. Fig.3 showstha tstrongly ,a swel la sweakly ,hybridisin gplaque scoul deasil yb edetecte d against a completely negative background. Differences in signal strength may reflect differences inplaqu esize ,difference s inth esiz eo fth ehybridisin g fragment cloned ina particular phage or differences in the extent of homology with the probe. The latter reasoni sver yprobabl econsiderin gth eresult so f theSouther nblot s(Fig .2) . u& • • ••I Fig. 3. Plaquescreening of the B. cinereaSAS56 genomic library using theA. nigeras a probe. Duplicatefilters are shown. DNAfro m stronglyan dfro m weaklyhybridisin gphage swil lb efurthe r characterisedb y restriction analysis,subclonin gan dsequencing .Wit hth eclone dgene sw ewil lb eabl et o study the role of pectinolytic genes and enzymes of B. cinerea in pathogenesis with emphasis onth efollowin g threeaspects : Characterisation ofth eenzyme swhic har eencode db ythes egene s The enzymes may be produced inAspergillus, after multiple copies of the genes have been introduced into the genome by transfer mation, until a transformation system of 34 B. cinereabecome s available.I ti s important tocharacteris e theenzyme s toinvestigat e whether they differ for instance in substrate specificity, pH optimum or in their degradation products.Thi s will lead to abette r understanding of therol e of thevariou s pectindegradin g enzymesi nth einfectio n process. Studyo fth eregulatio no f pectinase encodinggene si nB. cinerea Sofar ,nothin gi sknow no fth ewa yth epectinolyti cgene sar eregulated : whetherthe yar e all regulated by the same activator and/or repressor or whether different genes are regulated indifferen t ways. Construction ofa B. cinerea strain with reduced virulenceb yinactivatio no f pectinolytic genes Wear eespeciall yintereste di nclonin g anddisruptin g thegen eencodin g aconstitutivel y produced endo-polygalacturonasewit h ahig h iso-electric point, which is thought tob e very important early in infection (Leone, 1990;va n der Cruyssen and Kamoen, 1992; Johnston andWilliamson , 1992). Genedisruptio n tostud y therelevanc e of pectinolytic enzymes in pathogenesis has been used with the bacterium Erwinia chrypanthemi and with Cochliobolus carbonum, thefunga l pathogen of maize. Inth e case ofE. chrysan- themi, inactivation of allfive pectatelyas e genes ledt o areductio n in virulence,bu tth e strain could still cause soft rot (Ried and Collmer, 1988). Only a secretion-defective strain was non-virulent. In C. carbonum, only one endo-polygalacturonase gene is present; itsinactivatio n had noeffec t on thevirulenc e of thepathoge n onmaiz e(Scott - Craig etal., 1990).Thes eresult s show thatpectinolyti c enzymes arehighl y unlikely to be theonl y pathogenicity factors, and it isunlikel y that inactivation of the pectinolytic genesi nB. cinereahav ea drasti ceffec t onit spathogenicity .Nevertheless ,the yd opla y animportan trol ei ninfection , andi ti stherefor ewel lwort hstudyin gth ecomple xproces s ofpecti ndegradatio nb yB. cinerea ata molecula rlevel . References Bussink, H.J.D., Kester, H.C.M. and Visser, J., 1990. Molecular cloning, nucleotide sequencean dexpressio n ofth egen eencodin gpr epro-polygalacturonas eI Io fAsper gillusniger. FEBSLetter s273:127-130 . Bussink,H.J.D. ,Buxton ,F.P . andVisser ,J. , 1991.Expressio n andsequenc ecompariso n ofth eAspergillus niger an dAspergillus tubigensisgene sencodin g polygalacturonase II.Curren tGenetic s 19:467-474. Bussink,H.J.D. ,va nde nHombergh ,H. ,va nde nUsse lP . andVisser ,J. , 1992. Character izationo fpolygalacturonase-overproducin g Aspergillusniger transformants .Applie d Microbiology andBiotechnolog y37:324-329 . Collmer, A. and Keen, N.T., 1986. The role of pectic enzymes in plant pathogenesis. AnnualRevie wo fPhytopatholog y 24:383-409. Crawford, M.S. and Kolattukudy, P.E., 1987.Pectat e lyase from Fusarium solani f.sp. pisi: Purification, characterization ,in vitro translatio no fth emRNA ,an dinvolvemen t inpathogenicity . Archives ofBiochemistr y andBiophysic s258:196-205 . DeGraaff , L.H.,va nde nBroek ,H.W.J .an dVisser ,J. , 1988.Isolatio n andexpressio no f theAspergillus nidulans pyruvatekinas egene .Curren tGenetic s13:315-321 . 35 Durrands,P.K . andCoope rR.M. , 1988.Th erol eo fpectinase si nvascula rwil tdiseas ea s determined by defined mutants of Verticillum albo-atrum. Physiological and MolecularPlan tPatholog y32:363-371 . Gysler,C , Harmsen,J.A.M ,Kester ,H.C.M. ,Visser ,J .an dHeim ,J. , 1990.Isolatio nan d structure of the pectin lyase-D encoding gene from Aspergillus niger. Genetics 89:101-108. Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J., 1990. Cloning and expression of a second Aspergillus nigerpecti n lyase gene (pelA): indications of a pectinlyas egen efamil y inAspergillus niger. CurrentGenetic s 18:161-166. Johnston, D.J. and Williamson, B„ 1992.Productio n invitro of polygalacturonases by Botrytis cinerea. Proceedings of the XthBotrytis Symposium, Crete,Greece ,Pudo c Wageningen p.59-62 . Khanh, N.Q., Ruttkowski E., Leidinger, K., Albrecht, H. and Gottschalk, M., 1991. Characterization andexpressio no fa genomi cpecti nmethylesterase-encodin g genei n Aspergillusniger. Genetics 106:71-77. Kusters-van Someren M.A., Harmsen, J.A.M., Kester H.C.M. and Visser, J., 1991. Structure of theAspergillus niger pelA gene and its expression inAspergillus niger andAspergillus nidulans. CurrentGenetic s20:293-299 . Kusters-van Someren M.A., Flipphi, M.J.A., de Graaff, L.H., van den Broeck, H.C., Kester, H.C.M., Hinnen A. and Visser, J., 1992.Characterizatio n of the Aspergillus nigerpelB gene: structure and regulation of expression. Molecular and General Genetics:i npress . Leone, G., 1990. Significance and role of polygalacturonase production byBotrytis cinerea in pathogenesis. PhD thesis, Wageningen Agricultural University, The Netherlandspp . 80. Movahedi, S. and Heale, J.B., 1990.Th e roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by different isolates of Botrytiscinerea Per s ex Pers. Physiological and MolecularPlan tPatholog y36:303-324 . Ried, J.L. and Collmer, A., 1988. Construction and characterization of anErwinia chrysanthemi mutant with directed deletions in all of the pectate lyase structural genes.Molecula rPlant-Microb e Interaction 1:32-38. Sambrook, J., Fritsch E.F., and Maniatis, T., 1989. Molecular Cloning, a laboratory manual.Col d Spring HarborLaborator yPress . Scott-Craig J.S., Panaccione, D.G., Cervone, F. and Watson, J.D., 1990. Endo-poly- galacturonase isno trequire dfo r pathogenicity of Cochliobolus carbonum onmaize . ThePlan tCel l2:1191-1200 . Van der Cruyssen, G. and Kamoen, O., 1992.Polygalacturonase s of Botrytis cinerea. Proceedings of the Xth Botrytis Symposium, Crete, Greece, Pudoc, Wageningen p. 69-75. 36 HOST-PATHOGEN INTERACTION AND PHSYSIOLOGY Botrytiscinerea: host-pathoge n interactions O. Kamoen Summary Theinteraction s ofBotrytis cinerea with acompatibl e hostar econsidere d here asthre e consecutivestages :(1 )germination ,(2 )initia lcolonisatio ndurin gpenetratio no fth ehos t and (3) expansion of the lesion. Incompatible combinations are not discussed. The morphologocial, physiological and biochemical factors described in the literature as involvedi nthes estages ,ar ereviewed . Introduction Thisrevie wo fdifferen t aspectso fth ehost-pathoge n interactionwil lb erestricte dmainl y to successful fungal infections that result in spreading lesions. It is meantt o provide a betterunderstandin g of theinfectio n process from published data,bu t somehypothese s areals omad ewhic hshoul db etested . Therevie w commences from theemergenc e of germtubes from conidia. Essentially thesam egrowt hprocesse soccu ri nmyceliu mg rowingfro m sclerotiaa s from asuccess ful penetrated germtube of a conidium. Therefore, the former will not be discussed. Infections derived from ascospores of Botryotiniafuckeliana o r from microconidia of Botrytis cinereaPers. :Fr .hav eno tbee ndescribe d inth eliterature . Threephase sca nb edistinguishe d during fungal infection of asucceptibl ehos tplan t andconsequentl y willb ediscusse d inthre esections . 1. Germination ofconidi ao nth eplan t surface. 2. Penetration ofth eepidermis . 3. Theexpansio n offunga l hyphaei nth ehos ttissue . In each section morphological, physiological and biochemical aspects of infection are discussed. Germination Inhibition of germination inB. cinerea mayb e due totoxi c substances released byth e plant, inhibition by the phylloplane microflora, competition with the phylloplane flora for nutrients,th eag eo f theconidi a and thenumbe r of conidia in theinoculu m droplet. Self-inhibition ofgerminatio noccur swhe nspore sar epresen ti nhig hdensity ;mor etha n 105conidia.m H leadst oa reductio n inth epercentag e ofgerminatio n andolde rconidi a alsosho wreduce d germination (Last, 1960). Alos so fendogenou snutrient s alsooccur s andthi sca nb ereverse d by addition ofnutrient s (Blakeman, 1980).However , infection using droplets containing high spore concentrations may be more effective than low inoculum densities, as was shown by Deveral and Wood (1961). They suggested that some of the conidia germinated and penetrated in the first instance; this resulted in nutrientleakag ean dsubsequen t stimulation ofth egerminatio n ofth eothe rconidia . 39 Stimulation of germination of conidia can be achieved by addition of nutrients and presence of pollen in the infection droplets or by presence of senescent or dead plant tissueso rwound s(Mansfield , 1980). Sugars have been examined for their ability to supplement for loss of endogenous reserves.Th e stimulation of germination by pollen, wounds or senescent or dead plant tissuescoul db eattribute d tonutrient s release,bu tno tentirel y (Vande n Heuvel, 1987). Spatialisolatio no fth efungu s andabsorptio no fth einhibitor sca nperhap spartl yexplai n these effects. Conidiao fspecialise dBotrytis spp .lik eB. fabae o nbroa dbea n(Mansfield , 1980)an d B. squamosao n onion (Clark and Lorbeer, 1976) germinated better than conidia of B. cinerea. Someo fthes especie swer eles ssensitiv et oth einhibitor so fthei rhost stha n B. cinerea (Rossall andMansfield , 1978). Germtubes ofB. squamosa growtoward sstomat a oranticlina l walljuncture s (Clark and Lorbeer, 1976) whereas germtubes of B. cinerea follow anticlinal walljunction s before penetration ofpetals .Appressori a often developa tth eape xo fa germtub e before penetration.Th eshape so fappressori avarie sfro m simpleswolle ntip st omany-branche d structures, e.g. on tomato leaves ( Verhoeff, 1980).Appressori a are mostly formed on anticlinalwal ljunction s (Ogawaan dEnglish , 1960; Clark andLorbeer , 1976). A gel, sometimes with microfibrils, often surrounds the hyphae and appressoria of B. cinerea and isprobabl y composed of aß(l-3 ) (1-6)gluca n (Dubourdieu, 1978).Th e largest molecules are insoluble compounds probably derived from the cell wall which mayb epartl ysee na sa gel ;th esmalle rmolecule s( < 106Da )ar emor esoluble . McKeen (1974) suggested, that the gel is necessary for adhesion of hyphae to the plant surface. Because age l makes good contact with the cell surface it might also affect the tropism towards anticlinal cell walljunction s to offer protection against desiccation or attract toxins and enzymes produced by the fungus to assist in penetration. This might beth e case in infections at high relative humidity in the absence of waterdrops on the leaf surface (e.g.Salina s etai, 1989). Glucansfro m B. cinerea areno tparticularl y toxict oplan tcells ,neithe rd othe ysee m toac ta selicito r for antifung alcompound s produced byplant s(Kamoe ne tal. , 1980). Degradation of the cuticle under germtubes has often been observed by scanning electron microscopy (e.g Pie and De Leeuw, 1991; Verhoeff, 1980). Whether this degradation of the cuticle is due toenzymes ,t o mechanical pressure, acombinatio n of both,o rt oothe rfactor s deservesfurthe r examination. When inoculum droplets are used, the plant cell turgor under the drops usually remainsintac tunti lpenetratio n occurs.W e mayconclude ,therefore , thatdegradatio no f the epidermal cell does not occur by the action of enzymes or toxins in the inoculum droplets on thesurfac e of theplant , though somecompound s secreted by thefungu s at, orshortl y after, thegerminatio n ofconidi ama yb eimportant . Cutinases seem tob eessentia l for penetration of thecuticl eb ydegradatio n thecuti n in some hostplants. Cutinolytic activity was detected in conidia of B. cinerea and in culture media in which the fungus had grown (McKeen, 1974; Salinas, 1992) and monoclonalantibodie sraise dagains tpurifie d cutinasegav eprotectio no fgerber aflower s toinfectio n byB. cinerea (Salinas, 1992).Pecti ndegradin g enzymes areals opresen ti n thegerminatio n fluid and aresecrete d early by conidia and young germtubes and both these enzyme activities do play a role during the penetration of germtubes into plant tissue. 40 Glucans and low molecular weight polysaccharides have been detected in the germinationfluid in vitro (Bowenan dHeale , 1987;Dubourdieu , 1978). Their influence ismainl yvisibl elate rdurin g hyphalexpansion . Penetration Penetrationca nb econsidere da sth ephas ebetwee nsurfac egrowt han dlesio n formation. Thefungu s penetratesth eintac tsurfac eo fth eepidermi sb ya narro winfectio n peg(Clar k andLorbeer , 1976)whic hma yresul tfro m anappressoriu m orth eti po fa germtube . On onions, B. squamosaan d B. cinereashowe d an intermediate swelling in the intracellular spaces after penetration, or the infection hyphae ramified within the outer periclinal cell walls of the epidermis (Tichelaar, 1967). Similar hyphal swellings have beenobserve di nothe rhosts ,e.g .tomat ofrui t (Verhoeff, 1970; Rijkenberg etal, 1980), andgerber ara yfloret s (Salinas,1992) . Theinfectio n pegmostl ypenetrate svi aanticlina lwal ljunction s(Ogaw aan dEnglish , 1960; Mansfield and Richardson, 1981). This may be due to a lower wax content (Lamarck,pers .comm. )perhap sb ya thinne rcuticle ,o rb yth eproximit yo fth everticall y orientedmiddl elamell a asa preferre d pectin-containing pathint oth etissue .Penetratio n inepiderma lcell sthroug hth eoute rpericlina lwall si nepiderma lcell sseem st ooccu rles s frequently. ' Duringth epenetratio nprocess ,th efollowin g changesca nb esee nb yelectro nmicros copy: the cuticle is further degraded (Verhoeff, 1980), the cell walls softened by the degradation of pectin (Pucheu-Planté and Mercier, 1983) and a thickening of the epidermal cell walls occurs (Pie and De Leeuw, 1991). During the first hours of penetration, plasmolysis of cells adjacent toth e infection peg may still occur (Geeraert andKamoen ,unpublishe ddata) . Thethicknes s of thecuticl e and epidermal cell layers has aneffec t upon penetration ofgrap eepidermi sb yB. cinerea (Prudetet al, seepag e 99). Botrytisspp . can penetrate the plant through natural openings. B. tulipae on tulips (Hopkins, 1921), B. gladiolorum on gladiolus (Bald, 1952), B. squamosa on onions (Clarkan dLorbeer , 1976)an dB. cinerea o nbroa dbea nan ddahli aca npenetrat estomat a (Louis, 1963). Instrawberrie san draspberries ,penetratio no fstigma soccur san dhypha e entertransmittin g tissueso fth ecarpel s (McNicolet al, 1985,Bresto n etal, 1986). Many types of wounds generally favour the penetration of B. cinerea and the sub sequentdevelopmen to flesions .Som eo fthes ehav eimportan tagronomi cconsequences , e.g. in grapes (Gärtel, 1970;Bessis , 1972;Fermau d and LeMenn , 1989)an d tomatoes (Wilson, 1963).Wound s favour the fungus in two ways:i t represents aread y access to theinterna l tissues; and it contains necrotic cells which mayhav e alowe r resistance to thefungu s andprovid ea saprophyti cgrowt hbas efro mwhic hfunga l enzymesan dtoxin s maydiffus e intoadjacen t healthytissues . Nutrients in the infection droplets also stimulate the penetration and the subsequent lesionexpansion .Phosphate sincreas eth epecti cenzyme si ninfectio n droplets(Va nde n Heuvelan dWaterreus , 1985)an dpurine s (Koet al., 1981 )stimulat e infection. Duringpenetration ,Botrytis spp .secret ea numbe ro fcompounds .Table s 1 and2 give a list of known secretions with comments about their possible importance for thehost - pathogen relationships. Pectindegradatio n associatedwit hth efirs tcel lnecrosi sseem st ob eth emos tmarke d phenomenondurin gth epenetration .Thes easpect so fearl ypecti ndegradatio nhav ebee n 41 Table 1: Listof enzymes secreted by Botrytis spp. withpossible importancefor thehost- pathogen interaction. Substrate Enzymes Selected references cutin (cuticle) cutinase Salinas 1990,1992 pectin (cellwall ) polygalacturonases Leone 1990 pectinlyase s Heale199 2 pectin methylesterase s Marcusan d Schejter 1983 protein (cellwall ) acid proteinase Movahedi andHeal e 1990 phenols laccase Marbachefa/. 1984 ß(l-3)glucans ß(l-3)glucanase Dubourdieu 1978 cellulose cellulases Verhoeff andWarre n 1972 phospholipids phospholipase Shepard andPit t 1976 (membranes) phosphatidase,lipase s Tseng etal. 197 0 Trofimenko etal. 197 5 Table 2:Possible foxinsproduced by B. cinerea. Class Secreted substances Selected references polysaccharides glucans Dubourdieu 1978 rhamno-galacto-mannans Aksenova 1962 Kamoenan dDubourdie u 1990 organicacid s citricaci d Ladygyna 1962 oxalicaci d Ladygyna 1962 Verhoeff etal. 198 8 othersubstance s thiourea Ovcarov 1937 urea Ovcarov 1937 reviewed previously. The classification of the pectic enzymes (Rombouts and Pilnik , 1972),thei r relationship withcel l necrosis(Basha m andBateman , 1975;Movahed ian d Heale, 1990), host defences against pectic enzymes by inhibitors (Collmer and Keen, 1986, Schlösser, 1983) and the role of pectic cell wall fragments in host defence elicitation(Collme ran dKeen , 1986)hav ebee nadequatel y described. In B. cinerea a pectin-degrading enzyme is present in the conidium (Verhoeff and Warren, 1972)an di nth egermtub e(Va nde nHeuve lan dWaterreus , 1985;Leone ,1990) . This first secreted isozyme does not require induction by pectin fragments (Van der Cruyssenan dKamoen ,1992) . 42 Expansioni nth ehos ttissue s Thenumbe r of penetrations orinfection s mayb erathe r low in relation toth einoculu m present in the air,bu t onceestablished , lesions can expand rapidly in compatible host- pathogen combinations. Inou rinstitut ew ehav ebee n studyingth eborde rzon eo fexpandin glesion san d from this we concluded that there are two possibilities for expansion. The hyphal tips may grow between the living cells and the cells are killed only after hyphae have grown beyond them.Thi sha s been observed in strawberry petals.Th eothe r possibility is that host cells arekille d ahead of the hyphal tips and the fungus grows within the necrotic tissue,e.g .i nleaves ,stems ,an dgree nfruits .Th eassumptio ni nth elatte rcas ei stha tth e fungus wasunabl et ogro wi nlivin gtissue . Toelucidat eth ekillin g ofth ecell sahea do fth egrowin ghypha ew ear eattemptin gt o answertw oquestions ;viz .whic hsecretion s(o rsubstances )kil lth ecell san dho wd othe y movethroug htissues .Examinin gKoch' spostulate sfo rth edifferen t secretions(Dimon d andWaggoner , 1953),firstl y thesubstance s which mayb eresponsibl e mustb eisolate d from infected tissues showing characteristic symptoms.Tw opolysaccharide s (PS)wer e isolatedfro m infected grapes(Kamoe nan dDubourdieu , 1990); andpecti cenzyme s(PE ) wereobtaine d from inoculum droplets asdescribe d by Leone(1990 ) andfro m infected tissue (Balasubramani etal., 1971) . Secondly, thesecretion s isolated from the infected tissues must be purified and identified. This was partly done for glucans and rahmno- galacto-mannan-polysaccharides and a number of pectic isozymes have been purified partly (Leone, 1990). Thirdly, each of the (partly) purified secretions must evoke a characteristic symptom. With respect to the last of Koch's postulate, the following conclusions can be drawn: with partly purified rahmno-galacto-mannans, subcellular disorganisation was observed and the ability of cells to Plasmolyse was lost only later (Kamoen etal., 1978) ;wit h glucans,hardl y any toxicity wasobserve d (Kamoen etal., 1980); with partly purified pectic enzymes, membrane rupture was the first symptom (inabilityt oPlasmolyse )an dafterward s subcellulardisorganisatio noccurre d(Kamoe net al.,1978) . Oneconclusio n is, therefore thatfraction s ofbot hth eP San dP Ecomple xar e partlyresponsibl efo rcel lnecrosis . Other secretions also have been studied: an acid proteinase (Movahedi and Heale, 1990); organic acids (Kamoen, 1972),lacca s es (Dubourdieu etal., 1984;Peze t et al, 1991;Viterb oet al, 1992)an da phospholipas e (Shepard andPitt , 1976). Forkillin go fcell sahea do fth ehypha ea signa lmus tprecee dth ehyphae . Thissigna l could be transferred either via the symplast (Kahl, 1982) or via the apoplast by intercellulardiffusion . Argumentsi nfavou ro fintercellula rdiffusio n are:th ehypha ltip s aremainl ygrowin gi nth eintercellula r spacesan dtoxin san denzyme sar esecrete d there (Kamoen, 1972); the expansion increases at low water vapour deficit, which increases intercellular water content; and similar symptoms can be obtained with intercellular infiltration of fungal secretions (Kamoen etal., 1978).Th e diffusion must result in a gradiento ftoxi csecretions .A tth eboundar y ofth elesio nth efollowin g zonesca n often bedistinghuishe d (Kamoen, 1989): zone0 clos et oth enecroti ccente ro fth elesion swit h thehypha l tips secreting toxins andenzymes ;zon eI :diffusio n zonewit h toxicconcen trations of toxinsan d enzymes; zoneII :diffusio n zonewit h sublethal concentrationso f bothtype so fcompounds ,an dzon eIII :health ytissue . Amode l for expansion oflesion sbase d ondiffusio n hasbee n proposed by Kamoen (1972).Diffusio n ofth esecretion s mayoccu rvi aintercellula r spaces;a tth esam etime , 43 the secretions may be taken up in the cell walls and cytoplasm. From diffusion laws (Jacob, 1962),i t appears that thedistanc e overwhic h thecell s arekille d ahead and the expansion ofth elesion smainl ydepen do nth eintercellula rwate rcontent ,an dt oa lesse r extend on the concentration of toxins and enzymes produced by the hyphal tips. The intercellular waterconten tdepend s onth erelativ e humidity orwate r vapourdeficit , the stomatalaperture ,etc .A nalternativ ehypo thesi sfo rth eexpansio no flesion si sbase do n dilutionan dha sbee npropose db yHarriso n(1980,1988) .A nalternatio no fhumi dnight s anddrie rday sma ycaus eth etypica l zonation ofexpandin gBotrytis lesions . Concluding remarks Therei s someagreemen t about themorphologica l aspects ofBotrytis diseases,bu tles s is known about the biochemical and physiological aspects. We need strong evidence aboutth erol eo fth edifferen t enzymesan dtoxin ssecrete db yth epathoge ni nth ediseas e process.Fo rexample : - Whichpecti ndegradin gisozyme sar eessentia lan dar eproduce di nsufficien t quantities for expansion oflesions ? - Isth eproteinas ean d thephospholipas e essential for pathogenesis? - Whati sth erol eo f otherenzyme ssecrete d byBotrytis spp.? - Are acids and rahmno-galacto-mannans important for necrosis or for host defence elicitation? References Aksenova, V.A., 1962. On the toxicity of the polysaccharide fraction of the toxin of Botrytis cinerea. Proceedings of the Academy of Sciences of the U.S.S.R. 147:496- 498. Balasubramani,K.A. ,Deverall ,B J . andMuiphy ,J.V. , 1971. 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Transactions of the BritishMycologica l Society. 43:673-680. Leone, G., 1990. Significance and role of polygalacturonase production byBotrytis cinereai n pathogenesis. Ph.D. Thesis, Wageningen Agricultural University, The Netherlandspp .8 0 Louis, D., 1963. Les modalités de la pénétration de Botrytiscinerea Pers. dans les plantes.Annale sde sEpiphytie s 14:57-72. Mansfield, J.W., 1980. Mechanismso fresistanc et oBotrytis. InTh ebiolog y ofBotrytis. Eds.Coley-Smith ,J.R. ,Verhoeff , K.an dJarvis ,W.R .Academi cPress ,York ,Londo n p. 181-218. Mansfield, J.W. and Richardson, A., 1981.Th e ultrastructure of interactions between Botrytis speciesan dbroa d beanleaves .Physiologica l PlantPatholog y 19:41-48. Marbach, I., Harel, E. and Mayer, A.M., 1984. Molecular properties of extracellular Botrytislaccase .Phytochemistr y 23:2713-2717 . Marcus, L. and Schejter, A., 1983. 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Chemical Rubber Company Critical Reviews in Food Technology 3:1-26. Rossall, S. and Mansfield, J.W., 1978.Th e activity of wyerone acid against Botrytis. AnnalsApplie d Biology89:359-362 . 46 Salinas,J. , Glandorf, D.C.M., Picavet, F.D.an d Verhoeff, K., 1989.Effect s of temper ature, relative humidity and age of conidia on the incidence of spotting on gerbera flowers causedb yBotrytis cinerea. NetherlandsJourna lo fPlan tPatholog y95:51-64 . Salinas,J. , 1990. Protectiono fgerber aflower s againstinfectio n ofBotrytis cinerea wit h anticutinasemonoclona l antibodies.Act aBotanic aNeerlandi a39:313-31 4(abstract) . Salinas, J., 1992. Function of cutinolytic enzymes in the infection process of gerbera flowersb yBotrytis cinerea. Ph.D. Thesis,Universit yo fUtrecht ,Th eNetherlands ,pp . 105. Schlösser,E. , 1983.Allgemein ePhytopathologie ,G .Thiem eVerla g Stuttgart pp.280 . Shepard,D.V .an d Pitt, D., 1976.Purificatio n of aphospholipas e from Botrytis cinerea andit seffec t onplan ttissues .Phytochemistr y 15:1465-1470. Tichelaar, G.M., 1967. Studies on the biology of Botrytis cinereao n Alliumcepa. NetherlandsJourna lo fPlan tPatholog y 7 3:157-160. Trofimenko, N.M., Scherbakov, M.A., Alman, A.V. and Zadubskoja, L.M., 1975. (AbstractMycolog y 10(9)10,1976). Tseng,T.C. ,Lee ,S.L . andChang ,L.H. , 1970.A nextracellula r phosphatidase produced byBotrytis cinerea in vitro. BotanicalBulleti n Academy Sinensis(Taipei ) 11:88-97. Van den Heuvel, J., 1987. Substances in dead plant tissue that stimulate infection of French bean leaves by Botrytiscinerea. Netherlands Journal of Plant Pathology 93:135-146. Van den Heuvel, J. and Waterreus, L.P., 1985. Pectic enzymes associated with the phosphate-stimulated infection of French bean leaves by Botrytiscinerea. Nether landsJourna l ofPlan tPatholog y91:253-264 . Van der Cruyssen, G. and Kamoen, O., 1992.Polygalacturonase s of Botrytis cinerea. Proceedingso fth eXt hBotrytis symposium ,Heraklion ,Greece . Pudoc,Wageningen , TheNetherlands ,p . 69-75. Verhoeff, K., 1970. Spotting of tomato fruits caused by Botrytis cinerea. Netherlands Journalo f PlantPatholog y76:219-226 . Verhoeff, K. and Warren, J.M., 1972. In vitro and in vivo production of cell wall degrading enzymes by Botrytis cinerea from tomato. Netherlands Journal of Plant Pathology78:179-185 . Verhoeff,K. , 1980.Th einfectio n processan dhost-pathoge ninteractions .I nTh ebiolog y of Botrytis. Eds.Coley-Smith ,J . R., Verhoeff, K. and Jarvis,W.R . AcademicPress , NewYork ,Londo n p. 153-180. Verhoeff, K., Leeman, M., Van Peer, R., Posthuma, L., Schot, N. and Van Eijk, G.W., 1988. Changes in pH and the production of organic acids during colonisation of tomatopetiole sb yBotrytis cinerea. Journal ofPhytopatholog y 122: 327-336. Viterbo,A. ,Ba rNun ,N . andMayer ,A.M. , 1992.Th efunctio n of laccase from Botrytis cinereai n host infection. Proceedings of the Xth Botrytissymposium , Heraklion, Greece.Pudoc ,Wageningen ,Th eNetherlands ,p . 76-82. Wilson, A.R., 1963.Som e observations on the infections of tomato stems by Botrytis cinerea. AnnalsApplie d Biology51:171 . 47 Activation ofhos t defence mechanisms inrespons e to Botrytis cinerea J.B. Heale Summary Evidence from a range of host-parasite interactions shows that fragments, often of a carbohydrate origin, released from fungal cell walls,ca n elicit active defence mechan isms in host cells. There is some evidence for such direct (primary) elicitors from B. cinere a,bu t of more significance probably isindirec t(secondary ) elicitation caused byhos tcel lwal ldegradatio nand/o rhos tcel lnecrosis ,releasin gendogenou selicitors . An aspartic proteinase (APr) as well as endo-PG and endo-PL enzymes secreted by B. cinerea areimplicate d inbot hlatte rprocesses ,althoug h themechanism sinvolve d are distinct.Manna n polysaccharides mayfunctio n either asprimar y elicitors orb ycausin g cell death leading to secondary, endogenous elicitor release. In carrot root slices, elicitation of induced resistance responses causes activation of the enzyme ACC synthase,a ke yste pi nethylen ebiosynthesis ;accumulatio n of thephytoalexi n 6-MM is triggered by ethylene but induced resistance is not; both 6-MM accumulation and inducedresistanc ear ei nhibite db yfre e radicalscavengers ,thu sinducin g susceptibility; fatty acidperoxidatio n productsgenerate ddurin g ethylenebiosynthesi s areimportan ti n thistyp eo finduce d resistanceresponse . Introduction Asa necrotrophi cplan tpathogen ,Botrytis cinerea Pers. :Fr .ha sa critica lrequiremen tfo r deadplan ttissue ,o rexogeneou snutrient ssupplie di na naqueou senvironmen te.g .'thin ' water films on wound sites,a favoured entry route. Direct penetration involving cutin esterasema yals ooccu r(Shishiyam aet al., 1970;Salinas , 1992). Rapidmultiplicatio no f thepathoge nlead st ohig hinoculu mlevel swit hincreasin gextracellula rreleas eo fphyto - toxiccomponent sfro m spores andgrowin ghypha l tips(Heale ,1987) . The macerating activity of the pectic enzyme complex in B. cinerea is well under stood, but the primary mechanisms under lying the necrosis-inducing activity of the pathogen are still a controversial area, largely because of the lack of critical studies. Theseactivitie sar eusuall yascribe dt opecti cenzyme so fth eendo-P Gtyp ei nB. cinerea, but in recent studies we have also implicated an endo-pectin lyase (PL) in both maceration and cell death in Botrytis infection of carrot tissue (Movahedi and Heale, 1990a,b).D iLenn aetal. (1981)an dmor erecentl yKail eet al. (1991)hav eals oreporte d on the role of endo-PL in pathogenesis for B. cinerea. More significantly, we have demonstrated forth efirs ttim ea widesprea drol efo ra nasparti cproteinas e(APr) ,bot hi n earlyhos tcel ldeat han di ninitia linfectio n stagesfo rBotrytis interaction sinvolvin geigh t different isolates of the pathogen with grape berries, strawberry, raspberry, cabbage, broad bean leaves and carrot slices (Movahedi and Heale, 1990a,b; Movahedi etal., 1991).Phytotoxi cmannan scontribut et ohos tcell-killin g inspreadin g lesions (Kamoen etal, 1978;Kamoen , 1984). 48 Manyo f thesepathogen-produce d components areassociate d withbot hnecrosi san d withsubsequen telicitatio no fresistanc eresponses ,bu tonl ya fe wstudie shav eattempte d a comprehensive analysis of the role of such individual components (Kamoen et al, 1978; Kamoen, 1984;Bowe n and Heale, 1987a).Thi s review will therefore attempt to clarify therol eo fdifferen t typeso felici to ri nBotrytis-host interaction si nrelatio nt oth e current interest in plant cell-signalling systems,an d themechanism s involved in active defenceresponse s 'triggered' underthes econditions .Finally ,'nobl erot ' ofgrap eberries , 'ghostspotting ' oftomat ofruits , aswel la slatenc yi ngeneral ,wil lb ebriefl y considered inrelatio nt osuc hmechanisms . Conditionswhic hfavou relicitatio no f activehos tdefenc e mechanisms Iffo rconvenience ,w esimplif y theoutcom eo fa potentia lBotrytis-plan tinteraction ,w e canconside rjus t twopossibl eresults :localise d infection andspreadin glesions . Due to a combination of two or more infection-negative conditions [e.g.lo w initial levelso f inoculum,lo w humidity orabsenc eo f free water,absenc e ofver yjuvenil e(o r senescing)hos ttissue ,absenc eo fwound so rexogeneou snutrients] , alocalise d infection occurs, with relatively little host cell necrosis. These conditions favour elicitation of defence mechanisms. Duet oth ecombinatio n of several infection-positive, predisposing factors [e.g.hig h initial inoculum, high humidity or presence of free water,juvenil e or senescent tissue, woundtissu eo rexogeneou s nutrients],th erat eo freleas eo fphytotoxi ccomponent san d their increasing effects overwhelm all responses by surrounding healthy cells, i.e. the latter arekille dbefor e theyca n reactt oth epresenc e ofpotentia l elicitors andspreadin g lesionsdevelop . Mechanismso felicitatio n In highly characterised systems, interactions between plant host and pathogen may be classed asrecognition ,signa l transduction andresponse .W e dono tye thav esuc hclear - cutobservation s for B. cinerea-host interactions.Fo r thepresent , weca n consider first: (a)direc telicitatio nb yprimar y fungal elicitors,an dsecondly :(b )indirec telicitatio n via either cell wall degrading enzymes (CWDEs) or other necrosis-inducing components from thepathogen ,causin g(c )th ereleas efro m thehos tcel lo fconstitutiv e(endogenous ) elicitor(s),whic hact(s )a s 'secondmessenger(s)' ,triggerin gactiv eresponse si n adjacent healthy cells.I n ordert odissec t thevariou s mechanisms,mos tresearcher s haveusuall y investigated singlecomponents ,whil eignorin g temporal aspects andth emultiplicit yo f interactionsbetwee nmolecula rcomponent so fpathoge nan dhost . Particularlyimportan t here areth e primary stages in theinteractio n between B. cinerea and its potential host, i.e. germination and the immediate 12 to 24 h post-germination period which usually decide the outcome between infection leading to spreading lesions or active resistance leading tolocalise d lesions.Als oconsidere d herei sth epossibl e roleo fethylen ewhos e synthesis invariably resultsafte r infection byB. cinerea. Direct elicitation Evidence from a range of host-parasite interactions suggests that fragments, often of carbohydrate origin, released from fungal cell walls, can elicit active defence 49 mechanisms in plants and cell suspension cultures (Templeton and Lamb, 1988). Elicitors include gl ycoproteins, simple and complex carbohydrates, certain fatty acids such as arachidonic acid, and glutathione. The best-known charact erised elicitors derived from fungal cell walls include a ß-linked heptaglucoside from Phytophthora megaspermaf.sp .glycinea activei nsoybean ,an dchitosa n oligosaccharides (Templeton andLamb , 1988).Th efac ttha tonl y 1 of30 0simila rhepta-ß-glucoside swa sactiv ea sa n elicitoro fP Aaccumulatio n suggeststh epresenc eo fa specifi c receptorfo rthi smolecul e (Hahnet al., 1989) .W eca nspeculat etha tsuc ha recepto rexist si nth eplasm amembran e of the host cell and that following a specific recognition event, a signal is somehow transduced to the nucleus and coordinated gene regulation results. Transcriptional activationo fhos t 'diseaseresponse 'gene sca nb edetecte dwithi n5 mi nafte r additiono f elicitort oplan tcel lsuspensio n cultures(Templeto n andLamb , 1988).Ho welicitor sac t atth enuclea rleve lt oregulat egen eexpressio n isstil lno tunderstood , althoughchitosa n is reported to interact directly with plant DNA and chromatin (Kendra et al., 1987). Whether such primary elicitors areimportan ti nB. cinerea interactions withit s hostsi s notclearl y established. Our previous studies with the carrot-5. cinerea interaction have revealed that heat- killed conidia or cell-free germination fluid induced various active defence responses including suberisation, lignification, and accumulation of phytoalexins including the polyacetylene falcarinol and theisocoumarin ,6-methoxymellei n [6-MM](Hardin g and Heale, 1981). Many of these responses can probably be explained by a secondary elicitationmechanism ,a sdiscusse dbelow .Kurosak iet al. (1987 ) onth eothe rhan dhav e shown that fungal mycelial preparations, chitin or chitosan, will induce chitinase in a strain of cultured carrotcells ,withou t any loss of host cell viability.Th ehos tchitinas e degraded the hyphal walls of the pathogen Chaetomium globosum and the liberated soluble fragments stimulated PAL activity and enhanced the biosynthesis of phenolic acids inth ecarro tcells . Other components possibly acting as primary elicitors in host interactions with B. cinereaa t sub-toxic levels include particular polysaccharides (mannan) fractions (Kamoen etal, 1978;Kamoe n 1984),bu ti f shown toposses seve nsligh tphytotoxicity , theyma yb ebette rregarde d asindirec telicitor s (seebelow) . Indirect elicitation viahos t cellwal l degradation and/or host cell necrosis In studies relating the time course of cell death in carrot root slices infected with B. cinereaspor e suspensions with the time of first detection of necrosis-inducing componentsi nplanta ,i tappeare d thattw ocel llayer swer ekille d after 3 h,increasin gt o eight layers after 12 h. Up to 8 h, the only in planta necrosis-inducing fraction was identified as having aspartic proteinase (APr) activity. Endo-PG and endo-PL activity werefirs tdetecte d at8 and 10h respectively ,followe d bypecti nmethyl esteras e(PME ) at 12 h, and the phytotoxic glycoprotein and polysaccharide components were not identified before 18h afte r inoculati n(Movahed i andHeale ,1990a,b) . In the same investigation, we showed that at relatively low concentrations, both purified endo-PL and theAP rfro m B. cinerea, caused celldeat h andinduce d resistance toth epathoge ni nsurfac ecel llayer so fcarro troo tslices .A thighe rconcentrations ,bot h causedextensiv ecel ldeat hi ncarro ttissu ean do fcarro tsuspensio ncells .Neithe renzym e lysed osmotically balanced carrot protoplasts unless carrot cell wall preparations were also present in the same suspension. Plasmolysed carrot cells were protected from the 50 cytotoxic activity of theendo-PL ,bu t not from APr-induced damage.I twa sconclude d that cytotoxicity of both endo-PL and APr was indirectly caused by toxic wall com ponents released separately by each enzyme, with evidence that different components wereinvolve d ineac hcase . The APr apparently releases a low molecular weight cytotoxic component from the hostcel lwal lwhic hca ndiffus e toth ehos tcel l plasmamembrane ,eve nwhe nth elatte r is no longer in direct contact with its wall (i.e. when the cell is plasmolysed). For the endo-PL,w ehav epropose d acytotoxicit y mechanism basedupo nfre e radicalsrelease d from enzyme-treatedcarro tcel lwalls ,whic hhav ea ver yshor thalf-lif e andrequir eclos e contactbetwee n the host cell wall and the vulnerable polyunsaturated fatty acids of its plasmamembran efo rperoxidation-typ ereaction st ooccur .W ehav eals oshow ntha tfre e radical scavengers inhibit the resistance responses of carrot tissue to this pathogen (Hoffman andHeale ,1989) . Alternatively,Kail eet al. (1991 ) havesuggeste d thatpecti cenzyme sfro m B. cinerea release a high level of Ca2+ ions normally bound to pectic substances in the middle lamella during host maceration (in this case Swede bulb tissue). As regards the mechanism of cytotoxicity operating here,the y proposed anincrease d flux of Ca2+int o cells from theapoplast , resulting in thelowerin g of H+/K+ATPaseactivit y and thehig h K+efflu x observed from diseasedtissues . That indirect elicitation can occur in the B. cinerea-cartot interaction was demon strated byth efac t thattriggerin g of the pathway toaccumulatio n of theisocoumari n 6- MMcoul db einduce db ysoluble ,non-cytotoxi celicitors ,release dfro m damagedcarro t cells by endo-PL and APr enzymes when tested separately (Movahedi and Heale, 1990a,b).Treatmen to fcarro tcel lwall swit hAP rreduce dthei rprotei nconten tb yc . 30% and increased the macerating activity of endo-PL by 40%. When APr activity was specifically inhibited in spore suspensions by 'Pepstatin', necrosis and infection were completelyprevente di nmos tcases ,eve nthoug hther ewer en oeffect s ongerminatio no r growth invitro (Movahedi andHeale , 1990a,b;Mohaved i etal., 1991). Kurosaki et al. (1984, 1985), investigating carrot suspension cells, reported that pectinase ortrypsi n released hostcel l wallfragment s thatelicite d the production of the carrot phytoalexin 6-MM.Th eelicitor s purified from pectinase-treated carrotcel lwall s weredistinc tfro m thoserelease db ytrypsin .Th eresult sar econsisten twit hou r findings regarding APr-treated as compared with endo-PL-treated carrot cell walls, which both resultedi nth ereleas eo fnon-cytotoxi celicitor so f6-M Maccumulation s(Movahed ian d Heale, 1990b). The phytotoxic and elicitor fractions of cell wall components released bothb yAP ran dendo-P Lwer edistinc tfro m eachother .Th eforme r areheat-labil esinc e autoclaving thesolubl e fraction ofcel l wallAPr - orendo-P L hydrolysates toinactivat e theenzyme sals oremove d thetoxi cactivity ,wherea sth eelicito rfraction s werestabl et o autoclaving for 20min . De Lorenzo etal. (1991 ) have shown that whereas an endo-PL from the bacterium Erwinia carotovoracause dmaceratio n inpotat otissu eonl ya thig h pH(abov e7.0 ,wit h anoptimu m at9.0) ,ther ewa sa nextende d half-life for oligogalacturonides withelicito r activity atlo w pH (5.75) causing avariet y of disease responses in potato. Such active oligogalacturonides,whic hhav ebee n shownt ob eliberate dgenerall yb yman y different microbial endo-PGs and PLs have 10 to 13 galactosyluronic acid residues. They are active at micromolar concentrations, but their activity can be completely destroyed by endo-PGan dP Lunde roptima lcondition sf or enzym eactivit y(Darvil let al. , 1989).Thi s islikel yt ob ea 'fine-tuning' mechanismfo r theinteractio nbetwee nhos tan dB. cinerea, 51 based upon apoplastic pH levels which change during development and maturation. During active infection byB. cinerea in carrottissues ,th ep Hvalu efall s fron>c. 6.0i n healthy tissue toc .3. 5 over 3day s (Movahedi and Heale, 1990a),thu s changing toth e optimal value for APr activity. The possibility that endo-PL from B. cinerea releases effective elicitorsfro m thehos tcel lwal la tlo wp Hrequire sthoroug h investigationbot h herean di nothe rsystem sinvolvin g thispathogen . PG-inhibiting proteins areknow n to be present in a wide variety of dicotyledonous plants.Cervon eet al. ( 1989 )showe dtha tove ra 2 4h period ,suc hinhibitor-limited ,endo - PGactivit y results inth eliberatio n of 10t o 12 residuegalactosyluroni c acid fragments, thus essentially turning a virulence factor of the pathogen (the PG enzyme) into an avirulence factor, determining thereleas e of ahighl y activeelicito r from the hosts'own cell wall. It was already known from earlier work (Deverall and Wood, 1961a, b) that such elicitation resulted in activation of a host phenolase enzyme, with the resultant oxidisedphenol scausin gfurthe r inhibition ofth emaceratin g pecticenzym eactivity . Thedifferentia l rateso freleas ean ddiffusio n ofphytotoxi ccomponent san delicitors , aswel l asrate so frespons ear ehighl ycritica lhere .Furthermore ,ther ei sevidenc efo ra synergisticdiseas eresistanc erespons eb yth eplan ti nth emutua lpresenc eo fbot hdirec t fungal elicitors aswel la sit s owncel lwal loligosaccharin s (DeLorenz oet al, 1989). It is still not generally recognised that cell death caused by B. cinereaoccur s before maceration (Tribe, 1955; Movahedi and Heale, 1990b), and that it can be caused indirectly by thetoxi c effects of hostcel l wall components released by pectic enzymes (endo-PGan d-PL )o rth eA P ra sdescribe dabove . Therei sclear-cu tevidenc etha tinjur y to the membranes of pectic enzyme-treated host cells occurs almost immediately after exposure,an dbefor ean ysignifican t losso fwal lstructura lintegrit ycoul dpossibl yoccu r (Hislop etal., 1979;Byrde , 1982;Cooper , 1984).Thus , the hypothesis put forward by Bateman (1976) that the death of plant tissues treated with pectolytic enzymes results from plasmamembran e physical damage,cause d byburstin g of theprotoplast s through the enzyme-degraded and weakened cell wall under osmotic stress, i.e. 'the bursting hypothesis',ca nn olonge rb eheld . Kamoen (1984) has described elicitation of phytoalexins by B. cinerea in the outer zone (zone III) of restricted lesions on leaves at low humidity, occurring as aresul to f sub-toxic concentrations of mannan polysaccharides (10-50 x 103Da ) diffusing tothi s zone.Inhibitio n of plasmolysis indyin g cells in zoneI ,jus t ahead of leading hyphaei n zone0 ,wa sattribute dmainl yt oslow-diffusin g pecticenzyme s(e.g .PG :3 4an d5 6x 10 3 Da for two isoenzymes respectively), whereas chloroplast degeneration in cells still capable of plasmolysis was attributed to the more rapidly-diffusing, low molecular weightmannans ,whic hreache drelativel y highconcentration s inzon eII .Thus ,th eleve l of water or high humidity in determining diffusion of cytotoxic components, and the particular concentrations reached in different zones, will decide if active defence responses are sufficiently 'in place' before even moredamagin g pathogen components reachth esam elocation ,shoul d highwate rcondition sresume . Anendogenou selicito r released as aconsequenc eo fcel ldeath in carrotroo tcells , inducesresistanc e toB. cinerea. Low molecular weight components described as constitutive or endogenous elicitors havebee nobtaine dfro m planttissue swithou temp loymen to fan yCWDE-treatmen te.g . from damaged French bean (Hargreaves and Selby, 1978)an d carrot rootcell s (Bowen 52 and Heale, 1987b).Th e latter authors reported that a peptide (c. 5x 103Da ) extracted from carrot root tissues damaged by slicing or freeze-thawing, induced active defence mechanisms in carrot root slices against B. cinerea. Endogenous elicitor activity was presenti nhomogenate so ffres h andfreeze-thawe d tissues,bu twa sabsen ti nautoclave d tissue.A simila rpeptid efractio n wasals oobtaine d from carrot tissue treated withcell - freegerminatio nfluid o fth epathogen .Th edetectio no fthi speptid eelicito rwithi n2 h o f cell damage indicates that it is released or activated during the early stages of cell necrosis.Th epossibl erelationshi pbetwee n thistyp eo fendogenou s elicitor,release da s a direct result ofhos tcel l death (irrespective ofcausation ,bioti c orabiotic ) whichma y resultfro m hostcel lwal lo rplasm amembran edegradatio nb ya self-induce d (autolytic) hostenzym eo nth eon ehand ,an dothe rindirec to rsecondar yelicitor si nth efor m ofhos t cell wall fragments released by the pathogen's pectic enzymes described above on the other,ha sno tbee n investigated. Activationo fdiseas eresponse san dth e roleo fethylen e Whether primary or secondary elicitors (or both, acting synergistically) are involved, healthy carrot root cells undergoing induced resistance responses to B. cinerea accumulate a complex of phytoalexins (including 6-MM). We have concentrated in recent studies on the relationship between the induced resistance response, 6-MM accumulation andth erol eo fethylen e(Hoffman n andHeale , 1987;Hoffma n etal, 1988; Hoffman and Heale, 1989). Induction of active responses here is associated with activation ofa controllin g step(AC Csynthase )i nethylen ebiosynthesis ,bu twherea s6 - MMaccumulatio n istriggere db yethylen eitself ,induce dresistanc ei snot ,i.e . ethylene- treated slices accumulate thephytoalexi n butremai n susceptible to an invasive levelo f sporeinoculum .Bot hth ephytoalexi naccumulatio n andinduce dresistanc ear einhibite d byth efre e radical scavengers salicylhydroxamic acid andpropy l gallate,thu s inducing susceptibility. This is explained by the fact that the conversion of the precursor 1- aminocyclopropane-1-carboxylic acid (ACC) to ethylene by the ethylene-forming enzyme(EFE) ,i sknow n toinvolv ea fre e radical.Th efatty-aci d composition ofcarrot s includes about 70%linolei cacid ;bot hgrowth ,an dgerminatio n ofconidi ao fB. cinerea areinhibite d in thepresenc e of lipoxygenase and linoleic acid. It appears therefore that the fatty-acid peroxidation products generated during ethylene biosynthesis are importanti nth einduce dresistanc eresponse . Research in other host-parasite interactions has yielded findings which possibly implicatefundamenta l underlying mechanisms . Esquerré-Tugaye etal, (1989)workin g with tobacco andPhytophthora parasitica nicotianae, reported that apathogen-derive d glycoprotein elicitor which caused slight browning in tobacco cell suspensioncultures , induced lipoxygenase after only 1.5 h. In intact plants, lipoxygenase induction was maximal 2 days after inoculation of the resistant cultivar. They suggested that the hydroperoxides produced from linoleic andlinoleni c acids (polyunsaturated fatty acids) intobacc ob yth eincrease dlipoxygenas eactivit ycoul dpla ya multipl erol ei na serie s of cascade reactions leading to hypersensitive cell death, as well as participating in secondary signalst oadjacen t healthycells .Thi selicito r alsoinduce d thesam epathwa y of ethylene biosynthesis as described above for carrot root tissue, with an increase in ACC synthase activity within only 15mi n of elicitor treatment, followed by ACCan d ethyleneproduction . These workers proposed the following steps operating in a cascade involving: 53 recognition atth eplasm a membrane level leading tomembran e depolarisation, quickly followed by ethylene synthesis; ethylene and membrane depolarisation could play the part of rapidly exported cell-to-cell signals with intracellular signals including phospholipid metabolitessuc h ascompound s resulting from lipoxygenase actionand/o r inositol phosphates;io n fluxes, particularly Ca2+an d phosphorylation involving protein kinases(Farme ret al., 1989 )coul doperat ea spar to fth eintracellula rsigna ltransductio n systemleadin gt odiseas erespons egen eactivatio n (Esquerré-Tugaye etal, 1990). Expression ofresistanc e Thevariou stype so factiv eresistanc eresponse si nplant soperatin g againstBotrytis hav e been considered in detail by Mansfield (1980)an d arebeyon d thescop eo f thisreview . Theyinclud ephytoalexin s (PAs)whic hperhap sar ebette rconsidere d ashavin g arol ei n lesion restriction, as well as host cell wall changes involving formation of callose, suberisation, lignification, and papilla formation. Inmos tinteractions ,ther ei sevidenc e of acomple x of such responses implying coordinated regulation by the type of signals previously discussed. Just oneexampl e willb equote d here;i n carrot root slices under going active defence responses induced by cell-free germination fluid, heat-killed conidia, or sub-invasive levels of live conidia (1 x 105conidia.mH) > several different phytoalexin pathways are induced (including those producing isocoumarins, and polyacetylenes), and both suberisation and lignification are strongly developed (Heale andSharman , 1977;Hardin gan dHeale , 1981; Healeet al. ,1982a) . Inth euppe rfou rcel l layers of induced slices,w eobserve d morefrequen t nuclear migration, and an increase in both nucleolar volume and uptake of tritiated uracil, as compared with control, wounded-onlyslice s(Heal eet al, 1982b). Thecel lwal lmodification s wereals osee ni n control, wounded-only slices, but the level of expression was greatly elevated in the induced slices as a result of the fungal invasion and increased endogenous signals discussed earlier. Thus, there is evidence of a synergistic elevation of response in the presenceo f acomple x ofbot hhost - andpathogen-derive d signals. Latency, 'ghost-spotting'an d 'noblerot ' Adiscussio no fsigna ltyp eresponse si nhost sinvade db yB. cinerea woul db eincomplet e without a brief consideration of several well-known examples where host necrosis is absent or at least is strongly reduced. Latency ('quiescence') in fruit crops such as raspberry,strawberr yan dgrap ei sa commonl y occurringphenomeno n(Verhoeff , 1980). Itca n be initiated in a number of ways including colonisation of necrotic flower parts after blooming, without any further development of obvious symptoms until fruit maturity, when extensive rotting often occurs in wet or damp environments. Here, the longperio do f 'balanced parasitism' withouthos tdamag ei slikel yt ob epartl yexplaine d by a highly-suppressed APr- and pectic enzyme activity in the pathogen, possibly accompanied by release of elicitor-active signals. McNicol and Williamson (1989) describedgerminatio no fB. cinerea sporeso nth ewe tstigmati csurface s ofblackcurran t flowersfollowe d bythei rsystemi cinvasio no fth ewhol estyl etissu ewithou tan yobviou s necrosis,leadin g toprematur e abscission ofbloom san ddevelopin g fruits.On epossibl e mechanism suggested heret oexplai n flower shedding wasth ei nductio n ofethylen ei n thehos ttissue s asa resul t ofth einfectio n byB. cinerea. Ghost-spotting causedb y theinvasio n ofyoun g tomatofruit s byconidia l germtubes 54 of B. cinereai s associated with localised lignification (van Maarschalkerweerd and Verhoeff, 1976; Glazener, 1982),agai nevidenc efo ra signal-mediate ddefenc eresponse . Iti sestablishe dtha ta glycoprotei ninhibito ro fendo-P Gactivit yi ngrap eberrie si sa n importantfacto r inresistanc et oB. cinerea. Thefactor s determining 'Noblerot ' ofgrap e are certainly complex, involving climatic and edaphic factors, but we can at least speculate that reduced water availability within the fruit tissues at critical periods significantly reduces the diffusion of several necrosis-inducing components from the pathogen. Acknowledgements Theautho racknowledge sth efinancia l supporto fth eAFR Cfo rth esubstantia lpar to fhi s researchinvestigation s reviewedhere . References Bateman,D.F. ,1976 . Plantcel lwal lhydrolysi sb ypathogens . In'Biochemica laspect so f Plant Parasite Relationships'. Eds. 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The induced resistance response of carrot root slices to heat-killed conidia and cell-free germination fluid of Botrytis cinereaPers .e xPers .I .Th epossibl erol eo fcel ldeath . Annalso fBotan y49:847-857 . Heale, J.B., Dodd, K.S. and Gahan, P.B., 1982b. The induced resistance response of carrot root slices to heat-killed conidia and cell-free germination fluid of Botrytis cinerea Pers.e xPers .II .Nuclea rmigration ,nucleola rvolum ean duptak e oftritiate d uracil.Annal s ofBotan y49:859-872 . HealeJ.B .an d Sharman, S., 1977.Induce d resistance toBotrytis cinerea in root slices andtissu eculture so fcarro t(Daucus carota L.) .Physiologica lPlan tPatholog y 10:51- 61. Hislop,E.C. ,Keon ,J.P.R . andFielding ,A.H. , 1979. Effects ofpecti nlyas efro mMonilia fructigenao n viability, ultrastructure and localization of acid phosphate of cultured applecells .Physiologica l PlantPatholog y14:371-381 . Hoffman, R.M.an d Heale,J.B. , 1987. Celldeath ,6-methoxymellei n accumulation,an d induced resistance to Botrytis cinerea in carrot root slices. Physiological and 56 MolecularPlan tPatholog y30:67-75 . Hoffman, R.M. and Heale, J.B., 1989. Effects of free radical scavengers on 6- methoxymelleinaccumulatio n andresistanc et oBotrytis cinerea incarro troo tslices . Mycological Research92:25-27 . Hoffman, R.M.,Roebroeck ,E .an dHeale ,J.B. , 1988. Effects ofethylen ebiosynthesi si n carrot root slices on 6-methoxymellein accumulation, and resistance to Botrytis cinerea. PhysiologiaPlantaru m 73:71-76 . Kaile,A. ,Pitt ,D .an dKuhn , P.J. , 1991.Releas e ofcalciu m andothe rion sfro m various planthos ttissue sinfecte d bydifferen t necrotrophicpathogen s withspecia l reference toBotrytis cinerea Pers.Physiologica l andMolecula rPlan tPatholog y 38:275-291. Kamoen,O. , 1984.Secretion sfro m Botrytis cinereaa selicitor so fnecrosi san ddefence . Revued eCytologi ee td eBiologi eVégétale s 7:-241-248. Kamoen, O., Jamart, G., Moermans, R., Van DePutte, L. and Dubourdieu, D., 1978. Comparative study of phytotoxic secretions ofBotrytis cinerea Pers.ex .Fr. ,Mede delingen FakulteitLandbouwwetenschappen , Rijksuniversiteit Gent43:847-857 . Kendra,D.F. ,Fristensky ,B. ,Daniels ,C .an dHadwiger ,L. , 1987. Diseaserespons egene s inplants :expressio n andpropose dmechanism s ofinduction .In 'Molecular Strategies forCro pProtection' ,Eds .Arntzen ,C . andRyan ,C , AlanR .Liss ,Ne wYork , p.13-24 . Kurosaki, F., Matsui, K. and Nishi, A., 1984. Production and metabolism of 6- methoxymellein inculture dcarro tcells .Physiologica l PlantPatholog y25:313-322 . Kurosaki,F. ,Tsurusawa ,Y . andNishi ,A. , 1985.Partia lpurificatio n andcharacterizatio n of elicitors for 6-methoxymellein production in cultured carrot cells. Physiological PlantPatholog y 27:209-217 . Kurosaki,F. ,Tashiro ,N .an dNishi ,A. , 1987.Secretio n ofchitinas efro m culturedcarro t cellstreate dwit hfunga l mycelialwalls .Physiologica lan dMolecula rPlan tPatholog y 31:211-216. Mansfield, J.W., 1980. Mechanisms of resistance to Botrytis. In 'The Biology of Botrytis'.Eds .Coley-Smith ,J.R. , Verhoeff, K. and Jarvis,W.R. , Academic Press,p . 181-218. McNicol,R.J . andWilliamson ,B. , 1989. Systemic infection ofblac kcurran tflower s by Botrytiscinerea an dit spossibl einvolvemen ti nprematur eabscissio no ffruits . Annals ofApplie dBiolog y 114:243-254. Movahedi, S. and Heale, J.B., 1990a. Purification and characterization of an aspartic proteinasesecrete db yBotrytis cinerea Per se xPer si ncultur ean di ninfecte d carrots. Physiological andMolecula r PlantPatholog y36:289-302 . Movahedi, S. and Heale, J.B., 1990b.Th e roles of aspartic proteinase and endopectin lyaseenzyme s in theprimar y stages of infection and pathogenesis of various tissues bydifferen t isolateso fBotrytis cinerea. Physiologicalan dMolecula rPlan tPatholog y 36:303-324. Movahedi, S.,Norey ,CG. , Kay,J . andHeale ,J.B. , 1991.Infectio n andpathogenesi so f cash crops byBotrytis cinerea; primary role of an aspartic proteinase. In 'Structure andFunctio n ofth eAsparti cProteinases :Genetics ,Structure s andMechanisms' .Ed . Dunn,B.M. ,Plenu mPress ,Ne wYor kp .213-216 . Salinas, J., 1992.Cutinolyti c enzymes and the infection process of Botrytis cinerea in gerberaflowers .Ph DThesis ,Universit yo fUtrecht ,Th eNetherlands ,pp .105 . Shishiyama,J. ,Araki ,F . andAkai ,S. , 1970.Studie so ncutin-esterase .II .Characteristic s ofcutin-esteras efro m Botrytiscinerea an dit sactivit yo ntomat ocutin .Plan tan dCel l Physiology 11:937-945. 57 Templeton,M.D .an dLamb ,C.J. , 1988.Elicitor san ddefenc egen eactivation .Plan tCel l andEnvironmen t11:395-401 . Tribe,H.T. , 1955.Studie s onth ephysiolog y ofparasitism .XIX .O n thekillin g of plant cells by enzymes from Botrytis cinerea and Bacterium aroideae. Annals of Botany 19:351-368. VanMaarschalkerwaard ,M .an dVerhoeff , K., 1976.Lignificatio n asa possibl e defence mechanism in tomato fruits after infection by Botrytis cinerea. Acta Botanica Neerlandica25:256 . Verhoeff, K., 1980. The infection process and host-pathogen interactions. In 'The Biologyo fBotrytis'. Eds .Coley-Smith ,J.R. ,Verhoeff ,K .an dJarvis ,W.R. ,Academi c Press,p .153-180 . 58 Production invitr o ofpolygalacturonase s by Botrytis cinerea DJ. JohnstonandB. Williamson Summary Fourpolygalacturonase s (PGs)wer eproduce d byB. cinerea in amodifie d Czapek-Dox liquid medium containing citrus pectin. Twoendo-PG s andtw o exo-PGs were purified to homogeneity from 10-day-old cultures and polyclonal antisera were raised to both endo-PGs. In Western blots both the antisera reacted positively with either endo-PG isozyme,bu tno twit hth eexo-PG so rothe rextracellula rproteins .Th eantiseru mt oendo - PGIwa s used to study the production of endo-PGs in liquid media containing various carbon sources.Endo-PG swer efoun d tob econstitutivel yexpressed , whereaswhe nth e fungus was grown on 0.01%galacturoni c acid a novel peak detected by preparative isoelectricfocusin g wasattribute dt oexo-P Ginduction .Th erole so fthes efou risozyme s in pathogenesis of raspberry fruits is being examined by molecular and ültrastructural techniques. Introduction Botrytis cinerea Pers.: Fr.attack s flowers, fruits and vegetables of awid erang ehos to f plantsi ntemperat eregion sa sa necrotroph ,bu ti tcommonl yestablishe slaten t infections whichculminat ei npost-harves tgre ymoul d(Coley-Smit het al., 1980; Williamsonet al., 1987). This pathogen produces several enzymes which macerate plant cell walls. Poly- galacturonses (PGs)ar eth efirs t cellwall-degradin g enzymesproduce db ysevera l fungi whengrow no nisolate dcel lwall s(Coope ran dWood ,1975 )bu tlittl eattentio nha sbee n givent oth einductio n ofthes ePG si nB. cinerea. Inman yplan tpathogen s thesynthesi s ofPG si sregulate db ygalacturoni caci d(Lorenz oet al, 1987).I nB. cinerea biochemica l studies have shown that endo-PGs are expressed constitutively in ungerminated and germinated conidia (Verhoeff and Liem, 1978), and in vitro both constitutive and inducible PGs have been described (Leone and Van den Heuvel, 1986). This paper outlinesth epurificatio n oftw oendo-PG san dtw oexo-PG sfro m apectin-enriche dliqui d mediumwhic hwa sfull y describedelsewher e(Johnsto n andWilliamson , 1992)an dals o briefly provides new information about the production of these enzymes derived from immunologicaltechniques . Material and methods Fungalisolat ean dgrowt hcondition s Sporulating cultures ofB. cinerea isolate34 7(M I 339491) weremaintaine d according toHarriso n(1978) .Fo rpurificatio n ofPGs ,fort y 250m lconica lflasks ,eac hcontainin g 100m lo fa modifie dCzapek-Do xliqui dmediu msupplemente dwit h 1%glucos ean d1 % 59 citruspecti nwer einoculate d withmyceliu m ona naga rdisk .Th eflask s wereincubate d withorbita lshakin g at 120rp ma t20° Cfo r 10days .Th efiltrate wa sdialyse dagains tth e appropriatebuffe r before purification. Forproductio n studies,th efungu s wasgrow na s above in a modified Czapek-Dox liquid medium, but lacking pectin. After 6 days,th e mycelium wasrecovere d and transferred tosimila rflask s containing either an identical medium or one in which glucose was substituted with galacturonic acid at various concentrations for afurthe r 48h . Purification, separationan dconcentratio n ofPG s Endo-PGI,endo-PGH ,exo-PGI ,exo-PGI Iwer epurifie d tohomogeneit ya sdescribe db y Johnston andWilliamso n (1992) andendo-PG Iwa suse dt oproduc epolyclona l antisera from arabbit .Th egamma-globuli n fraction from theantiser awa suse d inWester nblot s and PTA-ELISA. For unambiguous detection and quantification of endo-PGs from culturefiltrates, i t wasfirst necessar y to separate and concentrate theisozymes .Endo - PGI was concentrated by loading culture filtrate previously dialysed against 20mM acetatebuffe r atp H5. 2o nt oa nS-Sepharos eFas tFlo wcolumn . Thecolum nwa selute d withth esam ebuffe r containing 1 MNaCl . Theunboun dmateria lcontainin gendo-PGI J was applied to an identical column containing Q-Sepharose Fast Flow and eluted similarly. Further analysis of isozyme production involved preparative isoelectric focusing (prep.IEF )i nth ep Hrang e3. 5t o 9.5. Results Enzyme characteristics Purification revealedtw oendo-PG s(endo-PG Ian dendo-PGII )wit hp ivalue so f8. 8an d 4.9 and twoexo-PG s (exo-PGI andexo-PGII ) with pi values of4. 9 and 3.5.Endo-PG I andI Ibot h had apparentM rvalue s of3 6kDa ;exo-PG I andI Iha dM r values of6 5 an d 70kD arespectively .Eac h proteinconsiste d of asingl epolypeptid echain .Exo-PG Ian d II were shown to be glycoproteins by their affinity to concanavalin A. Their pH and temperature optimahav ebee ndetermine d (Johnston andWilliamson , 1992). PGinductio n with galacturonicaci d Filtrate from cultures grown in a modified Czapek-Dox liquid medium containing galacturonicaci drangin gfro m 0.01%t o1.0 %reveale dn oconsisten tincreas ei ntota lP G activity,relativ et omycelia ldr y weight after 48h .Subjectio n of thefiltrat e topre pIE F revealeda nove lisozym epea kwit ha p ivalu ebetwee n3. 7 and6. 3whe nth efungu s was growno na mediu msupplemente d withgalacturoni c acid,bu tno twit h 1% glucose. Antiserum raised against endo-PGI reacted positively with both endo-PGI and IIi n Western blots but no reaction was detected against any other extracellular protein. Fractionsfro m prepIE Fsubjecte d toWester nblo tanalysi sshowe dth epresenc eo fendo - PGI in the medium when the fungus was grown on either 1% glucose or 0.01% galacturonic acid.Endo-PGI Iwa sno tdetecte d in either case.Followin g separation and concentration ofendo-PG I andI Ib yio nexchang echromatography ,bot henzyme swer e identified on blots from media supplemented with either 1% glucose of 0.01% galacturonic acid. 60 B. cinerea grown in duplicate flasks supplemented with either galacturonic acid or glucose produced endo-PGI and endo-P Gil to similar amounts regardless of carbon source,confirmin g theresult sobtaine d forWester nblots . Discussion Mostfunga l pathogensstudie dsho winductio no fendo-PG swhe ngrow ni nth epresenc e of low levels of galacturonic acid (Cooper and Wood, 1975;Kee n and Norton, 1966; Lorenzo et al., 1987). A recent study of PG-production of B. cinerea revealed both constitutive and inducible endo-PGs using gel electrophoresis (Leone and Van den Heuvel,1986) .I nou rwork ,th ene wpea ko factivity ,produce donl ywhe nth efungu swa s growni nth epresenc eo fgalacturoni cacid ,coul donl yb eaccounte dfo rb yth einductio n ofeithe rendo-PGII ,exo-PG Io rexo-PGI Ibecaus eth ep io fth eisozyme sreside swithi n thisp Hrang e(Johnsto n andWilliamson , 1992). Western blots of the prep IEFfraction s corresponding toendo-PG I revealed aban d from filtrates from culturesgrow nwit hbot hgalacturoni caci dan dglucose . Thepresenc e of theseband s is indicative of constitutive production of this isozyme. Although endo- PGII was not detected in that test, its presence was later proved after further concen tration of filtrate from either medium, suggesting that constitutive production ofendo - PGIIoccur si nB. cinerea underthes econditions .Constitutiv eproduction'o f bothendo - PGssuggeste db yWester nblot swa sconfirme d byPTA-ELISA . Theappearanc eo fth enove lP Gactivit y observed during prep IEFca n therefore not beaccounte d for byendo-PGI Iwhic hwa sconstitutivel y produced.W esugges ttha tthi s peak can only be explained by the induction of one or both exo-PG isozymes. The significance ofexo-P Ginductio n andconstitutiv eexpressio n ofendo-PG s isno tunder stood. Endo-PGI willb e positively charged after release of acidic vacuolar fluids from mesocarp cells in ripening raspberry fruits andresult s in thebindin g of this isozymet o thenegativel ycharge d pectinwithi ncel lwalls ,henc efacilitatin g itsactions .Endo-PGI I has a relatively low specific activity compared with endo-PGI when using polygalac- turonic acid as a substrate. It is possible that this isozyme degrades particular pectin moieties in the cell wall with higher efficiency. The monomers produced by these isozymes would probably induce exo-PGs and the resulting combination of PGs may then actsynergisticall y toefficientl y degradecel lwall san dproduc ea carbo n source for fungal growth.Furthe rstudie sar erequire dt odetermin eth eexten to fsynergis mbetwee n variouscombination s ofthes epurifie d isozymesin vitro andin planta. Fewfunga l PGshav ebee nshow nt ob eglycoprotein san dw ear eth efirs tt orepor tth e presence of exo-PGs from B. cinerea; thephytopathogeni c significance of theseglyco proteins has not been elucidated but it has been suggested that their ability to form complexeswit hhos tprotein s islikel yt odepen d onth ecarbohydrat e moietyo fth ePGs . Ourcurren tinvestigation s areconcerne d withth eultrastructura l localisation ofendo - PGsdurin g infection ofraspberrie san dblackcurrant sb yB. cinerea. We areals oclonin g the genes encoding these isozymes with a view to negating each of these isozymes by site-directed mutagenesist oasses sthei reffec t onpathogenicity . Acknowledgement Wethan k theScottis hOffic e Agriculture andFisherie s Department for funding. 61 References Cooper, R.M. and Wood,R.K.S. , 1975.Regulatio n of synthesis of cell wall degrading enzymes by Verticillium albo-atrum and Fusariumoxysporum f.sp . lycopersici. Physiological PlantPatholog y 5:135-156. Coley-Smith, J.R., Verhoeff, K. and Jarvis, W.R., 1980. The biology of Botrytis. AcademicPress :London ,p . 318. Harrison,J.G. , 1978. Roleo f seed-borneinfectio n inepidemiolog y ofBotrytis j"abae on field beans.Transaction so f theBritis hMycologica l Society70:35-40 . Johnston, D.J. and Williamson, B., 1992. Purification and characterization of four polygalacturonases from Botrytis cinerea. Mycological Research96:343-349 . Keen,N.T .an dNorton ,J.C. , 1966.Inductio n and repression ofendo-polygalacturonas e synthesisb yPyrenochaeta terrestris. Canadian Journalo fMicrobiolog y12:443-453 . Leone,G . andVa nde n Heuvel,J. , 1986.Regulatio n bycarbohydrate s of thesequentia l invitro productio no fpecti cenzyme sb yBotrytis cinerea. Canadia nJourna lo fBotan y 65:2133-2141. Lorenzo, G., Salvi, L., Degra, L., D'Ovidio, R. and Cervone, F., 1987. Induction of extracellular polygalacturonase and its mRNA in the phytopathogenic fungus Fusariummoniliforme. Journal ofGenera lMicrobiolog y 133:3365-3373. Verhoeff, K. and Liem, J., 1978. Presence of endo-polygalacteronase in conidia of Botrytis cinerea before and during germination. Phytopathologische Zeitschrift 91:110-115. Williamson,B. ,McNicol ,R.J .an dDolan ,A. , 1987.Th eeffec t ofinoculatin gflower san d developing fruits with Botrytis cinerea on postharvest grey mould of red raspberry. Annalso fapplie d Biology 111:285-294. 62 r Significance ofpolygalacturonas e production by Botrytiscinerea i n pathogenesis G. Leone Summary During infection of plants B. cinerea produces different kinds of cell wall-degrading enzymes,includin g thepecti cenzyme s polygalacturonases (PGs).T oestablis h therol e ofPG si ninfection , thesignificanc e oftota lP Gproductio nb yth efungu s isdistinguishe d from theactivit yo fa singl eisoenzyme ,PG2 .I ti spropose d thatth eprimar y significance of thesequentia l total PG production is related to thedigestio n of thepecti n portion of theplan tcel lwall .Th ephysiologica lan dbiochemica lpropertie so fPG 2indicat etha tthi s enzymeha sa doubl erol esupportin g thepenetratio n ofprimar y cellwall san dth eonse t ofth echai nproductio n ofothe r isoenzymes involved inpecti n catabolism. Introduction The fungus Botrytis cinerea Pers.:Fr. is a saprophyte ubiquitous on weakened ordea d plant material (Blakeman, 1980).A sa pathogen , itusuall y first becomesestablishe d on dead ormoribun d parts of ahos t and then spreads into adjacent healthy tissues(Mans field, 1980). Direct penetration of germtubes into undamaged tissues has also been observedo ndifferen t hosts,amon gthes ear egrap eberrie s(McClella nan dHewitt ,1973 ) and gerbera flowers (Salinas et al, 1989). B. cinerea is also known as a secondary invader, attacking plants already infected by other pests or weakened by senescenceo r stressfactor s (e.g.Leon ean dTonneijck , 1990). The most common symptom induced by B. cinerea is the decay of infected tissues. Cell wall-degrading enzymes secreted by the fungus, have long been known to be involvedi nth einfectio n process(Hancoc ket al., 1964).Pecti cenzymes ,i nparticula rth e polygalacturonases(PGs) ,hav ebee nassociate dwit hth epenetratio nan dcolonizatio no f Frenchbea nleave sb yB. cinerea (Vande nHeuve l andWaterreus , 1985). The actual role of PGsdurin g infection by B. cinerea has notbee n fully established butth esignificanc e ofP Gproductio nb ythi sfungu s inpathogenesi s hasbee nexamine d (Leone, 1990; Leone and Van den Heuvel, 1987; Leone et al., 1990a,b). Some of the resultsar ediscusse dbriefl y here. Results and Discussion Significance oftota l PGproductio n Polygalacturonases produced by B. cinerea can be separated into a number of electro- phoretic variants referred to as isoenzymes. To understand the significance of PG production in pathogenesis, it is important to distinguish between total PG production resulting from thecombine d activity of multiple isoforms, and single PG isoenzymes. 63 This approach helps to establish the role of each enzyme at an early stage (e.g. penetration)o ri nmor eadvance dstage s (e.g.colonization ) of infection. Several studies showed that B. cinerea produces a number of pectic isoenzymes belonging toth eP Ggrou p(Cruickshan k andWade , 1980;Drawer tan dKrefft , 1978; Di Lenna andFielding , 1983; Magro etal, 1980).A clea rrelationshi pbetwee n growtho f thefungu s ona cel lwall-relate d polysaccharide (isolatedbea ncel lwalls ,citru specti no r sodium polygalacturonate) as the sole C source, and PG production was reported by Leonean d Vande nHeuve l (1987).Th etyp ean dconcentratio n ofth esubstrate ,an dth e concentrationo fconidi ai nth einoculum ,influence d growthan dtota lP Gactivity .Pecti c enzymes were produced in a consistent sequence. Isolate BC1 first produced the constitutive isoenzyme PG2. This degrades sodium polygalacturonate with an endo- modeo factio n(Leon eet al., 1990a).Afte r PG2,othe rpecti cisoenzyme swer eproduced , includingPG 1 whichi sinduced .Th epecti cenzyme sar enumbere daccordin gt oVa nde n Heuvelan dWaterreu s( 1985) ; thisnotatio nwa sbase do nth elocatio no fP Gband si ngel s after electrophoresis and not on the sequence of production of pectic enzymes. The regulation of two enzymes, PG1 and PG2, was found to be mediated both by the C sources, most likely through enzyme inhibition by pectic oligomers and/or feedback repression by D-galacturonic acid, and by the adenylate pool, probably through the metabolicenerg ystatu so fth efunga l cell(Leon eet al, 1990b). Catabolite repression was not involved in the regulation of some pectic enzymeso f B.cinerea (Leon ean dVa nde nHeuvel , 1987); theproductio no fPG2 ,PG 3 andPG 4wa s little affected byth epresenc e of D-glucose.Thi scharacteristi c mayb eimportan t inth e colonization of host tissues. The production of certain pectic enzymes will not be influenced by the increasing release of free sugars caused by the coordinated actiono f thewhol earra yo fcel lwall-degradin g enzymeso fB. cinerea. AlsoPG 1 didno tunderg o catabolite repression (Leone etal, 1990b).It s synthesis could neverb ederepresse d by addition ofcAM Pt oa mediu mcontainin g D-glucose asth eonl y Csource ,a swoul db e expectedwhe na ninducibl eenzym ei sgoverne db ycatabolit erepressio n(Cooper ,1983 ; Pall,1981) . Thepoint sdiscusse d aboveindicat etha tth esignificanc e ofth esequentia lproductio n of PGsb y B. cinerea lies in the provision of nutrients from pectin-related polymers of plantcel lwalls .Th epresenc eo fmultipl eisoform s ofPG si srelate dt oa nadvance dstag e of infection when plant material is degraded and digested by the fungus. This might suggest thatB. cinerea evolved from asaprophyti c ancestor whoseecologica l taskwa s therecyclin g ofdea dplan tmaterial . Roleo finorgani cphosphat ei nP Gproductio n The infection of healthy green leaves by germtubes of conidia of B. cinerea is usually dependent on the presence of external nutrients, probably because of the scarcity of availablenutrient s onleaves ,compare d with thesurface s of flowers orfruits , whichar e morecommonl y infected (Blakeman, 1980).Inorgani c phosphate (KH2P04)an d purine nucleotide derivatives stimulate infection (Kö et al, 1981).Th e phosphate-stimulated penetrationo fbea nleave sb yisolat eB C 1 ofB. cinerea reportedb yVa nde nHeuve lan d Waterreus (1983) was correlated with a concomitant phosphate-stimulated increase in PGactivity ,particularl yPG 1an dPG 2(Va nde nHeuve l andWaterreus ,1985) . Twelvedifferen t isolates of B. cinerea were strongly dependent on KH2P04 for PG production(Leone ,1990) .Th einvolvemen to fKH 2P04and/o radenin enucleotide si nth e 64 regulation ofPG 1 andPG 2wa sstudie d indetai l withisolat eBC 1(Leon eet al., 1990b). Experiments were performed with cultures lacking cell wall-related substrates to establish whether this regulation acted independently of that for carbohydrates. Cyclic AMP, ADP and ATP stimulated the constitutive production of PG2 as well as fungal growth,wherea sAM Pinduce dproductio no fPG 1 butno tgrowth .Th eeffect s of KH^Ot onth etw oisoenzyme s and ongrowt h wereintermediat ebetwee n thoseo f AMPan do f the other nucleotides. The KH2P04 or adenine nucleotide-dependent PG production involved de novo enzyme synthesis. Inhibition of the ATP production in the fungal oxidative phosphorylation resulted in a rapid detection of PG1i n the culture medium. Experiments affecting theintracellula r concentrations of adenine nucleotides alsogav e indicationstha tth eKH 2P04-dependentPG 1 andPG 2synthesi sb yBC 1 wasmediate db y adenine nucleotides.I twa stherefor e postulated thatPG 1synthesi s wascontrolle d bya pecticinduce ran d alo wmetaboli c energystatu so fth efunga l cells,a situatio nlikel yt o occur when germinating conidia initiate infection. Some factors and their interaction involved in theregulatio n of the synthesis and activity of PG1an d PG2 of isolateBC 1 areillustrate d inFig .1 . Although theimportanc e of phosphate for PGbiosynthesi s hasbee n established, the significance of aphosphat e sourcefo r the fungus tobecom e infective isstil l unclear.I t might be possible that inorganic phosphate mimic somehow the effect jof dead plant material,on eo f theprimar y substrates for B. cinerea, triggering themetaboli c activities of thefungu s necessaryfo r planttissu edegradatio n and transformation. rytis cinerea cell wall "^ PECTIC I N tl constitutive ADENYLATE POOL REGULATION ATP(-) -^ < AJP •AMPI+ 3STRATE ICTION (+) AND ED-BACK IESSION (-) Fig. 1. Schematic representation of the regulationby carbohydrates andadenine nucleotides ofthe synthesis ofPGl andPG2 ofisolate BC1 ofBotrytis cinerea. 65 Origino fvariabilit y ofP Gproductio n B. cinerea is known for its phenotypic variability, possibly caused by heterokaryosis and/or diploidy (Grindle, 1979). Variability between isolates also occurs in PG isoenzymes(Magr oet al., 1980).Th eorigi no fthi svariatio nwa sstudie dwit h 12isolate s differing bysourc eo fhos tplant ,yea ro fisolatio nan dgeographica lorigi n(Leone ,1990) . Thecultura lcondition s used tostud yregulatio n by KH2P04 ofP Gproductio ni nisolat e BC1, wereuse d tocompar e theisozym ecompositio n of these isolates. Electrophoresis showed thatal lisolate sproduce dbasi cisoform s ofPG s(p iabov e8 )in vitro andin vivo on bean and tomato leaves,whic h always included at leaston e PG 1-o r onePG2-lik e isoenzyme. Twosource so fvariabilit ywer edistinguished :variatio nwithin ,an dbetween ,isolates . Theforme r isrelate dt opecti ncatabolis mb yth efungu s andi sinfluence d byth etim e of samplingan dcultura lcondition sin vitro and/o rin vivo (Leone , 1990; Leonean dVa nde n Heuvel,1987) . Variability between isolates was small when cultural conditions known to prevent variabilitywithi nsingl eisolate swer eused . Inpreviou sstudie si ti sprobabl etha ta tleas t parto fthi svariabilit ycoul dhav ebee nbase do ndifference s duet oth emetaboli ccontro l ofth eenzymes .Som eo fth ecause so fth eremainin gvariatio n inP Gproductio nbetwee n isolates (Leone, 1990) may be found in the genetical instability for specific characters (e.g. the differences found in AMP involvement in PGl-like isoenzyme synthesis), to mutations and/ort o modifications of proteins attranslationa l orpost-translationa lleve l (reflected, for instance,i nth esmal ldifference s inth erelativ eelectrophoreti c mobilities ofsom ePGs) . Whenconidi awer esuspende di na solutio ncontainin gnutrient san duse dt oinoculat e bean and tomato leaves, all B. cinerea isolates were able to infect and showed no host specificity andn oisoenzymati chos tadaptatio n (Leone, 1990). Theseresults ,couple dt o a lack of association between the rateo f PG production and the level of pathogenicity, indicate that PG production should be regarded as only one of a complex of factors contributing toth eexpressio n of pathogenicity ofB. cinerea. PG2an dit spropertie s In the phosphate-stimulated infection of French bean leaves by B. cinerea, PG2 accumulated in inoculum droplets, with PG1, 6t o 12hour s after inoculation (Vande n Heuvel and Waterreus, 1985). PG activity remained negligible unless penetration occurred. The same occurred during the infection of tomato leaves (Leone, 1990). Because of the probable importance of PG2 in infection, the enzyme was purified to studyit sbiochemica lpropertie s (Leoneet al., 1990a). PG2 is a basic (pi 9.1) endo-enzyme able to degrade pectin and sodium poly- galacturonate.It saffinit y for sodium polygalacturonatewa sthre etime shighe rtha ntha t forpectin .Thes eresult sindicat etha tPG 2i sabl et obrea kdow nth epecti cportio n ofth e plantcel lwall .Th eenzym ewa sproduce dconstitutively ,i twa spresen ti nungerminate d conidiaan dwa sfirst detecte ddurin gth esequentia lpecti cenzym eproductio nin vitro an d invivo (Leone , 1990;Leon ean dVa nde nHeuvel , 1987). Therefore, PG2seem st osho w a double role in the infection process;firstly b y assisting penetration and secondly,i n triggering the onset of the chain production of other isoenzymes for pectin catabolism (seeals oFig . 1).Th elatte r function couldb emediate d byth ereleas eo fmonomer san d 66 oligomers from the pectic portion of the cell wall (Leone et al, 1990a). The rapid softening ofth eprimar ycel lwal lb yth eendo-activit y of PG2wil lfacilitat e mechanical pressureexerte db yth egrowin ghypha l tipdurin g penetration. References Blakeman, J.P., 1980.Behaviou r of conidia on aerial plant surfaces. InTh e biologyo f Botrytis. EdsColey-Smith ,J.R. ,Verhoeff , K.an dJarvis ,W.R .Academi c Press, New York,London ,p . 115-151. Cooper,R.M. , 1983.Th emechanis m andsignificanc e ofenzymi cdegratio n of hostcel l wallsb yparasites .In Biochemica l plantpathology .Ed .Callow ,J.A. ,Joh nWile yan d Sons,Ne wYork ,London ,p .101-135 . Cruickshank, R. and Wade, G.C., 1980. Detection of pectic enzymes in pectin- acrylamidegels .Analytica l Biochemistry 107:177-181 . Di Lenna, P.an d Field, A.H., 1983.Multipl e forms of polygalacturonases in applean d carrottissu einfecte d byisolate so fBotrytis cinerea. Journalo fGenera lMicrobiolog y 129:3015-3018. Drawert, F.an d Krefft, M„ 1978.Characterisierun g extrazellular Proteine und Enzyme ausPektinkulturfiltrate n vonBotrytis cinerea. Phytochemistry 17:887-890. Grindle, M., 1979. Phenotypic differences between natural and induced variants of Botrytis cinerea. Journalo fGenera lMicrobiolog y 111:109-120. Gross,K.C .an dMoline ,H.E. , 1986. Growth of twopathogen s onisolate d cellwal lan d polysaccharide fractions from tomatofinit . Phytopathology76:573-576 . Hancock, J.G.,Millar ,R.L .an d Lorbeer,J.W. , 1964.Rol eo f pectolytic and cellulolytic enzymesi nBotrytis leafbligh t ofonion .Phytopatholog y54:932-935 . Kö, K., Akutsu, K., Kobayashi, Y, Om, Y, Watanabe, T. and Misato, T., 1981. Stimulativeeffect s ofpurin ean dthei rrelativ ecompound so nlesio nformatio n ofgra y mouldo ncucumbe rplant .Annal so fPhytopathologica l Societyo fJapa n47:228-233 . Leone,G. , 1990.In vivo an din vitro phosphate-dependent polygalacturonase production bydifferen t isolateso fBotrytis cinerea. Mycological Research94:1039-1045 . Leone, G. and Tonneijck, A.E.G., 1990. Acute ozone exposure predispose Phaseolus vulgarisbean st oBotrytis cinerea. Netherlands Journal ofPlan tPatholog y96:65-74 . Leone,G . andVa nde n Heuvel,J. , 1987.Regulatio n bycarbohydrate s of thesequentia l invitro productio no fpecti cenzyme sb yBotrytis cinerea. Canadia nJourna lo fBotan y 65:2133-2141. Leone, G., Schoffelmeer, E.A.M. and Van den Heuvel, J., 1990a. Purification and characterization of a constitutive polygalacturonase associated with the infection process of French bean leaves by Botrytiscinerea. Canadian Journal of Botany 68:1921-1930. Leone,G. ,Overkamp ,A.N. ,Kreyenbroek ,M.N. ,Smit ,E .an dVa nde nHeuvel ,J. ,1990b . Regulation by orthophosphate and adenine nucleotides of the biosynthesis of two polygalacturonasesb yBotrytis cinerea in vitro. Mycologica lResearc h94:1031-1038 . Magro, P., Di Lenna, P., Marciano, P. and Pallavicini, C, 1980. Variability of polygalacturonase and protein isoelectric focusing patterns in Botrytis cinerea isolates.Journa lo fGenera lMicrobiolog y 120:105-109. Mansfield, J.W, 1980. Mechanisms ofresistanc et oBotrytis. InTh ebiolog y ofBotrytis. Eds. Coley-Smits, J.R., Verhoeff, K. and Jarvis, W.R. Academic Press, New York, London,p .181-218 . 67 McClellan,W.D .an dHewitt ,W.B. , 1973.Earl yBotrytis roto fgrapes :tim eo f infection and latency of Botrytis cinerea Pers. in Vais vinifera L„ Phytopathology 63:1151- 1157. Pall,M.L. , 1981.Adenosin e 3',5'-phosphat e in fungi. Microbiological Review45:462 - 480. Salinas, J., Glandorf, D.C.M., Picavet, F.D. and Verhoeff, K., 1989. Effects of temperature, relative humidity and age of conidia on the incidence of spotting on gerbera flowers caused by Botrytis cinerea. Netherlands Journal of Plant Pathology 95:51-64. Van den Heuvel, J. and Waterreus, L.P., 1983.Conidia l concentration as an important factordeterminin gth etyp eo fprepenetratio n structuresforme d byBotrytis cinerea o n leaveso f Frenchbea n(Phaseolus vulgaris). PlantPatholog y32:262-272 . Van den Heuvel, J. and Waterreus, L.P., 1985. Pectic enzymes associated with the phosphate-stimulated infection of French bean leaves by Botrytiscinerea. Nether landsJourna lo fPlan tPatholog y91:253-264 . 68 Polygalacturonases ofBotrytis cinerea G. van derCruyssen andO. Kamoen Summary Polygalacturonase(PG )enzyme sproduce db yon ehomokaryoti cstrai no fB. cinerea an d expressedi nth epresenc eo fdifferen t carbonsource swer eexamined .Th etim ecours eo f theexpressio no fth eP Gisozyme swa sstudie d andthei rzymogra mpattern scompared . One isozyme was constitutively expressed but subjected to end-product repression; the other isozymes were more or less subjected to catabolite repression, end-product repressionand/o rinduction . Aclassificatio n intofou r 'expressiongroups 'i spropose dfo r thedifferen t detectedisozymes . Introduction Acompariso no fth enumbe ro fpolygalacturonas e(PG )isozyme ssecrete db yth e fungus B. cinerea revealed that between onean d thirteen isozymes wereproduce d by different strains. The complexity of the PG profiles could not be attributed entirely to heterokaryosis, as we observed similar high numbers of PGs in heterokaryotic and homokaryoticstrains . Themai n factor influencing theexpressio n of theseparat e isozymes seems tob eth e growthmedium . Ourhypothesi swa stha tgroup so fP Gisozyme scoul db edefine d which were similarly regulated. The following experiments describe the effect of catabolite repression,substrat einductio nan dend-produc trepressio no nth eexpressio no fsingl eP G isozymes ofB. cinerea. Material and Methods All experiments were done with cultures of the homokaryotic Botryotiniafuckeliana, (deBary )Whetz .(Botrytis cinereaPers. :Fr. )strai nR16 , suppliedb ydr . F. Faretra,Bari , Italy (strainhereafte r called B.c. FR16). Cultures and Samples Experiment1 Erlenmeyer flasks (100 ml) containing 50 ml Richards' medium were inoculated with 106, 105an d 104conidia.ml" 1,an d incubated on arotar y shaker (100rpm ) for 4day sa t room temperature in darkness. The medium contained per 1000 mlRichards ' medium either 20 g glucose, 20 g galacturonic acid, 20 g peptone, 5 g pectin or 5 g sodium polygalacturonate andth emediu mwa sadjuste d top H5 .Th eassay slaste d24,48,7 2o r 96 h. 69 Experiment2 In a 'two step culture' experiment the fungus was pre-grown in a medium with ahig h concentration ofglucos e(2 0g.H ) during 3days ,th emyceliu m thenwashe d andgrow n on a fresh medium containing 20 g.H or 1g. H glucose or 5 g.H pectin for another 2days . Three Erlenmeyer flasks of each medium were sampled every 24 h and the culture filtrates obtained and themycelia l dry weigh tsdetermined . Thefiltrate s weredialyse d forperiod so f2 4t o4 8h an dcentrifuge d (8000rpm/1 0min )t oremov einsolubl ematerial . They were then frozen and lyophilysed, dissolved in 1m l water, passed through age l filtration column (PD-10 Pharmacia Biosystems), lyophilysed again and finally redissolved in 1 mlwater . IEF-PAGEan dPG-activit y staining Iso-electricfocusin g (IEF)wa sperforme d onampholin egel s(broad-rang e pH3. 5 - 9.5; Pharmacia Biosystems) using the Multiphor II system (LKB Pharmacia). Applied amountsrange dbetwee n0.00 4an d0. 2units ,on euni twa sdefine d asth eactivit ywhic h released 1 mm oligalacturonic acid.mnr1. The three samples from the same medium were used as distinct IEF samples in different amounts. The zymogram technique of Cruickshank andWad e(1980 ) wasmodifie d insom easpects .Th eIE Fgel swer e rinsed several times with Mcllvain buffer (pH 5.2) and incubated in the substrate solution (prepared by dissolving polygalacturonic acid in buffer, at pH 5.2) at 37°C for 15min . Following thoroughrinsing wit hta pwater ,th egel swer eimmerse d in0.02 %rutheniu m red solution for 2 h or longer, and rinsed with tap water. The gels were dried under vacuum(Bio-Ra dGe lDryer) ,th erelativ edistance s andintensitie s ofth edifferen t spots ofP Gactivit y weredetermine d andcompare d toth epattern sobtaine d inothe rgels . Results Examination ofglucos ea sa catabolit e repressor Because the PG activities measured in the glucose-containing medium were relatively high,repressio n wasno texpecte d toresid ei nman yP Gisozymes . In4 day-old cultures with anexces s of glucose only one PG(p i9 )wa s consistently present (Fig. 1,ro w 2)an d this wecalle d PG-A.I n somecase s of high loading ofgels , oneo rtw oaci disozyme s(pic .4.8 )an don eo rtw owit hbasi cp ivalues ,coul db edetecte d insmal lamounts . Thecontro lculture sgrow no npepton eshowe dmarke ddifference s byth efirst da ya s additional alkaline isozymes and two acidic isozymes (pi 4.9 and 4.7) were observed. Thechoic eo fpepton ea sa referenc e carbon sourcewa stherefor e justified. Toassess whethercatabolit e repression byglucos eoccurs ,th esecon dmediu m ofth e 'two step medium', either supplemented with a high or low level of glucose was examined after 1 and 2day sgrowt h inthes emedia .N oPG- Awa sdetected .Tw oacidi c isozymesp i4. 7an d4. 9bu tn oothe risozyme swer edetected ,bu tthes ewer eonl ypresen t atth elowe rglucos econcentratio n (Fig.2 ,ro w 1 and2) . 70 DAY 1 DAY2 DAY3 DAY 4 Pon 9,0 4,9 M'7 GA GA/G Pee Pee/ Fig. 1. Profiles of PG isozymes produced by B. cinerea daily in basalsalt medium supplementedwith different carbon sources athigh concentrations. Dashes ofdifferent width correlate with the differentintensities ofactivity. The relative distances along the length ofeach column arein proportion tothe distances of theisozymes along the pH gradient. 71 DAY 4 DAY 5 2 % 6 0,1 % 6 0,5% Pec — Fig. 2. ProfilesofB. cinereaPG isozymes produced during 2 days in thesecond medium oftwo-step cultures. Examination ofpecti nan dpolygalacturonat e asinducer s Byth efirs tday ,mor eisozymes ,alkalin ea swel la sacidic ,wer edetecte di nculture sfro m pectintha ni nthos eo npepton e(Fig .1 comparelane s 1,4 and5) .Afte rda y2 ,th ealkalin e forms disappearan dth enumbe ran dconcentratio n of theacidi cform s increased. When thefungu s wasgrow n on thecombinatio n pectin/glucose(Fig . 1,ro w 6),tw o acidic PGs seemed to be controlled by substrate induction, rather than by catabolite repression. The PG-A again disappeared, after adela y i.e. oneda y later than on pectin alone(F ig . 1,row s 5an d6) .A possibl eexplanatio n isth edelaye d uptake ofth epecti n degradation products in the presence of glucose. Similar results were obtained using sodiumpolygalacturonat e assubstrate . Comparedt oth epatter nobtaine dwit hglucose ,her eagai nth ePG- Awa sno tdetected . Moreacidi cenzymes ,o fhighe ractivity ,wer edetecte di nthes eculture scompare dt oth e culture on a small content of glucose (Fig. 2, row 3). Here again the PG-A was notdetected. 72 Examination ofgalacturoni c acida send-produc trepresso r Wehypothesize dtha tth eabolishe dexpressio no fth ealkalin ePG so nth epecti nafte rda y 2coul db emediate db ypecti ndegradatio nproducts .T ochec kthi spossibility ,w estudie d theP Gisozyme sproduce do ngalacturoni cacid .N oPG- Awa sdetecte ddurin gth e4 day s (Fig. 1,ro w2 )o fth eexperiment . Theacidi cisozyme sthe nappeare di nth emedium .Th e combined effect of galacturonic acid and glucose in the medium resembled that of galacturonic acid, except for the presence of alkaline isozymes in small quantities. Therefore theresult sobtaine d withpecti n andgalacturoni caci d aresimilar . Temporal regulation Asequenc ei nth eP Gexpressio nca nb edefine d bycompariso no fth eprofile s duringth e successive days of incubation (the four lanes of Fig. 1). One alkaline and two acidic isozymes were generally expressed earlier than the other isozymes. No PG-A was producedb ymyceliu m inth esecon d medium ofth etwo-ste pcultur esyste m (Fig. 2). pi GROUP -9,0 A I D ! II G H -4,9 B -4,7 C III I ! IV M Fig. 3. Schematic representation of all PG isozymes detectedin mediacontaining a varietyof carbon sources. Romannumerals indicate thegroup number, PG isozymesare indicated by letters (seealso Table 1).The pi values of three important isozymes are shown. 73 Table 1. Proposedgrouping and designation of the PG isozymes ofB. cinereaaccording tothe regulatory response todifferent carbon sources (G= glucose, GA = galacturonic acid, Pect= pectin). Group Designation pi Description I A 9,0 nocatabolit erepressio n(G ) noinductio n end-product repression (GA,Pect) earlyexpressio n II BB 4,4,9 9 cataboliterepressio n(G ) C 4,7 induction(GA.Pect ) butinductio nno tnecessar y early expression III D- G -G 8,98, 9 cataboliterepressio n(G ) to induction nots oclea r 8,5 end-productrepressio n(GA,Pect ) IV H- M -M 4,94, 9 cataboliterepressio n (G) to substrateinductio n necessary 3,5 noend-produc t repression(GA ) Groupingan ddesignatio n ofth edifferen t isoenzymes Weconclude dtha tth eisozyme scoul db eplace di non eo ffou r groupsaccordin gt othei r regulatory responses on the different substrates and to their temporal expression. The groups areidentifie d by Roman numerals (Fig. 3,Tabl e 1).Withi n thegroups , theP G isozymes arename db ymean so fa lette ranalogou st oth eclassificatio n ofpecti n lyases used forAspergillus niger(Harmse n etal. , 1990).I n this way thethre eisozyme s most often observed arecalle dA ,B an dC .Th echaracteristic so feac hgrou par esummarise d inTabl e1 . Discussion Basedo nth edifference s inth etempora lexpressio n andi nth eexpressio no nth e different carbon sources,fou r 'expression groups'coul db edistinguished . A single isozyme called PG-A was detected generally first; and this was the only isozymedetecte d onmedi acontainin g highglucose .I ti sprobabl y analogous toth ePG 2 detectedi nB. cinerea strai nBC- 1 byLeon ean dVa nde nHeuve l(1987) .W einterpre tthi s 'glucoseeffect ' ascatabolit erepressio n of someo f thePG sa tth egeneti c level.Furthe r evidencecoul db eobtaine db ymeas u ring theamount so fmRN Aderive d from different PG-genes, as was done for thePL- A ofAspergillus nidulans by Dean and Timberlake (1989). Theabsenc eo fPG- Aproductio nb ypre-grow nmyceliu m transferred tofres h culture fluids (containing 20g or 1 g.H glucose or 5g. H pectin) cannotb e readily explained. 74 Possiblythi sisozym ei srelate dt oth egerminatio no fconidia ,an di tma yb erelease d from theconidi aan dthe ni ti sn olonge r synthesised. Pectin was shown to be an inducer for the isozymes of groups II, IIIan d IV.Thi s induction seemed even to be more effective than the repression by glucose. As pectin degradation progressed, isozymesi nth egroup sI an dII Iwer en olonge rdetected ,whil e higheractivitie swer edetecte dfo rth eisozyme so fgroup sI Ian dIV .W esugges ttha tther e isstron gend-produc trepressio n ofth ebasi cisozymes ;eve nPG- Awa shighl y subjected toend-produc trepression . Thegroupin gw epropos ereflec tth ephenotypes . Perhapsi ti sunlikel ytha tal lo fthes e forms constitutetru eisozyme sencode db ydifferen t structuralgenes .A variet y ofpost - transcriptional events andpost-translationa lmodification s can alterth eisoelectri c point of aprotein . Thequestio n as to whether there could be aconcerte d regulation of some PGs(belongin g toth esam egroup )require sfurthe r research. The production sequence for PGisozyme s by B. cinerea during infection of a plant mayb ea sfollows : PG-Awhic hi simmediatel y presenta thig h activity in vitrocoul db e the first 'acting element' invivo. Depolymerisatio n of pectin in cell walls would then induceothe r PGs.Afte r sometime ,th ebasi c isozymes wouldb erepresse d andth eaci d onestak eove r as 'acting elements' during lesionexpansion . References Cruickshank, R., and Wade, G.C., 1980. Detection of pectic enzymes in pectin-acryl- amidegels .Analytica lBiochemistr y 107:177-181. Dean,R.A .an d Timberlake,W.E. , 1989.Regulatio n of theAspergillus nidulans Pectate LyaseGen e(pelA) .Th ePlan tCel l 1:275-284. Harmsen, J.A.M., Kusters-Van Someren, M.A. and Visser, J„ 1990. Cloning and expression of a second Aspergillus nigerpecti n lyase gene (pelA): Indication of a second pectin lyasegen efamil y in A. niger. CurrentGenetic s 18:161-166. Leone,G . and Vande n Heuvel,J. , 1987.Regulatio n bycarbohydrate s of thesequentia l invitr oproductio no fpecti cenzyme sb yBotrytis cinerea. CanadianJourna lo fBotan y 65:2133-2141. 75 Thefunctio n oflaccas efro mBotrytis cinereai n host infection A. Viterbo, N.Bar Nun andAM. Mayer Summary Invasion of plant tissue by a pathogenic fungus involves the secretion of 'attack' enzymes which allow the fungus to penetrate its host. The host responds with various defence mechanisms, which include the formation of phenolics and lignification. Pathogenic fungi, such asB. cinerea have developed mechanisms to overcome orby pass such defences, one of which may be the secretion of oxidative enzymes, such as laccases. Some fungal laccases, including that of B. cinerea havebee n characterised in great detail. Appearance of laccase is rigourously controlled by the level of specific phenolics,b y pectins orthei rdegradatio n products andb y copper. Wehav e shown that factors which prevent or repress laccase formation can also afford protection of ahos t against attack by B. cinerea. The appearance of laccase activity, but not that of other enzymes, is prevented by cucurbitacin. The prevention by cucurbitacins of laccase formation byB. cinerea,withou taffectin g fungal growtho f otherfunga l attackenzyme s shouldtherefor e afford protection ofth ehos tt oB. cinerea. Cucurbitacinswhic har eabl e torepres s appearance of laccase can also prevent invasion of thehos tb y thepathogen , probably because lignin formation can occur. We would like to suggest that laccase shouldb econsidere d aspar to fth ecomple x of 'attack' enzymesforme d byB. cinerea. Introduction Invasion of plant tissue by fungal hyphae is accompanied by secretion of anumbe ro f enzymesb yth efungu s whichpermi ti tt openetrat eit shos tan dt oovercom esom eo fth e hostplan tdefenc emechanism s(Hal lan dWood ,197 3; Dickma net al., 1982;Marcu san d Schejter, 1983;Va n Etten and Kistler, 1984). Fungal attack is often very specific, e.g. attacking somecultivar sbu tno tother s(Bell , 1981).Th ehos trespond sb yth eformatio n ofvariou scompound swhic hdefend s itagains tth einvader ,includin gtoxi ccompounds , enzyme inhibitors, phytoalexins (Ouchi, 1983;Darvi l and Albersheim, 1984; Horsfall and Cowling, 1984; Mayer, 1989) or by the formation of defensive barriers, such as ligninan dcuti n(Horsfal l andCowling ,1984) . Some of the plant defense mechanisms are based on the formation of specific chemical substances such as phenolics and tannins (Weinhold and Hancock, 1980; Horsfall and Cowling, 1980). Many other plant secondary metabolites, with marked biologicalactivit yar epresen ti nplan ttissue s(Cutler , 1988; Mayer, 1989),an dthes ema y find uses in agriculture as protectants against pathogens. Secondary metabolites have generallybee nregarde da sbein gdefensiv e agentsagains therbivore s(insects ,predators) , butlittl eattentio n hasbee npai dt oth efunctio n ofsuc hcompound s against fungi. Fungi aremetabolicall y veryversatil ean dsho w aconsiderabl e ability todetoxif y or to overcome plant defense mechanisms,e.g . by inactivating phytoalexins (Smith etal., 76 1982;Va nEtten , 1982).On eo fth eway si nwhic hfung i inactivate plantdefen s esi sb y oxidizingcompound s whichhav ea protectiv e roleusin g variousenzyme s (Smithe tal. , 1982)on eo fwhic hi slaccase . Laccasebelong st oa grou po fenzyme sgenerall y referred toa sth epolypheno l oxidasespresen tbot hi nhighe rplant s andi n fungi. Laccasesar eenzyme swhic hcatalys eth eoxidatio no fOrtho -o rparadihydroxyphenol s toth ecorrespondin g quinones in areactio n using molecular oxygen.Th eoxidatio n isa twoste pprocess ,involvin g apparentlyfre e radicalformation . Anumbe ro fpropertie so f fungal laccases aregive ni nTabl e1 . Glycosylation seems to be important for enzyme activity, as even partial deglyco- sylationreduce dactivit yan dcause dlos so fcopper ,a tleas ti nRhus vernicifera (Grazian i etal, 1990)an dth esam eseem st ob etru efo rB. cinerea. Table 1 Someproperties offungal laccases Composition glycoproteins Molecularweigh t 38000- 35000 0D a Number of subunits variabletw ot ofour , apparentlyequa l pHoptimu m 3.5-7.5,usuall y witha fairl y sharppea k Copperconten t 1 atomTyp eI Blu ecoppe r(ES R signal) 1ato mTyp eI ICoppe r(no tblue ,ES Rsignal ) 2atom sTyp eII Icoppe r(ES Rsilent ) Substrates monophenols, o-diphenols,p-diphenols ,syringaldazine , relatedcompounds ,ligni n Latency notreporte d Presence usuallylo wleve lo fconstitutiv eenzym e enzymeinductio n byvariou scompound s Induction often highly specific, andinducer sdiffe r in different specieso rgener a ofplant s Location cytoplasmican dperhap s ino ro nth ecel lwal l Table 2 Suggestedfunctions for fungal laccases - Involved inspor eformation , possibly increasing resistanceo fspores ,e.g . against bacterialo rothe rattack . - Degradation,solubilisatio n anddepolymerisatio n oflignin . - Oxidativepolymerisatio n oflignins . - Oxidation ofvariou ssubstrates ,possibl y for utilisation byth e fungus. - Oxidation ofphytoalexins . - Detoxification ofplan tdefenc ecompounds . - Prevention oflignification , byoxidatio n ofphenolic srequire d for theprocess . - Parto fth eattac ksyste m ofpathogeni cfunctio n (ourhypothesis) . Thepropose d functions forlaccas ei ndifferen t fungi areshow n inTabl e2 . Except for the assumed function of laccase in the degradation of lignin (Higuchi, 1989) other functions of this widespread fungal enzyme remain undetermined or unproven.W e wisht opresen tevidenc efo r ourhypothesi s ofa laccas e function. 77 Some fungal laccases have been well characterised and their genes cloned (Yelton etal., 1985 ; Germanet al., 1988; O'Haraan dTimberlake , 1989),bu thomolog ybetwee n thegene si spoo ran denormou sdifference s appeart oexis tbetwee nlaccas eprotein s from different organisms.Th eenzym ema yhav edifferen t functions indifferen t fungi. B.cinerea i sa widesprea dplan tpathogen ,capabl eo fattackin gmos tplant san dalmos t allplan ttissue s(Coley-Smit het al., 1980 )an dcause s 'greyrot ' ingrape swhic himpair s winequalit y(Duberne tetal., 1977)althoug h 'noblerot ' resultsi nth eformatio n ofhig h quality wines. This lead us to a detailed study of the fungal laccase and its character isation (Gigief al.,1980 , 1981;Marbac h efa/. , 1983,1984 ,1985) . Results and Discussion We have purified laccase of B. cinereausin g precipitation, gel filtration and native acrylamide gelelectrophoresi s followed by elution, togiv ea homogeneou s preparation (Viterboan dMayer ,unpublished) .Som eo fth epropertie s ofthes elaccase sar eshow ni n Table3 . Laccaseformatio n inB. cinerea i sunde rver ytigh tcontro lan dappear st ob eregulate d bycompound s originating in thehos ttissu esuc h ascertai n phenoliccompound s andb y pectinsa swel la sb yth eleve lo fcoppe rions ,b yCa 2+an db yth eadditio no fEDT At oth e growth medium. The effect of pectins is a highly specific one. Low molecular weight derivatives of pectins (obtained from Professor C.W. Nagel, Washington State University) were tested for their ability to induce laccase formation in the presenceo f gallic acid. Dimers or tetramers derived from pectin as well as dimers, trimers or tetramers of galacturonic acid suppressed fungal growth and there was no laccase formation. A methylated pectin trimer was a good inducer of laccase and permitted fungal growth,whil eth ecorrespondin g unsaturated trimersuppresse d growth. Table 3. Properties oflaccases produced by B.cinerea, inthe presence of gallic acid or grapejuice as inducers. Gallicaci d Grapejuic e pHoptimu m 3.5 4-4.5 Isoelectric point aboutp H2. 0 various forms Relativeactivit ytoward ssubstrates : quinol 100 100 p-coumaricaci d 50 4 p-cresol 87 12 Sugar% oftota li nglycoprotein : Mannose 39 55 Glucose 24 13 Arabinose 7.4 2.7 Aminoaci dcomposition : % Basic 6.0 7.7 %Acidi c 20.4 16 Molecularweigh t 37-39kDa & 67kDa -i) Bands onSD SPag e 2 2 Subcellularlocatio n cytoplasmic -i) ')no tdetermine d 78 Laccase formation and secretion are entirely distinct from the growth and developmento fth efunga l mycelium.EDT Aca nrepres slaccas eformatio n andthi seffec t isrelate dt ocoppe ravailability ,sinc ei tca nb ereverse db ycoppe rions .Althoug hcoppe r isrequire dfo renzym eformation , thisrequiremen ti ssmall . Additiono f 1 mMcoppe ra s thechlorid eraise dth ecoppe rconten to fth emyceliu mfiftyfold , yetenzym eactivit yonl y doubled.A nindicatio no fth eimportanc eo flaccas ei nth einfectio nproces swa sdetected , byth eus eo fEDTA . Pretreatmento fcucumbe rfrui t with 12 mMEDTA ,a concentratio n which still permitted fungal growth, prevented infection of such fruits by mycelium of B. cinerea. It ispossibl e toextrac t fractions from cucumber fruit which inhibit theformatio n of laccase under inducing conditions (Bar Nun et al., 1988). Similar compounds were isolated from acommo n weed Ecballium and identified as cucurbitacins (BarNu n and Mayer, 1989a,b, 1990).W eshowe dtha tpurifie d cucurbitacins(tetracycli ctriterpenoids - lanosterol derivatives) can prevent laccase formation by B. cinerea under conditions favouring enzymeformatio n (Table4) . Table4: Effectof different cucurbitacins I, D,B andE, and partially purified extracts (PPE) ongallic acid and pectin-induced extracellular laccase activity Lengtho fcultur eperio d / 7da y 5 14 days Activity %Inhibitio n Activity %Inhibitio n Control 1.96 _ 1.09 — EtOH 1.41 - 0.98 - I 0.54 62 0.27 72 D 0.54 62 0.31 68 B 0.38 73 0.21 78.5 E 0.24 82.7 0.1 78.5 PPE 0.38 73 0.27 72 Total activity (|xl C^.mH.min-1), was measured using an oxygen electrode. Partially purified extracto r3 m go fpurifie d cucurbitacins dissolved in0. 2m lEtOH ,wer eadde d to3 0m lcultur emediu m atth eonse to fth egrowt hperiod . Thecucurbitacin s hadn oeffec t onth edevelopmen t of thefunga l mycelium,an dth e samefina l fresh anddr yweigh to fmyceliu m wereobtained . Laccaseformatio n inB. cinerea show sa vei ystric tdependenc eo ncultur econditions . Inexperiment so nth emas scultur eo fth efungu s onorang epeel sa sth esourc eo fcarbon , wehav e shown thatcontro l of laccase and of polygalacturonase are distinct (Karmona etal., 1991) .Formatio n of polygalacturonase, pectin methylesterase,cellulase ,asparti c proteinasean dcarbox ypeptidas ewer eno ti nan ywa yaffecte d bycucurbitacins . Our hypothesis suggests that the ability of B. cinerea to secrete an extracellular laccase is an essential part of its infective mechanism, although insufficient alone to causeinfection .W epostulat etha ti nadditio nt oth esecretio no fa variet yo flyti cenzyme s whichar erequire d topenetrat eint oth ehos ttissu ean dt olyse ,decompos ean dmacerat e it,B. cinerea also secretes laccase.Th e function of this laccase ist o inactivate thehos t defence mechanisms by oxidation of some of the compounds present in the host as protectants. This is supported by the work on Cryphonectria (Rigling, 1989; Rigling 79 et al, 1989), which showed that its hypervirulence correlates with reduced laccase activity. Wehav eteste d thishypothesi sindirectly .Factor spreventin glaccas eformatio n byth e fungus e.g.EDTA ,als oprotecte dth ehos tagains tinfectio nb yB. cinerea (Ba rNu net al., 1988; Bar Nun and Mayer 1989a, 1990). The applications of cucurbitacin I or plant extracts enriched in cucurbitacins, to cucumber fruit, leaves of carrots, prior to their inoculationb yB. cinerea afforded totalprotectio n againstth einvadin g fungus. Underth etes tcondition sused ,th ehos ttissu ewa sabl et ofor m alignifie d layer,whic h seemst opreven tfunga l penetration. Inth eabsenc eo fth ecucurbitacins ,lignificatio n was apparently delayed and the fungal penetration, accompanied by laccase secretion, occurred before formation of a protective barrier. When protection was afforded by cucurbitacins, which are pharmacologically active (Johnson et al., 1989; Lavie and Glotter, 1971;Ne s and Patterson, 1981; Rhem etai, 1977),n olaccas e secretion byth e fungus could be detected (Table 5). Apreviousl y unsuspected role of cucurbitacins in planttissu e mayb e thato f antifungal agents,whic h assist thehos tplan t towar d off the invadingfungus .Apparentl yn ocas eo fspecifi c inhibitiono fth ebiosynthesi so fa singl e recognised defined enzymeinvolve di npathogenicit y hasye tbee nreported . Table 5. Infection ofcucumber fruit with B. cinerea, laccaseformation andthe effectof cucurbitacinI. Treatment Presenceo fligni n andlaccas e after 24 hr 48h r 96 hr Lignin Fungus Lignin Fungus Lignin Fungus Laccase Control ± + — + — _ Infected ++ + ++ + ++ + + Cucurb. - - + - ± - - Infected + + onlyo n + onlyo n - +Cucurb . surface surface Cucumberswer epretreate dwit hEtO Ho rwit hEtO H+ cucurbitaci n (cucurb.)fo r2 4h an dthe ninoculated . Atth een d of each period tissue wasremoved , stained for presence of lignin and mycelium and tested for laccase activity.± = very little lignification; + lignification of a cell layer of at least 2 cells;+ + marked lignification, severallayer sthick .Fungus :myceliu mpresen t inth e cucumber tissue:+ present ;- absent. Basedupo n ourresults ,i ti shypothesise d thatb yinhibitin g theformatio n ofa nenzym e orenzymes ,produce d andsecrete d byth efungus , iti spossibl et oprotec t plants against the pathogen. Laccase might constitute such an enzyme in the case of B. cinerea, although iti sclea rtha tth eattac ko fB. cinerea ona hos tinvolve sa numbe ro fenzymes , ofwhic hlaccas ei s onlyone . References Aramayo, R. and Timberlake, W.E., 1990. Sequence and molecular structure of the Aspergillusnidulans yA(laccase )gene .Nuclei c AcidsResearc h 18:3415. Bar Nun, N., Tal Lev, A., Harel, E. and Mayer, A.M., 1988. Repression of laccase formation in Botrytis cinerea and its possible relation to phytopathogenicity. Phytochemistry 27:2505-2509. 80 BarNun ,N .an dMayer ,A.M. , 1989a.Cucurbitacins - repressors of induction oflaccas e formation. Phytochemistry28:1369-1371 . Bar Nun, N. and Mayer, A.M., 1989b. Cucurbitacins protect cucumber tissue against infection byBotrytis cinerea. Phytochemistry 29:787-791. BarNun ,N .an dMayer ,A.M. , 1990.Regulatio no flaccas eformatio nb yBotrytis cinerea. Comptes Rendus 4e Symposium International d'Oenology, Bordeaux 1989,Dunod , Paris,p . 122-126. Bell,A.A. , 1981.Biochemica lmechanis m ofdiseas eresistance .Annua lRevie wo fPlan t Physiology 32:21-81. Coley-Smith,J.R. , Verhoeff, K. and Jarvis,W.R . (eds.), 1980.Th eBiolog y ofBotrytis. AcademicPress ,Ne wYork .pp .318 . Cutler,H.G .(ed.) , 1988. Biologically ActiveNatura l Products.AC S Symposium Series 380,America n Chemical Society,Washington ,D. C. Darvill,A.G . andAlbershei mP. ,1984 .Phytoalexin san dthei relicitor s- A defens eagains t microbialinfection s inplants .Annua lRevie w ofPlan tPhysiolog y35:243-275 . Dickman, M.B., Patil, S.S. and Kolattukudy, P.E., 1982. Purification, characterisation and role in infection of an extracellular cut inolytic enzyme from Colletotrichum gloeosporioides Penz. on Carica papayaL . Physiological Plant Pathology 20:333- 347. Dubernet, M., Riberaux-Gayon, P., Lerner, H.R., Harel, E. and Mayer, A.M., 1977. Purification andpropertie s of laccasefro m Botrytis cinerea. Phytochemistry 16:191- 193. Germann, V.A.,Muller , G., Junziker, P.E. an Lerch, K., 1988.Characterisatio n of two allelic forms ofNeurospora crassa laccase: amino andcarboxy l terminal processing ofa precursor .Journa l ofBiologica lChemistr y263:885-896 . Gigi,O. ,Marbach ,I .an dMayer ,A.M. , 1980.Inductio n oflaccas eformatio n inBotrytis. Phytochemistry 19:2273-2275. Gigi, O., Marbach, I. and Mayer, A.M., 1981. Properties of gallic acid induced extracellularlaccas eof Botrytis cinerea. Phytochemistry 20:1211-1213. Graziani,M.T. ,Antonelli ,L. ,Sganga ,P. ,Citro ,G. ,Mondovi ,B .an dRosei ,M.A. ,1990 . Biochemical and immunological studies ofd eglycosylate d Rhusvernicifera laccase. Biochemistry International 21:1113-1124. Hall,Y.A .an dWood ,R.K.S. , 1973.Th ekillin g of plantcell s bypectolyti cenzymes .In 'Fungal Pathogenicity antth ePlan tResponse . Byrde,R.J.W .an dCutting ,C.V. ,Eds . AcademicPress ,Ne w York,p .19-38 . Higuchi,T. , 1989.Mechanis m ofligni ndegradatio n byligni n peroxidasean dlaccas eo f white rot fungi. In 'Plant Cell Wall Polymers: biogenesis and degradation. Lewis, N.G.an dPaice ,M.G. ,Eds .AC SSymposiu m 399.America n Chemical Society. Horsfall, J.G.an dCowling ,E.B. , 1984. Epilogue anenta philosoph y ofplan tpathology . In 'PlantDiseas e -a nAdvance d Treatise',vol .5 .Horsefall , J.G. andCowling ,E.B. , Eds.Academi cPress ,Ne wYork ,p .433-447 . Johnson,L.B. ,Reese ,P.B. ,Roberts ,E.V. ,Lam , L.K. and Vederas,J.C. , 1989.Structur e elucidation ofcordifolin , anove lcucurbitaci n from Fevillea cordifolia using onean d two dimensional N.M.R. techniques. Journal of the Chemical Society Perkins Transactions12111-2116 . Karmona, A.,Ba rNun ,N .an dMayer ,A.M. , 1991.Utilisatio n deBotrytis cinerea Pers. comme source d'enzymes pour la clarification des vins. Journal International des Sciencesd el aVign ee td uVi n24:103-124 . 81 Lavie,D . and Glotter, E., 1971.Th ecucurbitanes , agrou p of tetracyclic triterpenes.I n Progressi nth eChemistr y ofNatura lProduct s29:308-362 . Marbach,I. ,Harel ,E .an dMayer ,A.M. , 1983.Induce ran dmediu mdependen tpropertie s ofextracellula r laccasefro m Botrytis cinerea. Phytochemistry 22:1535-1538. Marbach, I., Harel, E. and Mayer, A.M., 1984. Molecular properties of extracellular laccasefro m Botrytis cinerea. Phytochemistry 23:2713-2717. Marbach, I., Harel, E. and Mayer, A.M., 1985. Pectin, a second inducer for laccase productionb yBotrytis cinerea. Phytochemist ry 24:2559-2561. Marcus, L. and Schejter, A., 1983. Single step chromatographic purification and characterisation of endogalacturonases and pectines terases of the fungusBotrytis cinereaPers .Physiologica l PlantPatholog y23:1-13 . Mayer A.M., 1987. Polyphenol oxidases in plants - Recent progress. Phytochemistry 26:11-20. Mayer, A.M., 1989, Plant-fungal interaction: A physiologists point of view. Phyto chemistry28:311-317 . Nes, W.D. and Patterson, G.W., 1981.Effec t of tetracyclic triterpenoids on growth of Phytophthora cactorum. Journalo fNatura lProduct s44:215-220 . O'Hara,E.B .an dTimberlake ,W.E. , 1989. Molecularcharacterisatio n ofth eAspergillus nidulansy Alocus .Genetic s 121:249-254. Ouchi, S., 1983. Induction of resistance or susceptibility. Annual Review of Phyto pathology21:289-311 . Rhem, S., Enslin, P.,Meewse , A.D.J, and Wessels,J.H. , 1957.Bitte r principles of the cucurbitaceae.Journa lo f theScienc eo fFoo d andAgricultur e8:674-691 . Rigling, D.H., 1989. Hypovirulenz, dsRNA und laccase-activität bei Cryphonectria (Endothia)parasitica, de mErrege rde sKastanienrindenkrebses .Ph.D .Thesi sZurich . Rigling, D.H., Heiniger, U. and Hohl, H.R., 1989. Reduction of laccase activity in ds RNA-containing hypovirulent strains of Cryphon ectria (.Endothia) parasitica. Phytopathology79:219-223 . Smith, D.A., Harrer, J.M. and Cleveland, T.E., 1982.Relatio n between production of extracellular kievietone hydratase by isolates of Fusarium and their phytopatho- genicity onPhaseolus vulgaris. Phytopathology72:1319-1323 . Van Etten, H.D., 1982.Phytoalexin detoxification by monooxygenases and its impor tancefo r pathogenicity. In 'ThePhysiologica l Basis of PlantInfection . Asada,W.R. , Bushneil, W.R., Ouchi, S. and Vance, C.P., Eds. Japanese Science Society Press, Tokyo,p .3 15-327 . VanEtten ,H.D .an dKistler ,H.C. , 1984. Microbialenzym eregulatio nan dit simportanc e for pathogenicity. In 'Plant Microbial Interactions' Vol, 1. Kosuge, T. and Nester, E.W.,Eds .Macmillan ,London ,p . 42-68. Weinhold, A.R. and Hancock, J.G., 1980. Defense at the perimeter-extracellular chemicals. In 'Plant Disease - an Advanced Treatise'. Horsefall, J.G. and Cowling, E.B.,Eds .Academi cPress ,Ne wYork ,p . 121-138. Yelton, M.M., Timberlake,W.E . and Vande n Hondel, C.A.M.J.J., 1985.A cosmi d for selecting genes by complementation - Aspergillus nidulans: the selection of the developmentally regulated yA locus. Proceedings National Academy of Science. USA82:834-838 . 82 Relationship between greymoul d ofgrape s and laccase activity in the must J.Roudet, S.Prudet and B. Dubos Summary In 1991,a correlatio nexiste dbetwee nth elaccas eactivit yi nth emus tan dth epercentag e of berries, infected by B. cinerea. This correlation is highly significant during the last 10day sbefor e harvest. Introduction Grey mould of grapevine isa seriou sdiseas e not onlybecaus e it affects thequantit yo f the harvest but also, and even more important, the quality of the wine produced from grapeswit hinfectio n byBotrytis cinerea Pers.:Fr. .I ti sknown ,tha ti nmus t of infected grapes, laccase activity is high and the activity of this enzyme detiorates wine quality. Visual observation of grey mould in the vineyard does not allow evaluation of the potential damage to the quality of the wine. Therefore we determined the relationship between the development of grey mould and the concentration of laccase present in musts. Developmento fgre ymoul dwa sfollowe d byvisua lobservatio nfro m véraisont o harvest onbunche s of grapes treated with fungicides tocontro l B.cinerea and onnon - treatedones . Theactivit yo flaccas ewa sdetermine dfo reac hbunc ha tharvest . Laccaseactivit ywa s measured spectrophotometrically, with syringaldazine as substrate (Harkin and Obst, 1973a,b;Harki net al., 1974).Th eresult sconcernin gth edevelopmen to fgre ymoul dan d theactivit y oflaccas emeasure d wereanalyse d statistically. Table 1. Correlationbetween infection of grapes by B. cinerea and laccase activity in the mustat three different sampling dates. Sampling date % Infection Laccase activity September 10 9 0.216 17 36 0.461 20 57 0.522 Significance threshold of correlation coefficient (Fisher) at 1% for 300 bunches observed:0.148 . 83 ATTACK (%) 100 -, 90 • 80 • 70 • - UNTREATED • FUNGICIDE 1 60 - •B- FUNGICIDE 3 50 - 40 - 30 • 20 vn-V' Fig. 1. Developmentof grey mould from véraison tillharvest FREQUENCY (%) 100 - UNTREATED • FUNGICIDE 1 60 • e- FUNGICIDE 2 50 40 v v-j.» &•» 84 ATTACK (%) 100 ! DATES &* ^ Fig. 3. Developmentof grey mould in bunches already showing symptoms on August 13. LACCASE ACTIVITY 45 -, UNTREATED I 0 10 80 30 40 50 60 70 80 90 100 ATTACK% Fig.4. Estimation of damage bygrey mould. Comparison of visual observations and determination oflaccase activity. 85 Results and Discussion Theyea r 1991wa scharacterise d bya nexplosiv edevelopmen t ofth ediseas edurin gth e three weeks preceding harvest. There were two distinct phases in the development of grey mould, which was correlated to rainfaill and maturity of the berries (Fig. 1). Fungicide treatments reduced the number of bunches affected and delayed the appearanceo fth eeffect s ofgre ymoul d(Fig .2) .O nth eothe rhand ,onc eestablishe d on the bunch, B. cinerea developed at the same rate on treated and non-treated bunches (Fig. 3). Asignifican t correlationwa sobserve dbetwee nth eleve lo fgre ymoul da tharves tan d theactivit y oflaccass ei nth emus t( Fig . 4). Agoo dcorrelatio nbetwee n rot,cause db yB. cinerea andlaccas eactivit y inth emus t wasobtained .However ,th efindings o f 1991 weredifferen t from thoseo fpreviou syears . So more experiments, to be carried out under different climatic conditions have to be done. References Harkin, J.M. and Obst, JR., 1973a. Syringaldazine, an effective reagent for detecting laccasean dPeroxydas ei n fungi. Experientia29:381-383 . Harkin, J.M. and Obst, J.R., 1973b. Lignification in trees: Indication of exclusive peroxidaseparticipation . Science 180:296-297. Harkin, J.M., Larkin, M.J. and Obst, J.R., 1974.Us e of syringaldazine for detection of laccasei nsporophore s ofwoo drottin g fungi. Mycologia66:469-471 . 86 Enzymatic detoxication ofstilbene sb yBotrytis cinerea and inhibition by grape berries proanthocyanidins R.Pezet, V. Pont and K. Hoang-Van Summary On a pectin-containing medium, B. cinereaproduce s a hydroxystilbene-degrading enzyme, stilbene oxidase, identified as a laccase. Stilbene oxidase oxidizes both proanthocyanidins and resveratrol. This oxidation detoxifies the hydroxystilbenes. Stilbeneoxidas ewa spartiall ypurifie d onSephade xG-100 ,DEAE-Sepharos eF Fan do n Syringyl EAJ-Sepharose4B.I tbind s onCo n ASepharos e 4B indicating aglycoprotei n nature.O nIEF-PAG E(p H3-10 ) thep ivalue swer efoun d tob ebetwee n 3.5 and4. 7 for thedifferen t isoenzymes. Therelativ emolecula rweight s(SDS-PAGE , 10%)o fth ethre e major bands of stilbene oxidase detected after IEF-PAGE purification were 97.6, 86,2 and 65.4 kDa. Proanthocyanidins from skin of grape berries inhibit competitively the activity of stilbene oxidase. The level of this inhibition seems to depend on the constitution ofth etannins . Introduction Pterostilbene (trans 3,5-dimethoxy-4'-hydroxystilbene) and resveratrol (trans 3,5,4'- trihydroxystilbene) areconstitutiv e components of thewood y parts of many specieso f the Vitaceae (Hart, 1981;Poo l et al., 1981). However, in the leaves and fruits these compounds are only produced after exposure to UV-radiation or after fungal infection and so they could act as phytoalexins. If phytoalexins are important factors in the resistance of aplant ,th eabilit y of apathoge n todetoxif y thesecompound s could bea n important feature in the success of a fungal pathogen (Van Etten etal, 1989). On the other hand, proanthocyanidins ofgrap e berries arepoten t inhibitors of stilbene oxidase produced by B. cinerea. These tannins could contribute to the resistance of grapes to infection by B. cinerea by inhibiting stilbene oxidase, and preventing detoxification of thephytoalexin sa ssuggeste db yNyerge set al. (1975). Results and Discussion Botrytis cinereaPers. : Fr. isolate P-69, used in the experiments was obtained from diseased grape berries (cv. Pinot). The methods used in this work were described previously(Peze t etal, 1991). Proteins precipitated from liquid culture filtrate, as well as the IEF-PAGE purified stilbene oxidase possess the ability to modify the UV spectra of hydroxystilbenes (Fig. 1).Thi s modification islinke d tot odetoxificatio n of pterostilbene (Table 1).Th e enzymatic transformation of resveratrol was demonstrated but not its detoxification, becausei ti sno ttoxi ci nth econcentratio n used inthi swork . 87 1 (a) 1 J" 0.8 O z 5 0.6 m ce O 0.4 0) H m •**^T A < 0.2 J*' A 0 200 300 400 200 300 400 WAVELENGTH (hm) WAVELENGTH (pm) 200 100 200 300 TIME(sec ) TIME(sec ) Figure 1. UV spectra of the degradation products of resveratrol and pterostilbene produced by the stilbene oxidase activity in culture filtrate of B. cinerea. The basic reaction mixture consisted of 30 pi of ethanolic solution of stilbene (Img.mt1); (a) and (b)pterostilbene; (c) and (d) resveratrol in3 ml citrate-phosphate buffer (0.1M, pH 5.2) (a) and (c) spectra without enzyme (—) as after theaddition ofstilbene oxidase (—)(b) and (d). Kinetic studies were done at an absorbance of 300 nm during enzymatic reaction. Stilbene oxidase in culture filtrate of B. cinerea depends on the age of the culture (Table 2). The crude enzyme solution was partially purified through several steps described in Table 3. The purified active fraction obtained from the laccase affinity gel Syringyl-EAH Sepharose 4B, was applied on a IEF-PAGE (pH 3-10). Proteins degrading hydroxy- stilbenes and having laccase activity were stained by incubating with p-phenyl- enediamine. Six important bands were visualized between pH 3.5 to 4.7. The positions Table1. Detoxification ofpterostilbene by a crudeprotein extract of the culture filtrate ofB. cinerea Stilbene Inhibition of germination3)(% ) Dead conidia (%) pterostilbeneb) 100 100 pterostilbene1')+ CE° ) 0 0 a) Percentage inhibition was determined from observations on 300 conidia b) Concentration 5 x lCXHM c)CE , crude extract of proteins secreted by B. cinereai n a liquid culture medium Table2. Stilbene oxidase in culture filtrates ofB. cinerea as afunction of the age of the culture Age of thecultur e stilbene oxidase activity Protein (days) (units») (|xg rah1) Î Ö43 üxï 2 0.64 9.85 3 0.68 11.18 4 0.80 19.80 5 1.83 25.33 6 3.27 25.90 7 7.69 144.48 8 27/76 1175.12 a) determined with 50 ml of acrud e enzyme solution (5ml) . Table3. Partial purification of stilbene oxidase produced byB. cinerea Steps Total activity11) Proteinsb> Specific activityc> Factor Culture filtrate 4421.06 15.66 352.12 1.00 (NH4)2S04 fraction 7804.94 323.20 689.77 ' 1.95 Cone, to 5 ml 8235.00 1175.12 1401.70 3.98 DEAE-Sepharose 16552.00 378.80 5591.89 15.88 Syringyl EAH 28550.00 49.92 11653.06 33.09 a) units min-1 (= change in absorbance per min (x -100) to convert to a positive number) b) (xgml- 1 c) units min-'mg-1 proteins of the proteins were located on an unstained IEF gel, the bands (1 to 6) were then removed and suspended in 3 ml of citrate-phosphate buffer (0.1M , pH 5.2).Th e activity ofth eprotein s wasdetermine d using pterostilbene orsyringaldazin e assubstrat e (Fig.2) . The major bands 4 to 6 were also transferred to an SDS-PAGE (10%) in order to determine the molecular weight. Fraction 4 showed an important band situated at 86.2 kDa, fraction 5 at 64.5 kDa and fraction 6 at 97.6 kDa. Hydroxystilbenes were oxidized into several unidentified products. One of them, separated by HPLC from oxidized pterostilbene and analyzed by mass spectroscopy has a molecular weight of 512 Da compatible with a dimeric structure of this stilbene at an oxidation stage. Stilbene oxidase activity canb einhibite d by several known laccaseinhibitors ,suc h as sodium diethyldithiocarbamate and sodium azide. Recently we have shown that proanthocyanidins (tannins) extracted and purified from the skin of grape berries, using themetho d of Porter (1989),ar estron g inhibitors of stilbene oxidase and laccase (Fig.3) . The Lineweaver-Burke plot with pterostilbene as substrate indicates a competitive type of inhibition (Fig. 4). The Michealis-Menten constant (Km) of B. cinerea of stilbene oxidase for pterostilbene was calculated as 66.6, 80 and 111.1 (|imoles pterostilbene ml"1) in the presence of0, 5 and 15m g mH inhibitor proanthocyanidin, respectively. Theimportanc e of phytoalexins inth eresistanc e of aplan t topathogeni c fungi can be indicated by the ability of a pathogen todetoxif y thesecompound s asi t invades the host. 89 o o D È > < Ld B. cinerea is known to metabolize and thus detoxify phytoalexins from a number of plants(Va nEtte net al., 1989) . Apathoge n likeB. cinerea migh tb eexpecte d toregulat e thesecretio n ofit senzyme s inrespons et oth enatur eo fth ehos tresistanc emechanisms . B. cinereaca n adjust themolecula r structure of itsextracellula r laccase toth ep Ho fth e hostan dth enatur eo f phenolics presenti nthi shos t(Marbac h etal., 1985) .Suc h effects could explain theheterogenicit y of polyphenol oxidasepurifie d from culturefiltrate s of B. cinerea. The pi value, molecular weight and affinity of polyphenol oxidase for laccases produced by B. cinereao n various substrates given in the literature differ considerably according to the methods used to cultivate the fungus (Marbach et al, 1984).I f laccasei s anintegra l parto f theinfectio n process ofB. cinerea in somehosts , it does not appear to be the case for infection of unripe berries of Vitis spp.Th e poor developmento fB. cinerea i nunrip egrap eberrie sindicate sth enatura lresistanc eo fthos e organs to the fungus, and probably the inactivation of exoenzymes of the parasite. Proanthocyanidins are important constituents of grape berries. They complex strongly with carbohydrates and proteins (Porter, 1989), and thus inhibit enzymes. The competitive inhibition of proanthocyanidins with stilbene oxidase (laccase) and pterostilbenea ssubstrat eshow stha ttannin sextracte dfro m grapeberrie sd ono tcomple x strongly with oxidase but have an affinity for the enzyme. The inability of proan thocyanidinst oinhibi tpolygalacturonas eo fB. cinerea (result sno tshown )confirm s that theinhibitor y activity of these tannins isspecifi c tosom eenzyme s and isno tth eresul t of aprotei ndenaturation . 90 100 OD 150 CONCENTRATION PRA (jug ml ) Figure 3. Inhibitionof stilbene oxidaseactivity by variousconcentrations ofproan- thocyanidins(PRA) extractedfrom the skin ofgrape berries. _L o J L_ check Q_ v + 5 fj,q ml _ PRA ß 4 • +15 ßq ml PRA c 'E o ° Ó o £ =*, 0 > - *~ 1/S (/imoles x 10 ptero.) Figure4. Lineweaver-Burke plot of the enzyme kinetics of stilbene oxidase in the presence of0.0, 5and 15 /igmt 1 ofPRA. K values were calculatedfrom this plot. 91 Proanthocyanidins, present in the skin of grape berries are mainly cyanidin and delphinidin. Theproportio n ofeac h differs according toth edevelopmenta l stage ofth e berries from whichth etannin swer eextracted .Th einhibitor y activity ofthes eproanth o cyanidinsextracte dfro m matureberrie st ostilben eoxidas ei slowe rtha ttha tfro m unripe berries. Therefore, proanthocyanidins could play an important role in the processes involvedi nresistanc eo fgrap eberrie st oth edevelopmen t ofB. cinerea. Acknowledgements Wethan kMrs .I .d eGroot efo r heressentia ltechnica lassistance . References Hart,J.M. , 1981.Rol eo fphytostilbene si ndeca yan ddiseas eresistance .Annua lRevie w ofPhytopatholog y 19:457-458. Marbach,I. ,Harel ,E .an dMayer ,A. ,1984 .Molecula rpropertie so fextracellula rBotrytis cinerealaccase .Phytochemistr y 23:2713-2717 . Marbach, I., Harel, E. and Mayer, A., 1985. Pectine, a second inducer for laccase productionb yBotrytis cinerea. Phytochemistry24:2559-2561 . Nyerges, P., Szabo, E. and Donko, E., 1975. The role of anthocyane and phenol compounds inth eresistanc eo fgrap eberrie sagains tB otrytis infection. ActaPhyto - pathologica Academiae ScientiarumHungarica e 10:21-32. Pezet, R., Pont, V. and Hoang-Van, K., 1991.Evidenc e for oxidation detoxication of pterostilbene and resveratrol by alaccase-lik estilben e oxidase produced by Botrytis nnerea.Physiologica l andMolecula rPlan tPatholog y39:441-450 . Pool,R.M. ,Creasy ,L.L .an dFrackleton ,A.S. , 1981.Resveratro lan dth eviniferins ,thei r application to screening for disease resistance in grape breeding programmes. Vitis 20:136-145. Porter L.J., 1989.Tannins .In 'Methods in Plant Biochemistry'. Vol 1 Plant Phenolics, p.389-419 . VanEtten ,H.D. ,Matthews ,D.E . andMatthews ,P.S. , 1989.Phytoalexi n detoxification: importance for pathogenicity and practical implications. Annual Review of Phyto pathology27:143-164 . 92 Differing biochemical and histological studies of twograp e cultivarsi n thevie wo f their respective susceptibility and resistance toBotrytis cinerea R. Pezetand V. Pont Summary Studies of resistance of grapevines toB. cinerea should consider allknow n parameters involvedi nth eprocess ,rathe rtha nfocu so non easpect .Wit hthi sperspectiv ew estudie d two contrasting Swiss grapevine cultivars, cv. Gamay which is susceptible and cv. Gamaret resistant to B. cinerea. Our analyses showed histological and biochemical differences between berries of thesetw ocultivars .Thei r respective ability tosynthesis e phytoalexins, the relative concentrations of glycolic acid, phenolic compounds and proanthocyanidins andth ethicknes san dstructur eo fth eski nar esom eo fth eparameter s whichprovid ea napproac ht oth eunderstandin g ofth ecause so fresistance/o fgrap et oB. cinerea. Introduction Inrecen tyear sth emechanism so fresistanc eo fgrap eberrie st oBotrytis cinerea Pers .: Fr. have been studied intensively . Jeandet and Bessis (1989) reviewed this subject and concluded that numerous mechanisms were probably involved in active and passive defence of grapes. The synthesis of phytoalexins (Langcake and Price, 1977), the inhibition of exoenzymes of B. cinerea by proanthocyanidins (Bachmann and Blaich, 1977; Pezet and Pont, 1992) and by unidentified glycoproteins (Grassin, 1987) or the toxicity of glycolic acid (Pezet and Pont, 1988), anthocyans and other penolic compounds (Nyerges et al., 1975) towards B. cinerea are some of the active defence systems involved. Passive defences could also be effective through the synthesis of lignified barriersproduce dfro m oxidizedphenol s(Mansfield , 1980),th estructur eo fth e epidermis (Bernard, 1976)an d thickness andcompositio n of thecuticl e (Radier, 1968). This papercompare s two grape vinecultivars ,cv .Gama y which is susceptible andcv . Gamaretwhic hi sresistan tt oB. cinerea inth eligh to fsom eo fthes eparameters . Results and Discussion Synthesiso fresveratro l After UVradiatio n (256nm) ,grap eberrie swer epeele dan dth efres h weighto fth eskin s determined. They were then extracted according to the method of Southerton and Deverall (1990) for free phenolic acids. Of the final methanol extracts, 30 \û was analyzedb yHPL Ci n thesyste m described byth esam eauthors ,excep t thatth esolven t systemwa sa gradien to fmethano li nformi c acid (50mM )( 45 %MeO Ht o80 % MeOH in 25 min). In this system resveratrol had a retention time of 5.65 min. Resveratrol 93 • GAMAY v GAMARET 0 5 10 15 20 HOURS AFTER IRRADIATION (UV 256 nm) Fig. 1. Synthesis of resveratrol after UV irradiation (256nm) by theberry skin ofcv. Gamay (susceptible) andcv. Gamaret(resistant). The skin was separatedfrom the pulp atdifferent times after UV irradiation andresveratrol was extractedimmediately. content of berries was determined from 0 to 20h after irradiation. It appeared thatcv . Gamaretsynthesise d moreresveratro l thancv .Gama y after 20h (Fig . 1 ). Natural amounts of resveratrol in berry skins at véraison and at harvest time are determined by thesam emethod .Th egrapevin ecv .Gamare t contained moreresveratro l thancv . Gamay(Tabl e1) . June 06 GLYCOLIC ACID CONCENTRATION (mg g dry weight) Fig. 2. Glycolic acid concentration in berries of cv.Gamay and cv.Gamaret atfive developmentalstages. Measurements were made onextract of whole berries. 94 Table 1. Contentofresveratrol in the skin of grape cvs Gamay and Gamaretat véraison, andat the endof the season. Resveratrol (ng.g-1dr yweight ) Gamay Gamaret Véraison 1.72 3.53 Harvest 3.24 16.66 Concentration ofglycoli caci d Organic acids wereextracte d with water from freeze-dried and powdered grapeberrie s (500 mg powder in 50 ml water); 5 ml of this extract was applied to a polyvinyl polypyrolidone (PVPP) column (1x6 cm),whil e 10|x l of the eluate was analysed by HPLC isocratically (H3P04, 0.1%) on a Supelcogel C-610H column (300 x 7.8 mm, detection at21 0nm) .Th eretentio n timeo fglycoli c acidwa s 16.62min .Periodically ,a t different developmentalstages ,th econten to fglycoli caci d inberrie swa sdetermine di n both cultivars.Th econcentratio n ofglycoli c aciddecrease d substantially in September, butth econcentratio n wasalway shighe ri ncv . Gamarettha n incv .Gama y(Fig .2) . Epicuticular waxes The amount of epicuticular wax on berries of both cultivars was determined after measuringthei rsurfac earea .Berrie swer esoake di nchlorofor m for3 0s , thesolven twa s evaporated to dryness under vacuum and theresidu e weighed. Higher densities ofwa x wereextracte d from cv.Gamare ttha ncv .Gama y(Tabl e 2). Table 2. Density ofepicuticular waxes ongrape berries ofcvs Gamay andGamaret in late September. Cuticularwaxe s(mg/cm 2) Dates, 1991 Gamay Gamaret September2 5 0.11 0.14 September 30 0.08 0.17 Histological observationo fepidermi s Atth evéraiso nstage ,halve so fberrie swer efixed, embedde di nparaffi n andcu tint othi n sections. After staining they were studied by light microscopy. There were no fundamental differences visible between the two cultivars in the first layers, but the inside layer of theepidermi s was much thicker in cv.Gamare t and the thickness of the epidermis asa whol ewa sgreate ri ncv .Gamare ttha ni ncv . Gamay. Phenolicacid s Phenolic acids were extracted and separated into four classes according to Southerton and Deverall(1990) .Extraction s weremad efro m entirebenie s untilJul y 12 1991an d 95 600- GAMAY . 500- - 400- _40 0 300- -300 * - 200- - 200 100- 1 100 0- M m' 0 <ü o o_l free phenolic acids JULY 30 SEPT.09 phenolic acids from glucosides GAMAY phenolic acids from esters GAMARET bound phenolic acids M PULP total phenolic acids • SKIN iimHSKIN Fig. 3. Concentrations ofphenolic acids Fig. 4. A. Proanthocyanidin concentra in grape berries of cvs Gamay and tionsin pulp and skinof two grapevine Gamaret, presentas free phenolic acids, cultivars, Gamayand Gamaret. glycosides, este rs and bound forms, B. Inhibitionof B. cinerea oxidase by determinedat five developmental stages. proanthocyanidinsextracted from skin andpulp of grape berries ofGamay and Gamaret (enzymatic reaction medium: 3 mlcitrate-P0 4buffer [0.1 M, pH5.2]; 30 pi crude enzyme [1.49 mg prot.mt1]; 20 pbl pterostilbene [1 mg.mt1 EtOH solution]; 20 ml proanthocyanidins [1.5 mg.mh1, aqueous solution]) from the epidermis from July 30 1991. Predominant phenolic acids of each class were identified andquantifie d by HPLC analysis (gradientI ) according toth esam eauthors . The total phenolic acid content was determined by adding concentrations ofidentifie d standard compounds which absorb at27 5 nm ineac h class. Six compounds, mainly predominant inth efre e phenolic acid class butals o present in other classes, were identified asstilbene sb ythei rU Vspectr aan dthes ewer esubsequentl yquantifie d using external peak standardisation forresveratro l at 275 nm. The level ofphenolic s was highesti nth eesterifie d form, exceptfo r cv.Gama ya tth een d of flowering time(Jul y8 1991).A tth een d ofth eseaso n (September9 1991),th etota lphenoli c acidswer etwic e ashig hi ncv .Gamare ttha ni ncv . Gamay(Fig . 3). 96 Proanthocyanidins Condensed anthocyanidins are important components of both the skin and the pulpo f grapeberries .The ywer eextracte db yth emethod so fCzochansk aet al. (1980)an dPorte r (1989)an dthei rconcentration sdetermine dgravimetrically .Delphinidi nan dcyanidi nw e re released from grape proanthocyanidins by acid hydrolysis. These anthocyanidins represent c. 2% of the weight of the proanthocyanidins extracted from grape berries. According to Czochanska et al. (1980) a still unsolved problem is the separation of polymeric proanthocyanidins from hydrolysable tannins.W econclude d thatth etannin s extracted from the skin and the pulp of grape berries were composed of condensed tannins and hydrolysable tannins (Fig. 4A). Is was clear that the concentration of proanthocyanidinswa salway sgreate ri nth eski ntha ni nth epulp .Highes tconcentration s were found in cv. Gamaret and they decreased in this period from July 30 until September9 1991.Th einhibitio nb ythes etannin so fstilben eoxidas efro m B. cinerea is showni nFig .4B .Fo rth esam econcentratio no fproanthocyanidins ,inhibitio no foxidas e was alwaysgreate r for thoseextracte d from cv.Gamaret . Atvéraison ,th etannin s from pulp of cv.Gamare t showed themos toxidas e inhibition.Thes eresult s indicate thatth e quality oftannin si nberrie si simportan tfo rth einhibitio n ofoxidase . Conclusions ' Allparameter sexamine di nberrie so fcv . Gamaretshowe dcondition sles sfavourabl e for development of B. cinereatha n those in cv. Gamay. These results accord with the observed resistance of cv. Gamaret to B. cinerea in thefield. Th e importance of each parameteri nth eresistanc eproces si srelative .I nthi smode li ti simpossibl et odesignat e asingl eresistanc efacto r asessential ;al lcontribut etoward sunfavourabl e conditions for growth ofB. cinerea, and are exhibited atdifferen t levels related to the developmental stages of the berries, climatic condition and grapevine cultivais. Aggressiveness of B. cinerea populations,a sye til ldefined , wasno ttake nint oaccoun ti nthi sstudy . Acknowledgements Wethan kMrs .I .d eGroot efo rproficien t technicalassistance . References Bachman, O. and Blaich, R., 1979. Vorkommen und Eigenschaften kondensierter Tanninei nVitaceen .Viti s 18:106-116. Bernard,A.C. ,1976 .Résistanc emécaniqu ede sbaie sd eVitis vinifera a uBotrytis cinerea Pers.Franc eViticol e 8:301-307. Czochanska, A., Lai Yap Foo, Newmann, R.H. van Proter, L.J., 1980. Polymeric proanthocyanidins. Sterochemistry, structural units and molecular weight.Journa lo f theChemica l SocietyPerkin ,I p .2278-2286 . Grassin, C, 1987. Recherches sur les enzymes extracelluliares sécrétées par Botrytis cinerea dans la baie de raisin. Applicati ons oenologiques et phytopathologiques. Doctoralthesi sUniversit éd eBourdeau xII . Jeandet, P.an d Bessis, R., 1989.Un e réflexion sur les mécanismes morphologiques et biochimiques del'interactio nvign eBotrytis. Bulletind el'OI V703-704,637-657 . Langcake, P. and Pryce, R.J., 1977. A new class of phytoalexins from grapevines. 97 Experientia 33(2):151-152 . Mansfield,J.W. ,1980 .Mechanis mo fresistanc et oBotrytis .In 'Th eBiolog yo fBotrytis ' eds.Coley-Smith ,J.R. ,Verhoeff , K.,an dJarvis ,W.R .p .181-218 . Nyerges, P., Szabo, E. and Donko, E., 1975. The role of anthocyan and phenol compounds in the resistance of grapes against Botrytis infection. Acta Phyto- pathologiaAcademi aScientia e Hungaria 10:21-32. Pezet, R. and Pont, V., 1992.Enzymati c detoxification of stilbenes byBotrytis cinerea and inhibition by grapeberrie s proanthoc yanidins.Proceeding s of theXt h Botrytis Symposium,Heraklion ,Greece .Pudoc ,Wageningen ,Th eNetherlands ,p . 87-92. Pezet,R .an dPont ,V. ,1988 .Activit éantifongiqu edan sle sbaie sd eVitis vinifera: effects d'acidesorganique se td uptéros tilbène .Revu eSuiss ed eViticulture ,Arboricultur ee t Horticulture20:303-309 . Porter.L.J. , 1989.Tannins .In 'Methods inPlan tBiochemistry ' Vol. 1. PlantPhenolics , éd.Harborne ,J.B. .Academi c Pressp .389-412 . Radler, F., 1968. La cire cuticulaire des grains de raisins et des feuilles de la vigne. Connaissanced el aVign ee td uVi n3:271-2 94 . Southerton, S.G. van Deverall, B.J., 1990. Changes in phenolic acids levels in wheat leaves expressing resistance to Pucciniarecondita f.sp. tritici. Physiological and MolecularPlan tPatholog y37:437-450 . 98 Some characteristics ofresistanc e of grape berries to grey mould caused byBotrytis cinerea S.Prudet, B.Dubos and R. LeMenn Summary Acompariso nb yligh tan delectro nmicroscop yo fth edevelopmen twa smad eo fth eski n in the two grapevine cvs Arriloba and Sauvignon, which differ in susceptibility to B. cinerea. The skin of cv. Arriloba (resistant) was thicker because of a higher number of cell layers, and had more dispersed cuticular striations than that of cv. Sauvignon (susceptible). Developmento fthes estructure s atth een do ffrui t maturation seemedt ob erelate dt o theirsusceptibilit y toB. cinerea, particularly thedevelopmen t ofdee phypoderma lcell s into mesocarp parenchyma (pulp) cells. In cv. Arriloba, the thickness of the skin is maintained longer at theen d of maturation owing toles s degradation of the numbero f celllayers . Thecontinue d presence of anendo-polygalacturonas einhibito r in resistant cultivars after véraison may also be involved. The pectic substances of the skin become more susceptiblet odegradatio nb ypectolyti cenzyme so fB. cinerea durin gmaturatio nan dthi s trend isgreate ri ncv .Sauvigno ntha ni ncv .Arriloba . Introduction Thesusceptibilit y ofgrape st oBotrytis cinereaPers. :Fr .varie saccordin g toth estag eo f development ofth eberries ;immatur egrape sbecom esusceptibl et ofunga l development after véraison. The discovery of an inhibitor of endo-polygalacturonase produced by B, cinerea in grapes(Grassin , 1987)gav ea fres h impetust ostudie so nth eresistanc eo fth evin et othi s pathogen.Th eprobabl elocatio no fthi sinhibito ri nth eepiderma lan dhypoderma llayer s (hereafter called 'skin')whic hconstitut eth efirs t barriert ofunga l penetration,raise sth e questiono fth erol eo fthes elayer si nresistanc eo fth egrap eberrie st oB. cinerea an dth e relationshipbetwee ndecrease si nth eleve lo fth einhibito ran dth emodificatio n ofthes e structures duringripening . Wetherefor eperforme d ahistologica lstud yo fth e 'skin' incultivar s Sauvignon(ver y susceptible) and Arriloba (resistant). First, we used light and electron microscopy to examine development of the 'skin' during maturation. Secondly, we investigated the changes in digestibility of pecticsubstance s from the 'skin' by pectinolytic enzymeso f B. cinerea andAspergillus niger. Material and Methods Fromflowerin g untilmaturatio no fth eberries ,sample so f thebenie s ofth ecv sArrilob a andSauvigno nwer etake nweekly . 99 By light, scanning and transmission electron microscopy, the following parameters werestudied :th esurfac e pattern andthicknes so fth ecuticle ,th eshap eo f theepiderma l cells,numbe ran d thickness of celllayer s underth eepidermi s and the thickness of the inner andoute rpecto-cellulos ewall so fepiderma lcells . The patterns on the cuticle surface of cvs Sauvignon and Arriloba were compared whenth eberrie s hada diamete ro f 3,11an d 16 mm. To study the breakdown of pectins from the 'skin' of berries of both cultivars, pectinolyticenzyme sproduce db yB. cinerea andb yA. nigerwer eused .Th epecti nwa s obtained after crushing theberrie s and dipping them in ethanol at 100°C.Th eethanol - insolublemateria l containing thepectin s wereuse d assubstrate .Th equantit y of pectin wasdetermine d by themetaphenyl-pheno l methods,a sdescribe d by Blumenkrantzan d Abroe-Hansen (1973).Neutra lsugar swer edetermine d byth esulphuri c phenolmetho d (Montreuil and Spik, 1963). Pectinolyticenzyme so fB. cinerea (polygalacturonas ean dpectinases )wer eextracte d from a liquid culture medium of the fungus and assayed by the method of Somogyi- Nelson (Somogyi, 1952). Polygalacturonase from A. nigerwa sobtaine d commercially andsimilarl y assayed. Digestionwa sperforme db yplacin gth esam equantit yo fpecti nwit heac henzym efo r 2h a t37°C . Results and Discussion No differences were observed between the two cultivars in cuticle surface patterns at thesestage so fberr ydevelopmen tusin ga scannin gelectro nmicroscope .Thi sparamete r probablyplay sn orol ei nth edifference s insusceptibilit y ofcultivar st oJS . cinerea. Both cultivars had similar patterns in the increases of cuticle thickness during muturation.B yligh tmicroscop e(Fig .1 )i twa sshow ntha tth ecuticl ei ncv .Arrilob awa s 4.75 (Jimthic k and that of cv. Sauvignon only 3.5 |xra. This greater thickness in cv. Cuticular thickness (urn) 5 1 -•- Sauvignon -o Arriloba Veraison Berry diameter Y (mm) 3 4 5 6 7 8 9 10 11 13 13 14 15 16 Fig. 1. Changes inthickness of the 'skin ' in berries of the cvsArriloba and Sauvignon. 100 Thickness (Mm) Berry diameter (mm) 3 5 7 9 11 13 15 Fig. 2. Changes inthickness of the cuticle. Arrilobawa svisibl ewhe n theberrie sha d attained adiamete r of 9mm .However ,whe n theberrie s were 16 mmi ndiameter ,th ecuticula r thickness incv .Arrilob awa sreduce d to4. 4 (im andtha to fcv .Sauvigno n to2.6 5|xm . 'Skin'thicknes sdevelope dsimilarl yi nbot hcultivars ;unti lvéraison ,it sthicknes swa s relatively high,bu tthe ndecreased .I ncv .Arriloba ,th efina l 'skin' thicknesswa s6 2|x m andtha to fcv .Sauvigno n 33 ijum. Thechange si nthicknes s of the 'skin' mayb erelate d toth echang ei n thenumbe ro f celllayers .Befor e véraison,cv .Sauvigno n hadc . 14 celllayer scompare d to 12.5i ncv . Arriloba, but after véraison, the layers decreased inbot hcultivars ,bu t was stillgreate r (6)i ncv .Arrilob aa tth elas ttw ostage so fmaturatio n than incv .Sauvigno n(3.5) . Using transmission electron microscopy (TEM),w e studied thickness of the cuticle and the inner and outer walls of epidermal cells.Th e change in size of these cells was expressedb yrati oR (lengt hL t oheigh tH o fcells) . TheTE M studies of thecuticl e confirmed the results from light microscopy thatth e cuticleo fcv .Arrilob a wasthicke rtha ni ncv .Sauvigno n (Fig. 2). Theoute rcel lwall so fth eepidermi swer eo fth esam ethicknes si nbot hcultivars ,wit h a steadyincreas et o 1.45 jim whenth eberrie sha da diamete ro fc . 16 mm,fo r theinne r walls,ther ewa s asimila rdevelopmen t inbot hcultivar s with athicknes s ofc .0.7 5|x m inberrie s of> 9 m mdiameter . The development of the epidermal cell ratio R, as shown in Table 1, indicated the tendency for thecel l pattern tochange .R increase d from 1.33 to4.4 3 in cv.Sauvigno n and from 1.30 to 2.68 in cv. Arriloba. This indicates that these cells flatten during maturation, and contribute to the reduction found in the final 'skin' thickness, a phenomenonmor epronounce d incv .Sauvigno ntha ni ncv .Arrilob a(Tabl e1) . On the basis of these observations, the change in 'skin' thickness at the end of maturation probably plays an essential role in the varying degrees of susceptibility to B. cinerea. Clearly, the physical barrier offered by the 'skin', decreases during 101 maturation,particularl y incv .Sauvignon .Thes echange si ncel l wallstructur eindicate d that there may be chemical changes in the pectin composition which may affect their degradationb ypectinases . Table 1. Developmentof the epidermalcell ratio R(= length tothe height of the cells) inberries ofcvs Sauvignon andArriloba. Diametero f R theberrie s(i nmm ) cv.Sauvigno n cv.Arrilob a 3 1.33 1.30 5 0.99 1.22 7 1.48 1.64 10 1.56 1.78 12 3.52 2.14 13 3.70 2.43 14 4.43 2.68 Thequantitativ e changes in pectic substances present in the 'skins',an d their levelso f neutral sugars and acids in the period of July 10 to September 14 1990 are shown in Fig. 3. Thechange s inpecti c substancesdurin g maturation of 'skins' ofcv sSauvigno n and Arrilobawer esimilar ,particularl yregardin g acidpectin swhic hwer eexamine dfo r their digestibility. Anincreas ei nth edigestibilit y of 'skin' pectinsdurin g maturation wasfoun d inbot h cultivars (Fig. 4) when pectinesterases and polygalacturonases of B. cinerea and poly galacturonase of A. niger were used. There was no difference before véraison, but Concentration (fg/mg of A.I.M.) Pectinic 400-, substances 350- Total 300 250 Neutral 200 150 100 Acid Sampling 50 ^date 10.7 26.7 07.8 20.8 10.9 24.9 Fig. 3. Quantities of neutral and acidsugars in grapeberries of cvsSauvignon and Arriloba. 102 Optical density O.a -, B.cinerea enzymes A.niger enzyme Sampling date 10.7 26.? 07.6 20.8 10.9 24.9 Fig. 4. Changesin digestibility of pectin during maturation of berries of the cvs Arriloba andSauvignon. / thereafter pectin from cv.Sauvigno n wasmor edigestibl eb y pectinases from B. cinerea thanpecti nfro m cv.Arrilob aan dthi sdifferenc e increasedunti l harvest. Inbot hcultivars ,pecti n digestibility increased during maturation; butwa sgreate ri n cv.Sauvigno n than in cv.Arriloba . Biochemical analysis of thesegrap e pectins should nowb eperforme d toimprov eknowledg eo fthi sresistanc emechanism . In the cv. Sauvignon susceptible to B. cinerea, the decrease in the mechanical and biochemical performance ofth e 'skin ' during maturation wassignificantl y greatertha n inth eresistan tone .Th evariou spattern so fdevelopmen to fth e'skin 'comple xma y affect thepolygalacturonas e inhibitorpresen ti nthes ecells . While these results to some degree explain the increased susceptibility of cv. Sauvignonberrie s at véraison,the yexamin eonl y one aspect of finit resistance.There fore they should be considered only as part of the complex defence system of the grapeberries againstB. cinerea. References Blumenkrantz, N. and Abroe-Hansen, G., 1973. New method for quantitative determination ofurani cacid .Analytica lBiochemistr y 54:484-489. Grassin, C, 1987. Recherches sur les enzymes extracellulaires sécrétées par Botrytis cinereadan sl abai ed eraisin .Application soenologique se tphytopathologique .Thès e deDoctora td el'Universit éd eBordeau xI ,France . Montreuil,J .an dSpik ,G. , 1963,.Method scolorimétrique sd edosag ede sglucid stotaux . Monographied ulaboratoir ed eChimi eBiologiqu ed el aFacult éd eScience sd eLille . Somogyi, M., 1952. Notes on sugar Determination. Journal of Biological Chemistry 195:19-23. 103 In vitro bioassays toevaluat e the relationship between grapevines and Botrytis cinerea R.Bessis, D. Vanneland M. Barbier Summary Culturefiltrates fro m media inwhic hB. cinerea wasgrow ncontai n substances thatar e toxic tograpevin e invitro. The level of toxicity varied with cultivar and thespecie so f Vitis. Theheteropolysaccharide-containin g fraction wasfoun d tob eth emos t toxic.Th e screeningo fVitis germplas mfo rsusceptibilit yt otoxi cmetabolite so fB. cinerea ca nno w beperforme d rapidly invitro undercontrolle dconditions . Introduction Cultivars of grapevines differ in susceptibility toB. cinerea; some ares o sensitive that they are being grown less (e.g. cv. Folle blanche), others are more resistant (e.g. cv. Cabernet-Sauvignon).Variatio n alsooccur si nth edevelopmen t ofB. cinerea during the growing season on different Vitis spp. Grape berries must be wounded, split open, or being fully ripened tob einfected . Thetim eo f ripening ofbunche s isimportant .O nth e samevine ,onl yth ebunche so fth efirs tgeneratio n aresusceptibl ea tharvest ,whil ei nth e otherbunches ,althoug h underth esam eclimati cconditions ,diseas e symptomsma yno t appear. Therefore thefactor s responsiblefo rgrap eresistanc ear ecomple xan ddepen do nth e species,stag eo ffrui t development, andcultura lconditions . Consequently, it isdifficul t toscree ngrapevin e cultivars for resistance toB. cinerea inth efiel d anda nin vitro approachfo r theseevaluation swa ssought . Material and Methods Plantswer ecultivate d invitro incontrolle dcondition s (Barbier andBessis , 1987)whic h provided reproducible results throughout theyear .Th eplan tculture s used in thiswor k werederive dfro m asingl ebudde dmicro-cuttin gtake nfro m themiddl epar to fth eplant . Cellsuspension swer eprepare dfro m leavesa sdescribe delsewher e(Barbie r andBessis , 1990). Fourisolate so fBotrytis cinerea Pers .: Fr.wit hsimila rgrowt hrat ean dtoxicit yo fthei r culturefiltrates wer eprovide db yM .Lerou x(INRA ,Versailles) .Onl yisolat eN wa suse d in ourstudy .I twa smaintaine d by subculturing aconidia l suspension onsoli d orliqui d malt-extract media. The medium used for micropropagation of grapevine also proved excellentfo rgrowt ho fth efungus . After4 week sincubatio nth emediu mwa sfiltere d andth eprotei nfractio n precipitated byammoniu msulphat eaccordin gt oth etechniqu eo fKamoe n( 1984 )an dKamoe net al. (1978).Th epolysaccharid efraction s werethe nprecipitate di ntw ofraction s accordingt o themethod sdescribe db yDubourdie u (1982);th efirst fractio n (P05 )wa sprecipitate db y 104 ,Bfel, 1800 1400 4100/2 4400 1800 1400 4400/2 4400 4800 TREATMENT TREATMENT CH95 1400 4400/2 4400 4800 1800 1400 4400/2 4400 TREATMENT TREATMENT CH96 7 •• ~ 6 §5 ~ 4 I— X 3- s2 X 1 F^tfv* ,«PTJ ^ , i .TE»' , K*^3 0 1800 1400 4400/2 4400 -1 J TREATMENT Fig. 1 Height(cm) ofmicropropagated plants ofV. riparia (R),cvs Cabernet-Sauvignon (CS), Pinot(P) and Chardonnay clones 95 (CH95) and 96 (CH96) after 2 months culture oncontrol medium (T) oron media containingfiltrates obtained after 1 week of incuba tion in800 ml (1800) and 400 ml (1400) or after 4 weeks of incubation in800 ml (4800) and400 ml (4400). The medium 4400/2 equals thefiltrate 4400 twice diluted. 105 onehal f volume of ethanol and thesecon d (P4)wa s obtained by adding 3.5 volumeso f ethanolt oth esupernatant . Results and Discussion After 2week sincubatio n onth emediu m used forgrapevin emicropropagatio n (Barbier and Bessis, 1987), B. cinerea produced toxic compounds causing necrosis on micro- cuttings ofc vChardonnay . The phytotoxicity of the culture medium after growth of B. cinereadiffere d for different species and cultivars. All theChardonna y plantlets showed necrosis, whereas theselectio n 41B (Vfvinifera xV. berlandieri) grown under the samecondition s grew satisfactory. Weconclude d from thesetw oexperiment s thatth efungu s released substances inth e medium which were toxic to grapevine and that germplasm of grapevine differed in sensitivity tothes ecompounds . Subsequenttest sexamine dth erelationshi pbetwee n thelengt ho fexposur et ovariou s phytotoxic materials from B. cinerea and the species of the grapevine. The results are giveni nFig .1 . Thisbioassa y allowed rapid discrimination between species andcultivars :V. riparia showed littleo rn osensitivit y toth efiltrate ;V. viniferacvs .Pino tan dChardonna y were very sensitive, whereas the cv. Cabernet-Sauvignon was slightly less sensitive. This resulti simportan t becausei nvineyards ,cv .Cabernet-Sauvigno n isknow n tob eon e of thecultivar s leastaffecte d byB. cinerea. Toxicity of thecultur e medium increased with thelengt h of theincubatio n and after 1wee kth etoxicit ypronounce d easily( Fig .1) . The fraction containing heteropolysaccharides (Dubourdieu, 1982) washighl y toxic (Fig.2) , but thegluca n fraction washardl ytoxic .Afte r dialysing thefiltrate , itstoxicit y wasreduce d slightly. CH96 7 •• 6 • 55 4 • X 3 CD « 2 Ld X 1 0 KS^T^N D PO.5 P4 TREATMENT Fig. 2 Height(cm) of micropropagated plants ofV. riparia(R), cvs Cabernet-Sauvignon (CS), Pinot(P) and Chardonnay clones 9 5 (CH95) and 96 (CH96) after 2 months growth on controlmedium (T) or on media with dialysedfiltrate (D) or filtratefractions containingglucan (P05)or heteropolysaccharides (P4). 106 S90 40 80 120 160 TIME (HOURS) Fig. 3 Destruction of chlorophyll pigments in leaves of micropropagatedplants ofV. vinifera cvsPinot (P),Chardonnay (CH),Cabernet-Saugvignon (CS) andV. riparia (R) incubatedfor 6, 24and 48 h or 1 week inundiluted fungal culture filtrates. Results are expressedas percentagesof the chlorophyll content of controlleaves. Thepoints representthe mean of three experiments with twosamples each. Variabilityfor eachpoint wasalways less than 7%. This bioassay allowed ust oestablis h alin kbetwee n the susceptibility of grapevines in the vineyard toB. cinerea and the sensitivity of micropropagated plants to die culture filtrates of the fungus. It was shown that the heteropolysaccharide fraction was highly toxic. However, this plant bioassay requires large volumes of filtrate and a 2 month cultivation period. Therefore, more rapid assays were evaluated. Excised leaves of micropropagated plants of various grapevines were incubated with culture filtrate of B. cinerea. The leaves turned brown rapidly and this alteration was measured quant itatively by destruction of chlorophyll (Fig. 3). The control leaves submerged in uninoculated mediumshowe d nodecreas ei nth echlorophyl l content.N odiscriminatio n wasfoun d between grapevinegermplasm . Forexampl eV. riparia, which is resistant in thefield , wasver ysusceptibl eunde rthes econditions . The study of the activity of thevariou s fractions on excised leaves showed that the fraction P05 wa sneve rphytotoxic ,th efractio n FPwa sphytotoxi c toal lcultivar san dth e fraction P4 destroyed chlorophyll in allV. vinifera cultivars, but not inV. riparia. This fraction therefore contains a factor which may be useful in discrimination between genotypes. Naked protoplasts from Vitisvinifera cv. Chardonnay and from V. riparia, or protoplastswit hregenerate dcel lwall sexpose dt ocrud ecultur efiltrat e ofB. cinerea die d rapidly. With V.riparia, protoplast s were more sensitive than cells. Using selected fractions of the culture filtrates with protoplasts or cells it was shown that the hetero- polysaccharide-containing fraction wasth emos ttoxic . This study demonstrated the possibility of devising rapid screening tests for measuringth esensitivit y ofgrapevine s toB. cinerea, but for practical usemor ewor ki s needed to examine more cultivars, the correlation of symptom development and the 107 susceptibility ofth ecultivar sunde rfield condition san dt oidentif y thetoxi ccompound s involved.I tshoul d nowb epossibl e toscree nne wcultivar s of zygotico rsomati corigi n and establish testprocedure s for usei n vitro.Othe r technical aspects of this work have beenpublishe d elsewhere(Vanne letal, 1991a,b). References Barbier,M .an dBessis ,R. , 1987. Isolemente tcultur ed eprotoplaste sd efeuille sd evign e (Vitis viniferacv . Chardonnay).Bulleti nd el'O.I.V . 679/680:765-775. Barbier,M .an dBessis ,R. , 1990.Isolatio n andcultur eo fgrapevin ecv .Chardonna ylea f protoplasts.Euphytic a47:39-44 . Dubourdieu, D., 1982.Recherch e sur les polysaccharides sécrétés parBotrytis cinerea dansl abai ed eraisin .Thès eDoctorat ,Universit éBordeaux ,France . Kamoen, O., Jamart., G., Moermans, R., Vandeputte, L. and Dubourdieu, D., 1978. Comparative study of phytotoxic secretions of Botrytiscinerea Pers. : Fr.. Mede delingenFacultei tLandbouwwetenschappen , Rijksuniversiteit Gent43:847-857 . Kamoen,O. , 1984.Secretion sfro m Botrytis cinereaa selicitor s ofnecrosi san ddefence . Revued eCytologi ee tBiologi eVégétale ,Botaniqu e7:241-248 . Vannel,D. ,Barbier ,M .an dBessis ,R. , 1991a. Etuded el atoxicit éd ufiltra td ecultur ed e Botrytiscinerea su rde svitropla nt sd evignes :I Effe t dufiltra t brut.Viti s30:167-175 . Vannel,D. ,Barbier ,M .an dBessis ,R. , 1991b. Etuded el atoxicit éd ufiltra td ecultur ed e Botrytiscinerea su rde svitropla nt sd evignes :I IEffe t desdifférente s fractions isolées àparti rd ufiltrat brut .Viti s30:213-218 . 108 The use ofphytoalexi n induction and ofin vitro method s as a toolfo r screening grapevines for resistance to Botrytis cinerea Ph. Jeandet, M.Sbaghi and R. Bessis Summary The ability of in vitro cultures of grapevine (Vitisvinifera L.) to synthesise the phytoalexinprecurso r resveratroli nrespons et oultraviole t lightirradiation swa sstudie d todevelo pmethod sfo r screeningfo rresistanc et ogre ymoul dcause db yB. cinerea. The concentration of resveratrol in leaves of 150-day-old plantlets cv.Caberne t Sauvignon exposedt o40 0ixW/cm 2irradiatio na t25 4n mfo r7 mi nwa stwic etha tfo rcv sPino tNoi r and Chardonnay.Thi s ranking accordswit h therelativ e susceptibility of thesecultivar s togre ymoul d after leaf inoculation inth e field. Introduction Grey mould caused byBotrytis cinerea Pers.:Fr . is an important disease which causes seriouslosse st oa wid erang eo fplants .I nvineyard sth ediseas eaffect s bothth eyiel do f grape berries and thequalit y of wine. Because of itslac k of host specificity and of the difficulty of protecting thecrop ,w edescrib e hereprocedure s devised for screening for resistancet oB. cinerea ingrapevine sproduce db ysomati cembryogenesis . Invitro methodsusin g vegetativemultiplicatio n andsomati cembryogenesi s(Bessis , 1986)ar enecessar y becauseconventiona l hybridisation techniques areno tpermitte d in traditional wine producing areas where the production of great wines is based upon a limitednumbe ro fcultivar swhic hhav eassure dth ereputatio no fthes evineyards .Indeed , invitro technique spresentl yuse do na larg escal efo rclona lpropagatio n ofa wid erang e of crop plants have also proved tob e of interest in grapebreedin g programmes where greater variabilities can be obtained and used in the selection of improved disease resistant cultivars (Bessis, 1986). However, no method is available to identify soma- clones derived by somatic embryogenesis with increased levels of resistance to B. cinerea. Traditionalfield screenin g is unsuited to the selection of alarg enumbe ro f somatic embryos;t oreduc e thetim etake n toidentif y resistant individuals,w edescrib e herea techniqu edevise d tous eth eassessmen t of thephytoalexi n precursor, resveratrol as a selection criterion to aid the screening of in vitro-grown plantlets for grey mould resistance. Previous results (Langcake, 1981;Poo l etal., 1981 ) have shown that the speed and intensity of resveratrol synthesis are positively correlated with resistance of grapevine cultivars to grey mould. Therefore the production of resveratrol, induced by various stressfactors , hasbee nuse da sa screenin g procedurefo r hostresistanc et oB. cinereai n thefiel d (Jeandetan dBessis ,1989) . Unfortunately, resveratrol formation by field-grown leaveswa shighl ysensitiv et oenvironmenta lchange s(Barlas set al., 1987),thu slimitin g itseffectiveness . Useo fin vitro techniques canovercom ethi styp eo fvariation . 109 Thispape r describes experimentsdesigne d to measuresynthesi s resveratrol in vitro andt ocompar ethre ecultivar so fV. vinifera showingdifference s insusceptibilit yt ogre y mould, namely cvs Pinot Noir and Chardonnay, which are considered to be very susceptible toB. cinerea, and cv.Caberne t Sauvignon, which is least affected bygre y mould. (a) CJ 1 3 1.5 1.0 O> 0.5 » i I I 1 C) 5 10 15 20 25 (b) (c) 40 120 100 30 80 20 60 40 10 20 JIJUWJ*VL/ ^ —I- 10 15 20 25 10 15 20 RETENTIONTIME S(MINUTES ) Fig. 1:HPLC analysis of stilbene-type phytoalexins Chromatographic conditions were as follows: linear gradient elution within 25mi n from 40% to 100% methanoli nwate ra ta flo w rateo f 1 ml.mhr1.Th eseparatio nwa sperforme d ona Beekma nC1 8colum n (Ultrasphere ODS,4. 6 mm x 25cm , 5 |xm).Detectio n was at 307 nm. (a) Synthetic standards; (b)UV - irradiated leaves (cv. Cabernet Sauvignon); (c) Photochemical isomerisation (within 1 h of daylight exposure) of trans-resveratrol to the cis-isomer in a grape leaf extract; 1, trans-resveratrol; 2, cis- resveratrol; 3, trans-pterostilbene. 110 Results and Discussion The efficiency of three different procedures for elicitation of resveratrol in plantlets grown in vitro were tested: viz. inoculation with conidial suspensions of B. cinerea; induction by galacturonic acid; and induction by short wave UV-irradiation (254 nm). The abaxial leaf surface of the three cultivars wereteste d by inoculating a single drop (50 (xl) of a conidial suspension of B. cinerea containing c. 4 x 104 conidia.mH. In all material this resulted in the development of large necrotic areas and dense sporulation over most of the leaves after 72 h of incubation in darkness. No fluorescence indicative of the presence of resveratrol was detected in the apparently healthy tissues adjacent to theinfecte d zones 48 h after inoculation. This suggested that invitro plantlets were very susceptible to infection even when alo w density conidial inoculum was used. Galacturonic acid,whic h isrelease d by pectinolytic enzymes from pecticpolymer s in cell walls may stimulate phytoalexin accumulation (Bachmann and Blaich, 1980). Higher galacturonic acid concentrations (0.5 and 1%) were phytotoxic, inducing large necroses onth elea f surfaces. Solutionscontainin g 0.1% galacturonic acid showed potent elicitor activity for resveratrol formation, but this result was not reproducible. Our observations therefore indicate that these first two procedures are unsuitable for studies of the elicitation of resveratrol by grapevine in vitro. Radiation with awavelengt h of 260-270n m has aselectiv e andreproducibl e effect on resveratrol synthesis by grape leaves (Langcake and Pryce, 1976) and by grape berries (Jeandet, 1991; Jeandet et ai, 1991) under field conditions. In contrast Barlass et al. (1987)reporte d thati nvitro-grow n plantletsshowe d agenera l necrosis andn o associated resveratrol formation when irradiated under the same conditions, i.e. at 254 nm with a fluence rateo f60 0jjiW/cm 2fo r 10mi n(0.3 6J/cm 2).Thes eauthor s therefore implied that the technique could notb euse d with inv/fro-grow n leaves.I n order to avoid undesirable effects of UV-irradiation on this material, our experiments were performed with the energy fluence rate and exposure time reduced. A fluence rate of 400 |xW/cm2 with Table1. Production of resveratrol in leaves of different grape cultivars grown in vitro. Trans-resveratrol (mg.g-1 fresh weight +SE) a> Pinot Noir Chardonnay Cabernet Sauvignon Nl») 88.6 ±5.3 174.8± 10.8 213.0+13.7 N2 102.3+ 6.1 not done 192.7± 10.6 N3 97.0+ 5.9 63.2 ±4.2 202.2 ±12.1 N4 89.7 ± 5.5 39.7 ± 2.9 110.0± 6.5 N5 37.0 ±2.9 70.0 ±3.9 138.6± 5.9 N6 42.2 ±3.1 70.0 ±4.5 77.0± 3.5 N7 48.2 ±3.4 78.2 ±4.9 138.4± 7.1 N8 35.0 ±2.8 79.6 ±4.1 138.4± 6.5 a) Each value represents the resveratrol production of three leaves taken at the same leaf position on different shoots of the same vine variety. All measurements were done in triplicate. SE= Standard Erroro fth ethre emeasurements .Resveratro l concentration in leaves was estimated by GLC (seeJeande t etal., 1991). b) Leaf position on shoot numbered from base. Ill exposureo f7 mi n(0.1 7J/cm 2) wassufficien t toinduc estilben eproductio n inleave so f thethre ecultivar s tested (Fig. 1)(Jeande tet al, 1992). The results obtained from assaying resveratrol from 150-day-old plantlets showed differences between cultivars evaluated by their ability to synthesize resveratrol in responset oUV-irradiation ,a sshow ni nTabl e 1. Theconcentratio n of resveratrol inth e leaves of cv. Cabernet Sauvignon, when expressed as a mean of the values given in Table 1(15 1 |xg.g_1 fresh weight), was twice as high as that for cvs Pinot Noir and Chardonnay (67 and 82 p-g.g-1 fresh weight, respectively). This accords with the grey mould resistance of thecorrespondin g cultivars assessed by leaf inoculation in the field in which cv.Caberne t Sauvignon hasintermediat e field resistance whilecv s PinotNoi r andChardonna yar ebot hmor esusceptible . References Bachmann, O. and Blaich, R., 1980. Vorkommen und Eigenschaften kondensierter Tanninei nVitaceen .Viti s18:106-116 . Barlass, M., Miller, R.M. and Douglas, T.J., 1987. Development of methods for screening grapevines for resistance to downy mildew. II. Resveratrol production. American Journal ofEnolog y andViticulture ,38:65-68 . Bessis,R „ 1986.Grapevin e physiology: thecontributio n ofcultur e invitro. Experientia 42:927-933. Jeandet, P., 1991. Interaction plante-pathogène: recherches sur la production de resveratrol (3,5,4'-trihydroxystilbène), une phytoalexine de la vigne. Ph.D. Thesis, Universityo fBourgogne ,France . Jeandet, P.an d Bessis, R., 1989.Un e réflexion sur les mécanismes morphologiques et biochimiques del'interactio n vigne-Botrytis. Bulletin de l'Office International del a Vignee td uVi n62:637-657 . Jeandet, P., Bessis, R. and Gautheron, B., 1991.Th e production of resveratrol (3,5,4'- trihydroxystilbene) by grape berries in different developmental stages. American Journalo fEnolog y andViticultur e42:41-46 . Jeandet, P., Sbaghi, M. and Bessis, R., 1992. The production of resveratrol (3,5,4'- trihydroxystilbene) bygrapevin e in vitro cultures andit s application toscreenin g for greymoul d resistance.Journa l ofWin eResearc h 3:47-57. Langcake, P., 1981. Disease resistance of Vitis spp and the production of the stress metabolites resveratrol, epsilon-viniferin, alpha-viniferin and pterostilbene. Physio logicalPlan tPatholog y 18:213-226. Langcake, P.an d Pryce, R.J., 1976.Th eproductio n of resveratrol by Vitis vinifera and othermember so fth eVitaceae a sa respons et oinfectio n orinjury .Physiologica lPlan t Pathology9:77-86 . Pool,R.M. ,Creas yL.L .an dFrackelton ,A.S. , 1981.Resveratro l andth eviniferins , their application to screening for disease resis tance in grape breeding programs. Vitis 20:136-145. 112 Ethylene production and growth ofBotrytis cinerea in kiwifruit as influenced by temperature and low oxygen storage N.D. Niklis, C.C. Thanassoulopoulosand E.M. Sfakiotakis Summary Theeffect s oftemperatur ean dlo w0 2concentration so nth edevelopmen to fgre ymoul d caused by B. cinerea, on ethylene production and tissue softening in kiwifruit were examined. Kiwifruit stored at 0°,5 °an d 10°Cproduce d significant amounts of ethylene 20-30 days after inoculation with B. cinerea, but only trace amounts weredetecte d inhealth y controls. In another experiment in which fruits were inoculated with B. cinerea and exposedt oa rang eo ftemperature s(-2° ,-1° ,0° ,5 ° and 10°, 20°,30°C) ,lo wtemperature s reduced ethylene production and softening of flesh. Infection was delayed for c. 24-48 days and fungal growth rates were reduced at 0°, -1° and -2° compared to higher temperatures.Storag eo fth einoculate dkiwifrui t atlo woxyge n( 1 % 0 2)an d0° Cstrongl y reduced thegrowt hrat eo fth efungu s andethylen eproduction . Introduction Serious losses in kiwifruit production occur because of grey mould caused by Botrytis cinerea Pers.:Fr. (Sommer, 1982;Somme r etal., 1983) .Severa l postharvest treatments have been used to control fungal growth during storage of fruits. Low temperature suppressed thepathogenicit y ofB. cinerea (Brooksan dCooley , 1928)an delevate d C02 concentrations maysuppres sothe rfung i (El-Gooranian dSommer , 1981).Lowerin gth e 02 concentration of the atmosphere from c. 21% of air to 2 to 3% suppressed the pathogenlittl ean donl yafte r the0 2ha dbee nlowere dt o< 1 % wa sth efungu s suppressed (Couey and Well, 1970;Foolstad , 1966).Thes e near anaerobic storage conditions may risk injury to the fruit. Several postharvest pathogens produce ethylene at high rates during growth oncitru s fruits orin vitro (Chou andYoung , 1973; DaSilva etal. , 1974 ; Tzeng andDevay , 1984) This paper examines, a) the possible involvement of B. cinerea in ethylene production, b) theeffec t of temperature andc )th eeffec t of low oxygen on greymoul d development,ethylen eproductio n andsoftenin g ofth einfecte d fruit. Material and Methods Kiwifruit (Actinidia deliciosa) cv. Hayward harvested from commercial orchards or takenfro m refrigerators ofPieri acount yo fCentra lMacedoni ai nNorther nGreec ewer e used. For inoculations,a n isolate of B. cinerea, obtained from kiwifruits in thiscount y wasuse di nal lexperiments .Th efungu s wasmaintaine d onpotat odextros eaga r(PDA) . Two parallel punctures were made in the stem end area of the fruit; the fruits were inoculated herewit ha piec eo faga r(c .5 mm )bearin g mycelium ofB. cinerea. 113 Experiment 1 Kiwifruits wereharveste d during October 1989fro m acommercia l orchard when their averagefirmnes s andsolubl e solidsconten twer e5. 5k g and 8.7% respectively.Hal f of thefruit s wereinoculate d andth eothe rhal fwer ekep ta scontrol .Th efruit s wereplace d into5 L jars ,eac hcontainin g 18 fruits,representin g oneplot .On eja r withhealth y fruits andon ewit hinoculate d wereuse dfo reac htemperatur etreatment .Th ejar swer eplace d inwaterbath sadjuste d to0° ,5 ° or 10°C.Continuou saeratio n(9 0mlmùr 1)wa suse dan d theethylen econcentratio n inth egas-phas ewa smeasure d inth eai rflow . Sampleswer e taken4 5(10°C )an d 175 (0°,5° )day safte r inoculationan dth eexperimen twa sreplicate d threetimes . Experiment 2 Kiwifruits were taken from the commercial refrigerator of the Union of Agricultural Cooperatives-Pieriawit haverag efirmnes s andsolubl esolid sconten to f3. 5k gan d 13% respectivelywer euse d tostud yfunga l growth,ethylen eproductio n andfles h firmness. Inoculated kiwifruits wereplace d in 5L jars , 18fruit s in eachjar , and thejar s were placedi nwaterbath so f30° ,20° ,10° , 5°, 0°,-1° ,-2° Ci nsi xreplications .Anti-freez e was usedi nth ewate rfor- 1 and-2° Ctreatments .Measurement swer emad ei nfou rsamplin g periods,fou r fruits eacho fth efirs t threesample san dsi xfo r thelast . Experiment 3 Fruitsfro m thesam esourc ea si nExpt . 2,wit hfirmnes s of3 kgan d 12.7%solubl esolid s were used. Inoculated fruits were placed in sealed 400 Lstee l chambers at 02 concen trations of 1%an d 21% (0°Can d97 %RH) ,2 0fruit s perplot .Th e0 2 concentrationso f the storage atmosphere was monitored with a paramagnetic gas-analyzer. This experiment was replicated four times. Measurements were taken six times in six subsequent samplingperiods ,fiv e fruits perplo ti neac hperiod . Foranalysi s ofgas-phas eethylen econcentration , the 'head-space' gassample s were removed by 5m lsyring e and ethylene concentration was analyzed by Varian 3300G C equipped withFI Di n samplestake nfro m 1 mlsyringe . Thesolubl esolid sconten twa sevaluate db yportabl eelectroni crefractomete r (Atago, model PR-1). The flesh firmness (two measurements per fruit) was measured after removalo fskin ,usin ga Chatillo nteste rfitte d witha 7. 9m mplunger .Infecte d flesh area wasmeasure db ya rule rfro m thepoin to finoculatio nt oth een dpoin to fth erotte d flesh. Statistical analysiswa sperforme d byM-STA T(Micro-static ) IBMprogra m and figures wereplotte d using theCRICKE T Appleprogram . Results and Discussion Expt. 1.Ethylene production from infectedand healthy kiwifruits attemperatures 0°, 5° and10°C. The ethylene concentration in the gas-phase in the non-inoculated fruit was low(Fig . la,b ,c) .Ethylen eproductio n at 10°C wasc .twic etha ta t0°C ,bu ti n thelatte r the decline in ethylene levels was slower. Infected kiwifruits showed significant increasesi nethylen econcentratio ni nth egas-phas ei nth eperio dfro m 20t o3 0day safte r inoculation at all temperatures (Fig. 1),bu t then decreased as the fungus colonized the tissuean dsoftene d it. 114 A o°c 100 120 140 160 1S0 B Infected fruit 5"C 180 C 10-C Infected fruit 40 60 80 100 120 140 160 180 DAYS (after inoculation) Fig. 1. Ethylene concentration inthe gas phase ofthe control(non inoculated kiwifruit) andwith Botrytis cinerea infectedfruit, kept under continuous airflow (90ml.min~') at temperatures0°C (A), 5°C (B) and10°C (C). Expt. 2.The influenceof different temperatures in ethylene production and the growth of greymould. Attemperature s of 30°,20 °an d 10°Ca rapi dgrowt ho fth efungu s occurred and by the 15thda y the fruit wascompletel y destroyed. Ethylene production, firmness andth einfecte d areaar eshow ni nfigur e 2.A t30° Cther ewa sstil lsignifican t growtho f mycelium,an dproductio n ofspore san dscleroti ainsid eth efruit . At5° , 0°,-1 °an d-2° C thela gphas ewa sc .2 0day sbu tth egrowt hrate swer elowe rtha na thighe rtemperatures . The ethylene production decreased at -2°C but the small amount of ethylene at -2°C originated from the grey mould infection. At -1° and -2°C, mycelial growth did not 115 |ISD 5% • (10*, 20*. 3CC) -1*C -2'C 30 40 SO 60 DAYS (alter Inoculation) Fig. 2. Growthof Botrytis cinerea in kiwifruit (A), softening (B) and ethyleneproduction ofthe infectedkiwifruit at constant temperatures of-2°, -1", 0°, 5°, 10°, 20''and30°C(CI and C2). 116 sor A E J. 40 S '• ffi / E 30 - j/ .,-' 1%Oxyg«n < / ..-*' a 20 ,#«* UI i- .•* ü „***^* .» * ïï 10 .-••* z LSD 5% 1I i—'-t^- _,—«^1 —i i_ . 0 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 30 40 50 60 70 80 90 100 DAYS (after Inoculation) Fig. 3. Growthof Botrytis cinerea in kiwifruit (A), softening(B) and ethylene production (C) ofthe infectedfruit undernormal and low oxygen (1%) atmosphere storage at0°C. appear on thefrui t surface, but insideth e flesh; both flesh and skin wereinfecte d at 0°, 5°an d 10°C. Expt. 3.Effect of O 2 concentrations onethylene production andfungal growth. Low 02 concentrations reducedproductio no fethylen ean dfunga l growthfo r4 5 days,bu tha dn o effect onfirmnes s ofth efrui t (Fig.3) . Iti seviden tfro m thisstud ytha tb ystorin gkiwifrui t atlo wtemperature s(0° ,5 ° and10°C ) ethyleneproductio nwa snegligible .However ,th efrui tinfecte d withB. cinerea produce d 117 significant amounts of ethylene 20-30 days after inoculation at these temperatures. In subsequentexperiment sfruit s inoculatedwit hB. cinerea andexpose dt o5° , 10°,20 ° and 30°Cshowe dma tth egrowt h rateo ffungu s wassimila rbu twit ha nincrease dla gphase . This trend was most marked at lower temperatures whererottin g wasdelaye d for >2 4 days,bu tth egrowt h rate of thefungu s waslower .Ethylen e production wasreduce d at low temperatures; even at -2°C infected fruit produced enough ethylene to induce ripening.Storag eo fth einoculate dkiwifrui t atlo woxyge nconcentratio n (1%0 2)an da t 0°C, reduced the growth rate of fungus and ethylene production, but did not stop the pathogen completely andthu sdi dno treduc esoftenin g ofth e fruit. References Brooks, C.A. and Cooley, J.S., 1928.Tim e temperature relations in different types of peachro tinfection . Journalo fAgricultura lResearc h37:507-543 . Chou,W.T .an d YoungF.S. , 1973.Th ebiogenesi s of ethylene inPénicillium digitatum. Archiveso fBiochemistr y andBiophysic s157:72-83 . Couey,H.M .an dWell ,M.J. , 1970.Lo w oxygen orhig hcarbo n dioxide atmospheres to controlpostharves tdeca yo fstrawberries .Phytopatholog y60:47-49 . DaSilva, J.E. and Herinksson, E. and Herinksson, E.L., 1974. Ethylene production by fungi. Plant SciencesLetter s2:63-66 . El-Goorani, M.A. and Sommer, N.F., 1981. Effects of modified atmospheres on postharvest pathogenso f fruits andvegetables .Horticultura l Review 3:412-461 . Foolstad,N.M. , 1966.Mycelia lgrowt hrat ean dsporulatio n ofAlternari atenuis ,Botrytis cinerea,Cladosporium herbaruman d Rhizopus stoloniferi n low oxygen atmos pheres.Phytopatholog y56:1098-1099 . Sommer, N.F., 1982. Postharvest handling practices and postharvest diseases of fruit. PlantDisease ,Ma y 1982 p.357-364 . Sommer,N.F. ,Fortage ,R.J . and Eduards,D.C. , 1983.Minimizin g postharvest diseases ofkiwifruit . California AgricultureJan.-Feb . Tzeng,D.D . andDevay ,E.G. , 1984. Ethyleneproductio n andtoxigenicit yo fmethionin e and its derivatives with riboflavin in cultures of Verticillium, Fusarium and Colletotrichum speciesexpose d tolight .Physiologi aPlantaru m62:545-552 . 118 Influence ofplan t growth regulators onmycelia l growth, germination of conidia and pathogenicity of Botrytis cinerea S.Benliojgu and D. Yilmaz Summary The effect of the plant growth regulators 4-CPA, gibberellic acid, Na-salts, N-m tolypthalamic acid and ethephon on mycelial growth, spore germination and pathogenicity ofthre eB. cinereaisolate swa sinvestigated . Five concentrations of each growth regulator were tested invitro and pathogenicity testswer eperforme d ontomat ocv . Supermarmande.Th erecommende dconcentratio no f 4-CPAa t 10fjLg.mh 1 inhibitedmycelia lgrowt han dconidia lgerminatio n butgibberelli c acid, Na-salts, and N-m tolyphtalamic acid had no effect. Ethephon slightly affected mycelial growth ofB. cinerea. On thebasi s of these results invitro, theeffec t of plant growth regulators onpathogenicit y ofB. cinerea isolates isdiscussed . Introduction Turkeyproduce sannuall yc .5. 6millio nton so ftomatoe si ngreenhouse san di nth efield ; 74% of the total production comes from southern and south-western coastal areas. Despiteintensiv espraying ,tomatoe sgrow ni ngreenhouse si ncoole ran dhumi dseason s suffer from several diseases, especially from grey mould caused by Botrytis cinerea Pers.:Fr. .Grower sappl ygrowt hregulator s(PGRs )t oincreas efrui t settingan dyiel dan d thesema yinfluenc e theseverit y ofth edisease . Theeffect s of some widely used plant growth regulators on mycelial growth, spore germination andpathogenicit yo fB. cinerea aredescribe di nthi spaper . Material and Methods Threeisolate s of B. cinerea from theprovince s of Antalya andMugl awer euse d inth e experiments. Thesensitivit yo fthes eisolate st oth ePGR swa steste do npotat odextros eaga r(PDA ) amended after autoclaving with0.3,1,3 , 10,30mg.m H of4-CP A(Tomaton e0.145%) ; 0.5,1,10, 50,100 ixg.mF ofgibberelli c acid (Berelex 9.6%);0.1,0.3 , 1,3, 10 fig-mF of Na-salts (sodium ortho-nitrophenolate 0.2%, sodium para-nitrophenolate 0.3%an d sodium5-nitroquacalat e0.1% ;Atonik) ; 1,2,10 , 20, 100jig.m H ofN- mtolyphtalami c acid(Tomase t20% )an d 10, 50, 100,500 , 1000(xg.m Fo fethepho n (Ethrel48%) . The sensitivity of isolates to PGR was determined by transferring mycelial plugs (4m mi ndiameter )fro m 3t o4 day-ol dculture so nPD At ofres h PDAplate scontainin g theabov econcentration s ofPGRs ,thre eplug spe risolate .Unamende dPD Aplate swer e inoculated as controls. After 3day s incubation at 20°C in darkness, colony diameters were measured and the growth of isolates calculated as a percentage of rates on unamended PDAplate s(Georgopoulo san dDekker , 1982). 119 Table 1. The effectof five growth regulators infive different concentrations on mycelial growthofB. cinereaisolates after 3 days of incubation at 20°C. Concentrationsare given inppm a.i. Untreated control =100. (*is the concentration recommended) Isolates Mycelialgrowt hrat e( %o fcontrol ) 4-CPA(pp m a.i.) 0.3 1 3 10* 30 90/3-D 90 90 82 13 0 90/3-T 99 90 79 38 0 90/8-1 102 94 84 31 0 gibberellicaci d 0.5 1 10 50* 100 90/3-D 107 109 112 112 106 90/3-T 95 95 97 94 90 90/8-1 98 102 100 100 96 Na-salts 0.1 0.3 1 3* 10 90/3-D 102 98 95 90 83 90/3-T 99 103 97 95 82 90/8-1 101 98 93 88 78 N-mtolyphtalami c acid 1 2 10 20* 100 90/3-D 95 96 89 88 88 90/3-T 106 97 98 97 94 90/3-1 102 102 104 105 98 Ethephon 10 50 100 500* 1000 90/3-D 104 102 99 87 63 90/3-T 99 99 97 91 66 90/8-1 102 104 102 94 68 Todetermin eth eeffec t ofPGR so ngerminatio no fconidi aa suspensio nwa sprepare d from sporulating colonies onPD Aan dadjuste d to 103conidia.m H with steriledistille d water and 100 ml of suspension was spread on plates in each treatment. After 24 h incubation at20°C ,th epercentag egerminatio nwa sdetermined . The effect of PGRs on the pathogenicity of B. cinerea was evaluated using tomato plantscv .Supermarmand ea t5- 6 truelea f stage.Plant swer espraye d withth e following 120 100 • 90/3-D (r - -0.80) 90/8-1 (r=-0.82) 90 • 90/3-T (r=-0.87) 80 • .1 70 • 'S c E 60 « en - 50 .2 o ^ 30 o 20 10 0.3 1 3 10 30 pg/ml Log c one. Fig. 1 The effectof4-CPA on conidialgermination of three B. cinereaisolates commercially advised concentrations of PGRs: 10ixg.m H a.i. 4-CPA; 20 |xg.mH a.i. N-m tolyphtalamic acid; 3 ixg.mH a.i. Na-salts; 50 (xg.ml1 a.i. gibberellic acid and 500 |Ag.mH a.i. ethephon. A conidial suspension of B. cinerea(10 5conidia.mH ) produced on autoclaved carrot bouillon (300 g grated carrot in 500 ml distilled water + 1%gelatin )wa sspraye do nplant safte r2 4h .Th eplant swer ethe ncovere dwit hplasti c bagsan dincubate d at2 0± 1°C. Thebag swer eremove d4 8h later .Diseas eseverit ywa s evaluatedb ymean so fa scal e(Dele net al., 1986)wher e0 i sn oinfectio n and5 th ewhol e plantcovere d with sporulating lesions6 day safte r inoculation. Results The effects of different concentrations of 4-CPA, gibberellic acid, Na-salts, N-m tolyphtalamic acid and ethephon on mycelial growth of threeisolate s of B. cinerea axe, showni nTabl e1 . The percentage inhibition of growth of the three isolates on PDA containing 10 (xg.ml-1 4-CPA were 87,62 and69 .Gibberelli c acid,Na-salts ,N- mtolyphtalami c acid and ethephon had no significant effect on these isolates, even when applied at recommended and higher rates. Only ethephon at 1000 ppm inhibited growth of B.cinerea b y40% .Simila rresult swer eobtaine di nth eexperiment swhic hexamine dth e 121 effect of PGRs on conidial germination. Only 4-CPA influenced the germination at concentrations in the range0. 3 jig.mF to 30mg.mF . Anegativ e correlation between concentration of4-CP Aan dpercentag egerminatio n ofconidi awa sfoun d (Fig.1) . In pathogenicity experiments,plant s sprayed with4-CPA ,gibberelli c acid,Na-salts , N-mtolyphtalami c acid andethepho n showed 50%, 62%,62% ,64 %an d 80%infectio n respectively,compare d to60 %o fth econtrol . Discussion In our assays, gibberellic acid had no effect either on the mycelial growth or conidial germinationo fB. cinerea, wherea sOzbe kan dDele n( 1989 )reporte dtha tgibberelli caci d stimulated the mycelial growth of this fungus invitro. Brecbuhle r (1982) and Pearson (1982), however claimed that application of gibberellic acid to vineyards reduced B.cinerea infections .I nou rpathogenicit ystudies ,plant sspraye dwit hth erecommende d concentrations ofPGR sshowe d similar levels ofdiseas eseverit y asth econtrol .Excep t for4-CP Athes efinding s confirmed ourin vitro results.Nevertheless ,growt h regulators may decrease or in crease the susceptibility of different plant species and cultivars to B. cinerea. Furtherinvestigation si nth efield o ri ngreenhouse sar erequire dt oclarif y the effect ofPGR so nthi s fungus andth edevelopmen t ofgre y mould in glasshouse-grown tomatoes. References Brecbuhler, C, 1982.Relatio n beteen stalk necrosis andBotrytis cinerea ongrapevine . European andMediterranea n PlantProtectio n Organisation Bulletin 12:29-35. Delen, N.,Yildiz ,M . and Benlioglu, S., 1988.Botrytis cinerea izoladarinin Captan ve Dichlofluanid'e Duyarliliklari Üzerinde Çalismalar. DOGA, Turkish Journal of Agriculture andForestr y 12:348-358. Georgopoulos, S.G. and Dekker, J., 1982. Detection and measurement of fungicide resistance, General principles-FAO Method no.124 , FAO Plant Protection Bulletin 30:39-42. Ozbek, T. and Delen, N., 1989. Sebze Seralarinda Kullanilan Oskin Grubu Bzi Hormolarin KursuniKü fHastaligini n{Botrytis cinereaPers. :Fr. )Gelisimin eEtkileri . Ege Üniv. Fen Bilimleri Enstitüsü, Bitki Koruma Ana Bilim Dali, Bornova - Izmir, 1989p p2 8( MS c .Thesis) . Pearson, R.C., 1982. Chemical Control of Botrytiscinerea on grapes in New York. European andMediterranea n PlantProtectio n Organisation Bulletin 12:101-104. 122 The physiology ofconidiatio n inBotrytis squamosa F.J. BalisandJ.W. Lorbeer Summary Onion foliar extract agar supplemented with glucose (4 g.H) as a carbon source and Ca(N03)2.4H20( 4g.H ) andterme dcalciu mnitrat eaga rprovide d asuitabl emediu mfo r theproductio n of conidia ofB. squamosa. When isolateBS82- 1wa sgrow n oncalciu m nitrateaga ri nPyre xPetr idishe si na nincubato ra t 18°Cunde r 14h photoperiod so fnea r UVradiatio nmassiv econidiatio noccurred . Thissyste mconsistentl yprovide dconidi ao f thatisolat eo fB. squamosa forepidemiologica l investigations andstudie so nth econtro l of thisfungu s inNe wYork . Introduction Botrytissquamosa Walker ,th ecausa l agento fBotrytis lea f blighto fonion ^i son eo fth e mostdestructiv e pathogens of oniongrow n onorgani csoil si nNe wYor k(Hancoc kan d Lorbeer, 1963; Lorbeer, 1992). Epidemiological studies of the fungus have been complicated by the difficulties of producing high levels of conidial inoculum with regularity andth efactor s regulating theproductio n ofconidi a of thisfungu s invitro are not well understood (Bergquist etal., 1972).Thi s study had two objectives: firstly, to develop a reliable technique for the consistent production of conidia, and secondly, to identify andelucidat ethos efactor s whichpla ymajo rrole si nregulatin gth ereproductiv e behaviour of B. squamosain vitro.Th e first part of the study involved an invitro investigationo fth eeffect s ofnear-ultraviole t(NUV )radiatio nan dmoistur estres so nth e production of conidia of the fungus. The second part examined invitro th e effects of carbon-nitrogen nutrition onconidiatio n and therol eo fNa +,K +an dCa 2+cation s inth e production of conidia and sclerotia. Isolate BS82-1 of B. squamosa was used in all experiments. Results and discussion Effects ofNU Vradiatio n Effects of NUV radiation were studied with cultures of B. squamosa grown on onion foliarextrac taga r(OEA )i nPyre xPetr idishe san dexpose dt o0,8,14,1 6o r2 4h o fNU V radiation. This was supplied by a Sylvania F20T12/BLB, black light blue, 20 Wbul b emitting radiation between 310-420nm ,wit h apea kemissio n at35 6n m suspended 45 cm above the cultures. Pyrex glass is an efficient transmitter of NUV radiation. All experimentswer econducte d in incubators at 18°Cfo r 12 days. TheOE Awa sprepare d byresproutin g onionbulb s (cv.Downin g YellowGlobe ) for 6 to 7 weeks at which time the foliage was harvested, washed and dried at 50°C. Desiccated foliage was stored in the laboratory at room temperature until used. To prepareth emedium , 15 go fdesiccate d tissuewa ssteame di n 11 ofdeionize d water for 123 1h ;th eresultan tslurr ywa spasse dthroug hfou r layerso fcheeseclot han dcentrifuge d at 300rp mfo r 3mi nt o remove leaf debris.Bacto-Difc o agar (2% w/v) was added to the centrifuged extractt oproduc eth eOEA . A similar study made use of onion leaf sections infected with B. squamosa at high humidityi nPyre xPetr idishes .Th elea f sectionswer eexpose dt ocontinuou sdarkness , a 14h photoperio d ofNU Vradiation ,o rt ocontinuou sNU Vradiatio na t 18°Cfo r 12days . Asphotoperiod s of exposuret oNU Vradiatio n wereincrease d from 0t o2 4h ,i twa s found thatth eproductio no fconidi awa sincrease dwhe nth efungu s wasgrow no neithe r OEA or the onion leaf sections. Production of conidia was greatest after continuous exposureo fculture st oNU Vradiation . Effects ofmoistur estres s Theeffec t of transient drying (10h )o f4 day-ol d cultures of thefungu s grown onOE A andb ysimila rtransien tdryin g ofinfecte d onionlea f sectionswa sstudied .Lea f sections were prepared by inoculating and incubating them in a moist chamber for 4-5 days, during whichtim elesion s andblightin gdeveloped .The nthe ywer edrie d for 14 hunde r acoinciden t photoperiod ofNU Vradiatio n inchamber sa t 100,87,7 6o r44.5 %relativ e humidity.Thes ehumiditie s wereestablishe d by using 20m l ofdeionize d H20(100%) , or2 0m lo fa saturate d slurryo fKN atartrat e(87%) ,NaC l(76% )o rK 2HP04(44.5% )i n eachchamber .Followin gdrying ,th elea fpiece swer eplace di na 100%relativ ehumidit y chamberfo r 36h befor e theproductio n ofconidi awa smeasured . In another series of experiments, B. squamosa was grown in Pyrex Petri dishes containing7 m lo fOE Aadjuste d from-1. 5ba rt o-9 , -27,-3 6o r-4 5ba rwate rpotentia l 1 wit' KCl,NaN0 3,sucrose ,o rPE G4000 .Th eculture swer eincubate da t18° Cwit ha 1 5h photoperiod ofNU Vradiatio n andconidi awer equantifie d after 12 to 14 days. Although conidiation was induced by transient drying of cultures of B. squamosa growing onOEA ,transien tdryin g ofblighte dlea fsegment sdi dno tincreas econidiatio n compared to the non-dried control. No significant increase in conidiation occurred on OEAadjuste d from -1.5 to-4 5 barwate rpotential . Effects ofcarbon-nitroge n nutrition Productiono fconidi aan dscleroti awa sexamine dwhe nth efungu s wasgrow no nOEA , with or without enrichment with nitrogen as NaN03 at4 g.H. These media were then supplementedwit hcarbo na sglucos ea t0,2, 4o r6 g.H .Th eculture swer egrow na t18° C under a 14h photoperio d ofNU Vradiatio n for 12 to 14 days. Conidiationb yB. squamosa o nOE Aenriche dwit h0 o r4 g. Hnitrogen ,usin gNaN0 3, and supplemented with0 ,2, 4 or6 g. H carbon,usin gglucose ,wa sstimulate d inmedi a containingbot hnutrients .O nOE Aalon e4.0 6x 106conidi ape rdis hwer eproduced ,bu t withth eadditio no feithe r4 g. H nitrogeno r2 g. H carbon,conidiatio nwa sreduce dt oa meano f2. 6x 106o r3.0 5x 106conidi ape rdish ,respectively ,an dconidiatio n decreased further with increasing supplements of carbon. On the addition of both 4g. H nitrogen and2 g. H carbon,conidiatio nincrease d to4.6 3x 106conidi ape rdish ,an dcontinue dt o increasewit hgreate rsupplement s ofcarbon . Production of sclerotia increased greatlyfro m zero on OEA alonet o356. 8 sclerotia perdis h onOE Asupplemente d with6 g. H carbon. Sclerotial production wasdepresse d at all levels of supplemented carbon, in the presence of 4 g of nitrogen, compared to 124 treatments with equalcarbo n supplements butwithou t anitroge n enrichment. Increases in numbers of sclerotia with increases in carbon supply were less in the presence of nitrogen enrichment. Because the addition of nitrogen using NaN03 also depressed thewate r potentialo f eacho fth eOE Amedia ,i twa snecessar yt odetermin ei fNaN0 3wa sactin gmerel ya s an osmoticum.Therefore , anexperimen t wasdevise d inwhic hOE Awa seithe radjuste d or not adjusted to -15 bar water potential using either NaN03, KCl, a 5:3:2 molar ratio mixture of NaCLKŒNajSO,,, or PEG 4000. All media were then supplemented with 2.5g. H carbona sglucose .PE G400 0containin gmediu mwa ssolidifie d with 1%Gelrit e gellamgu mbecaus ePE G400 0inhibit s solidification ofstandar dlaborator yagars . Inth epresenc eo fNaN0 3,sporulatio nwa stwic etha ti nan yothe rtreatmen tbu tther e weren osignifican t differences insporulatio no nunadjuste d OEAan dth emedi aadjuste d to -15 bar with KCl and the NaCl:KCl:Na2S04 mixture. Sporulation was depressed significantly on the medium adjusted to-1 5 bar with PEG4000 . Sclerotial production wasgreates twhe n KClwa sused . These results provided evidence for a strong NaN03 effect on B. squamosa reproductive behaviour, most likely as a result of providing nitrate nitrogen, and not because of osmotic effects. When KCl was present in high concentration, sclerotial production wasinduced . Conidial andsclerotia lproductio nwer eals ostudie d ona nenriche ddefine d medium. Themediu mconsiste do f 15g glucos e( 6g carbon),0.5 5g KH 2P04,0.5g MgS0 4.7H20, 0.02 gCaCL,.2H 20, 0.02 gNaCl ,0.9 7 mgFeCl 3.6H20,0.1 5 mgMnS0 4.H20, 0.88m g ZnS04.7H20,3 m gH 3B03,2 m gH 2Mo04.4H20,4m gCuS0 4.5H20, 20g Bacto-Difco agar in 1 1 deionized water. In these experiments which also concerned the role of carbon:nitrogenratio so n thereproductiv e behaviour ofB. squamosa, NaN03 wasuse d toprovid e0 , 1, 2,3,4 , 5o r6 g. H nitrogen.Cultur emedi awit h0 t o6 g o fnitroge npe r litre had water potentials of -2.4, -5.7, -9.2, -12.7, -16.4, -19.0 and -22.9 bar, respectively,an dp Hreading so f 5.98,5.87,5.79,5.72 , 5.68,5.6 3an d5.60 ,respectively . When the defined medium, containing 6g. H carbon was supplemented with 0 to6 g.1-1nitrogen , sclerotia were produced at high carbon:nitrogen ratios and conidia were most abundant at low carbomnitrogen ratios.Whe n themediu m contained nonitrogen , conidiawer eno tproduce d andscleroti awer eminute ,poorl ydelimite d andonl yweakl y pigmented. With 1 and 2 g. H nitrogen and 6:1 and 3:1 carbon nitrogen ratios, conidiation was minimal while normal sclerotia were abundant. At 3 g.1"1 nitrogen, a carbon:nitrogen ratio of 2:1, sclerotial production was sharply depressed while conidiation was stimulated. At 4, 5 and 6 g.H nitrogen, sclerotia were absent while conidiationincrease dt oa maximu mo f3.6 9x 105 conidiape rdis ha t5 g.1-1nitrogen ,an d thendecrease d to2.1 5x 105conidi ape rdis h at6 g.1" 1nitrogen . The effect of different cations associated with nitrate nitrogen on reproduction was also studied. OEA was used as the base medium and was supplemented with 4 g.H carbon asglucos efo r alltreatments .Th ecarbon-supplemente d OEAwa senriche d with 1 4 g.1- nitroge n using eitherNaN0 3, KN03o rCa(N0 3)2.4H20(CN A= Calciu mNitrat e Agar), or with it lacked nitrogen; aconcentratio n of each cation equal to that provided with each nitrate source, respectively, using NaCl, KCl or CaCl2.2H20 was provided. Two identical experiments with 3x2 factorial designs with three cations (Na+, K+ or 2+ _ Ca ) and two anions (N03 or Ch) were conducted. The medium enriched with Ca(N03)2.4H20 supported the greatest conidiation. Conidiophores were exceptionally elongated on the media enriched with Ca2+. On the Na+ and K+ enriched media, 125 conidiophoreswer eshor twit hlarg etermina lcluster swhe nN0 3-wa sused ,bu telongat e withsmalle rbu tmor enumerou sintercalar ycondia lcluster swhe nth eCI -wa sused .Th e difference in sporulation on the CaCL,.2H20 and Ca(N03)2.4H20 enriched media was greater than for the NaCl and NaN03 or the KCl and KN03 enriched media, with extremely heavy conidial production on CNA. On this medium, a dense covering of conidiophores developeda reddish-brow ncoloratio nwit h 12.68an d 16.84x 106conidi a produced per dish in trial 1an d trial 2, respectively. This was more than double the conidialproductio no nan yothe rtreatmen ti neac htrial .Comparin gth emonovalen tNa + and K+cation s to the divalent Ca2+ cation, was significant (at P = 0.0001), providing further evidence for the strong conidiation stimulus provided by CNA. Sclerotial productioni nbot htrial swa smos tnumerou si nth eK +enriche dmedia . Inaddition ,o nK + enriched media, sclerotia were mostly 1-1.5 mm in diameter, while on media enriched with either the Na+ orCa 2+cation , sclerotia were mostly slightly larger at 1-1.5 x 1.5- 2m mi ndiameter . Inconclusion ,th etw opart so fth estud ygav eth efollowin g results. The first part of the overall study showed that NUV radiation stimulates the production of conidia ofB. squamosa invitro whilemoistur e stress does not.Althoug h B.squamosa produce sconidi aprimaril yo ndried ,necroti cfoliag e tissuei nth efield , iti s unlikely thatdryin g ofth etissu epe rs ei sa nimportan t stimulusfo r conidiation. Forth esecon dpar to fth eoveral lstudy ,th efollowin g relationshipswer erevealed .(1 ) On the variously supplemented OEA, carbon and nitrogen, provided as glucose and NaNOj interacted in a manner that when both nutrients were provided at favourable levels (high carbon, high nitrogen = low carbon:nitroge n ratios) an increase in sporulation by B. squamosa occurred. Conversely, on amended OEA, sclerotial productionwa sinhibite db yNaN0 3supplement s atal llevel so fcarbon .(2 )Similarl yo n thedefine d medium at high carbon:nitrogen ratios, sclerotial production was favoured, whilea tlo wratios ,conidiatio nwa sfavoured . (3)Th eCa 2+catio nspecificall y interacted withnitrat enitroge nt oresul ti nth ehighes tlevel so fconidiation .Th eK +catio n favoured sclerotial formation byB. squamosa. References Bergquist, R.R., Horst, R.K. andLorbeer ,J.W. , 1972.Influenc e of polychromatic light, carbohydratesource ,an dp Ho nconidiatio no fBotryotinia squamosa. Phytopatholog y 62:889-895. Hancock, J.G. and Lorbeer, J.W., 1963. Pathogenesis ofBotrytis cinerea, B. squamosa, andB. allii ononio nleaves .Phytopatholog y53:669-673 . Lorbeer, J.W., 1992. Botrytis leaf blight of onion. In Plant Diseases of International Importance. Eds. Chaube, H.S., Singh, U. S., Mukhopadhyay, A.N. and Kumar, J. PrenticeHall ,Englewoo dCliffs , NewJersey ,U.S.A. ,p . 186-211._ 126 Detection ofBotrytis cinereao ngerber a flowers usingmonoclona l antibodies J.Salinas and A. Schots Summary Monoclonal antibodies (MAbs) have been developed against whole conidia and extracellular proteins from conidia of B. cinerea. Both types of MAbs react in an immunofluorescence test.Th eMAb ssho wn oreactio nwit hhealth ygerber aflower s and sporesfro m othercommo nairborn efungi .Th ereactivit ywit hothe rBotrytis specie sha s been tested.Mor e than 75% of conidia from ten selected isolates ofB. cinerea reacti n theimmunofluorescenc e test.O nth ebasi so fthes eresult sa routin etes twil lb edevelope d for thedetectio n ofB. cinerea conidiao ngerber a flowers. Introduction Botrytiscinerea Pers. :Fr .ha sbecom ea nimportan tlimitin gfacto rfo rth eproductio nan d export of gerbera flowers in the Netherlands. Conidia produced by the fungus spread easily through theair .Afte r landing onth eflowers , conidia remain dormantunti l athi n water film, for instance through condensation, isavailabl e for conidial germination and flowers becomeinfecte d within afe w hours (Salinas etal., 1989) .Especiall y inautum n and in spring, lesions caused by the fungus lead to serious economic losses (Bakker, 1986).Symptom sca nb evisibl ei ngreenhouse sdurin gth egrowt ho fth eflowers ,bu tca n also develop during storage, transport and shipment of the flowers, when changes in temperature occur leading to high humidity conditions and subsequent infection when conidia of B. cinerea are present (Salinas et al, 1989; Verberkt, 1986). Therefore, a serological test is now being developed based on specific monoclonal antibodies to monitor the presence of dormant conidia on flowers at any stage of production and handling. Material and Methods Fungalculture s Isolates of B. cinerea were obtained from field infected plants (Table 1).Th e isolates werekep ta ssporulatin g cultureso nX-mediu m at4° Ci ndarknes s(Salina set al., 1989). Dryconidi awer eharveste d from fungal culturesgrow n ontomat oaga r(Salinas ,1992) . Antigenpreparatio nan dimmunisatio n Twosource s ofantige nwer eused .Whol econidi awer ewashe deigh t timeswit h2 0m M piperazine-HCl,p H6. 0 toensur e thatmos t of theextracellula r proteins wereremoved . Micewer einjecte d four times,wit h4 week-intervals , using about 5.106conidi a inPB S 127 Table 1. Isolatesof Botrytis cinereaused in the experiments. Isolatenumbe r Isolated from Be- 7 tomato Be- 8 taxus Be- 9 carnation Be-10 cyclamen Bc-12 gerbera Be-13 gerbera Be-14 gerbera Be-15 gerbera Be-18 gerbera Bc-25 rose using Freunds Incomplete Adjuvant (FIA) for the first three immunisations and no adjuvant for thelas timmunisation .Th esupernatan t from thefirs t washstep ,containin g mosto fth eextracellula rproteins ,wa sconcentrate d ona nAmico nYM1 0ultrafilte r until aprotei nconcentratio no f 1 mg.mHwa sreached .Mic ewer eimmunised ,fou rtime swit h 4 week-intervals, with 100 |ig of these extracellular proteins in PBS. For the first immunisation Freunds Complete Adjuvant was used, for the second and third immunisation FIA,whil eadjuvan t wasomitte d for thelas timmunisation . Production ofmonoclona l antibodies Monoclonalantibodie s wereproduce d asdescribe db y Schots etal. (1992). Immunofluorescence Millipore Multi Screen-HV 96-well filtration plates, pore size 0.45 (im, were blocked using5 %dono rhors eseru mi nPB S+ 0.1 %Twee n2 0(PBST )fo rtw ohour sa t37°C .Th e plateswer ewashe d four timeswit hPBS Twhereafte r 100 |JL1 of asuspensio n of conidia 6 (5. 10 conidia.mH inPBS )an d 100 JJLI of hybridomacultur e supernatant wasadde d to eachwell .Th eplat ewa sincubate dfo r 1 ha t37°C .Th eplat ewa swashe dfou rtime swit h PBST. Subsequently 50 |xl of a goat anti mouse IgG(H+L)-FTTC conjugate (Sigma), diluted 1:100 in PBST + 0.1% BSA was added to each well and incubated for 1 hr.a t 37°C. The plate was washed twice with PBST and once with ultrapure water; 15 |xl antiquench solution(Johnso n andNogueir aAraujo , 1981)wer eadde d toeac hwell ,th e conidiawer eresuspende d and2 |x lfro m eachwel lwa stake n andpu to na 2 4wel lmult i test slide (Nutacon). Subsequently, the reaction of the conidia was monitored using a fluorescence microscope. Isotypedeterminatio n Thesubclas so fth eimmunoglobulin sproduce db yth ehybridoma swa sdetermine dusin g anELIS Aa sdescribe db y Schotset al. (1992). 128 Protein concentration Protein concentrations of the extracellular proteins were determined according to Bradford (1976)usin gbovin eseru m albumina sa standard . Results and Discussion Fusion experiments carried out with splenocytes from mice immunised with whole conidia or extracellular proteins resulted in eight hybridomas producing monoclonal antibodies(MAbs ,Tabl e2 )whic hwer ereactiv ei nth eimmunofluorescenc e assay(Fig . 1 and 2).T oensur e that we would only obtain MAbs which were specific for thegenu s Botrytisw e used a mixture of spores from saprophytic fungi isolated in greenhouses (Pénicillium spp.,Aspergillus spp., Alternaria spp., Cladosporium spp., Trichoderma spp.an dMucor spp. ) as negativecontrols .Non eo f theMAb sreacte d with spores from thesefung i (Table2) .A furthe r characterisation wascarrie d outwit hconidi afro m other Botrytisspp . and healthy flowers. Several MAbs (Table 2) reacted with conidia from B. aclada and B. squamosa. However, the reaction pattern with conidia from B.squamosa differe d considerably from thatobserve dwit hB. cinerea a sonl ypatche so n thesurfac e ofth econidi afluoresce d (Fig.2 )an dno tal lMAb sreacte d withthi sBotrytis species.N oreactio n ofth eeigh tMAb swa sobserve d withhealth ygerber a flowers. Themic euse di nthes efusio n experimentswer eimmunise dwit ha mixtur eo fconidi a orextracellula r proteinsextracte d from conidiao fte nB. cinerea isolates(Tabl e 1).Thi s was done to avoid too specific MAbs, as was observed by Hardham etal. (1986 ) with MAbs against zoospores and cysts of Phytophthora cinnamomi. Indeed, all ourMAb s Fig. 1. Reaction of Botrytis cinerea conidia with monoclonalantibody 9E11. The immunoreaction wasvisualised using agoat anti-mouse-FITC-cojugate. Bar= 10 p.m. 129 reacted with conidia from the ten isolates used. In most cases at least 75%, and often moretha n90% ,o fth econidi awer epositiv e(Tabl e3) .Th e absence of areactio n canb e a consequence of either changes in the cell wall or disappearance of the extracellular proteinsfro m deado rdyin gconidia . Most of the prerequisites for the development of a routine test have been met. The MAb sdi dno treac twit hothe rfung ian dmicroorganism sgenerall ypresen ti ngreenhouse s (data notshown) .Also ,n oreactio n with tissuefro m gerbera flowers wasobserved .Th e final prerequisite is that MAbs havet o react with all B. cinerea isolates to avoid false negatives.Concernin g this point the reaction was quantified with conidia from isolates usedfo rimmunisatio n only.A tes twit hothe risolate si spresentl yi nprogress . A routine test based on monoclonal antibodies can replace tests based on selective media(Kerssies , 1990)o rth eus eo fpolyclona lantisera .Polyclona l antisera(e.g .Ricke r et al., 1991) are not specific, while it has often been shown that cross absorption to improve specificity, decreases sensitivity dramatically. Disadvantages of the use of selectivemedi aar etha tmos tselectiv emedi a areno tcompletel y selectivean dworkin g with such media is usually rather time consuming. Furthermore, some taxonomical knowledgei srequired . Ates tbase d onMAb si sspecifi c andca nb eperforme d withina few hours and the reaction pattern of the MAb used, concerning specificity and sensitivity,i sexactl yknow n andwil lno tvary . The test for the detection of B. cinerea on gerbera flowers will be based on the immunofluorescence assay described in this paper. The advantages of an immuno fluorescence assayar eth esensitivit y andth especificity ; evena singl econidiu mca nb e detected.Surprise swhic hca noccu rbecaus eo fpresenc eo fcross-reactin gspore so fothe r fungi, whichstructur ean dreactio npatter n mostlikel ydiffe r from conidiao fB. cinerea, Fig. 2. Reactionof Botrytis squamosa conidia withmonoclonal antibody 4H10. The immunoreaction wasvisualised using agoat anti-mouse-FITC-cojugate. Bar= 20fjjn 130 Table 2. Immunofluorescence reactions of monoclonal antibodies with saprophytes, B. aclada and B. squamosa. Antibody Isotype Negative control1) B. aclada2* B. squamosal l-4-'91 1-10-'91 l-l-'92 9E11 IgGl -4) +5) 4H10 IgM - - + 4A3 IgM n.d.6> + n.d 9C7 IgM n.d. + n.d 10G1 IgM n.d. + n.d 7B9 IgM n.d. - n.d 9A12 n.d. n.d. + n.d 2G11 IgM n.d. + n.d 1)mixtur e of saprophytes isolated from gerbera plants in the 91-92 season. 2) mixture of four B. aclada isolates 3) mixture of two B. squamosa isolates 4) no visible immunofluorescence 5) immunofluorescence visible 6) not done / Table3. Immunofluorescence reactions of monoclonal antibodies with ten different isolates ofB. cinerea. Antibody Isolates Bc7 Bc8 Bcl3 Bcl5 Bcl6 Bcl7 Bc21 Bc25 Bc27 Bc29 4H10 99D 67 83 70 99 99 70 80 95 80 9E11 83 99 74 99 99 96 89 86 99 99 4A3 82 70 99 99 99 94 70 75 80 99 9C7 93 42 54 99 99 99 99 99 90 99 10G1 99 79 93 99 91 75 99 90 99 99 7B9 79 75 75 79 99 73 99 90 92 91 9A12 91 50 80 56 86 92 80 78 57 58 2G11 99 73 50 50 90 90 89 80 80 80 ') percentage of conidia showing fluorescence are excluded. Trials of this test are presently in progress using gerbera flowers obtained from a flower auction. Preliminary results show that more than 90% of the conidia of B. cinerea on infected flowers can be washed off and reacted positive in the immuno fluorescence assay. References Bakker, A.G.M., 1986. Najaarproblemen in de gerberateelt. Vakblad voor de Bloemisterij 41:20-21. Bradford, M.M., 1976.A rapid and sensitive method for thequantificatio n of microgram quantities of proteins utilizing the principle of protein clye binding. Analytical Biochemistry 72:248-254. 131 Hardham, A.R., Suzaki, E. and Perkin, J.L., 1986. Monoclonal antibodies to isolate species and genus specific components on the surface of zoospores and cysts of the fungus Phytophthoracinnamoni. CanadianJourna lo fBotan y64:311-321 . Johnson,G.D .an dNogueir aAraujo ,CM.d eC , 1981.A simpl emetho d ofreducin g the fading of immunofluorescence during microscopy. Journal of Immunological Methods43:349-350 . Kerssies,A. , 1990.A selectiv e medium for Botrytis cinerea tob e used in aspore-trap . NetherlandsJourna lo fPlan tPatholog y96:247-250 . Ricker, R.W., Marois, J.J., Dlott, J.W., Bostock, R.M. and Morrison, J.C., 1991. Immunodetection and quantification of Botrytis cinerea on harvested wine grapes. Phytopathology 81:404-411. Salinas, J., Glandorf, D.C.M., Picavet, F.D. and Verhoeff, K., 1989. Effects of temperature, relative humidity and age of conidia on the incidence of spotting on gerberaflower sb yBotrytis cinerea. Netherland sJourna lo fPlan tPatholog y95:51-64 . Salinas,J. , 1992.Functio n ofcutinolyti c enzymesi nth einfectio n ofgerber aflower s by Botrytis cinerea. PhDThesis ,Universit y ofUtrecht ,Th eNetherland s p. 85-93. Schots, A., Pomp, R. and van Muiswinkel, W.B., 1992. Production of monoclonal antibodies.In Technique s inFis hImmunology .Stolen ,J.S. ,Fletcher ,T.C. ,Anderson , D.P.,Kaattari ,S.L .an dRowley ,A.F . eds.SO SPublications ,Fai rHaven ,NJ .p : 1-18. Verberkt, H., 1986. Tijdens verwerking, bewaring en transport waken voorBotrytis. Vakblad voord eBloemisteri j41:14-15 . 132 The effect of gamma irradiation onBotrytis cinereaan d B. acladacausin g rot of pear and onion respectively M.Giirer and O. Tiryaki Summary Doseso f3 kGydelaye d rottingo fpea rcv .Ankar acause db yBotrytis cinerea,bu tfaile d to inhibit theproces s completely. Controls developed rots with an average diametero f 25.6 mm, in irradiated rots pears had an average diameter of 13.2m m after irradiation with3 kG y 10day safte r inoculation. Irradiation ofB. cinerea wit h3 kG yin vitro cause d heterogenous sporulation and mycelial growth. Similar results were obtained with B. aclada inonions . Introduction Botrytis cinerea Pers.:Fr . on pear fruits andBotrytis aclada Walker on onion bulbs are the main causes of storage rot in Turkey. Nelson et al. (1959) stated that gamma irradiationo fth edose so f 1.105 repan d2.10 5re pha da fungistati c effect onB. cinerea o f grape,whil ewit hdose so f4.10 5re p( =3. 7kGy )o rhighe rfungicida l effects occurred. Tiryaki (1990) described the sensitivity of a number of fungi to gamma irradiation invitro at3-4° Can dB. cinerea wasth emos tsensitiv eo fth efung i tested. Material and Methods B. cinerea was isolated from pear cv. Ankara, and B. aclada was isolated from onion bulbsan dthes efung i wereculture do npotat odextros eaga r(PDA ,Difco ) for irradiation studies.A ^ o gammairradiato r (10kGy )wa suse di nal lexperiments . Agarplate sinoculate dwit hB. cinerea andB. aclada wer eincubate d at22±1° Cunde r fluorescent light during a 12h photoperio d for 7days .Activel y growing cultures were irradiatedwit h 1,2an d3 kGyan dthe n6 m maga rdisk sremove dan dtransferre d tofres h PDA.Aga rplate sinoculate dwit hB. cinerea wer eincubate da t3-4° Cafte r pre-incubation at 23°C for 4 h, whileB. aclada was incubated at 22+l°C. These exposure treatments werereplicate d four times for eachisolate .Th ediamete r of colonies was measured and themos teffectiv e irradiationdos edetermine d after 7 days. Forin vivo studies,fruit s weresurface-sterilise d with0.5 % NaOCl for 2t o3 mi nan d rinsed with water.Tw otissu edisk s with adiamete r of6 m m were removed from either side of each fruit. The holes werefilled wit h disks of 7-day-old cultures of B. cinerea. After incubation at23° Cfo r 21h (Berah a etal., 1959 )fruit s wereexpose d to 1,2 an d3 kGyirradiatio nan dstore da t0° Can d85-90 %relativ ehumidit yfo r6 0days .Th ediamete r of rot on each fruit was measured and the most effective dose determined. The same inoculationmetho dwa suse dforÄ .aclada o nonio nbulbs .T otes tth epossibl elong-ter m effects of irradiation on pathogenicity of B. aclada,7-day-ol d culture of the fungus irradiated previously with the most effective dose invitro, wa s used as inoculum and 133 compared to non-irradiated inoculum. Inoculated onion bulbs were covered with polyethylenebag san dincubate da t22±1°C .Afte r7 day sincubation ,th elesio ndiameter s were measured. Ten fruits or bulbs were used in each of the three irradiation and inoculation treatmentsi nthes eexperiments . Results and Discussion After irradiation with 3 kGy, B. cinereashowe d initially no growth after 7 days incubation at 3-4°C, compared to 24.5 mm in the control, B. acladawa s much less affected, even at 3kG y (Table 1).Thes e results were similar to those of Barkai-Golan etal. (1967).Nelso n etal. (1959)reporte d thatmycelia l growth ofB. cinerea irradiated with the equivalent of 3.7 kGy, was slight after 12day s at 3 to 4°C whereas mycelial growthwa sinhibite d completelywit hth eequivalen t doseo f7. 4kGy . Table1. Colonydiameters of irradiated and non-irradiated cultures ofB. cinereaand B. acladaon PDA7 days after inoculation. Irradiation Colonydiamete r(mm ) dose(kGy ) B.cinerea B. aclada 0 24.50a* ) 63.5a 1 7.00b 60.0a b 2 6.75b 56.0b c 3 6.00b 53.0 c *)figure s followed bydifferen t lettersdiffe r significantly atp < 0.00 1 Table 2. Developmentof mould on pear fruits wound-inoculated with B. cinerea, exposedto irradiation dosesand cold-stored. Irradiation Rotdiamete r(mm ) dose(kGy ) Daysafte r irradiation 10 20 30 35 40 0 25.63a * 35.04a 40.58a 46.29b 48.58b 1 22.83b 34.54a 49.46a 53.33a 57.21 a 2 15.79 c 25.88b 43.58b 50.63a b 59.04a 3 13.21 d 20.88c 32.13 c 37.54c 44.54b *figure s followed bydifferen t lettersdiffe r significantly atp < 0.00 1 Irradiation (1kGy ) stimulated sporulation ofB. cinerea. B. cinereare-isolate d from pears after irradiation with 3 kGy, showed atypical sporulation andmycelia lgrowth .Th emos tinhibitor ydos efo rpea rro tafte r 10day swa s 3kG y(Tabl e2) . Themos tinhibitor y irradiationdos efo r mycelialgrowt h ofB. aclada was3 kG ybu t irradiated B. aclada isolates still caused rot of onions, viz. irradiated 27.6 mm and unirradiated, 31.4mm . Nelsonet al. (1959 )observe dtha tth emycelia lgrowt ho fB. cinerea di dno tstar tunti l 12day s after irradiation ,bu tw efoun d pronounced effects after 10day s(Tabl e2) . Our resultswer esimila ra sthos edescribe db yBehar aet al. (1960). 134 References Barkai-Golan,R. , Gorodoeiski,N .an d Kahan, R.S., 1967.Effec t of gamma irradiation on development of fungi, Botrytiscinerea and Rhizopus nigricans, causing rot in strawberry fruits. FoodIrradiatio n8:34-36 . Beraha,L. ,Ramsey ,G.B. ,Smith ,M.A .an dWright ,W.R. , 1959.Factor sinfluencin g the use of gamma irradiation to control decay of lemons and oranges. Phytopathology 49:91-96. Beraha, L., Smith, M.A. and Wright, W.R., 1960.Gamm a radiation dose response of somedeca yi npathogens .Phytopatholog y 50:474-476. Nelson, K.E., Maxie, E.C. and Eukel, W., 1959. Some studies in the use of ionizing radiation to control Botrytisro t in table grapes and strawberries. Phytopathology 49:475-480. Tiryaki, O., 1990. Inhibition of Pénicilliumexpansum, Botrytis cinerea, Rhizopus nigricans and Alternaria tenuissima, which were isolated from Ankara pears after gammairradiation .Journa l ofTurkis hPhytopatholog y 19:133-140. 135 Impedance mycology, a newrapi d method for theevaluatio n ofmetaboli c activity ofBotrytis cinerea and B. acladaa sinfluence d by environmental factors P.V Nielsen andJ. Kampp Summary Studies of spoiling capacity of fungi as affected by environmental factors, such as substrate composition, temperature, humidity and atmosphere, can advantageously be performed by impedance mycology, as this method is easy, rapid and produces information unattainable by traditional methods. Results from a study of the effect of storagecondition s ongrowt ho fBotrytis sp.ar egiven . Introduction Theus eo frapi d methodsi nmicrobiolog y hasincrease d substantially duringth elas ttw o decades,especiall yi nth eare ao fclinica lan dfoo d microbiology.Th eobjective s ofthes e methodshav ebee nt oobtai nfas t andreliabl eresult si nclinica l diagnosis andi ncontro l during food manufacturing. These methods are now being used in bacteriological and mycological research,t oobtai n newinfomatio n whichwoul dno thav ebee npossibl eb y traditionalmethods . Impedance mycology is probably themos tpromisin g new method for studies of the effect of environmental factors on fungal growth. The method is based on the measurement of metabolic activity rather than secondary characteristics such as colony diameter,mycelia ldr yweigh to rinhibitio nzones .Th emetho di ssimple ,rapi dan da hig h numbero ftest sca nb eperforme d simultaneously. The technique has been established as a tool for rapid detection of bacterial conta mination of a wide range of foods, and now it is entering the disciplines of food technologyan dgenera lmycology .Beside sth efundamenta l workb yWilliam san dWoo d (1986), most emphasis has been directed towards detection of yeasts in fruit mixtures (Fleischer etai, 1984),orang ejuic e(Zindulis , 1984)an dwin e(Henschk ean dThomas , 1988).A recen t study of impedance changes induced bymould s causing food spoilage resulted in thedevelopmen t ofa mediu m for generalimpedimetri c examination of food products(Watson-Crai k etal., 1990). Thispape rdescribe sth eprinciple so fimpedanc emycolog y andthe ngive sth eresult s of a study of thecombine d effect of temperature, humidity and controlled atmosphere storageo ngrowt h ofB. cinerea andB. aclada. Results and Discussion Microbial metabolism alters the electrical properties of the growth medium as large moleculesar edegraded ,othe rexcrete dan dsom esubstrat ecomponent sassimilate d from the medium. Theseelectrica l changes are monitored by an impedimeter by sending an 136 alternatingelectrica lcurren tthroug hth emediu man dmeasurin gth eimpedanc ethi s flow encounters. Impedance (Z) isth eresistanc e towardsflow o f anelectrica l current inth e substrate.I t consists of twocomponents ,conductanc e (G) which is associated with the mobility and number of ions in the substrate, and capacitance (C) which is associated withchange si n closeproximit y toth eelectrode .A detaile d explanation ofthi si sgive n byFirstenberg-Ede n andEde n(1984) . Severalstudie shav eshow ntha tth ecapacitanc esigna li ssuperio rt oconductanc ean d impedance for monitoring fungal growth ,wherea s conductance is preferable for most bacterial studies(Nielsen , 1992;Watson-Crai ketal., 1989; Zindulis,1984) . Theimpedanc ereading sar etake nb yth eBactomete ran dpresente da scurve sshowin g thechang ei nimpedanc ewit htim e(Fig . 1 ).Detectio ntim efo rgrowt h (DT)i srecorde d whena significan t changei nimpedanc eha soccurred . **Institutte tfo rBioteknologi ,DT H** - Ö.0 7.5 15.0 ££.5 30.0 37.5 45.0 5£.5 60.6 67.5 75.0 h Fig. 1. Impedence values obtained whenBotrytis cinerea isolate 1BT 7036 from an Italian peach wascultured on PYG,a medium specially designedfor the Bactometer. PYGcontains 10.0 g potassium phosphate; 7.5g yeast extract; 30.0g glucose and 20.0 g agarper 1000 ml distilled water. Withina certai nrang eo fth einitia lspor enumbe r(10 2- 106cfu.measurin g cell"1)a goo d correlationexist sbetwee nlog(cfu ) andth edetectio ntim e(DT) ,a sshow ni nFig .2 . This relationship is species-specific due to differences in growth rate (Nielsen, 1991).Th e metabolicgrowt hrate ,|x ,ma yb ecalculate dfro m theslop eo fth erigh tlin eportio no fth e curve.Growt h rate: |x= ln(10)Alog(cfu)/ADT-2,30/a. Lownumber so fcf u resulti nincrease dscattering ,whil edetectio na thighe rlevel swil l occur immediately after germination. The curve will level off and the detection time converget oa commo npoin ta ssee ni nFig .2 .Consequentl ya nestimat eo fth elengt ho f lagphas ei sth eintercep tbetwee n regressionlin ean dth elin elog(cfu ) =7. 0 As part of a study of the role of storage conditions and antagonists on thekeepin g qualityo fonions ,apple san dcarrot si nDenmark ,th ecombine deffec t oftemperatur ean d humidity,controlle d atmosphereso ngrowt ho fB. cinerea an dB. aclada wa sstudied .Th e experimental domain was: temperature (5-25°C), humidity (aw 0.970-0.996), 02 (1- 137 O) 40 E •*-> 30 c o 20 o °-*->3£w Fig. 3. Responsesurface plot of detection time for growthof B. cinereaIBT 7036 cultureson PYG, aw0.983, pH5.0 at 19.2°C. The multiple linearregression isbased on 30measurements, andR2 = 0.93. 138 21%),C0 2(0-10%) ,N 2(balanced) ,p H(3-6.5) .Fo rthes estudies ,a specia lchambe rwa s developed. The response surface plot inFig . 3show s thepredicte d values of detection time for B. cinerea isolateIB T703 6culture d onphosphat eyeas tglucos e agar(PYG) ,a t 19.2°C withwate r activitya, ,0.98 3a tp H5.0 . Otherresult sfro m thisstud y(dat ano tshown )indicate dtha tB. acladai smuc hmor e inhibited by decreased water activity. As a linear relationship existed between the logarithmt ocf u anddetectio ntime ,a respons esurfac emode lshoul ddetec ti fth e fungal growth ratewa s affected byth efactor s examined.I nthi sexperimen t growth rateswer e generallyno taffected ; onlytha to fB. aclada wa ssomewha tdecrease d whenth eoxyge n level was decreased. Consequently the difference in detection time is primarily duet o changesi nla gtime . References Firstenberg-Eden, R. and Eden, G., 1984. Impedance microbiology. New York: John Wiley & Sons. Fleischer,M. , Shapton, N.an dCooper , P.J., 1984.Estimatio n of yeast numbers in fruit mixfo r yogurt.Journa l ofth eSociet yo fDair yTechnolog y37:63-65 . Henschke,P.A .an dThomas ,D.S. , 1988. Detectiono fwine-spoilin gyeast sb yelectroni c methods.Journa l of Applied Bacteriology44: 1 23-133. Nielsen, P.V., 1991.Preservativ e and temperature effect ongrowt h of three varietieso f theheat-resistan tmold ,Neosartoryafischeri a smeasure db ya nimpedimetri cmethod . Journalo fFoo d Science56:1735-1740 . Nielsen, P.V., 1992. Rapid detection of heat-resistant moulds in fruit juice by an impedimetric method. In 'Modern Methods in FoodMycology' , eds.Samson ,R.A. , Hocking,A.S. ,Pitt ,J.I .an dKing ,A.D. ,Amsterdam :Elsevier .I npress . Watson-Craik, I.A.,Aidoo ,K.E .an dAnderson , J.G., 1989.Introductio n ofconductanc e andcapacitanc echange sb yfood-born e fungi. FoodMicrobiolog y6:231-244 . Watson-Craik,I.A. ,Aidoo ,K.E .an dAnderson ,J.G. , 1990. Developmentan devaluatio n of amediu m for themonitorin g of food-borne mould by capacitance changes.Foo d Microbiology7:129-145 . Williams,A.P .an dWood , J.M., 1986. Impedimetricestimatio n ofmoul dIn 'Method so f Mycological Examination of Foods', eds. King, A.D., Pitt, J.I., Beuchat, L.R. and Cory,J.E.L. ,Ne wYork :Penu mpress ,p .230-238 . Zindulis, J., 1984. A medium for the impedimetric detection of yeast in foods. Food Microbiology 1:159-167. 139 Nuclear magnetic resonance (NMR)microimagin g of soft fruits infected byBotrytis cinerea B. Williamson, B.A. Goodman, J.A. Chudekand D.J. Johnston Summary Healthytissue an dtha tinfecte d byB. cinerea havebee ndistinguishe d inbot hraspberr y andblackcurran t fruits byNM R microimaging using agradien tech opuls esequence .I t waspossibl et ofollo w non-invasivelyth eprogressiv e development of infection inbot h fruits overa 4-da y period. Introduction Fruits of red raspberry (Rubus idaeus L.) andblackcurran t (Ribes nigrum L.) arepron e to grey mould caused byBotrytis cinerea Pers.:Fr . (Williamson etai, 1987;McNico l and Williamson, 1989; McNicol et al., 1990). Mature fruits are particularly difficult subjects for conventional histological studies; artifacts caused during fixation, dehy dration andembeddin g occurbecaus e themesocar p consists mostly of extremelylarge , thin-walled parenchyma cellswhic h surround thetoug h endocarp oflignifie d sclereids. The tissue changes associated with the onset of disease caused by release of fungal enzymes, toxins and organic acids further exacerbate attempts to visualise events as infection proceeds. NMRmicro-imagin gno woffer s analternativ eapproac ht oth estud y ofinfecte d fruits. The recent development of microimaging attachments for conventional 'high field' NMR spectrometers (>7 T fiel d strength)ha smad ei tpossibl et operfor m non-invasive histology in plants and fruits (e.g.Walte ret al., 1989 ;Bowtel l etal, 1990;Williamso n étal, 1992). By making the correct choice of imaging pulse sequence and experimental para meters,i t is possible todiscriminat e between water molecules in different chemical or structural environments (Wehrli etal, 1988; Faust etal, 1991).Thi s paper describes howth etechniqu eca nb euse dt ofollo w infection ofraspberr yan dblackcurran tfruit sb y B.cinerea. Parto fthi swor kha sbee nreporte dbriefl y elsewhere(Goodma netal, 1992). Material and methods Inoculation offruit s Glasshouse-grown fruits of raspberry cv.Autum nBlis s andblackcurran t cv.Be nAlde r were harvested by cutting the pedicel. Single fruits were incubated at high RH in polyethylene boxes at 20°C before and after inoculation to follow the progress of infection byNM Rimaging . An inadequately fertilized raspberry fruit, which developed only three drupelets attachedt oth ecentra lreceptacle ,provide dmateria lfo rinitia linoculations . Onedrupele t 140 was inoculated with dry conidia from a culture of B. cinerea isolate 347 grown on medium-X plus 10%sucros e (Harrison, 1978);on edrupele t waspierce d with a sterile needlea sa woun dcontrol ;an dth ethir dwa slef t intacta sa health ycontrol . In a completely fertilized ripe raspberry consisting of c. 100 drupelets, a single drupeletwa ssimilarl ywound-inoculate d andth efrui t examinedove r4 days . Arip eblackcurran twa swound-inoculate di nth emedia ntransvers eplan e(equatorial ) and left until it was completely mouldy and then examined in comparison to that of a healthyunwounde d fruit ofth esam eage . Anotherrip eblackcurran twa swound-inoculate d similarlyan duse dfo ra tim ecours e studyo fth e infection. NMRmicroimagin g NMRimage swer eacquire dusin g aBruke rmicroimagin g unitfitte d witha 2 5m mcoi l attachedt oa Bruke rAM300/W BFourie rTransform spectromete r(7. 2T ; 300MH z1H) . Datawer ecollecte d with astandar d gradientech o sequence as25 6x 51 2uni tmatrices , whichwer etransforme d into 256x 25 6pixe limages .Accumulatio n parameters for the raspberrymeasurement swere :puls eangle ,10 ° (Hermite pulse) ;tim eo fecho , 13.24ms ; repetitiontim e22 0ms ;dephasin ggradient ,-0, 3 mT/cm;rea dgradient ,0. 3 mT/cm;phas e encoding gradient, 0.25 mT/cm (2 x 10"3mT/c m increment); slice select gradient, 0.2 mT/cm.Thes eyielde dpixe ldimension s of7 0x 7 0m mwit ha slic ethicknes so f50 0|xm . Thirty-two accumulations were used for the generation of each image, which thus Fig. 1. Gradientecho NMR image ofa three-drupelet raspberry fruit. DrupeletA was inoculated with conidia ina single wound; drupeletB was wounded but notinoculated; drupeletC was ahealthy unwoundedcontrol. 141 Fig. 2. Gradientecho NMR imagesof blackcurrant fruit. (a)Health y (b)Badl yinfecte d withB. cinerea 142 required approximately 30 min for total acquisition. Accumulation parameters for the blackcurrant measurements were: dephasing gradient, 0.28 mT/cm; read gradient, 0.28 mT/cm;phas eencodin ggradient ,0.2 1 mT/cm(1. 8x 10~3mT/c mincrement) ;slic eselec t gradient,0.1 8 mT/cm; allothe rparameter s wereth esam ea sfo r theraspberr ymeasure ments. Theseproduce dpixe ldimension so f6 0x 6 0|x man da slic ethicknes so f30 0|xm . Results and Discussion In NMR images of healthy raspberry fruit using a gradient echo pulse sequence, pronounced radial striations are observed in the mesocarp (Williamson et al., 1992). Similarstructure s havebee nexplaine db y Bowtell etal. (1990 ) asbein g causedb yth e largedifference s inmagneti csusceptibilit y thatexis ta tth ejunction sbetwee nth ewater - filledcell san dth egas-fille d intercellularspaces .Th eNM Rimag eo fth eundamage dan d wounded drupelets in thethree-drupele t raspberry fruit showed similar structure,wher e asthi swa scompletel ylos tfro m theinfecte d drupelet(Fig .1) . NMRimage sproduce dfro m asingl etransvers eslic eo fa fully-forme d raspberry fruit in the plane of the wound inoculation showed boundaries between the infected and uninfected tissuewit ha stead ymovemen to fthes eboundarie sacros sth efrui t duringa 4 - day period. Even after 4 days there was a distinct boundary between infected and uninfected tissue.Thi sexperimen t wasreporte d briefly byGoodma n etaU (1992) ,wh o confirmed from histologicalstudie stha tth eboundarie sbetwee nstriate dan dnon-striate d components in theNM R images correspond toth e limits of progression of theinterna l fungal mycelium. Asimila rse to fexperiment swa sperforme d withblackcurran tfruit s andimage s from healthyan dseverel yinfecte d fruits areshow ni nFig .2 .Th eovar ywall ,th eparenchym a derivedfro m theplacent aan dth eseed swer eclearl ydelimite di nth ehealth y fruit. Each seedwa ssurrounde db ya brigh tlaye rwhic hcorresponde dt oth egelatinou sepidermi so f the seed coat. Dark fibrillar structure was present throughout the parenchyma of the healthyfruit , butwa sabsen tfro m theimage so fth einfecte d fruit. In thefinal blackcurran t experiment, images from eightslice swer eacquire d simul taneously,s otha tth edevelopmen tcoul db efollowe d essentiallyi nthre edimensions . As withraspberries ,th esprea do finfectio n couldb esee nclearl yove ra 4-da yperiod ,bu tth e boundariesbetwee ninfecte d anduninfecte d tissuewer eles swel l defined. Conclusions NMR microimaging represents a powerful method for non-invasive observation of the developmento fplan tdiseas eprocesse san dpromise st ofacilitat e significant advancesi n understanding diseaseprocesse s inplant sa tth etissu elevel . Acknowledgements Wethan kth eScottis hOffic e Agriculturean dFisherie s Departmentfo r funding. 143 References Bowtell, R.W., Brown, G.D., Glover, P.M., McJury, M. and Mansfield, P., 1990. Resolution of cellular structures by NMR microscopy at 11.7 T. Philosophical Transactionso f theRoya l SocietyLondo n(A )333:457-467 . Brown, J.M., Thomas, J.F.,Cofer , C.P. and Johnson, G.A., 1988.Magneti c resonance microscopy of stem tissues of Pelargonium hortorum. Botanical Gazette 149:253- 259. Faust, M., Liu, D.,Millard , M.M. and Stutte,G.W. , 1991. Bound versus free water in dormantappl ebud s- a theor yfo rendodormancy .Horticultura l Science26:887-890 . Goodman, B.A., Williamson, B. and Chudek, J.A., 1992.Non-invasiv e observation of thedevelopmen to ffunga l infection infruit . Protoplasma 166:107-109. Harrison, J.G., 1978. Role of seed borne infection in epidemiology of field beans. Transactionso fth eBritis hMycologica l Society7 0:35-40. McNicol,R.J .an dWilliamson ,B. , 1989.Systemi cinfectio n ofblackcurran t flowers by Botrytiscinerea an dit spossibl einvolvemen ti nprematur eabscissio no ffruits .Annal s ofApplie dBiolog y 114:243-254. McNicol,R.J .Williamson ,B .an dDolan ,A. ,1990 .Effect s ofinoculation ,woundin gan d temperatureo npost-harves tgre ymoul d(Botrytis cinerea) ofre draspberry .Journa lo f Horticultural Science65:157-165 . Walter,L .et ai, 1989.Nuclear-magnetic-resonanc e imaging ofleave so fMesembryan- themum crystallinum L.plant sgrow n athig h salinity.Plant a 178:524-530. Wehrli,F.W. , Shaw,D .an dKneeland ,J.B .(eds.) ,1988 .Biomedica lMagneti cResonanc e Imaging,Principles ,Methodolog y and Applications.Publishe d by VCHPublishers , NewYork . Williamson, B., Goodman, B.A. and Chudek, J.A., 1992.Nuclea r magnetic resonance (NMR)micro-imagin g ofripenin gre draspberr yfruits .Ne wPhytologis t 120:21-28. Williamson,B. ,McNicol ,R.J .an dDolan ,A. , 1987.Th eeffec t ofinoculatin gflower san d developing fruit with Botrytis cinerea on post-harvest grey mould of red raspberry. Annalso fApplie dBiolog y 111:285-294. 144 EPIDEMIOLOGY Epidemiology of grey mould, caused byBotrytis cinerea invegetabl e greenhouses Y Elad, D.Shtienberg, H.Yunis and Y Mahrer Summary Controlo fgre ymoul ddepend so nfungicid e applications,becaus esever eepidemic sca n be expected when conditions in greenhouses favour the disease. Microclimatic conditions which affect epidemics were characterised in unheated polyethylene green housest oinvestigat eth epossibl e introduction of non-chemicalcontro l methods forus e withcucumbe rcrops .Fro m 14t o9 day sbefor e theappearanc eo fsymptoms ,plant swer e predisposedt oth ediseas eb ylo w(<9°C )an dhig h(>24°C )temperature san db ydryness . Infection occurred 7-8 days before symptoms were visible and was promoted by high humidity(>91 %r.h. )an dtemperature si nth erang eo f9- 24°C .Afte rinfectio ntoo kplace , theinfectiv e mycelium was relatively protected by the host tissue and temperature and r.h. requirements were less stringent. A model based on qualitative analysis of the relationshipsbetwee n microclimatic factors andfrui t orste minfection s wasdeveloped . Thetw oimportan tparameter s associated withoutbreak s ofepidemic swer eth eduratio n of leaf wetness and the duration of night time temperatures between 9° and 21°C.Th e daily averages for thethreshold s were7 h pe rda y for thewe tperio d and 9.5 hpe rda y for theduratio n of specific temperatures. Thenutritiona l andhormona l statuso fhos tplant s alsoaffecte d their susceptibility to development of grey mould epidemics. High humidity restricted movement of calcium to upper plant parts and calcium enrichment ofhos ttissu e reduced the susceptibility of pepper,tomat oan deggplan tt ogre ymould .Applicatio n ofauxi nt oenhanc efrui t setting ofeggplan treduce d theirsusceptibilit y toth edisease . In order to reduce dependence on fungicides to which the pathogen has become resistant,alternatio nwit hth ebiocontro lagen tTrichoderma harzianum wit hfungicid e is suggested. This method should reduce exposure of the pathogens' population to fungicides. Thesurviva l ofB. cinerea ininfecte d stems andth eeffec t ofgre y mouldo n cucumber production were studied during high summer temperatures. The outcomeo f theseepidemiologica lstudie sha sbee nth eintroductio no fa workabl econtro lprogramm e for theintegratio n of non-fungicidal measures into theproductio n system toreduc eth e proportion offungicide-resistan t isolatesan dt omak ebette rus eo favailabl echemicals . Introduction Inwar m regions production of manyvegetabl ecrop s canb edon ei n non-heated green housesdurin gwinter . Parthenocarpiccultivar so fcucumbe r(Cucumis sativus L.) ,tomat o (Lycopersiconesculentum Mill.) ,peppe r(Capsicum annuum L. )an deggplan t(Sola num melongena L.) are examples of these crops and they are all seriously affected by grey mould caused by Botrytis cinerea Pers.: Fr. Control of grey mould in vegetable crops dependso nfrequen t fungicide applications,bu tresistanc eo fth ecausa lagen tt ocommo n 147 fungicides is widespread. At the beginning of the 1980s resistance of the B. cinerea population to benzimidazoles was already widespread in Israel, and resistance to dicarboximide fungicides had been identified in some greenhouses (Katan, 1982). In recentyear sresistanc et odicarboximide s becamecommo n (Yunisan dElad , 1989). The mixture ofdiethofencar b withcarbendazi m wasteste d recently for greymoul d control, but unfortunately resistance was already found (Katan etal., 1989).Sinc eresistanc e to currently available fungicides is common in greenhouses, alternative methods to suppressB. cinerea areo fgrea timportanc et ocommercia lgrowers . Microclimaticcondition s whichaffec t greymoul d Information on theeffec t of microclimatic conditions on grey mould is available from manystudie swit hvariou scrops ,bu ti ti shighl yvariable .Hunte ret al. (1972 )foun d that infection of racemes of macadamia by B. cinerea was correlated with duration of leaf wetnessa ttemperature sbetwee n 18°an d22° Co r>95 %r.h. .Jarvi s(1980 )reporte d that optimumtemperature sfavourin g infection ofothe rhost swer ebetwee n 15° and20°C ,bu t infection couldoccu reve na t2°C .Simila rrange so ftemperature ssuitabl efo rgre ymoul d development were reported for flower crops, but the r.h. requirements varied in the different studies. For gerbera and rose flowers the optimal r.h. was >95%, but disease developed on the flowers at 80% r.h. (Marais etal., 1988; Elad, 1989; Salinas et al, 1989). In Israel non-heated growth structures arecovere d with a new type of polyethylene covers which are opaque to infrared (IR-thermal) radiation; differences between night timelea fan dai rtemperature sar eno wusuall ysmalle rtha nunde rth epolyethylen ecove r previously used (Elad et al., 1988).I t was suggested that high temperatures, near the optimum for development of grey mould, compensate for the reduced duration of wetness. Microclimatic parameters were monitored in cucumber crops grown in non-heated greenhouses during the winter of 1987/88.Ai r and leaf temperatures, r.h., rainfall, and radiation,wer erecorde d hourlyb yC21 Xdat alogger s(Campbel lScientifi c Inc.,Logan , UT, USA). Air and leaf temperatures were measured using copper-constantan thermocouples andr.h .wit hPCRC-1 1 electro-humidity sensors(Yuni set al, 1990). The 1987/88 winter season wascharacterise d by arelativel y largenumbe r of rainy days.I n the greenhouses r.h. is usually high for several hours of the day,dependin g on outside humidity, e.g. during and after rain showers. Air temperatures inside the greenhouses rangedbetwee n3 ° and38° Can dexceede dth eoutsid etemperatur eb y2-10° Cthroughou t theday .A tnight ,ai rtemperature sinsid eth egreenhous ewer eequa lt o 1° Co rhighe rtha n those outside. Location, orientation and position of the main entrance dictated the conditions inside eachgreenhouse . Persistance of temperature in defined ranges,r.h .i n certainranges ,an dth elea fwetnes swer ecalculated .Multipl eregressio n andcorrelatio n analyses (according toR 2values )betwee n thesetemperatures ,r.h .an dwetnes sduratio n parameters in certain ranges, and disease incidence (square root transformed), were conducted using SASprocedures . Diseasewa scharacterise db ytw ostages ,accordin gt oth erat eo fit sdevelopmen tan d the microclimatic conditions influencing it. In the first phase of the epidemic, a high correlation was found between infected fruits and air temperature in the range of 11- 25°C, and r.h. in the range of 97-100% or leaf wetness. In the second phase, disease incidence was better correlated with air temperature in the range of 11-16°C and r.h. 148 Table 1. Rangesof microclimatic parameters having a significant (P<0.05) effecton cucumbergrey mould in winter. Daysbefor e Ranges of R2 diseaseassessmen t microclimatic variables Temp (°C) r.h. (%) 1989 1990 FRÜHINFECTIO N Diseaseestablishmen t period 1- 2 9-18 61-91 0.8415 0.9234 2- 4 18-21 0.9568 4- 5 76-82 0.8912 6- 7 9-24 >91 0.9734 0.8683 5- 8 >24 79-85 -0.8929 -0.8928 10-14 27-30 61-85 0.9127 0.8378 Epidemic outbreakperio d 7- 9 15-21 79-94 0.8489 0.8277 9-12 24-30 61-94 0.8511 0.9296 11-14 0-9 82-88 0.8699 , 0.8528 Epidemicdeclin eperio d 3- 5 24-27 52-79 0.944 0.831 5- 7 6-12 70-99 0.802 0.91 9-14 18-24 61-79 0.8827 0.8829 10-14 79-91 0.8977 STEMINFECTIO N Epidemic outbreak period 1- 2 9-24 76-91 0.8469 0.9255 4- 7 12-24 79-97 0.9375 0.799 8-11 >30 61-67 -0.894 -0.8157 8-11 82-91 0.8157 8-11 wetness 0.8157 14 58-76 0.9255 Epidemicoutbrea k period 1- 4 70-90,49-58 0.8677 0.8318 3- 5 24-27 52-79 0.944 0.831 7- 8 15-21 91-100 0.8401 0.8281 7- 8 wetness -0.9326 10-14 61-85,79-91 0.8977 0.8409 10-14 wetness -0.8328 >85% (R^O.681); there was no correlation between disease and leaf wetness at this stage.Developmen t of stem infections wascorrelate d with airtemperatur e in therang e of 11-16°C during the first phase of the epidemic. By contrast, the second phase was characterised bya clos ecorrelatio nbetwee n steminfection s andr.h .i nth erang eo f80 - 149 100%,bu t alsowit h air temperature in the rangeo f 11-16°C,o rwit h airtemperatur ei n therang eo f 11-25°Can dr.h .80-100% ,an dlea fwetnes s(Yuni set al, 1990). Inaddition , itwa sreveale d that in a 'wet' winter season,r.h .an dlea f wetnessinfluenc e thediseas e lesstha ntemperatur ei nth eoptima lrange ,tha tis ,i na 'wet ' winterth elimitin gfacto r for disease development is temperature (Elad etal., 1988;Yuni s etal, 1990).A n attempt wasmad et odetermin eth econdition swhic har emos timportan tfo rdiseas edevelopmen t in greenhouses. An example of the microclimatic conditions on certain dates before disease assessment, which were positively correlated with fruit or stem infections of cucumber, is presented in Table 1.Th edat areflec t thecondition s which affect various stageso fdiseas edevelopment .Th econdition swhic hprevai l7 t o8 day sbefor e symptom appearance and which are significantly correlated with grey mould incidence are high humidity and temperatures around the optimum for germination of conidia. Infection probably takes place during that time,befor e grey mould is observed macroscopically. During the week between infection and symptom appearance the factors which are correlatedwit hincidenc ear elowe rr.h .(91% )an dlea f wetness,probabl ybecaus ea ttha t time the mycelium of B. cinerea is located inside the water-soaked tissue, where it is protected from external dryness. An interestingfinding i s thecorrelatio n between high temperature (>24°,27 ° or 30°C),lo w temperature (<9°C), or low r.h. on the one hand, and, on the other hand, disease incidence 9-14 days after the occurrence of such conditions. These conditions do not affect germination, infection or mycelium devel opment insideth ehos ttissue ,bu trathe rinfluenc e thehos tsusceptibilit y togre ymould . Indeed,i twa sfoun d incontrolle dexperiment si ngrowt hchamber stha tpredispositio nt o infection of cucumber plants by grey mould occurred when the plants were incubated under these conditions for one week before inoculation. During the next stages of an epidemic, once disease is established in the greenhouse, the microclimatic conditions which arecorrelate d withdiseas e incidencear emor ebroa d andcompensatio n probably occursbetwee n humidity and temperature.Compensatio n for reduced exposure tohig h humidityb y temperatures around theoptimu mfo r greymoul dwa ssuggeste d in testso f different greenhouses(Ela d etai, 1988). Developmento fa mode lfo r predictingoutbreak s ofepidemic s ofgre ymoul d Empirical forecasting systems aredevelope d from the observation and analysis of data ondiseas ean dothe rbioti can dabioti cfactors . Suchsystem sca nb edevelope di non e of twoways .Th e first is aquantitativ e approach, which isbase d on statistical analysiso f observed data and model development of epidemics. The second is a qualitative approach, involving the development of prediction criteria (rules) without any formal statistical analysis(Madde n andEllis ,1988) . Thenex tste po fou rresearc hwa saime da tcharacterisin gth emicroclimati ccondition s inducing outbreaks of grey mould epidemics. Disease incidence of infected fruits or nodesan dmicroclimati c parametersrecorde di nth e 1989an d 1990season swer euse dt o develop models for predicting outbreaks of grey mould epidemics. The precision and predictive ability of the model were verified and validated. Verification was accomplished using the same data sets (1989 and 1990) that had been utilised for constructing the models. Models that produced reasonable predictions were examined further, and validated with the 1988 data set. The 1988dat a set was considered as an independentone ,becaus ei twa sno tuse di nth econstructio n ofth emodels .Th eprecisio n ofth eregressio nmodel swa sdetermine dusin gsevera lstatistics ,includin gth e coefficient 150 of determination (R2),th e error variance (MSE) and the significance of the regression modela sdetermine db yth eF-test .Th eprecisio no fth equalitativ emode lwa sdetermine d viacarefu l examination ofth emodel' sprediction swit hactua ldiseas eobservations . Temporalprogres scurve s ofgre ymoul d epidemics werequit edifferen t between the 1989 and 1990seasons .I n 1989th ediseas e appeared in the second part of the season, whereas in 1990 it developed very rapidly early in the season and the percentage of infected fruits diminished gradually thereafter. Thepopulatio n sizeo f thepathoge nwa s not areliabl eindicato ro f diseaseincidence ,becaus ether ewa sn ocoincidenc ebetwee n thetim eo fpeak so rth enumbe ro ftrappe dspore san dth epercentag eo finfecte d fruits or nodes. Initial efforts weredevote d todevelopin g quantitative, statistically-based prediction models. Simple, multiple and stepwise regression analysis techniques were used to quantify the relationship between disease incidence and microclimatic parameters. In some analysis non-linear regression techniques were used. The percentage of infected fruits wasth edependen tvariable ,an dvariou smicroclimati cparameter swer euse da sth e independentvariable si nal lanalyses .Th efollowin g parameters,alon eo ri ncombination , were used as independent variables in different runs: mean, minimum ormaximu m air temperature; mean, minimum or maximum r.h.; duration of wet foliage and of temperature ranges of <6\ <9\ 9-15°, 15-21°,9-21° ,>21 °an d> 24°C .Th eduratio no f parameters werecalculate d indifferen t analyses over a singlenigh t (18.004O08.0 0h) , 1day ,3 dayso r7 days .Th eduratio no fth eabov eparameter swa sals oaverage dove rth e following timeintervals ,prio rt oth edate so fdiseas eobservation :0-3,0-7,3-5,5-7,7-9 , 9-12o r 12-15 days. Although avas t amount of time and effort was devoted toconstructin g quantitative models using formal statistical techniques, the overall results were very disappointing. Statistically significant, biologically sound predictions were not obtained for even a single regression model. The very few significant regression equations (based on the 1989an d 1990dat a sets) produced insignificant, insufficient predictions when utilised for the 1988dat aset . Alternatively,a qualitativ eapproac h wasemploye dfo rdevelopin g amode lt opredic t grey mouldepidemics .Th edevelopmen t procedureconsiste d of thefollowin g steps:(i ) diseaseincidenc e andmicroclimati c parameters weredelineate d onth esam etim escal e (i.e.diseas eprogres scurve san dmicroclimati c parameters curves,respectively) ;(ii )th e timeo foccurrenc e ofmarke d changesi n therat eo fdiseas edevelopmen t wasindicate d on all graphs; and (iii) microclimatic parameter curves were examined carefully in an attemptt oelucidat eth ecause sfo rth emarke dchange si nth erat eo fdiseas edevelopment . The qualitative approach yielded an accurate, biologically sound prediction model. Marked changes in the rate of disease development occurred twice in each growing season:durin gweek s 14an d 17 in 1989,an dweek s 10an d 17i n 1990. Verticalline swer e drawn at these time points on the disease progress curves. Among the microclimatic parameters that were examined in this study, the coincidence of two of the parameter curves wasobserve d atthes etim epoints :th eduratio n of persistence ofwe tfoliage , and theduratio no ftemperatur ei nth erang eo f9-21° Cdurin gth enigh t(18.0 0t o08.0 0h) .Fo r bothparameters ,thes ethreshold swer e7 h pe rda yfo rth ewe tperio dan d9. 5 hpe rda yfo r theduratio n of optimal temperature.Th evalue suse dwer edail y averages overth ewee k thatprecede d eachdiseas e observation.Thes emicroclimati c parameters willb e referred tohereafte r asth ewe tperio d andth eduratio n ofoptima ltemperature ,respectively . Thenex tste pwa st ovalidat ethi smode lwit ha nindependen tse to fdata .I n 1988 there 151 were twodiseas e outbreaks, during thefirst hal f and atth een d of theseason . Thetw o occasionswer epredicte daccuratel yb yth emodel .Th eoccurrenc eo fnod einfection swa s recordedi nal lexperiments . Peaksi nth enumbe ro finfecte d nodespe rplan tappeare d5 - 7 weeks after the outbreak of infected fruits. A similarity was found between micro climaticcondition swhic hwer ecorrelate d withfrui t infection, andthos ecorrelate d with node infection. The germination of spores of B. cinerea, tested in controlled environment exper iments,wa smarkedl y influenced byth epersistenc eo ffre ewater . Germination occurred only when spores were exposed tofre e water. In this casegerminatio n was initiated at 22°Cafte r 4h incubation .Dryin g thespore s attha ttim ehalte dth egerminatio nprocess . When free water was eliminated, germination was not observed for at least 24 h, regardlesso fth er.h. .However ,germinatio ndi doccu ri nth eabsenc eo fwate rafte r 30h ofincubatio n at 100%r.h. .A t< 95 %r.h. ,germinatio nwa sdelaye dunti la tleas t5 2h o f incubation. Theinfectio n processinvolve sthre econsecutiv ephases :germination ,penetratio nan d establishment (Goodman et al, 1986).Durin g the two first processes the conidia and newlygerminate dconidi aar eextremel ysensitiv et omicroclimati cinfluences . Inth elas t process,however , themyceliu m isexpose d toth econdition s prevailing within thehos t tissue.Th emai ndifferenc e between thesetw oenvironment s is the accessibility of free water.Wherea sfre e wateri sforme d onth efoliag e surface only occasionally,gree nhos t tissuema yb econsidere d asconstantl y water-soaked. Theproces sfollowin g germinationi spenetration .Thi sproces si nB. cinerea last sc . 2- 3h .Thus ,th epathoge n isexpose dt oenvironmenta l microclimatic influences for 9-10h (7h fo rgerminatio nplu s2- 3h fo r penetration). Thisfigur e resemblesth eduratio n ofth e optimal temperature threshold in thefield. Onc e the pathogen has penetrated the host tissue,a shortag eo ffre e wateri sn olonge ra limitin gfacto r for infection. Similarresult s (7h )wer ereporte d for germination andpenetratio n ofconidi a ofB. squamosa inonio n (Aldermanan dLacy ,1983 )an do fB. cinerea ( 5h )i ngerber aflower s (Salinaset al, 1989) Wear e confident of the potential benefits of the model proposed here as a tool for preventing outbreaks of epidemics. Similarly, Vincelli and Lorbeer (1988) based their prediction system for B.squamosa ofonio no ntemperatur ean dlea f wetnessduratio ni n order to predict infection potential when inoculum is present. In Israel, a recom mendation togrower s asa noutcom e ofth epresen t study is asfollows : onnight s when temperatures between9 an d21° Car eexpected ,th eduratio n ofth ewe tperio d shouldb e reduced to <7 h. This can be done by forced ventilation or by ventilation through openings in the greenhouse. In heated structures, heating can also be beneficial in suppression ofgre ymould . Theeffec t offertilisatio n onseverit yo fepidemic s Nutritionalregime sca ninfluenc eth esusceptibilit yo fplant st odisease s(Goodma net al., 1986). The effect of the nitrogen source depends on the plant species and on the conditionsunde rwhic hi ti sgrown . Forinstance ,Verhoef f (1965)foun d adecreas ei nth e susceptibility oftomat ot ogre ymoul dwhe nth ecro pwa sgrow ni nsoi lwit ha hig hleve l of N. Hobbs and Waters (1964) reported the opposite results with chrysanthemum flowers undersuc hconditions .So l(1967 ) found thatplant s of Viciafaba fertilised with aammoniu msourc eo fN wer emor esusceptibl et ogre ymoul dtha nplant sfertilise d with a nitrate source.I nexperiment s tostud y theeffec t of nitrogen sourceo ngre ymould ,i t 152 was found that the N source did not influence the susceptibility of eggplant or pepper plants to grey mould. In contrast a higher proportion of nitrate in the fertiliser was associated withreduce d incidenceo fth ediseas ei ncucumbe rplants . One of the significant environmental features in greenhouses is the high r.h. under whichcrop sar egrown .Decrease dtranspiratio ndu et ohig hr.h . reducedth etranslocatio n of some ions from the roots to the canopy (O'Leary and Knecht, 1972). Calcium is generally immobile in the phloem and moves almost exclusively in the xylem. The amounto fcalciu menterin ga norga ni stherefor edependen to nth evolum eo fwate rflo w through thexylem .Environmenta l factors sucha s atmospheric humidity whichdirectl y influence transpiration can cause or aggravate calcium shortage in young tissues and fruits. Calciumca nals ob etranslocate di nrespons et oroo tpressure ,whic hi ssensitiv et o atmospherichumidit y andt oth esalinit y ofth eroo tsolutio n (Hobbsan dWaters ,1964) . Negative effects of high humidity have been reported for crops such as cucumber, tomato,peppe ran deggplan t(Adams , 1984; Bakker, 1988,1990). Calcium nutritionwa s shown to reduce grey mould of rose flowers (Volpin and Elad, 1991),tomatoe s (Elad, unpublished; Stall, 1963),peppe r andeggplant ,bu tit seffect s oncucumbe r greymoul d wasno tobviou s(Yuni set al., 1991). Calciumi susuall yfoun di nhig hconcentration si nth eplan tcel lwal l(Ressigno let ai, 1977).I t is responsible for the integrity of membranes and production of the cell wall (Simon, 1978; Baydounan dNorthcote ,1981) .Calciu mi sinvolve di nstabilisatio no fth e cell walls because of thehig hbindin g of calciumions topecti n (Demarty etai, 1984). The presence of thiselemen t is associated with delayed senescence of plant tissues by reducing respiration rate and ethylene production, and delaying the softening of fruit flesh (Ferguson, 1984), thus making it also less susceptible to degradation by the enzymeso fB. cinerea. Prevention ofmembran edeterioratio n canresul t alsoi nreduce d leakageo fnutrient st oth esurfac eo fth ehos ttissue ,thereb yrenderin gthe mles savailabl e toth epathoge n (Volpinan dElad , 1991). The presence of calcium in the root zone solution does not necessarily ensure high assimilationi nth ecanopy .Provide dtha tcalciu mi spresen ti nth eroo tzone ,transpiratio n ratea swel la sroo tpressur ear eresponsibl efo rit smovemen tt oth ecanop y(Palzkil lan d Tibbitts, 1977; Kirkby, 1979). Calcium-related disorders usually occur when there is interference with the uptake and distribution of the ion (Kirkby, 1979). In our work calcium fertilisation of the cucumber crop resulted in higher concentrations of the element in the upper, young leaves.However , the r.h. dictated the differences between calcium and other treatments.Th eeffec t ofcalciu m ongre ymoul d reduction wasmor e pronounced incucumbe r plantsgrow n under high,tha n underlowe rr.h. ,i n spiteo f the fact thathighe rr.h .i sassociate dwit hmor esever eepidemic so fgre ymoul di ncucumbe r (Yuniset ai, 1990). Theus eo fcalciumnitrat ei sno tth eonl ywa yt oenric hplan ttissue swit hth eCa 2+ion . Gypsumca nb eusefu l for supplyingth eplan twit hcalciu man dfo rreducin gth eseverit y ofgre ymould .I tca nb ea ninexpensiv ewa yo fsupplyin g theio nwhe nplant sar egrow n insoi lo ri nsoilles ssubstrates . Effect ofhormon etreatment so ngre ymoul ddevelopmen t Hormonetreatment sca n affect thedevelopmen t of anepidemi c ingreenhouses .Auxin s areapplie d toensur e fruit setting and development in eggplant or tomato greenhouses. Auxin translocation from meristems, flowers and young fruits is associated with 153 movemento fcalciu mtoward sthes eorgan san dit sincorporatio nint ocells .Auxin ssee m toaffec t thesusceptiblit y of youngfruit s togre ymould . Theeffec t of sprays of Ca(N03)2 with or without auxin, on grey mould of eggplant fruits wasteste di nnon-heate dan dheate dgreenhouses .I nth elatter ,th eincidenc eo f fruit rotwa sreduce db yspray so fcalciu man dN-M- T(Tomaset ,N-metatolyphthalami cacid ) butno tb yth eauxi n4-CP A(Tomatoton ,4-chloro-phenoxyaceti cacid) ;th ecombinatio n of calcium with either of the auxins was better than either treatment alone. The only treatmenteffectiv e in anon-heate d greenhouse was4-CPA .I n anotherexperiment ,2,4 - Dmethano l aminesal t [AlbarSuper ]wa seffectiv e undernon-heate d conditions (Yunis etai, 1991). Integrationo fchemica l andbiologica lcontro l Biocontrol of plant pathogens in integrated pest management programmes is a worth while goal,bu t despite considerable research, only rarely have workable strategies for consistantbiologica l control of plantdisease s havebee n implemented. There areman y reasonsfo r this,bu tmos to f thedifficultie s indevisin gbiologica lcontrol sar erelate dt o the complexities of the systems being managed. Nevertheless, there are increasing economic and social pressures to develop usable biological control strategies (Elad, 1990). Trichoderma harzianwn has proved to be a reliable biocontrol agent of grey mould in cucumber.I n most of theexperiment s thepreparatio n significantly decreased the severity of the disease. In some cases, when the biocontrol was applied with the dicarboximide fungicides iprodione or vinclozolin, there was a trend toward better control in comparison with either agent alone. The slightly improved efficacy of the mixture ofT. harzianum with these fungicides may be due to the presence of resistant B. cinerea strainsi nth egreenhous e(Ela d andZimand , 1991). Theultimat e aim of ourwor kwa st oreduc eth eapplicatio n of fungicides during the growing season without loss of control. For this reason we alternated T. harzianum application with fungicides, which resulted ina leve l ofcontro l similar totha t obtained withth efungicid e treatmentsrecommende d for greymoul dcontro lwithou tan y failures as occurs commonly with Trichoderma treatments alone. Alternation of chemical and biologicalcontro lha dtw oeffects : first,ther ewa sa reductio ni nfungicid e residueso nth e fruits. Second, thepathoge n wasles sexpose d toth efungicide s andtherefor e underles s pressure to develop resistance. Similar results were obtained by Gullino etal. (1991) , whospraye ddicarboximid e+ thira mi nalternatio nwit hTrichoderma fo rcontro lo fgre y mould intomatoe s andstrawberries . Theleve l of theT. harzianum population onleave s andfruit s ofcucumbe rremaine d highi nplant streate dwit hth ebiocontro lpreparation .Th esurviva lo fTrichoderma o nth e phylloplane is a key factor for achieving effective control (Elad and Zimand, 1991). Similarly, McKenzie et al. (1991) concluded that the poor activity often shown by Trichoderma againstgre ymoul d undersever ediseas epressure ,ca nb epartl y explained by its poor survival in thephylloplan e under field conditions. Our preparation ensured satisfactory survivalo fa nefficien t populationfo r 1 monthunde rcommercia lgreenhous e conditions,an da nadequat epopulatio no fth eantagonis twa sals oestablishe di nth enon - treatedcontro l plots.Thi sma yb edu et osecondar ydistributio n ofth epropagule s ofth e fungus in the air. The secondary dispersal may beimportan t for the establishment and survivalo fth ebiocontro l agent,bu tunde rou rcondition si tdi dno treduc egre ymoul di n thecontro lplot s sufficiently. 154 Calculations of relationships between microclimatic conditions and disease control achieved by Trichoderma revealed anegativ e effect of wetness and apositiv e effect of temperatures above 20°C.Thi s mayexplai n the failure ofT. harzianum tocontro lgre y mouldi non eexperiment . Asearc hcoul db econducte dfo risolate so fTrichoderma whic h effectively control the disease under awid erang e of conditions.Meanwhile , effective nessi sachieve db ya combinatio n ofth ebiocontro l agentwit h fungicides. Effect ofreduce dpopulation so fB. cinerea onyiel do fnon-infecte d fruit Totes twhethe rth ediseas ecause dyiel dloss ,experiment swer econducte di ntomat oan d cucumbergreenhouse swit hfungicide s appliedweekly .Significan t controlwa sachieve d with tebuconazole (with or without dichlofluanid), fenbuconazole and diethofencarb + carbendazim. Linear regressions were produced todetermin eth erelationshi pbetwee n greymoul d incucumbe ran dfrui t yield. Datao ndiseas eincidenc ean dyiel do findividua lplot swer e analysedi nal lexperiments .Th efollowin g correlationwer eexamine dfo reac hsamplin g date: (a) %infecte d fruit versus yield of cucumbers in the following week (the week followingdiseas eassessment) ;(b) numbe ro finfecte d fruits versusyiel di nth efollowin g week;(c )cumulativ enumbe ro fdisease d fruits versuscumulativ eyiel di nth e following week; (d)numbe r of infected stem nodes versus yield in the following week; and (e) cumulative number of infected stem nodes versus cumulative yield in the following week. No significant negative correlations were obtained between any disease parameters and yield. The lack of correlation between diseased senescening female flowers of cucumber and decreased yields is due to the nature of the development of fruits in parthenocarpic cultivars of cucumber (Yunis et al., 1991). Abortion of the senescing female flowers is anatura lphenomeno n andcompensatio n in fruit development occurs when some flowers abort in the greenhouses. Only some of the flowers develop to producea full-siz e cucumbereve nwhe nther ear en odiseas eepidemics .Th eplant sreac t similarly when senescing flowers become infected by B. cinerea; in both cases, other healthy flowers develop. It appears that the optimum temperatures for grey mould epidemics in cucumber are in the range (11-25°C) in which fruit production is not affected. An r.h. >85% which promotes epidemics of grey mould, is also a condition underwhic hsatisfactor y cucumberyield s areproduced . Sincether e was nocorrelatio n between low disease level and cucumber yield under these experimental conditions, it can be recommended that fungicides aimed against B. cinerea should beapplie d lessfrequently . Under severeepidemi c conditions,usuall y at the end of the season, grey mould lesions may occlude translocation in the stems causing wilt and death of upper parts of the plant. Therefore, there is only a need for frequent sprays of effective fungicides when a severe epidemic is expected. Plant compensation for lost fruits does notsee m tooccu r in tomato and thequalit y andyiel d of this crop were significantly reduced by grey mould epidemics. Similar results have alsobee nobtaine d withpepper ,eggplant ,strawberry ,bea nan d cut-flowers. Survivalo fJ? ,cinerea duringth ewar msumme r Surveysi ngreenhouse s havebee nconducte d sinceth efirs t detection of dicarboximide- resistant isolates in Israel. At the beginning of each season, the relative incidence of 155 resistantisolate s was lower than that at theen d of the previous season. An increaseo f resistantisolate soccurre d inrespons et ospray swit hiprodion eo rvinclozoli ndurin gth e season, a phenomenon reported also from covered crops in other areas of southern Europe.W ehav enote d failures ingre y mouldcontro li ncucumbe rgreenhouse seve na t thebeginnin g ofth eseason . Although considerable information is available about grey mould, limited attention hasbee ngive nt oth esurviva lo fB. cinerea i na war mclimate .I nregion si nEurop ewher e theclimat e is cool and humid, the pathogen is active during summer in vineyards and greenhouses and in greenhouses during winter and the limiting factor for survival is therefore low temperatures. In warm regions with hot, rainless summers grey mould epidemics occurs in greenhouses during winter. We found that B. cinerea survives in plant debris,particularl y in infected stems.Fo r thefungus'surviva l infected hosts were thebes tecologica lniche ,th epathoge nsurvive di nu pt o 18%o fste msamples .Althoug h survival was poor outside the infected tissue and in fruits and flowers. Conidia on the surfaceo fdebri sprobabl ycanno tsurviv edurin gth esummer .Coley-Smit h(1980 ) found thatunde rles sadvers econditions ,th econidi adi dno tsurviv emor etha n5 3days .Interna l stem tissues perhaps provide an isolated niche which may protect the mycelium from extreme conditions or from other microorganisms. There is also a possibility that dicarboximide-resistant isolates have undergone selection for survival under high temperatures and dry conditions. Therefore, there now may exist in greenhouses a population of B. cinerea different from that in the early 1980s when resistance first appearedi nIsrael .B. cinerea coul db eisolate dfro m varioussusceptibl ecrop san dweed s undersumme rcondition s andthes eplant sma yoffe r anothermean s of summer-survival for the pathogen. Grey mould is seen occasionally in rose greenhouses during the surrmer. In these cases there are probably borderline conditions of humidity and temperature in whichB. cinerea candevelop .Remova l of infected plants at theen do f eachgrowin g seasoni simportan tt oreduc eth eamoun to fsurvivin g inoculum anddela y inth eonse to fepidemic si ngreenhouses . Themai nreaso n for thedeclin ei nB. cinerea indebri s during summeri stha t under such conditions survival of B. cinerea might be poor, as was shown for B. convoluta (Mass, 1969). According to earlier work and our results, no importance should be attributed to survival of B. cinereaa s sclerotia; the latter cannot survive at high temperatures under dry conditions. Moreover, in Israel, sclerotia are found rarely on plants infected by B. cinerea and are never produced during summer (Yunis and Elad, 1989). Concluding remarks Use of the measures dictated by the developed model to reduce or delay grey mould epidemics of fruits or stems will decrease the need for fungicide applications. Future worko nth epredictio n ofgre ymoul depidemic s shouldb eaime d atth edevelopmen to f modelsfo r othercrop sgrow ni nnon-heate d orheate dgreenhouses ,suc ha stomatoes . 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Epidemiology ofBotrytis blighto fmacadami aracemes .Phytopatholog y 62:316-319 . Jarvis, W.R., 1980.Epidemiology . InTh e Biology ofBotrytis. Eds.Coley-Smith , J.R., Verhoeff, K.an dJarvis ,W.R .Academi cPress ,London ,p .219-250 . Katan, R., 1982. Resistance to 3,5 dichlorophenyl-N-cyclicimide (dicarboximide) fungicides in the grey mould pathogen Botrytiscinerea in protected crops. Plant Pathology 31:133-141. Katan,R. ,Elay ,Y andYunis , H., 1989.Resistanc et odiethofencar b (NPC)i nbenomyl - resistantfiel d isolates ofBotrytis cinerea. PlantPatholog y38:86-92 . Kirkby, E.A., 1979. Maximizing calcium uptake by plants. Communications of Soil Sciencean dPlan tAnalysi s10:89-115 . 157 Madden, L.V. and Ellis, M.A., 1988. How to develop plant disease forecasters. In Experimental Techniques inPlan t Disease Epidemiology. Eds.Kranz ,J .an dRotem , J.Springer-Verlag ,Berlin ,p . 191-208. Marois,J.J. ,Redmond ,J.C .an dMacDonald ,J.D. , 1988.Quantificatio n ofth eimpac to f environmento nth esusceptibilit yo fRosa hybrida flower s toBotrytis cinerea. Journal ofth eAmerica n Society ofHorticultura l Science 113:842-845. Mass, J.L., 1969. Effect of time and temperature of storage on viability ofBotrytis convolutaconidi aan dsclerotia .Plan tDiseas eReporte r53:141-144 . McKenzie, K.L., Benzin, D., Dellavalle, D. and Gullino, M.L., 1991.Surviva l on the phylloplane of strains of Trichoderma spp. antagonistic to Botrytiscinerea. Petria 1:133-134. O'Leary, J.M. and Knecht, G.N., 1972. Salt uptake in plants grown at constant high relativehumidity .Arizon aAcadem yo f Sciences7 : 125-128. Palzkill,D.A . andTibbitts ,T.W. , 1977.Evidenc e thatroo tpressur eflo w isrequire d for calcium transportt ohea dleave so fcabbage .Plan tPhysiolog y60:854-856 . Ressignol,M. ,Lamant ,D. ,Salsac ,L .an dHeller ,R. , 1977. Calciumfixatio n byth eroot s ofcalcicol ean dcalcifug e plants:Th eimportanc eo fmembran esystem san dthei rlipi d composition. In Transmembrane Ionic Exchange in Plants. Eds. Thellier, M., Monnier, A., Demarty, M. and Dainty, J. Editions du CNRS, Paris et Editions de l'Université, Rouen,p .483-490 . Salinas, J., Glandorf, D.C.M., Picavet, E.D. and Verhoeff, K., 1989. Effect of temperatures, relative humidity and age of conidia on the incidence of spotting on gerbera flowers caused by Botrytis cinerea. Netherlands Journal of Plant Pathology 95:51-64. Simon, E.W., 1978. The symptoms of calcium deficiency in plants. New Phytologist 80:1-15. Sol,H.H. , 1967.Th einfluenc e ofdifferen t nitrogensource so n(1 )th esugar san damin o acidsleache dfro m leavesan d(2 )th esusceptibilit y of Viciafaba toattac kb yBotrytis fabae. Mededelingen Fakulteit Landbouwwetenschappen, Rijksuniversiteit Gent 32:768-775. Stall, R.E., 1963. Effects of lime on Botrytis grey mould of tomato. Phytopathology 53:149-151. Verhoeff, K., 1965. Studies of Botrytis cinerea in tomatoes. Mycelial development in plantsgrowin gi nsoi lwit hvariou snutrien tlevels ,a swel la si ninternode so f different age.Netherland sJourna lo f PlantPatholog y71:167-175 . Vincelli, P.C. and Lorbeer, J.W., 1988. Relationship of precipitation probability to infection potential ofBotrytis squamosa ofonion .Phytopatholog y 78:1078-1082. Volpin, H. and Elad, Y., 1991. Influence of calcium nutrition on susceptibility of rose flowers toBotrytis blight .Phytopatholog y 81:1390-1394 . Yunis, H. and Elad, Y., 1989. Survival of calcium nutrition on susceptibility of rose flowers to dicarboximide-resistant strains of Botrytis cinerea in plant debris during summeri nIsrael .Phytoparasitic a 17:13-21. Yunis,H. ,Elad ,Y . andMahrer ,Y , 1990. Effect ofai rtemperature ,relativ ehumidit yan d canopy wetness on grey mould of cucumbers in unheated greenhouses. Phyto parasitica 18:203-215. Yunis,Y , Elad, Y and Pressman, E., 1991. Calcium enrichment of fertiliser to reduce grey mould of greenhouse-grown eggplant, pepper and cucumber. Phytoparasitica 19:246(abstract) . 158 Epidemiology ofBotrytis cinereai n gerbera and rose grown in glasshouses A.Kerssies Summary Thedispersa l of conidia of B. cinerea and theirhorizonta l and verticaldistributio n was studiedi ngerber aan dros ecrops ,grow nunde rglass .Conidi awer ecaugh ti nsimpl etrap s based on impaction placed at different heights in each glasshouse and their numbers derived from colonycount safte r incubation. Lesionsdu et oconidia linfectio n wereals o counted on the flowers. No seasonal pattern was found in the numbers of colonies developedfro m trappedconidia ,bu tth enumber so flesion so nth eflower swer eseasonal . Insprin g andearl ysumme rfe w lesionswer eproduced ,wherea sman ylesion s appeared in other seasons. Linear regression accounted for 77 and 81%o f the variation in the numbero flesion so ngerber aflower s inth etw oglasshouse si nterm so frelativ ehumidit y (positively correlated), total incident solar radiation outside the glasshouse (negatively correlated) andag eo fth ecro p(positivel yc orrelated) . Therol eo f waxan dcuticula rmembran ei nth esusceptibilit y offlower s wasunclear . Nocorrelatio n wasfoun d between theamoun t of wax andcuticula r membrane percm 2 andth enumbe ro flesion s ongerber a andros eflowers . Introduction Botrytiscinerea Pers.: Fr. causes damage to ornamentals like gerbera, rose, chrysan themum and several other flower species such as Saintpaulia grown as pot plants (Kerssies, unpublished data).Conidia , ascospores,mycelia l fragments and sclerotiaar e important for dispersal (Jarvis, 1980), although only conidia seem to play a role in diseasedispersa li nglasshouses .Conidi aar edisperse db yai rcurrents ,wate rdroplet s and insects. Necrotic lesions ('spotting') on flower buds and petals are caused by early infections of plant tissue. Studies on dispersal of plant pathogens in glasshouses are scarce(Flunkin gan dSchölte ,1983) .Hirs t(1959 )wa sth efirs tt omonito rdensitie so fair bornespores ,an dB. cinerea conidiawer eamongs tthos etrapped .Frinkin g etal. (1987) studied the dissemination of particles in a glasshouse divided into three bays using Lycopodium sp. Little is known about the factors influencing dispersal of spores of B. cinerea in ornamentals in glasshouses. Frinking and Schölte (1983) showed that the complex dispersalproces sinvolve saspect so fth epathogen ,host ,environmen tan dth eactivit yo f man.Th eai mo fthi sstud ywa st oinvestigat eth epattern so fconidi adispersa li na gerber a and rose crop growing in glasshouses and the effects of environmental factors on dispersal andinfectio n ofth eharveste d flowers during storagean d transport. 159 Materials and methods Measuremento fenvironmenta l conditions Therelativ ehumidit y(r.h. )an dth etemperatur ei nan dabov eth ecro pwer emeasure dan d meanedove r 1 hperiod susin gpsychrometer scouple dt oa dat alogger . Incidentradiatio n outside theglasshous e(Jcm-^day- 1)wa smeasure db y aKip psolarimete r 8m abov eth e ground atth eResearc h Station for Floriculturei n Aalsmeer.Th ewindspee d outsideth e glasshousei nAalsmee r wasmeasure d andmeane d over 1 hperiod susin g atachomete r 8.5 mabov eth eground . Dispersalan ddistributio no fB. cinerea The dispersal and distribution of B. cinerea in a gerbera crop grown on rockwool was studied in two glasshouses, one of 100m 2 in Aalsmeer (glasshouse A;48 0 plants,cv . 'Terrafame') andon eo f35 0m 2i nVleute n(glasshous eV ; 2000plants ,cvs . 'Rosamunde' and 'Maria').Th edensitie so fB. cinerea conidi ai nth eai ro fth eglasshous ewa sstudie d betweenApri l 1988an dOctobe r 1989usin gtw omethods . Inmetho d 1,spor etrap swer e distributed withinan dabov eth ecrop si na regula rpatter n (Kerssies, 1990); inmetho d2 , thenumbe ro f lesionso npetal so fgerber aflower s wascounte d after harvest. In glasshouse A, spore traps were exposed 63 times over a period of 545 days at .100 .200 A 300 400 500 t î t t SU AU Wl SP Time in days after t=0 Fig. 1. Numberof colonies per spore trap(n=24) placed within the crop from 8.30-16.30 inglasshouse A in 1988-1989. SP,SU, AU, WI: beginningof spring (21 March), summer (21 June),autumn (21 September) andwinter (21 December), respectively. 160 daytime and 55times ove r a period of 545 days at night. Rowers were harvested and lesions were counted 46 times over a period of40 5 days.I n glasshouse Vspor e traps were exposed 49 times over a period of 439 days, at daytime only. Flowers were harvested andlesion swer ecounte d4 5time sove ra perio d of37 1 days. Statistical analysis The mean numbers of colonies on 53 trapping occasions over 545 days and mean numbers of lesions ongerber a petals in 36sample s over 371day s (glasshouse A)wer e the dependent variables [ln(N+l) transformed] in regressions against environmental variables.Th evariable suse di nth ebes tequatio ndetermine dwit hth edat ao fglasshous e Awer eals ouse dfo rth edat afro m glasshouseV (onl yfo rth enumber so flesions) ,wher e lesionso npetal swer ecounte d3 7time sove ra perio do f39 0days . Results Colonies Figure 1show s thenumbe r of colonies perspor e trap within thegerber a cropbetwee n 08.30 and 16.30ove r aperio d of 545day s in glasshouseA .Th enumbe ro f colonieso n — D— Aalsmeer Vleuten (n-24) (n-30) 500 100 200 300 400 500 t t t Time in days after t=0 Fig. 2. Number of lesionsper flower formed on ten petals of gerbera flowers in glasshouseA (n=24flowers) and V(n=30flowers, t=t -llOdays), in1988-1989. SP, SU, AU,WI: beginning of spring (21 March), summer(21 June), autumn(21 September) and winter(21 December), respectively. 161 e o w ID .O E c c CO O 100 200 300 Time in days after 01-01-1991 Fig. 3. Numberof lesions formed on rosepetals (n=100flowers) in 1991. spore traps 0.5 m and 1.5 m above the crop showed the same patterns. High and low numberso fcolonie salternate drapidl yove rtime .Th enumbe ro fcolonie si nth edaytim e was significantly correlated with thosea tcorrespondin g nights,bu t about 3 to 4 times higher. The mean number of colonies over time trapped in glasshouse Vbetwee n 8.30 and 16.30 was low compared to those in glasshouse A. Peaks could hardly be distinguished.Mea nnumber sfluctuate d overtim efro m 0- 7 colonie spe rspor etrap . Lesions Thenumbe ro flesion sforme do nte npetal so fa singl egerber aflowe rvarie dfro m0 - 37 5 inglasshous eA (n=2 4flowers ,Fig .2 )an dfro m 0- 45 0i nglasshous eV (n=3 0flowers , Fig. 2). During spring and early summer (t=350 -1=460 in glasshouse A and t=200- t=330 in glasshouse V) very few lesions occurred on petals, though many lesions appeared atothe rtimes .Toward sth een d ofth erecordin g period thenumbe ro f lesions was still increasing and the pattern was similar for the two glasshouses. The mean numbero flesion sove rtim ewa sles svariabl etha nth emea nnumbe ro fcolonies . Regressionanalysi s Agoo dlinea rregressio n modelexpressin g fluctuations inth enumbe ro fcolonie scoul d notb emad ewit hth eindependen tvariable sused .Th eadjuste d R2value swer eal lbelo w 0.4.Th ebes tlinea r regression model for thenumbe r of lesions (glasshouse A) utilised threevariables :MRH ,M S andt andgav ea n adjusted R2o f 0.81 (P<0.05).MR Hi sth e 162 meanR Hfo rday s6, 7an d8 befor eth eda yo fharvestin ggerber aflowers ;M Si sth emea n incidentradiatio nfo rday s1, 2an d3 befor eth eharves tday ;whil et i sth eag eo fth ecrop . Thismode lfo rth enumbe ro flesion so ngerber apetal sexplaine d 77%o fth evariatio ni n glasshouse V (adjusted R2 of 0.77; P<0.05). The levels of the two linear regression modelsar esignificantl y different atP<0.05 .Thi ssuggest stha tth etyp eo fglasshous eha d a significant effect onth elinea rregressio n model.Th ebes tlinea rregressio n model for the number of lesions on gerbera petals for the combined glasshouses is (adjusted R2=0.77; P<0.05): Y= -6.6(±1.5 )+ 0.12(±0.03)*MR H- 0.00074(+0.00017)*MS +0.0042(±0.001)* t +0.98(+ 0.23)*G(3) , inwhic hG i sth eglasshous eeffec t (J-Jcm2),G i s0 o r 1. Thisi sth eeffec t of differences betweenglasshous eA an dV i nsize ,height ,productio n system,locatio netc . Preliminaryexperiment s withros eflower s Astud yo fth edispersa lan ddistributio no fB. cinerea i na ros ecrop ,wher ecro pstructur e is totally different from a gerber acro p on rockwool was started in January 1992i n a glasshouseo f 300m 2i n Aalsmeer (2400plants ,cv . 'Sonia') andend s inJanuar y 1993. Observation dones ofar , showed that inth eros ecro phig h and lownumber s oftrappe d sporesals oalternate drapidly .Th enumber so flesion so nros epetal sar elo wcompare dt o thoseo ngerber a petals (<3,Fig .3 )an d onlyi n September andOctobe r(1991 ) 10 to4 0 lesionswer ecounte d onpetals . CM E o 20 <0 "™üz*!* iwtta^wffw*. in - TpspS HM |»|H*F E MWMMWlhWH* 3 163 Discussion In the spore traps of glasshouse A, the increasing number of colonies over time was attributed to the increasing quantity of dead plant tissue which accumulated as the gerberacro p aged (> 6months ) andth erise i nr.h .i n thecro pt obetwee n 85an d 100% whenth ecanop yclosed .Change si nth enumber so fcolonie sove rtim ecoul db ecause d bychange si nenvironmenta lcondition si nth eglasshouse .Difference s inth enumber so f coloniesbetwee n samplestake nb yda yan dnigh ti nglasshous eA coul db eexplaine db y increasedai rmovement si nth eglasshous ei ndaytim edu et olabour ,ope nwindows ,ope n doors and higher temperatures. The differences in the numbers of colonies between glasshouseA an dV wer edu et oth edifferenc e inproductio nsystems .Th eexposur etim e of gerbera flowers (7 - 14days ) was longer than for the spore traps (< 16hours ) and therefore thelatte rwa smor esensitiv et orapi d changesi nconditions .Althoug h Salinas etal. (1989)showe dtha ton econidiu mca ninduc ea lesio no na gerber apetal ,significan t correlations (P<0.05 ) between thecolonie s and lesion numberswer e notfoun d inthi s study.Th elac ko fcorrelatio n mayb edu et odifference s inexposur etim ebetwee n traps andflowers , todifference s inth epositio n oftraps(vertical )an dflower s (horizontal)an d to variation in unfavourable conditions for germination. This is in accordance with the findings of Salinas etal.(1989) . In both glasshouses, the numbers of lesions depended more on the season than the numberso fcolonie sobtaine d(Fig . 1 and2) .Th enumber so flesion sfro m thesam eday s ofth eyea ri nglasshouse sA an dV a tlocation s thirtykilometre sapar twer e significantly correlated and not significantly different (r>0.30 at P<0.05 and n=41). Frinking et al. 0.40 0.30 O) E, CM Eo Pi ,_ UJ 0.20 (B "-" 1 ' Q- X <0 , 1 1 1 v * 0.10 -- - ! 1 • 1 . 0.00 l I !.: • . Sonia Madelon Frisco Mercedes Motrea Cultivar Fig. 4b Amountof wax per cm 2rose petal (mg, n=6)of five cultivars. '> ')determine daccordin gt oth emetho ddescribe db ySilv aFernande set al., 1964. 164 0.40 O) E £ 0.30 - o Q. (D S 0.20 e '""•".•;ï"i (1987) suggested that spores of B. cinerea could be transported rapidly throughout the glasshouse. Zadoks (1967) and Frinking (1991) stated that fungi can enter and leave glasshouses veryeasil ybecaus eo fa continuou sexchang eo f airbetwee n theglasshous e andth eoutsid eenvironment . Theseresult ssugges ttha tth edensit yo fspore san dth er.h . areno tth emos timportan tvariable sregulatin g numbero flesions ,bu t otherfactor s such asa combinatio no fradiatio n andr.h. ,th estructur ean dcompositio n ofth ecuticle ,o rth e water relations (water potential, osmotic potential) in the flower. In spring and early summer,flower sma yfor mthicke rcuticle san dmor ewa xdu et ohig hvalue so fradiatio n and low r.h. (Baker, 1974; Kolattukudy, 1985). Germinated spores of B. cinerea may havemor edifficult y topenetrat eth ecuticl eo fgerber a petals in spring ansumme rtha n inautum nan dwinter .Additionally ,conidi ao fB. cinerea onth eflowe r surfacema yhav e beenaffecte d byradiatio n andrelativ ehumidity . Therol eo f waxan dcuticula r membrane inth e sensivity of flowers for B. cinerea is still unclear. So far no correlation is found between the amount of wax and cuticular membranepe rcm 2an dth enumbe ro flesion so ngerbera -an dros eflower s (Fig. 4a,b,c) . References Baker,E.A. , 1974.Th einfluenc e ofenvironmenta l onlea fwa xdevelopmen t inBrassica oleraceavar . gemmifera. NewPhytologis t73:955-966 . Frinking,H.D. , 1991.Aerobiolog y of 'closed' agricultural systems.Gran a30:481-485 . 165 Frinking,H.D . and Schölte,B. , 1983. Dissemination of mildew spores in aglasshouse . PhilosophicTransaction s ofth eRoya l Societyo fLondo nB 302:575-582. Frinking, H.D., Gorissen, A. and Verheul, M.J., 1987. Dissemination of spores in a glasshouse:Patter n orchaos ?Internationa lJourna l ofBiometeorolog y 31:147-156. Hirst, J.M., 1959. Spore liberation and dispersal. In Plant Pathology: Problems and progress 1908-1959. Ed. Holton, CS. University of Wisconson Press, Madison p.529-538 . Hollaway, P.J. and Baker, E.A., 1968. Isolation of plant cuticles with zinc chloride- hydrochloricaci dsolution .Plan tPhysiolog y4 3:1878-1879 . Jarvis, W.R., 1980. Epidemiology. In The Biology of Botrytis. Coley-Smith, J.R., Verhoeff, K.an dJarvis ,W.R. ,eds .Academi cPress , 1980p .219-250 . Kerssies,A. , 1990.A selectiv e medium tob euse d in aspore-trap .Netherland s Journal ofPlan tPatholog y96:247-250 . Kolattukudy,P.E. , 1985.Enzymati cpenetratio no fth eplan tcuticl eb yfunga l pathogens. AnnualRevie wo fPhytopatholog y23:223-250 . Salinas,J. , Glandorf, D.C.M., Picavet, F.D.an d Verhoeff, K., 1989.Effect s of temper ature, relative humidity and age of conidia on the incidence of spotting on gerbera flowers causedb yBotrytis cinerea. Netherland sJourna lo fPlan tPatholog y95:51-64 . SilvaFernandes ,A.M. ,Baker ,E.A .an dMartin ,J.T. , 1964.Studie s onplan tcuticle .VI . Theisolatio n andfractionatio n ofcuticula rwaxes .Annal s ofApplie dBiolog y53:43 - 58. Zadoks, J.C., 1967. International dispersal of fungi. Netherlands Journal of Plant Pathology 73, Supp.1:61-80 . 166 BIOLOGICAL CONTROL Biological control ofBotrytis: state-of-the-art B. Dubos Summary In 1951, it was shown that fungi, bacteria and actinomycetes were able to limit the infection ofB. cinerea onlettuc eleave sthoug hi twa sno tunti lth e 1970stha tbiologica l control of this pathogen on grapevine, apple and strawberry was developed using Trichodermaspp . The number of target crops has now expanded greatly and the range of antagonists widenedt oinclud eyeast san dbacteria .Thoug hsometime sunsuccesful , thefiel d results obtained havebee n sufficiently encouraging tohav ele dt oa nexaminatio n of themode s ofactio no fantagonisti corganism s towardsth epathoge n anda nevaluatio no fth e effect ofenvironmenta l conditions onthei rbehaviour .Thes estudie smad ei tpossibl et oselec t andgeneticall yimprov eth eantagonisti cstrains .Industria lproductio no fbiopesticide si s nowestablishe d andresearc hha sbee nundertake n toimprov eproduction s Nevertheless,th eapplicatio n ofbiologica l controlagent sagains tB. cinerea i sonl yi n itsinfancy . Todetermin eth eprincipa lresearc hprioritie swhic hwil llea di nth efutur e to theregula rus eo fbiologica lcontro lfo rmanagemen to fgre ymoul dcause db yB. cinerea, arevie w ofpresen tknowledg ei sgive nhere . Introduction Agriculturewil lhav et ob emor eeconomical ,bu tals omor erespectfu l toth eenvironmen t in the next century and during this transition period biological control will probably become a recognised alternative or complement to chemical control if microbiologists andplan t pathologists are sufficiently determined andconvincin g in dealing withthos e whoshap eagricultura lpolicies . Asa polyphagou s andubiquitou s fungus Botrytis cinerea Pers.:Fr .i sresponsibl e for various disease symptoms in a wide range of cultivated plants of great economic importance.I ti sa facultativ eparasit ean dcause sseriou sdisease so ndirectl yconsumabl e products, such as fruits and vegetables and it is therefore a perfect candidate for biologicalcontrol . This overview has two aims: firstly, to review the state-of-the-art knowledge and secondly to define priority research directions for establishing the practical use of biologicalcontrol . State-of-the-art knowledge Thefirs tstep si nbiologica lcontro lwer etake nb yWoo d(1951 )wh oinoculate dsenescen t lettuce leaves with antagonists {Fusarium sp., Pénicillium claviforme) to prevent the primary establishment ofB. cinerea. Positiveresult sprovidin g effective diseasecontro l undercold-fram e conditions were obtained.Woo dbelieve d that saprophytic activityo n dead lettuce tissue was,t o a largeextent , responsible for disease control under natural L 169 conditions. Using the same principle, Newhook (1957) controlled grey mould on tomatoes grown in a glasshouse by spraying a spore suspension of Cladosporium herbaruman dPénicillium sp .o nth efloral debri sattache dt oth efruit . Bhattan dVaugha n (1962)obtaine d nearly40 %reductio n ofstrawberr ygre ymoul d throughth eapplicatio n ofC. herbarum tosenescen tflower s underglasshous econditions . Ruinen (1961)dre wattentio n toth elac ko fresearc h onth esaprophyti cmicroflor a of aerial plant surfaces. Subsequently, the role of the saprophytic microflora in the biological equilibrium was established and the dangers of its destruction, particularly throughth eirrationa lus eo ffungicide s becameknown .Blakema nan dFrase r(1971 )an d Blakeman (1972) pointed toth e antagonistic function of bacteria againstB. cinerea on chrysanthemum andbeetroo tleaves ,bu talthoug ho ftheoretica limportance ,thes eresult s wereno tfollowe d bypractica l applicationsi nth econtro lo fB. cinerea. Paradoxically, further research on biological control of B. cinerea from the 1970s concerned thefunga l genus Trichoderma whose antagonistic properties had long been known,bu twhic hwa sno tusuall y acomponen t ofth emicroflor a of thephylloplane .A t the Botrytis symposium in Aberdeen, Scotland in 1982, Tronsmo and Dubos respectively, presented the first results concerning control of dryro t of apples andgre y mould ofth egrapevin eb ystrain so fTrichoderma. Sincethen ,wor ko nthi santagonisti cgenu sha sprogresse d rapidlyan dth enumbe ro f Botrytishost svastl yincreased .I nparallel ,th eantagonisti cpropertie so fmicroorganism s found as part of the phyllosphere microflora have been studied. The interaction of pathogenic fungi and antagonists in vitro,particularl y Trichoderma spp., have been studiedextensively ,bu tthes ewil lonl ybriefl y mentioned. Three methods of antagonism are usually studied: 1) antibiosis - production of diffusing substances withfungistati c orfungicida l action;2 )competitio n- theabilit yt o exploit environmental factors at the expense of the parasite; and 3) mycoparasitism - destruction or alteration of thehypha e of the pathogen, involving physical contactan d prédation,followe d byenzymati clysis . It should benote d thatreaction s invitro between antagonists and B. cinerea depend greatly on the growth conditions and especially the nutrient status of the medium, temperaturean drelativ ehumidity .Th eimportan teffec t oftemperatur ewa sshow ni nth e interactionsbetwee n Trichodermaan dBotrytis (Duboset al., 1983) . MostTrichoderma strains possess antagonistic properties between 15° and 25°C, but strains with similar properties above and below these values are relatively rare; the same occurs in anta gonistic yeasts and epiphyticbacteria .Temperatur etherefor e isa ke ylimitin gfacto r in thechoic eo fstrain ,particularl y whenth elatte ri sfo r usea spost-harves ttreatment . Othertest sar eappropriat ewhe nselectin gstrain sfo r aparticula rphas eo fth ediseas e cycle.In vitro,Koh l and Schlösser (1989) selected strains of Trichoderma, capable of destroying sclerotia of B. cinerea at low temperature, with a view to destroying the pathogen during the survival phase. Peng and Sutton (1990) assessed the efficacy of antagonists by inoculating strawberry leaf disks first with the antagonist, then with B. cinereaan dsubsequentl yassesse dsporulatio no fth epathoge n onth edisks . Initial in vitro screening, however impressive the candidate antagonist, is often misleading;strain s whichma yb econsistentl yefficien t invitro ma yb eunsatisfactor y in thefield , and theconvers e is also true.Thi s observation may indicate theexistenc eo f othertype so fantagonisti cactio noperatin ga tth etarge ttha nthos egenerall yinvestigated . 170 Variousmodel sstudie dan ddifferen t modeso fapplyin gantagonist s Bio-preparations of antagonists sprayed to protect herbaceous plantpart s and fruits from infection by B. cinereaar e shown in Table 1. Postharvest treatment of fruit, vegetablesan dflower s bydippin go rsprayin gwit hantagonist sar esummarise di nTabl e 2. Soil-born inoculum of B. cinerea requires a different approach. For damping off diseases,th eantagonis tma yb eapplie da sa see dcoatin go rincorporate ddirecd yint oth e soil. A compost rich in Trichoderma is often used to destroy thepathoge n or limit its saprophyticdevelopmen t oncro pdebri so rplant swhos esensitiv e organsar ei ncontac t withth esoil .Fo rsoilles scrops ,particularl ycarnation ,th eantagonist sar esupplie di nth e nutrient solution (Table3) .Metabolite s produced by antagonistic microorganisms have alsobee napplie d for biocontrol (Table4) . Themos textensiv ean dprolonge d work onbiologica lcontro l ofB. cinerea hasbee n performedi nvineyard si nFrance ,Italy ,Spain ,Israe lan dothe rcountries . Thisexperienc e willtherefor e beth ebasi sfo rou rfurthe r discussion ofbiologica l controlo fgre ymould . Table 5 shows the results of nine years of experimentation on cv. Sauvignon in the Bordeaux region (Dubos, 1987).Th e protection we obtained proved excellent in some Table 1. Biologicalcontrol ofB. cinereaand other Botrytis spp. byspraying antagonists on aerialplant parts Hostplant s Antagonistic microorganisms Strawberry Trichoderma spp.,T. viride, Gliocladium roseum, Pénicillium spp.,Gladosporium herbarum, compostextrac t Apple Trichoderma spp., Bacillussubtilis Cucumber T. harzianum Grape T. harzianum, epiphyticbacteria ,Bacillus subtilis Cyclamen T. harzianum Beans (B.fabae) T. viride Onion Gliocladiumspp., Trichoderma spp., Aureobasidiumpullulons Table2. Biologicalcontrol ofB. cinereaduring storage Hostplant s Antagonistic microorganisms Apple Cryptococcusspp. ,Candida guilliermondii, T. viride,Acremonium breve Kiwi Trichodermaspp. ,Gliocladium spp.,Paecilomyces spp. Pear Trichoderma spp.,Gliocladium spp., Paecilomyces spp. Carrot T. harzianum Rose Exophialajeanselmei, Coryneform-type bacterium 171 Table 3. Biologicalcontrol ofB. cinerea, usingantagonists applied to soil and as a seed- coat. Hostplant s Antagonistic microorganisms Tulipbulb s Gliocladium roseum,T. hamatum,Streptomyces spp. Pinus massoniana Pseudomonasspp. ,Bacillus cereus Lettuce Streptomyces spp. Carnation Bacillussubtilis, Streptomycesspp . Strawberry Trichoderma spp. Miscellaneous Aphelenchoi'des composticola*) *)Nematod e Table4. Biological control of B. cinerea usingmetabolites produced by antagonistic microorganisms. Hostplant s Metabolitean dsourc e Rapeseedlin g Pyrrolnitrin (Pseudomonas cepacia) Kiwifrui t Metabolites of Trichoderma spp.,Paecilomyces spp., Gliocladium spp. Pear Metabolites ofTrichoderma spp.,Paecilomyces spp., Gliocladium spp., Pyrrolnitrin (Pseudomonas cepacia) Apple Pyrrolnitrin (Pseudomonas cepacia) years, or inadequate (1983 and 1984). In all cases the efficacy was lower than that provided by dicarboximides in the absence of strains of B. cinerea resistant to these fungicides. Similar results havebee n obtained in other regions,an d for other crops (strawberry, apple,tomato ) andfo r otherantagonists . Theseinconsisten t results rapidly guided research toinvestigation s about themode s ofactio no fantagonist san dth erelationship sbetwee nhost ,pathoge n andantagonist . Modeo factio no fantagonist si nplant a Apart from the phenomena of competition, antibiosis and mycoparasitism which have beenestablishe d experimentally, theinductio n ofdefenc e mechanisms in thehos tplan t shouldb econsidere d inthi scontext . Competition Competition is probably one of the most studied modes of action and is the most important for control ofgre y mould.B. cinerea often first develops as asaprophyt e on senescentleave s and flowers of strawberry,tomat o andgrapevines .Fro m these nutrient bases,i tproduce sa myceliu m whichinvade sth ehealth ypar to fth eplant . 172 Table 5. Summary ofresults ofbiological control ofB. cinerea ongrape cv. Sauvignon inthe Bordeaux area over 9 years. Year roti ntreate d roti ncontro l efficacy of plots(% ) plots(% ) treatment(% ) 1976 9.4 33.6 70 1977 9.5 31.4 71.7 1978 7.6 24.6 69.3 1979 4.2 22.5 81.1 1980 7 15.9 57 1981 9.7 24 59.6 1982 4 27 84 1983 25.2 34 26 1984 27 35 22.9 It has been shown in many crops that Trichoderma is a pioneer coloniser of senescing floral caps on developing grapes and prevents the saprophytic colonisation by the pathogen. Such competition for nutrients is thought to occur in postharvest control of apple by Cryptococcus laurentii, a yeast which rapidly colonises wounds using the nutrients necessary for thedevelopmen t ofB. cinerea (Roberts, 1990).Competitio n for carbon and nitrogen sources between cells of C. laurentii and B. cinereacoul d be responsiblefo rcontrol ,a sconidia lgerminatio no fsom estrain so fB. cinerea i sknow nt o beaffecte d byth ecarbohydrat e andnitroge n statuso fthei rimmediat e environment. Production ofantifunga l andfungistati c compounds Whileth eproductio no fantifunga l andfungistati c compoundsin vitro i swel lknown ,thi s typeo fantagonis m israrel ymentione d inbiologica lcontro ltrials .Thi sma yb ebecaus e it is difficult to demonstrate this activity in vivo. These metabolites were always consideredt ob eantibiotics ,an dbecaus eth eus eo fantibiotic si nagricultur ei sprohibite d inmos tcountries ,researcher stende d nott oselec tstrain sexpressin g metabolitesfo r use on the aerial parts of plants. In the late 1980s, there was a shift in attitude, since metaboliteswer ebein g used for biologicalcontro lo f fruit storagedisease s (Janisiewicz andRoitman , 1988). Ferreira(1990 )achieve d ahig hleve lo fprotectio n ofgrap eberrie s by applying certain strains of epiphytic bacteria and the mode of action in this casei s thoughtt ob ea nantibioti cwhic hinhibit sgerminatio n ofB. cinereaconidia . Mycoparasitism Thisphenomeno nha sbee n described hardlyfo rbiologica l control ofB. cinerea, except inth ecas eo fgre ymoul do fgrapevine .Th eincidenc eo finfecte d bunchesi ntreate dan d controlplot susuall yd ono tdiffe r statisticallybu tth eseverit yo fro ti nth ebunche si sles s in the former. This probably indicates that from the time ofripening onwards , when inoculum is abundant, Trichoderma does not reduce the number of infection sites but prevents their development. Microscopical observations have shown that on rotting grapes the antagonist sporulates on the margin between the necrotic and healthy area, thus preventing spread of B. cinereat o healthy grapes in the bunch. Trichoderma therefore behaves asmycoparasite . 173 Improvingth eefficac y ofbiologica l controlagent s When using T. harzianum to control grey mould of grapes, all the data suggest that a conidial concentration in the order of K^.ml1 is themos t suitable for bio-preparations (Dubos etal., 1983,Gullin o etal., 1983).Belo w this concentration, arapi d declinei n efficacy is observed, and no particular improvement seems to be achieved above this inoculum density. An identical result was obtained by McLaughlin etal. (1990 ) with Candidasp .t oreduc elesio ndevelopmen ti napple scause db yB. cinerea durin gstorage . Adjuvants are added to bio-preparations to enhance the intrinsic properties of the antagonists,o rt oassis ti nit smaintenanc ei nth ephyllosphere . McLaughlinet al. (1990)note dtha tth eadditio no fsal tsolution simprove dth e efficacy ofCandida sp .o napple si nstorage ;CaCl 2wa sth emos teffective , andwit hcertai nstrain s it was possible to reduce the concentration of the inoculum 100-fold. This effect was unrelatedt oth eosmoti cpotentia lo fth esolutions .Gullin oetal. (1989 )studie dnutritiona l effects onth eantagonisti c activityo f Trichoderma spp.o ngrape . Tronsmo(1986 ) found that Trichoderma was moreefficien t against grey mould in apple and strawberry, if the conidialsuspensio n sprayeddurin gblossomin gcontaine d solublecellulose . Strains selected for use on grape, are generally ineffective on strawberry (Gullino, 1983)an dTronsm o(1986 )cam et oth esam econclusio nregardin gcontro lo fdisease so n applean dstrawberry . Asingl estrai nma ygiv ehighl yvariabl econtro lfro m onevineyar dt oanother ,o rfro m one year to another. Favourable results obtained with a strain of T. harzianum against greymoul di n avineyar d inth eAtlanti cregio n willno tb eapplicabl e inmor enortherl y vineyards, such as in the Champagne region because the antagonistic potential of Trichoderma strainsi shighl ytemperatur edependent ,an doperate sbes tbetwee n 15° and 25°C; in northern vineyards, rot often develops at temperatures lower than 10°C. Similarly, theinferio r results obtained in 1983an d 1984i n the Bordeaux area with the T. harzianumreferenc e strams were due to very dry climatic conditions and to an explosion of grey mould before harvesting. Theefficac y ofbiologica l control hasbee n subsequently improved from 20t o70 %b yus eo fa 'thermophilic' strain. Whereprimar y inoculum,whic h arises from within thecrops ,play s anessentia lrol e on the development of an epidemic, as with B. squamosa in onions, Fokkema etal. (1991) selected strains of Trichoderma or Gliocladium inhibiting sporulation of the pathogen by colonising dead leaf parts and other crop residues. Similarly Kohl and Schlösser (1989) selected Trichoderma strains for destruction of sclerotia of B. cinerea whichar eth eprimar yfocu s ofth ediseas ei nman yplants . To offer a mixture of antagonistic strains for controlling the same disease is an unattractive option andtherefor e attemptshav ebee nmad et oimprov estrains . Strainenhancemen t Mutagenic agents have been used by Davet et al. (1991) to obtain mutants of T. harzianum having either ß-1-3glucanas eo ra chitinas eactivity ,o rboth .I twa snote d that although theantagonisti c activity wasimprove d in thelaboratory , noimprovemen t in efficacy was detected in the vineyard, probably because these strains were less competitive in the phylloplane than wild type strains. Similar results with mutants resistantt obenomy lhav ebee nobtaine d (Mighell etal., 1991). Protoplast fusion techniques combinedesirabl e attributes from two ormor eparenta l 174 strainsi nth eprogenies .B ythi smethod ,Mighel let al. (1991)obtaine dsevera lsomati c hybrid strains of T. harzianum which combined colour and/or fungicide resistance markers. The use of these fusion products in the vineyard gave variable degrees of survivali nth ephylloplane ,bu tth eaptitud eo fwil dtype swa salway sbetter . Cloning andcharacterisatio n ofgene sinvolve d indi egenera lmetabolis m contribute to the development of expression vectors for these antagonistic species. With this objective Heidenreich and Kubicek (1991) havestarte d to cloneth eT. harzianum gene involved in mycoparasitism of B. cinerea but atpresen t no practical results havebee n obtained. Invie wo fth ecomplexit yo fth eproblem scurrentl yassociate dwit hchoic eo fsuitabl e strains, the need to control B. cinerea more effectively, and the desire to conserve the advantageso fbot hchemica lan dbiologica lcontrol ,integrate dcontro lprogramme shav e beendevised .Thi swa sachieve dfo r greymoul d ofgrap eb y alternating biologicalwit h T. harzianum and chemical treatments,eithe r by use of thefou r standard sprays,o rb y using the data derived from a predictive model; it was also achieved by mixing the antagonists with lowlevel s of cyclicimide s (Dubos etal., 1983 ; Gullino, 1983).Thes e strategies have been used successfully in many grape growing countries and make it possible to decrease the amount of fungicides used, and minimise the risk of the development of resistant strains.Result s on strawberry, cucumber and tomato obtained with theT. harzianum strain T39 selected by Elad etal. (1991 ) were identical, and a formulation of this strain has been produced industrially by the Makteshim company (Israel). Limited information is available about industrial production. Liquid culture media are generally used for bacteria and yeasts but these may not be appropriate for filamentous fungi because their physiology is often highly disturbed. In the case of T. harzianum, chlamydospores were mainly produced (Arteconi et al, 1991),bu t this typeo f propagule hasno tbee n field-tested. Jim etal. (1991)develope d mediayieldin g high levels of T. harzianum in submerged cultures, but only 1t o 10%o f the conidia survived drying. Addition of osmoticants to the medium allowed production of dessication-tolerant conidia. Usually,4 0t o 50%o f conidia from osmotically-enhanced mediasurviv edrying .Via let al. (1991)studie dth eproductio no fconidi ao fT. harzianum in a solid medium (beet pulp). They showed a relationship between decreases in respiratory activity and yield of conidia exhibiting good storage activity after 120 h. Ultrastructural studies also revealed that these conidia had reached astat e of advanced maturity. Isbiologica l controlo fgre ymoul dcommerciall yviable ? Despite the numerous positive results, biological control of B. cinereai s not used extensively. Nevertheless, the company Makteshim has been developing Trichoderma since 1985. T.harzianum has been tested in Israel, Europe, South America and New Zealand on grapevine, cucumber, tomato, strawberry, bean, carrot and kiwi fruit and resultswer econsisten t incontrollin gB. cinerea. Theirbio-preparatio n calledTriehode x is registered in Israel and Yugoslavia. Another biofungicide called Mycostop (White et al., 1990) based on Streptomyces griseovirides should obtain registration for the controlo fB. cinerea inlettuc e inFinlan d after toxicologicaltrials . By present knowledge, the use of antagonistic biocontrol agents in association with conventionalfungicide s seemst ogiv esatisfactor y controlo fgre ymoul di na wid erang e ofcrops ,an dthi sdevelopmen tmus tb edemonstrate dconvincingl yt oth epolicy-makers . 175 Concluding remarks Chemicalcontro li sstil luse droutinel yan donl yrarel yi sconsideratio ngive nt oit s effects on saprophytic microflora, whose vital role in the biological equilibrium is more fully appreciated. Present-day management practices for controlling diseases, particularly greymould ,i s stillfa rfro m theconcep to fintegrate dcontrol ! Theepidemiologica lapproac hi nfield condition sperforme dunti lno wo ngre ymould , is a very conventional approach which is limited to the transient observation of phenomena. SinceB. cinerea attack sa wid erang eo fhos tplant san di subiquitous ,epidemiologica l studies ofgre y mould should bedon eo n awide r scalean dcomparison s madebetwee n different climatic regions. During their development cycle, pathogens move through various physiological states which are not receptive to the action of an antagonist. Vulnerablephase si nth elif ecycl eo fth epathoge nmus tb e identified. Noneo f themodel sinvolvin gcompetition ,mycoparasitis m andantibiosi shav ebee n analysed in detail and there is awid eresearc h areafo r studies of nutrient sources,th e enzymesinvolve d andth etyp eo fmetabolite shavin g afungicida l orfungistati c action. Inth epas tmos twor ko nBotrytis spp .ha sbee nperforme d bymycologist swh ohav e studied thefilamentou s fungi, theyeast san dbacteri ahav ereceive d lessattention . Work onthes eantagonist s hasno wstarte d andrequire smuc hmor eresources . Although new methods in molecular biology do exist to improve antagonistic activities, the genes involved in antagonism are still to be identified. Studies of the preciseeffec t of antagonists onth epathoge n aretherefor e of greatimportance . Industrialproductio no fa biopesticid ei sdifficul t andth eorganis mmus tb ei na stabl e physiological state to survive in the formulation until it is used. Transmission electron microscopyha smad ei tpossibl et oasses sth equalit yo fa biopesticid e(Via let al., 1992), andthi smetho dcoul db efurthe r developedi nqualit yassessments . The future perspectives for biological control in general, and especially against grey mould, lie in a wide-ranging approach, which should include research organisations, publican dinterprofessiona l bodies andindustrialist sfro m thecro pprotectio n sector,s o that techniques may be standardised and models validated. It seems incomprehensible that, ata tim ewhe n ourgovernment s arebecomin g moreawar eo f thenee dfo r anagri culture system whichi seconomica l andmor erespectfu l toth eenvironment , biological controli sstil lno ta playe ro nth eteam ! References Arteconi, M., Bergonzoni, P., Perrone, L., Mallegni, C, 1991. Trichoderma and Gliocladiumproductio n in submerged culture with the use of and automatic fermenter.Proceeding s Trichodermaan dGliocladium Workshop ,Belgirate ,Italy ,wp . 136. Bhatt,D.D .an dVaughan ,E.K. , 1962.Preliminar yinvestigation so nbiologica lcontro lo f greymoul d(Botrytis cinerea) ofstrawberries .Plan tDiseas eReporte r46:342-345 . Blakeman,J.P .an dFraser ,A.K. , 1971.Inhibitio n ofBotrytis cinerea sporesb ybacteri a onth esurfac e ofchrysanthemu m leaves.Physiologica l PlantPatholog y 1:45-54. Blakeman, J.P., 1972. Effect of plantage on inhibition of Botrytiscinerea spores by bacteria onbee troo tleaves .Physiologica l PlantPatholog y2:143-152 . 176 Davet, P., Lal, B., Lung-Escarmant, B. and Gallet, J.F., 1992. A method to screen Trichoderma isolates against Sclerotinial fungi and Armillaria root rot. Biological Controlo fPlan tDiseases .Progres san dChallenge sfo rth eFuture .NAT OAS ISeries . SeriesA ,Lif e Sciences230:427-430 . Dubos,B. , Roudet, J. and Bulit,J. , 1983. Influence de la température sur les aptitudes antagonists des Trichoderma à l'égard du Botrytis cinerea Pers. Les antagonismes microbiens.Le scolloque sd el'INR A 18:81-90. Dubos,B. , 1987.Funga l antagonism in aerial agrowbiocenoses. Innovative approaches toplan tdiseas econtrol .J .Wile yan dSon sp . 1 07-135. Fokkema, N.J., Gerlach, M. and Köhl, J., 1991. Biological control of Sclerotinia sclerotiorum and Botrytis spp.In Biologica l control of Plant Disease. Eds. Tjamos, E.C., Papavizas, G. and Cook, R.J. NATO ASI Series. Series A, Life Sciences 230:267-271. Ferreira,J.H.S. , 1990. Invitro evaluation ofepiphyti cbacteri a from tablegrape sfo r the suppression of Botrytis cinerea. South African Journal of Enology and Viticulture 11:38-41. Gullino,M.L. , 1983.Situatio n actuelle et perspective d'avenir de la lutte biologique et intégrée contre la Pourriture grise en Italie. Les antagonismes microbiens. Les Colloquesd el'INR A18:91-98 . Gullino,M.L. ,Mezzalama ,M .an dGaribaldi ,A. , 1986.Biologica lan dintegrate dcontro l ofgre ymould :result san dperspectives .Quadern idell aScuol ad iSpecializzazion ei n Viticolturae dEnologi a9:299-308 . Gullino,M.L. ,Aloi ,C .an dGaribaldi ,A. , 1989.Evaluatio n ofth einfluenc e of different temperatures,relativ ehumiditie san dnutritiona l supportso nth eantagonisti cacitivit y of Trichoderma spp.agains tgre ymoul do fgrape .Influenc e ofenvironmenta l factors on the control of grape pests, diseases and weeds. Proceedings of the EC Expert's Group.Thessaloniki ,Greec ewp . 100-104. Heidenreich, E. and Kubicek, A., 1991. Towards cloning of biocontrol genes of Trichoderma harzianum.Proceeding s Trichoderma and Gliocladium Workshop, Belgirate,Ital yp . 164-167. Jin, X., Harman, G.E. and Peruzzotti, B., 1991. Production of quality biomass of Trichodermaharzianum for biocontrol using liquid fermentation. Proceedings Trichoderma andGliocladium Workshop,Belgirate ,Ital y p.84-85 . Janisiewicz,W.J .an dRoitman ,J. , 1988.Biologica l controlo fblu emol dan dgre ymol d of applean dpea rwit hPseudomonas cepacia. Phytopathology78:1697-1700 . Köhl,J . and Schlösser,E. , 1989.Deca yo f sclerotiao fBotrytis cinereab y Trichoderma spp.a tlo wtemperatures .Journa l ofPhytopatholog y 125:320-326. Mighell,Q. ,Fioretta ,C , Cavallarim,L .an dGullino ,M.L. , 1991.Protoplas tpreparatio n and fusion in antagonistic Trichoderma spp. Proceedings Trichoderma and GliocladiumWorkshop ,Belgirate ,Ital y p. 112-115. McLaughlin, R.H.J., Wisnicwski,M.E. , Wilson, C.L. and Chalutz, E„ 1990.Effec t of inoculum concentration and salt solutions on biological control of postharvest diseases ofappl ewit h Candida sp.Phytopatholog y80:456-461 . Newhook, F.J., 1957.Th erelationshi p of saprophytic antagonism tocontro l ofBotrytis cinerea Pers.o ntomatoes .Ne wZealan d Journal of Science andTechnolog y38:473 - 481. Peng,G . andSutton ,J.C. , 1990. Biologicalmethod st ocontro lgre ymoul do fstrawberry . BrightonCro pProtectio nConference , Pestsan dDisease s p. 233-240. 177 Roberts, R.G., 1990.Postharves t biological control of grey mold on apple by Crypto- coccus laurentii. Phytopathology 80:526-430. Ruinen, J., 1961.Th e phyllosphere. An ecologically neglected milieu. Plant and Soil 15:81-109. Tronsmo,A. , 1986.Trichoderma use da sa biocontro lagen tagains tBotrytis cinerea rot s ofstrawberr yan dapple .Meldinge rFr aNorge sLandbrukshogskole ,65:1-22 . Vial, I. and Dubos, B., 1992.Qualit y criteria for abiopesticid e based on Trichoderma harzianum. Biological Control of Plant Diseases. Progress and Challenges for the Future. NATOAS ISeries .Serie sA ,Lif e Sciences230:367-370 . White,J.G. ,Linfield ,CA. ,Lahdenpera ,ML . andVoti ,J. , 1990.Mycosto p- anove lbi o fungicidebase do nStreptomyces griseoviridis. BrightonCro pProtectio n Conference, Pestsan dDisease s p.221-226 . Wood,R.K.S. , 1951.Th econtro lo fdisease so flettuc eb yus eo fantagonisti corganisms . I.Th econtro lo fBotrytis cinereaPers .Annal so fApplie d Biology38:203-216 . 178 til Biological control ofBotrytis cinerea o n grapevine by compost extracts and their microorganisms in pure culture N.Ketterer, B.Fisher and H.C. Weltzien I Summary Compostextract sreduce d infection ofgrapevin eb yB. cinerea. Detached leaf bioassays demonstrated thatth esuppressio ncoul db eenhance d to90-95% .Fo rprovin g fungicidal effects ofcompos textracts ,cattl emanure ,hors emanur ean dgrap emar ccompos twa si n vestigated after afermentatio n period of8 days.Thes econdition spromote da numbe ro f bacteria of the genus Pseudomonas and spore forming genera. Previous studies have evaluated the microbial composition of the extract. The highest number of completely mesophilic aerobic bacteria was found in the cattle compost. Microorganisms were therefore isolated from extracts oftha tcompost ; acollectio n of4 5strain swa sobtained . Eightisolate s reduced thediseas e levelo fB. cinerea tomor etha n 98%. Subsequently a field trialwa sperforme d withth ere dvin ecv . 'Domina' inth eAhr-valley ,Germany .Du e to prevailing weather conditions in 1991 there was a late infection of B. cinerea on berries. The treatments with compost extracts reduced the infection significantly and after addition of casein (0.5%) and pine needle oil (0.05%) before application of the extracts,th eresult swer esimila rt othos eachieve d withconventiona l fungicides. Introduction Due to the increasing environmental problems concerning the use of fungicides, alternative methods of disease control have to be studied. Biological control of plant diseases depends on antagonistic competition, antibiosis or hyperparasitism. Appli cations of suspensions of Trichoderma- or Gliocladium spp. have been tested with success, using their antagonistic and parasitic potential for disease suppression (Papavizas, 1985).Schönbec k andDehn e(1986 ) found areductio n indisease ,possibl y asa resul to finduce dresistanc et ovariou sobligat eparasite safte rapplicatio no fsoilborn e microorganisms andthei rcultur efiltrates . Forpratica l useo fthes emechanisms ,substrate s withhig hmicrob epopulation s such as compost have been explored. Composted organic soil amendments have been used successfully to suppress soilborne pathogens. Hoitink et al. (1977) described the suppressiono fPhytophthora cinnamoni b ycomposte dhardwoo dbark . Hoitinkan dFah y (1987) concluded that the microflora of composts is responsible for the suppression of soilborne plant pathogens. Soil amendments with composted manures also have depressive effects on Erysiphe graminis of wheat and barley if compared with plants cultivated in field soil (Budde and Weltzien, 1988). Direct application of aqueous extracts to composted manure-straw-soil mixtures to plant surfaces by dipping or spraying was first reported by Weltzien and Ketterer (1986). In these experiments the extracts reduced thedown y mildew Plasmopara viticola ondetache d grapevineleaves . These promising results lead to thetestin g of watery extracts from composted organic 179 wastean dth eculture so f microorganisms isolated from thatextract s againstB. cinerea inlaborator yan dfield conditions . Material and Methods Extractiono fcompost s The composts consisted of animal manure (horse and cattle) with cereal straw and amendments of top soil of fermented compost. Aqueous extracts of compost were obtainedb ymixin gcompos twit hta pwate ri nrati o1:1 0(v/v) .Th esuspensio nwa sstirre d once and kept for extraction fermentation under laboratory or field conditions with temperatures ranging between 16 and 21"C. The extraction time during which the compostwa scovere d withwate rwa s 1,3,7 and 14 days.Befor e usefo r diseasecontro l theextract swer efiltere d throughchees ecloth . Isolationo fmicroorganism s Toisolat ebacteria , thecompos t extracts wereshake n vigorously in water (1g compos t orextrac ti n9 m lta pwater) .Dilution st o 10~9wer eprepare dan d0.0 5m lo feac hdilutio n waspoure d intoPetr idishe swit h thefollowin g selectivemedia :Cas oAgar ,Merc kNo . 5458/-Bactilli;GS PAgar ,Merc kNo . 10230/'Pseudomonades. Growtho fmicroorganism san dinoculu mproductio n Themicroorganism s weregrow na t2 1° C in 100m lErlenmeye rflask s containing 25m l nutrientsolution .Afte r 4day seac h suspension wasdilute d withta pwate r (1:5,v/v )an d the antagonistic efficiency of the isolates to B. cinerea was tested on detached vine leaves. Applicationo fcompos textract s andmicroorganism s Astandar dbio-assa y wasuse dfo r screening compostextract s andmicroorganisms .Th e bio-assay comprised a detached leaf test with vine leaves kept in a moist chamber. Detached leaves were inoculated with eight droplets (20 |xl containing 2 x 106 conidia.mH) on the adaxial side.Mal t extract (1%) was added as anutrien t source for inoculation. In the screening tests, droplets contained 5 |JL1 of the compost extract, or microorganisms suspended in a nutrient solution (15 |xl malt extract). Successful infections were recorded after 2 to 3day s incubation at 21°C and a 14h photoperiod. Grape berries, cv. Blauwer Spätburgunder, were dipped into compost extracts. After 3day sthe ywer einoculate dwit ha conidia lsuspensio no fB. cinerea. Th ediseas eseverit y wasassesse d asth epercentag eo fdisease dberries . Fieldexperimen t Thefiel d trial was located in avineyar d atth eAhr-valley ,Germany ,wit h the red vine cultivar Domina. Thevineyar d was split into 27plot s of about 30plant s each with the following treatments: horse manure compost extracts two month-old (CH2) and four month-old (CH4); CH2 + dextrose (0.5%); CH2 + casein (0.5%) + pine needle oil 180 Table 1. The effectof extraction times usedto prepare compost extracts onthe disease intensityofB. cinereaon detached grapevine leaves. Treatment Classo flesio nsize 1) Extractiontim e(days ) 1 3 7 14 Control (water) 4.5a » 4.75a 4.6 a 4.95a Horsemanur e 2.8 c 1.70c 0.4 c 3.0 b Grapemar c 3.8b 2.5 b 1.25 b 3.5 b Cattlemanur e 2.5 c 1.45 c 0.3 c 2.0 c ') 1 =ver ysmal llesion ; 5 =larg elesio n 2)Differen t lettersindicat estatistica l significance at5 %level . (0.05%); CH2 + oil (0.05%); oil; fungicides and untreated check. Oil and casein were added immediately before application of compost extract. Theseeigh t treatments were replicatedthre etime san dassigne dt oplot sa trandom .Application swer ecarrie dou twit h a motor backpack sprayer at a rate of 1000-1500 1 per hectare at c. 12 day intervals, beginning in May until the end of August. The disease severity was assessed as percentage of diseased foilage or berries using the index of the official guidelines for pesticideevaluatio n (Flick etal, 1989). Results Studieso ndetache d vineleave san dberrie s Theeffect s ofdifferen t compost extracts were studied invivo o ndetache d grapeleave s and berries. In the first experiment the extraction times varied between 1an d 14days . Theresult ssho w aninhibitor yeffec t ofcompos t extracts onlesio n sizes (Table 1).Th e importance of a suitable extraction time for an effective disease suppression was apparent, the optimum timebein g 8days .Th e suppression was enhanced by upt o60 - 90%b ysprayin g compostextracts .Simila rpromisin gresult swer eachieve d inth eberr y test(Fig .1) . When rinsing off the extracts from the leaf surface 2 days before incubation of the pathogen theeffec t ofsuppressio n waslos tcompletel y (Fig. 2). In the mycelial growth test with B. cinerea the extract-water agar mixtures were coveredwit h 8 mlstandar dmal taga ran dinoculate dwit ha mycelia ldisk .Extract s ofal l threecompost s reducedth emycelia lgrowth .Eve nth eextract s obtained after eightday s extractiontim einhibite dmycelia lgrowt hstrongl y(Fig .3 )an dinhibitio n ofgerminatio n ofconidi awa sobserve db yligh tmicroscopy . Inothe rexperiment s wefound , after sterilefiltratio n orhea tsterilisatio n ofth ehors e manurecompos textrac ttha tth ebrot hremaine dineffectiv e (Fig. 4). Fig. 5 summarises the microbial composition of the extracts. The total number of colony forming units (cfu) increased from 1 to7 day s ofextractio n timean d decreased thereafter. Thehighes tcoun twa s 108 cfu.mH after 7 days. Toexamin eth eantagonisti ceffect s further weisolate dmicroorganism sfro m different composts andcompos textract s andteste dthei refficac y againstB. cinerea ongrapevin e 181 attack oi berries (%) 3 days extraction time 8 days extraction time IH control KMI grape marc I'M cattle manure ÜÜ horse manure Fig. 1. Effectof three extractsproduced intwo extraction times ondisease intensity of B. cinerea ongrape berries. Differentletter sindicat esignifican tdifferences ,p = 0.0 5 class of lesion size treatments I control cattle manure ÜH horse manure I grape marc Fig. 2. Effect on disease intensityof removing the extractsbefore inoculation with B. cinerea. Different lettersindicat esignifican tdifferences ,p = 0.0 5 182 area of mycel growing (mm) 1 day 4 days 8 days control compost base: extraction time Hi cattle manure horse manure lüü grape marc Fig. 3. Effect of different compost extract-water agar mixtures on mycelial growth of B. cinerea. % Efficiency of Botrytis suppression 100 4 days extract 8 days extract •iansterll KB milk filter Ül steril nitration H! heat steril Fig. 4 Effect ofsterilised horse manure compost extracton infection ofgrapevine leaves by B. cinerea. Different letters indicate significant differences, p= 0.0 5 183 clu/ml 1.000E+09 l.OOOE+08 10000000 1000000 1 day 2 days 4 days 7 days 14 days extraction time compost base: ^^ HH cattle manure KWhors e manure HU grape marc Fig. 5. Number of colony forming units obtained from three compost extracts after different extraction times. class ol lesion size Pseudomonas Enterobacter Bacilli control Fig. 6. Effect ofmicrobial isolates onB. cinerea on grapevine leaves. 184 benies attack in % bonitation 2.10.91 •• untreated IM CH2 extract EÜÜ CH2+Deztrose iü CH4 extract EiÜ CH2+Cas.+OU • Oll Hi Casein MM Euparen Fig.7. Control of grey mouldof grapes by compost extracts containing nutrients and pineneedle oilin comparison tofungicides. leaves (Fig. 6). Eight isolates, namely four species of Pseudomonas, three isolates of Enterobacteriaceae andon especie so fBacillus wer ehighl yinhibitor y toB. cinerea. Fieldexperimen t Duet oth eprevailin g weathercondition s in 1991ther ewa sa lat einfectio n ofB. cinerea onberries .Treatment s withcompos textract s reducedthi s attacksignificantl y compared tocontrol s(Fig . 7).Whe nCH2-extrac twa sadde dwit hcasei n(0.5% )an dpin eneedl eoi l (0.05%) before spraying,th eresult s weresimila r tothos e achieved after application of conventional fungicides. There was no significant difference in efficacy between the treatments of CH2-extract, CH4 extract and CH2 amended with dextrose. The yield increased from 1.1k g pergrapevin eplan to nth euntreate d controlt o 1.8 kgpe rplan ti n theplot s treated with CH2+ casein + oil. CH4 , CH2 and CH2+ dextrose produced a yield increaseo f 1.4 - 1.5k gpe rplant . Discussion The suppressive potential of extracts was largely based on the presence of active microorganisms. Consistent results were obtained with detached leaves and berries in laboratoryexperiment s andunde rfiel d conditions.Th etota lnumbe ro f microorganisms and the efficiency of the extracts was increased and results were more consistent after addition ofnutrients .A ne wsyste mfo rbiologica l plantprotectio n seemst ob epossible , butmor einformatio n isrequire d aboutth emode(s ) of actionan dmethod so f producing theextracts . 185 References Budde, K. and Weltzien, H.C., 1988. Untersuchungen zur Wirkung von Kompost extrakten und Kompostsubstraten im Pathosystem Getreide-Echter Mehltau (Erysiphegraminis). Mitteilunge n der Biologische Bundesanstalt für Land- und Forstwirtschaft 245:366. Flick, G.,Holz , B.,Huber , W., Kassemeyer, H.H., Kast,W.K. , Lipp,H.P. ,Lorenz , D., 1989.Prüfun g vonFungicide n zurBekämpfun g vonBotrytis cinereaPers .a nRebe n (Ertragsanlagen). Richtlinie Prüfung Pflanzenschutzmitteln. Zulassungsverfahren BiologischerBundesanstal tfü r Landun dForstwissenschafte n 2(22-1.4):1-9 . Hoitink, H.A.J., Van Doren, D.M. and Schmitthenner, A.F., 1977. Suppression of Phytophthora cinnamoni in a composted hardwood bark potting medium. Phyto pathology67:561-565 . Hoitink,H.A.J .an dFahy ,P.C. ,1986 . Basisfo rcontro lo fsoilborn eplan tpathogen swit h composts.Annua lRevie wo fPhytopatholog y24:94-114 . Schönbeck,F .an dDehne ,H.W. , 1986. Useo fmicrobia lmetabolite sincludin gresistanc e againstpathogens .In Microbiolog y ofphyllosphere . Eds.Fokkema ,N.J .an dVa nde n Heuvel,J .Cambridg eUniversit yPres sp . 361-375. Papavizas,G.C. , 1985.Trichoderma andGliocladium biology ,ecolog y andpotentia l for biocontrol.Annua l Review ofPhytopatholog y23:23-54 . Weltzien, H.C. and Ketterer, N., 1986.Contro l of downy mildew,Plasmopara viticola (de Bary) Berlese et de Toni, on grapevine leaves through water extracts from composted organicwastes .Journa lo fPhytopatholog y 116:186-188. 186 Effect of substrate, temperature and time of application on the effectiveness of three antagonistic fungi against Botrytis cinerea N.E. Malathrakis andO. Kritsotaki Summary Theinfluenc e ofsubstrate ,temperatur ean dtim eo fapplicatio no fth eantagonisti c fungi Trichoderma sp., Pénicillium sp. andAcremonium alternation against B. cinerea were studied using potatodextros eagar ,disc so fcucumbe rfruit s andyoun gbea nplants .Th e antagonists wereapplie d asa spor esuspensio n upt o5 day sbefor e theinoculatio n with B. cinerea. Testswer eperforme d atfou r temperaturesan dgrowt ho fB. cinerearecorde d after 2t o4 days .I twa sfoun d thatth eearlie rth eapplicatio n of theantagonist s andth e higherth eincubatio ntemperature ,th estronge rwa sth einhibitio n ofB. cinerea. Antagonists needed less time for growth on PDA and young beanplants , to be effective againstB. cinerea tha no ncucumbe rdiscs .O ncucumbe rdisc scomplet econtro l wasno t achieved,eve ni f theantagonist s havebee n applied 5day sbefor e thepathogen . Pénicilliumsp .wa smor eactiv ea tlo wtemperature stha nth eothe rtw oantagonists . Introduction We found previously that Trichoderma harzianum, Pénicillium sp. and Acremonium alternatum were effective against Botrytis cinerea Pers.: Fr. invitro (Malathraki s and Klironomou, 1992), but in greenhouse experiments only the first was moderately effective. This is a common phenomenon with biological control agents and several factors areprobabl y involved (Dubos, 1987);thre e of these,substrate , temperature and timeo fapplicatio n werestudie d toenhanc eth eperformanc e ofth eabov eantagonists . Material and Methods Testswer ecarrie dou to npatot odextros eaga r(PDA) ,disc so fcucumbe rfruit s andyoun g beanplants .Inoculu m ofT. harzianum, Pénicillium sp.an d A. alternatum were applied atdifferen t timesbefor eo rafte r theinoculatio no fth esubstrat ewit hB. cinerea.Test s on PDAwer ecarrie dou tsimultaneousl ywit hth ethre eantagonist sa t10° ,15° ,20 °an d2 5 °C buto ncucumbe rdisc san dyoun gbea nplant sth etest swer ecarrie d outseparatel y atth e same temperatures. The inoculum of the antagonistic fungi was prepared as follows: stock cultures of each fungus were transferred onto PDAplate s and incubated at2 2° C for 4days .Th espore s produced wereharveste d in steriledistille d water,centrifuge d at 400 rpm for 10min ,th esupernatan t removed and thepelle t resuspended in water. The spore concentration was determined with an haemocytometer and spores were kept at 5°Cbefor e usei neac hexperiment . 187 Antagonism onPD A To examine the effect of time of establishment of the antagonist on B. cinereafiv e treatmentswer euse di nwhic h 16 Petridishe scontainin g PDAwer eevenl y seededwit h 0.5m lo f sporesuspensio n ofeac hantagonis tcontainin g 106spores.mF ,3, 2an d 1 day before theinoculatio n oro nth esam eda yan d 1 dayafter . Fourdishe so feac hantagonis t were incubated at each of the four temperatures. Four other unseeded dishes were similarlyincubate da scontrols .Al lth eplate swer einoculate dcentrall ywit ha 5 m mplu g of young mycelium ofB. cinerea on day zero.Th ediamete r of themycelia l growtho f B. cinereawa s recorded in each Petri dish 2 days later and the mean and standard deviation for eachtreatmen tcalculated . Antagonismo ncucumbe rdisc s Fresh Dutch type cucumber fruits (Cucumis sativusL. ) were surface-sterilised with alcohol and cut into discs c. 5m mthick . Seven discs wereplace d in each 15c m Petri dish.Th edisc si nfou r disheswer ethe nseede d evenlywit hth eantagonis t 5,4, 3,2 an d 1 daybefor einoculatio nwit hB. cinerea, ando nth esam eda yan dth eda yafter . Foreac h disk 40 |xl of a spore suspension containing 2x 106spores.m H were used. As for the PDAexperiment ,th edisc swer eincubate da tfou rtemperature san do nda yzer oal ldishe s were inoculated. The diameter of therottin g zone on the cucumber discs was recorded 2day slater . Antagonismo nbea nplant s Plants of bean (Phaseolus vulgaris L.) grown in 6 cm pots with two fully developed leaves wereuse d in theseexperiments .Te npuncture s weremad ewit h aneedl ei neac h leafbefor eth eantagonist swer eapplied .A spor esuspensio no fantagonis tcontainin g10 6 spores.mH wasspraye d onto2 4o fthes eplant sunti lru n off on4 ,3 ,2 an d 1 day before inoculation with B. cinerea and on the same day of inoculation. Six plants for each antagonist treatment were placed in growth chambers at 10°, 15°, 20° and 25°C on a 12/12 h photoperiod and 100 r.h. Six plants untreated with antagonists were also in eluded in each chamber. All plants were spray-inoculated with B. cinerea on day zero witha suspensio ncontainin g 106spores.mH .Th einfectio n ofwounde dbea nleave swa s recorded 3-4 days later on a 0 to 5 scale (0 = no infection, 5 = infection of c. 1 cm diameter)an dth emea n andstandar ddeviatio n ofeac htreatmen twa scalculated . Results PDAtest s Theantagonist sseede d3, 2o r 1 daysbefor einoculatio nb yB. cinerea wer ealread ywell - developed and2 day safte r theinoculatio n withB. cinereath egrowt ho fPénicillium sp. and Acremonium alternatum, but not that of Trichoderma sp., was inhibited near the inoculum. It was found that the higher the temperature and the earlier the application of the antagonists, the greater the inhibition of B. cinerea(Tabl e 1). To obtain complete inhibition allthre eantagonists ,wit ha fe wexceptions ,neede d tob eapplie d moretha n3 188 Table 1. GrowthofB. cinereaon PDA seeded by spores of three antagonists, 2days after incubationat four temperatures Pénicilliumsp. A. altematum Trichodermasp. 10°C Control 5.7 +0. 5 -30 0.0±0.0 0 2.8± 0.45 4.0±0.9 0 -2 1.5 ±0.30 2.2+ 0.2 5 2.3± 0.25 -1 2.3± 0.2 5 6.0±0.6 0 2.8± 0.50 0 3.5 ±0.65 5.3±0.2 5 4.0±0. 0 +1 6.3± 0.75 5.5± 0.30 5.7± 0.50 15°C Control 20.0±0.7 0 -2 0.0± 0.0 0 0.0±0.0 0 0.0±0.0 0 -1 0.0 ±0.00 11.0±0.4 0 2.3± 0.50 0 16.7±1.1 5 19.2±1 0 15.5±1.6 5 +1 15.8±0.7 5 18.0±1 0 19.8± 0.25 20°C Control 33.7±0.8 5 -1 0.0± 0.0 0 1.0±0.0 0 0.0±0.0 0 0 0.0±0.0 0 26.7± 10 15.5±1.6 5 +1 30.7± 1.45 35.7± 0.50 34.7+1.05 25°C Control 41.0±0. 4 -1 0.0±0.0 0 0.0±0.0 0 0.0±0.0 0 0 3.5 ±0.85 19.2±2.2 0 6.5± 0.3 0 +1 33.7±4. 1 35.2±0.8 0 35.2±1.0 0 •) Mean± S.E., n= 4 .Tim e(days ) antagonistpresent .Minu s andplu sindicate s thatth e antagonistswer eapplie dbefor e orafte r theda yo finoculatio nb yB. cinerea (day0 ) days before the inoculation with B. cinerea at 10°C,2- 3 days at 15°C and only 1 day previouslya t20 °an d25°C .A. altematumwa sgenerall yles sinhibitor ytha nth eothe rtw o species and this was evident through the binocular microscope 24 h after their application,a t20 °an d25°C . Cucumberdisc s Theinfectio n of cucumber discs byB. cinerea wasinhibite d by allth e antagonists;th e higherth etemperatur e andth eearlie rth eapplicatio no fth eantagonists ,th estronge rth e inhibition ofinfection . Forcomplet einhibitio na t 10° and 15°Cal lth eantagonis tneede d moretha n 5day s establishment. Pénicillium sp.wa sth emost ,an d Trichoderma sp.th e least effective. Beanplant s Theantagonist s developed onth edea d tissuearoun d thewound so nleave s after 1 to3 days depending on the fungus and development was faster at 25°C and than at lower temperatures (Table 2). Trichoderma sp.develope d more rapidly than Pénicillium sp., which developed faster than A. altematum. Tob ecompletel y effective at 10°an d 15°C Pénicillium and Trichoderma needed to be applied 2 days before inoculation with 189 Table 2. Effectof time of application of antagonist and temperature oninfection caused byB. cinerea %Lea f woundsinfected 1) Pénicillium sp. A. alternatum Trichoderma sp. 10°C Control 11.6±2.7 0 75.3 ± 9.80 16.7±3.3 0 -22) 0.0±0.0 0 12.7± 4.10 0.0±0.0 0 -1 2.2±1.0 0 33.5 ± 7.40 0.0 ±0.00 0 8.4± 0.70 48.7± 8.50 10.6±3.4 5 15°C Control 46.5±4.5 0 100.0± 0.00 92.8±3.1 0 -2 1.8 ±0.90 6.7 ± 4.60 0.5± 0.32 -1 2.1±2.1 0 20.7±11.4 0 11.2±3.8 0 0 30.5±4.4 0 84.7±11.9 0 91.3±4.9 8 20°C Control 78.4±12.1 0 88.7± 7.20 0.0±0.0 0 -2 0.4± 0.4 0 0.0± 0.00 0.0±0.0 0 -1 0.4± 0.4 0 0.0± 0.00 0.0 ±0.00 0 22.2± 9.7 0 32.0±13. 3 7.5 ±3.60 25°C Control 76.7± 6.0 0 33.0± 7.70 26.7± 5.90 -2 0.0± 0.0 0 0.0± 0.00 0.0±0.0 0 -1 0.0±0.0 0 0.0± 0.00 0.0 ±0.00 0 17.6± 2.7 0 2.7 ± 1.90 0.0±0.0 0 !)Mean±S.E.,n =6 . 2) Time (days) antagonist present. Minus and plus indicates that the antagonists were appliedbefor e orafte r theda yo f inoculationb yB. cinerea (day0) . B.cinerea, bu tfo rsimila refficac y at20 ° and25° Cal lth eantagonist sneede d only 1 day. Antagonists applied the same day as B. cinerea (day 0) were still effective at 20° and 25°C,les ss oa tlowe rtemperatures . Discussion Thesetest s suggested that substrate,temperatur e andtim eo f application of antagonists relativet oinoculatio no fth epathoge nconsiderabl yinfluence d theperformance .Al lwer e lesseffectiv e oncucumbe rdisc stha no nPD Ao ryoun gbea nplants ,and ,irrespectiv eo f thesubstrate ,th eearlie r theapplicatio n ofth eantagonis t thegreate r itseffectivenes s at alltemperatures .A sgre ymoul di smos tseriou si ngreenhouse si nGreec edurin g winter, withtemperature s inth erang eo f 12°-17°C (Malathrakis, 1989),biologica lcontro lusin g theantagonist sPénicillium sp. , A. alternatumo rTrichoderma sp .wil lno tb eeffective , as theynee da highe rtemperatur et odevelop . Fresh wounds without resident mycelium of B. cinerea may be easily protected by antagonistsi f theyar eapplie d soonafte r thewound shav ebee nmad e(Fokkema ,1991) . However grey mould usually develops after the pathogen has become established on dying orsenescen t tissues.O n presentevidenc eprotectio n against this typeo f infection 190 may be difficult unless colonisation of these substrates has been inhibited (Newhook, 1957). Antagonists should be selected which developrapidl y onfres h wounds or senescent tissues at low temperature and are more adaptive to conditions prevailing in protected cropping(Dubos ,1992) . References Dubos, B., 1987.Funga lantagonis m inaeria lagrobiocenoses .In Innovativ e approaches toplan tdiseas econtrol .Ila nChe t(ed.) .Joh nWile yan dSons .Ne wYor kp .107-135 . Dubos,B. , 1992.Biologica l control ofBotrytis: Stateo f theart .Proceeding s of theXt h Botrytissymposium ,Crete ,Greece .Pudoc ,Wageningen ,Th eNetherland sp .169-178 . Fokkema, N.J., 1991. The phyllosphere as an ecological neglected milieu: A plant pathologistspoin to fview .In Microbia lecolog yo fleaves . Andrews,J.H .an dHirano , S.S.(eds.) .Springe rVerla gp . 3-18. Malathrakis, N.E., 1989. Development and control of plant diseases in greenhouses. Bulletin ofth eHelleni cPhytopathologica l Society2:42-54 . Malathrakis, N.E. and Klironomou, E.J., 1992.Contro l of grey mould on tomatoes in greenhouses with fungicides and antagonists. Proceedings of the Xth Botrytis symposium,Crete ,Greece .Pudoc ,Wageningen ,Th eNetherland s p. 282-286. Newhook, F.J., 1957. The relationship of saprophytic antagonism to control Botrytis cinerea Pers.o ntomatoes . NewZealan d Journal of Sciencean dTechnolog y38:473 - 481. 191 Biological control ofBotrytis leaf blight of onions: significance of sporulation suppression J.Köhl, W.M.L. Molhoek, C.H. van derPlas, G.J.T. Kesseland N.J. Fokkema Summary Saprophytic antagonists can be applied to suppress the sporulation of Botrytis spp.o n necrotictissue .Th eeffec t ofsporulatio nsuppressio no na nepidemi co fBotrytis spp . was studied in a field experiment with onions. When 30-50% of the necrotic tissue was removed from the field, the content of Botrytis spores in the air above the crop was reducedb y 34%an dth eepidemi cdelayed .I nlat eAugust ,th enumbe ro flesion s onth e leavescause db yB. cinerea andB. squamosa wa s 1.1 lesionscnr 2 inth econtro lan d0. 6 lesionscnr 2 after removalo f thenecroti ctissue . The potential microbial antagonist Gliocladium roseum, was sprayed as a conidial suspension ontoth eplant sweekly ,bu tthi s did notresul t incolonisatio n of thenecroti c tissuean dha dn oeffec t onth eBotrytis epidemic.Furthe rscreenin g of antagonistswit h emphasis onthei recologica lcompetenc ei snecessary . Introduction Botrytisspp .sporulat eprimaril yo ndea dtissu eo fth ehos tplants .Th enecroti ctissu ema y alsoprovid ea suitabl esubstrat efo rsaprophyti cantagonists .I nthi ssubstrate ,antagonist s mayinterfer e with thesaprophyticall y growing mycelium ofBotrytis spp.an dsuppres s sporulation of the pathogen. An effect of introduced antagonists on the epidemic of Botrytisca nonl yb eexpecte dwhe nmos to fth einfection s duringth eepidemi car ecause d by spores produced within the crop and inoculum produced outside the crop is less important.A fiel d experimentwit honion swa suse dt omeasur eth eeffec t ofsuppressin g sporulation onepidemic s ofB. squamosa andB. cinerea, causing onion leaf blightan d leaf spot respectively. Gliocladium spp. have suppressed sporulation of Botrytis in bioassaysalmos tcompletel y (Fokkemaet al., 1992),an dtherefor e astrai no fG. roseum wasuse d asa nantagonis t inth efiel d experimentdescribe dhere . Materials and Methods Onionswer esow ni nplot so f9 x 12m i na randomise dbloc kdesig nwit hsi xreplicate s pertreatment .Plot swer eseparate db y 12 mwid esuga rbee tstrip st ominimis e interpiot interference. Toobtai n ahomogeneou s infection withB. squamosa atth ebeginnin g of theepidemic ,scleroti a of thepathogen , grown on sterile onion leavesi n wetchambers , were spread over the plots at the end of April. Sclerotia are regarded as the primary inoculum ofB. squamosa (Ellerbrockan dLorbeer , 1977). Tostimulat eth eeffec t of anantagonist , allonio nleave swhic hwer enecroti c overa t least 50%o f their length werecu tan d removed from the plots weekly such thatdurin g the experiment c. 30-50% of dead leaf tissue was removed. In a second treatment, 192 G. roseum was applied weekly as a conidial suspension. Conidia were produced on autoclavedwhea tkernel san dsuspension swer eprepare db yaddin gta pwate rwit h0.01 % Tween 80 to the cultures. After filtration through a double layer of cheesecloth, the conidialconcentratio nwa sadjuste d toc .1 x 106conidia.mH .N ofurthe rtreatment swer e carried outi nth econtro lplots . Rotorods (Sampling Technologies, Los Altos Hills, Ca, USA) were used to trap airbornespore s(Edmonds , 1972).Tw orotorod s wereuse d simultaneously inth ecentr e ofeac ho ftw oplot so feac htreatmen ta ta heigh to f0. 3m .Th econcentratio n ofairborn e sporeswa scalculate d from thenumbe ro f sporestrappe ddurin g 15 min. Tenplant spe rplo twer esample dweekl yfro m June 11 untilAugus t20 .Lesion swer e counted for all leaves except those with more than 50% the leaf length necrotic. The surface of the green leaves was measured and the number of lesions per cm2 was calculated.Th elengt h ofgree nan dnecroti clea f partswa sals orecorded . Samples of necrotic leaf tissue were washed thoroughly to remove adhering spores andhomogenised , anddilution s ofth ehomogenat ewer eplate d onmal taga rcontainin g 15 |xg tetracycline.mH to determine the colonisation of the tissue by the applied antagonist. Airtemperature ,relativ ehumidit y andlea f wetnesswer econtinuousl y monitored. Results Duet oa dr yperio da tth ebeginnin go fMay ,n osporulatio no fB. squamosa wa sfoun d on the introduced sclerotia. Within 21 days, many of the sclerotia were attacked by collembola (e.g. Sminthurus spp.). Thus, it can be assumed that most infections of Botrytis spp.durin g thefiel d experimentwer ecause d bynatura l inoculum.Compariso n 0) O c 0) D Ö" (1) LL Length of conidia (urn) >. 1. Lengthof conidia of B. cinerea (-•-) andB. squamosa (-a-) grown on sterile on leaves andof conidia produced onnecrotic onion leaftips (-O-) ortrapped with torods (-0-) inthe field. 193 1 1:05 1 1:58 750 m 500 conidia < 15 urn conidia 250 > 15 urn u & // Fig. 2. Concentration of airborne conidia of Botrytis cinerea (< 15 /xm) and B. squamosa(> 15 um) onAugust 16 after removal of necrotic leaf tissue orapplication of Gliocladium roseum. Columns with the same letterdo no differ significantly according to LSD-test(p = 0.05). of thesiz eo fth eBotrytis conidia (Ellis, 1971)collecte d with theRotorod s orproduce d ondea d onionlea f tips,wit h those produced byisolate s ofB. cinerea andB. squamosa onsteril eonio nleave sshowe dtha tth emajorit y ofBotrytis conidi aproduce di nth efiel d belonged toB. cinerea (Fig.1) . Onfive day si nAugust ,spore swer etrappe d whenabundan tsporulatio nwa sfoun di n thecrop .Eac hday ,tw ot ofou r runs of 15 minwer ecarrie d outbetwee n 10.00a.m .an d 1.00 p.m. during which period a peak of spore release could be expected (Lacy and Pontius, 1983). Thespor eloa dabov eplot swher eth enecroti ctissu eha dbee nremoved , was always lower than in the control plots. Reduced spore loads measured at high concentration levels of airborne conidia weresignifican t but, not atlo wlevel s (Fig.2) . The results of all spore trappings are summarised in Fig. 3. Spores were trapped in between asuga rbee tbuffe r cropan da plo ttreate dwit hth eantagonis t averaged 50% of theconcentratio n measured aboveth econtro lplo ta tth esam etime .Botrytis spp .di dno t sporulate on sugar beet tissue. Most of the trapped spores had the size of conidia of B. cinerea (Fig. 1).Sprayin g withG. roseum had noeffec t on the spore load aboveth e treated plot. Thefirst Botrytis lesions occurred onJun e 11 but theepidemi c was interrupted bya period of hot and dry weather. In themiddl e of July,ne w lesions were found andthei r number increased exponentially until late August. B. cinerea was frequently isolated from surface sterilised lesions, whereas B.squamosa was never found. In plots where necrotic leaf tissueha dbee n removed, theslop eo fth eregressio n linewa s significantly lower( p< 0.001 )tha nth econtro l(Fig . 4).I nlat eAugust , 1.1lesion scm- 2wer ecounte d inth econtro lplots ,wherea sonl y0. 6lesio ncnr 2wer efoun d inplot swher ea nantagonis t hadbee n simulated by theremova l of 30-50%o f thenecroti c tissue.Th etreatment s in 194 750 dead leaves ro removed GliocladJum 500 a ro control •o 'c 250 - o U Fig. 3. Concentrationof airborne Botrytis conidia during 5 days in August after removal of necrotic leaf tissue or applications of Gliocladiumroseum (means of 2-4 measurementsper day). 1.50 A uni control • - - Gliocladium • — dead leaves removed E o t 1.00- cat g IS) 0.50 0) e Z zs & pHiwiMiwiudmiwimiHgl*11*'- 0.00 100 1 10 120 130 140 150 Days after sowing Fig.4. Numberof lesions caused byB. cinerea orB. squamosa ononion leaves after removalof necrotic tissue orapplications with Gliocladium roseum. 195 whichG. roseumwa sapplie ddi dno taffec t thenumbe ro flesion san di twa sno tpossibl e to isolate G.roseum from necrotic leaf tissue sampled 6day s after theantagonis tha d been sprayed. Discussion Aclea rrelationshi pbetwee nth eamoun to fsuitabl esubstrat efo rsporulatio n ofBotrytis spp.,th espor e load inth eai rabov eth ecro p andth enumbe r oflesion s onleave s was found inth efiel d experiment. Therefore, anepidemi c ofBotrytis spp.i nonion s mainly dependso nspore sproduce d insideth efiel d onhos tplants .Thi sseem stru efo rth ehos t specific pathogen B. squamosa andth e non-specific, ubiquitous B. cinerea whichwa s dominanti nou rfiel d experiment. Sinceconidi awer eals o trapped between theplots ,i t canb eassume dtha tsom einteraction sbetwee nplot soccurred .I na larg efield ,th eeffect s of sporulation suppression willb emor edistinct . Necrotictissu ei sa muc hmor eattractiv esubstrat efo rsaprophyti cantagonist stha nth e surface of an intact leaf. Consequently, control ofdisease s caused byBotrytis spp. by suppression of sporulation seems to be more feasible than the useo f antagonists to preventinfection s onth egree nleaf .Alon g thisline ,Pen g andSutto n(1990 ) found that spraying with G. roseum wasequall y effective asfungicide s inreducin g sporulationo f B. cinerea ondea dleave s andth eincidenc eo ffrui t roti n strawberry fields. G. roseum didno tcolonis eth enecroti c leaf tissueunde r ourconditions .Thi sca nb e duet oth esprayin g technique,t oth equalit y ofth e conidia applied ort o the ecological competence ofth e antagonist which isprimaril y asoi l inhabitant. Further screeningo f antagonistsamongs tsaprophyte snaturall yoccurrin go nnecroti clea ftissu ean do fothe r strainso fGliocladium spp .an dTrichoderma spp .i nbioassay san dunde rfiel d conditions isnecessary . References Edmonds, R.L., 1972. Collection efficiency of Rotorod samplers for sampling fungus sporesi n theatmosphere .Plan tDiseas eReporte r56:704-708 . Eilerbock, L.A. and Lorbeer, J.W., 1977.Source s of primary inoculum ofBotrytis squamosa. Phytopathology 67:363-372. Ellis,M.B. , 1971.Dematiaceou s hyphomycetes.Commonwealt h Mycological Institute, Kew. Fokkema, N.J., Gerlagh, M. and Kohl, J., 1992. Biological control of Sclerotinia sclerotiorum andBotrytis spp.In Biologica l control of plant diseases Progressan d challengefo rth efuture , Eds.Tjamos ,E.C. ,Papavizas ,G .an dCook ,R.J .Proceeding s NATOAdvance d ResearchWorkshop .Serie sA :Lif e Sciences230:267-271 . Lacy,M.L . andPontius ,G.A. , 1983.Predictio n ofweather-mediate d release ofconidi a ofBotrytis squamosa fro m onionleave si nth efield . Phytopathology73:670-676 . Peng,S .an dSutton ,J.C. , 1990.Biologica lmethod st ocontro lgre ymoul do fstrawberry . British CropProtectio n Conference -Pest s andDiseases ,p . 233-249. 196 Biological control ofBotrytis ro t of apple M.L.Gullino, D.Benzi, C. AM, A.Testoni and A. Garibaldi Summary Twoyeas tisolate s identified as Trichosporon sp.an d Candida sp.effectivel y controlled rot of apple caused by B. cinerea. Wounded fruits (cv. Golden Delicious), treated with 106cells.m H of the biocontrol candidates and inoculated with the pathogen showed a significantly lower disease incidence as compared with control fruit at both 2-4° and 22 °C.Th e biological agents were effective when disease incidence was moderate and remained satisfactory eveni f thediseas e wasmor esevere . Introduction Postharvest losses due to rot caused by Botrytis cinerea Pers.: Fr. can be serious on several perishable crops, even in production areas where the most advanced storage technologies are available.Wher e permitted, fungicides are aprimar y control measure for postharvest rots (Eckert and Ogawa, 1985).Durin g recent years, a combination of biologicalan dregulator yfactor s hasincrease d interestan deffort s inth edevelopmen to f agricultural production, storage and marketing systems that utilise reduced chemical inputs. Public awareness of fungicide residues in food has increased, and government regulatoryagencie sar erespondin gb yreassessin g theus eo fman ypesticides .Moreover , development of fungicide resistance, common in the case of postharvest pathogens (Romano and Gullino, 1983),ha s stimulated efforts to develop alternative systems for diseasecontrol . Under these perspectives, the search for biological control agents (BCA) effective against postharvest disease looks challenging (Wilson and Wisniewski, 1989). The presentwor ksummarise sresults ,previousl y published (Aloiet al., 1991 ;Gullin oet al., 1991,1992),obtaine d inItal yb ysearchin g for BCAactiv eagains tBotrytis roto fapple . Material and Methods Potential antagonists were isolated from the surface of apples harvested in unsprayed orchards(Gullin oet al., 1991).Thei rbiocontro l activitywa steste do napple ,cv . Golden Delicious, surface sterilised in sodium hypochlorite (8% as chlorine),rinsed wit h tap wateran dpuncture da tth eequatoria lregio n(thre epuncture spe rapple) .Th eantagonist s wereapplie d at 106cells.ml- 1fo ryeast ,o r 108cells.ml" 1fo rbacteri ab yusin g 10\d cell suspensionpe rwoun d (Tables 1 and2 )o rb ydippin g fruit for 2mi ni na cel lsuspensio n (Table3) .Afte rtreatmen twit hth eantagonists ,fruit s werekep ta t20-22° Cfo r 12t o2 4h , theninoculate d witha conidia l suspension ofB. cinerea (5x 105conidia.mH ) using 10 IJL.1 suspension perwoun d orb ydippin g fruits for 2mi ni nthi ssuspension .A mixtur eo f fourt osi xisolate so fB. cinerea,sensitiv e(S ,Table s 1 and2 )o rbenzimidazole-resistan t 197 Table 1. Effectiveness ofdifferent microorganisms againstBotrytis rotof apple at two temperatures. Treatment Rotincite db yB. cinerea( % ofth econtrol ) 24°C 2TC 2.33 Y»> 13 abc2) 21b 4.4Y 10a b 9a 1.16B* 45 ede 41 d 1.7 B 40abe d 13 ab 4.42B 45 ede 62e 5.50B 48d e 93 f 11.31 Y 45 ede 33 c 11.33 B 72 e 40c d TBZ(lg.H) 13 abc 5a Control 100 f 100 f (27)3) (49)3) ')Y = Yeasts ,use d at 106cells.mH ;B = Bacteria ,use da t 108 cells.mH 2) Values of the same column, followed by the same letter do notdiffe r significantly following Duncan's Multiple Range Test (p= 0.05) 3) Diameter (mm) oflesion s incontro l fruit Table2. Effectiveness of two biocontrol candidates against Botrytis rot of apple cv. Golden Delicious, caused byisolates which were benzimidazole-sensitive (S)or -resis tant (RB) (Gullino etai, 1992). Treatment Rot incited by B. cinerea (%o fth e control) S RB 2-4°C 22°C 4°C 22°C Candida sp.4. 4 10a « 9a 16 a 37a Trichosporon sp.2.3 3 13 a 21a 34a 25a TBZ(lg.H) 13 a 5a 62b 99b Control 100b 100b 100b 100b (27)3) (49)3) (22)3) (33)3) 2)an d 3) seeTabl e 1 (RB, Tables 2an d3) ,wer e used for inoculations. After inoculation with the pathogen, apples werekep t at20-22° C orstore d at2-4°C .I nth elatte rcase , apples were transferred from 2-4° to 20-22°C for 10day s before measuring thediamete r of the decayed tissue around the wounds. Results and Discussion The screening ofhundred s ofbacteria l andyeas t isolates yielded some microorganisms that markedly inhibited Botrytis rot.Mos t microorganisms which were active against 198 Table3. Effectiveness of several treatments against rotof apple after wound inoculation withB. cinerea (fruit kept 30 days at2°C+ 8 days at 20-22°C) (Abi et al., 1991). Treatment B. cinerea Rotdiamete r (mm) in trial i ii in 1)No t wounded - 0.1 a» 0.0 a 0.0 a 2) Control2) - 1.7 c 20.2 e 7.8d 3) Control3) + 24.2 f 55.6g 34.7 g 4)TBZ(lg.l->) + 11.5e 61.4 h 33.2f 5) 2.33 - 0.2 a 1.6 b 2.5 b 5)4.4 - 0.7 b 3.4 c 10.2 e 7) 2.33 + 2.1c 19.3d 6.7 c 8)4.4 + 4.8d 29.4f 7.5d 1)se e table 1,bu t p = 0.01 2) not treated, not inoculated with B. cinerea 3) not treated, inoculated with B. cinerea B. cinerea also showed activity against Pénicillium expansum. Yeasts generally showed a higher activity than bacteria. Twoyeasts ,code d as 2.33 and4. 4 and identified as Trichosporon sp. and Candida sp. respectively (Gullino et al., 1992) showed effective and consistent biocontrol against B. cinerea at 2-4° and 20-22°C (Table 1). The two biocontrol candidates showed an activity comparable totha to f thefungicid e TBZwhe n rotwa s caused by benzimidazole- sensitivestrain s ofB. cinerea.O n thecontrary ,i nth epresenc eo f benzimidazole-resistant B. cinerea, TBZ failed to control Botrytis rot (Table2) .Th eantagonist s did notcaus e any significant damage to apple fruit. The best biocontrol activity was observed in the presence of attacks of medium intensity (Table 3,tria lI) . In conclusion, the effectiveness of Trichosporon sp. 2.33 and Candida sp.4.4 , looks promising. The levels of the efficacy of these two BCA is similar to that reported for other biocontrol candidates (Wilson and Wisnieswki, 1989; Roberts, 1990). The good activity shown by the two yeasts against B. cinerea suggest that they are worth to be appraised for use on other crops commonly affected by Botrytis rot during storage and transit, such as kiwi, grape and strawberry. Studies on the mode of action of these two antangonists are required todevelo p thebes t method of application. Acknowledgement This research has been supported by a grant from Ministero dell'Universita' e della Ricerca scientifica e technologica (MURST 40%: Nuove strategie di difesa a basso impatto ambientale). References Aloi, C, Benzi, D., Testoni, A. and Gullino, M.L., 1991. Biological control of postharvest diseases of apple: experimental results. Proceedings ANPP Illrd International Conference on Plant Diseases, Bordeaux p. 695-701. 199 Eckert, J.W. and Ogawa, J.M., 1985. The chemical control of postharvest diseases: subtropicalan dtropica lfruits .Annua lRevie wo fPhytopatholog y23:421-454 . Gullino, M.L., Aloi, C, Palitto, M., Benzi, D. and Garibaldi, A., 1991.Attempt s at biological control of post-harvest diseases of apple. Mededelingen Fakulteit Land bouwwetenschappen, Rijksuniversiteit Gent56:195-202 . Gullino,M.L. ,Benzi ,D. ,Ozino-Marletto ,I .an dAvataneo ,M. , 1992.Caratteristich ed i duepotenzial imezz ibiologic id ilott acontr oagent id imarcium ii npost-raccolta . Atti Giornatefitopatologiche 2:381-388 . Roberts, R.G., 1990. Postharvest biological control of gray mold of apple by Cryptococcus laurentii. Phytopathology 80:526-530. Romano, M.L. and Gullino, M.L., 1983. Evaluation of the sensitivity to several fungicides of postharvest apple pathogens in North-Western Italy. Mededelingen FakulteitLandbouwwetenschappen , Rijksuniversiteit Gent48:591-602 . Wilson,C.L .an dWisniewski ,M.E. , 1989.Biologica l control ofpostharves t diseaseso f fruits and vegetables: an emerging technology .Annua l Review of Phytopathology 27:425-441. 200 Biological control ofBotrytis cinereao ncol dstore d dutchwhit e cabbage C. Leifert, D.C. SigeeandHA.S. Epton Summary Bacterial antagonists ofB. cinereawer eisolate dfro m Dutchwhit ecabbag eo nth ebasi s of invitro inhibition inplat eassays .Al lspecie s showing antagonism at4° Cwer ei nth e generaPseudomonas andSerratia. Cabbag edisk stake nfro minterna lleave sneede dhea t treatment(50° Cfo r 15 min)t oovercom e theinitia l resistance of theplan ttissue ,whic h prevented spore germination and fungal spoilage for periods of at least 2 months.O n heated cabbagedisk s spores ofB. cinerea germinated andmyceliu m covered thewhol e diskwithi n 2weeks .Spoilag e ofheate d diskscoul db eprevente d bydippin g ofdisk si n suspensions ofPseudomonas andSerratia antagonists. Introduction Botrytis cinereaPers. :Fr .i son eo fth emos timportan tpost-harves t diseases offrui t and leafy vegetables. For many plant species B. cinereai s an opportunistic pathogen attacking weakened, wounded or senescenttissues . Healthy leaf tissues have,however , been described as being highly resistant to attacks of B. cinerea (Newhook, 1951).I n contrastt oman yothe rfungi , itca ncaus espoilag ea ttemperature s( 1 to 10°C)whic har e usedfo r long term storageo ffrui t andvegetables .Ro to fcabbag eb yB. cinereausuall y starts after 2-3 months of cold storage and is often confined to the outer, dried out, senescent cabbage leaves (Wale, 1980).Post-harves t treatment of fruit and vegetables with fungicides such as benomyl (Benlate) or iprodione (Rovral) greatly reduced the losses due to rot by B. cinerea and allowed cabbages to be stored for up to 9 months (Leifert etal., 1992a).However ,th epost-harves tus eo ffungicide s iso fgrowin gconcer n and this is likely toresul ti n legislation preventing theus eo f some or possibly allsuc h applications inth eEC .Thi s situation andreport so fincreasin gresistanc eo fmajo r post- harvest pathogens such as B. cinereat o fungicides (Wilson and Wisniewski, 1989) prompted a search for alternatives to fungicide control of post-harvest fungal diseases. One alternative to fungicides is the use of bacterial and yeast antagonists as potential biocontrol agents for pathogens of avariet y of stored vegetables and fruit (Wilson and Wisniewski,1989) . We have recently reported the isolation from Dutch cabbage of strains of Serratia liquefaciens, Serratia plymuthica and Pseudomonasfluorescens wit h in vitroactivit y againstB. cinerea an dAlternaria brassicicola, an dth eeffec t ofnutritio nan dtemperatur e on in vitroantagonism s in plate assays (Leifert etal, 1992a,b). This paper describes someinteraction s between plant resistance and antagonisticSerratia and Pseudomonas spp. observed when studying spore germination of B. cinereao n heat-treated and untreatedcabbag elea f tissuedisks . 201 Material and Methods Stock cultures of B. cinerea, previously isolated from diseased Dutch white cabbage, were maintained on cabbage agar 5 (50 g homogenized cabbage tissue, 10g agar, 1 1 distilled water) at 5°Ci n thedark . Forsporulation ,B. cinerea wasgrow n for 5day so n malt extract agar at 20°C in thedar k followed by 3day s under UV-light. Spores were harvested by pouring 10 ml of sterile Ringer's solution onto a fungal plate and suspending sporesusin g abacteriologica lloop .Spor esuspension s werefiltere d through a doublelaye r of muslin and adjusted to aconcentratio n of 4 x 106spores.ml- ' using a haemocytometer. Leafdisk s(1 5m mdiameter )wer ecu tfro m internalleave so fcabbage s(th eoutermos t five leaves werediscarded ) using acork-borer .Disk s weretransferre d to a2 5 mluniv ersalbottl ean deithe rheate d for 20mi na t50° Co ruse ddirectly .Disk swer elef t tocoo l and then placed in a bacterial suspension of Pseudomonas or Serratiaisolate s from Dutchwhit ecabbag e (Leifert etal., 1992a) .Afte r inoculation withbacteria ,disk s were transferred toa tissu ecultur eassa yplat e(8. 5x 13 cm)wit h2 4well so f 15 mmdiamete r (Fig. 1).A non-heated disk was placed under the inoculated heated disk to mimic the situation in a stored cabbage head where susceptible, senescent outer leaves overlay resistant metabolically active inner leaves. Assay plates were then placed open in a Fig. 1. Leafdisks inassay dish Assaydis hwit h leaf disksinfecte d withB. cinerea b yapplyin g2. 5m lo f afunga l suspension containing (104 spores) into the centre of the disk after 8 weeks of incubation at 4°C in the dark. Disks showing visiblefunga l growthhav ebee nheat-treate d (15mi na t50°C )prio r toinoculatio n withB. cinerea. Disks withoutfunga l growthha dno tbee nheated .Disk so nth erigh thal fo f thetra ywer ewounde di nth ecentr e and fungal spores wereapplie d toth ewound ;disk s in thelef t half wereno t wounded. 202 Table 1. Suppressionof B. cinereaby Pseudomonas and Serratia spp. on heated cabbageleaf disks. No.o fB. cinerea Bacterialinoculu m(c : fu.ml-i) sporesi m Dculated perdis k P. fluorescens S.plymu thica S. liquefaciens CL42,66,8 2 CL43 CL80 7 7 7xl0 7x10« 7xl0 5x10s 5xl07 5x10« 4 10 + V V V 103 2 + + + + V 10 + + + + + + V = variable results (inhibition of fungal growth in 1o r 2 out of 3 experiments); + = inhibition of fungus until end of experiment in all3 experiment s (6week safte r control disks showedfunga l growth). Eachexperimen t wasrepeate d 3times .- =n oinhibitio n offunga l growth. laminar flow cabinet until the disk surfaces were dry. Afterwards 2.5 jxl of fungal suspensioncontainin g 102, 103o r 104 sporeswa splace d inth ecentr eo fth edisk .Assa y plateswer eplace dove rwate ri na see dtra y(1 5x 2 0cm )an dth etra yseale dwit hplasti c film. Trayswer eincubate d at4° Ci nth edar kfo r 10weeks . Fig. 2. Non-heated, non-woundeddisks 203 Fig. 3. Non-heated, woundeddisks Results and Discussion When spores of B. cinereawer e inoculated onto non-heated disks they failed to germinatean dn ospoilag eo flea fdisk soccurre dfo rperiod so fu pt o3 months(Fig . 1 and 2). When leaf disks were wounded in the centre with a sterile pipette tip some spores germinated in thevicinit y ofth ewoun dbu t again failed toestablis h andcaus e spoilage onth elea f disk(Fig . 1 and3) .Thi ssuggest stha tplan tresistanc emechanism sar eactiv e and prevent spore germination and fungal establishment. Heating of cabbage disks resulted in rapid fungal growth and 14 days after inoculation the fungal mycelium coveredth ewhol edis k(Fig . 1 and4) .Thi ssuggest stha theatin gha dabolishe dth eplant s resistance mechanism. Fungal establishment could be prevented on heated disks by dipping ofdisk sint obacteria l suspensions ofSerratia andPseudomonas whichshowe d invitro antagonis m in plate assays. All non-identified bacteria which did not show in vitroantagonis m could not prevent fungal development. After inoculation with some antagonistic Serratia and Pseudomonas strains (CL42,CL43 , CL66, CL80 and CL82) leafdisk sremaine dwithou tsign so fspoilag eunti lth een do fexperiments ,afte r2 month s (Table1) . SinceSerratia andPseudomonas antagonists canpersis to nuntreate d cabbageleave s in high numbers [103- 104cfu/cm ^ leaf surface (Leifert etal, 1992a)],an d increasei n numbers on damaged and heated cabbage tissue, they may be potentially useful bio- control agents against fungal pathogens when plant resistancemechanism s breakdow n atth eonse t of leaf senescence.W ear ecurrentl y testing this hypothesis by determining 204 Fig. 4. Heated,non-wounded disks Figure 2-4: Electron micrographs of cabbage leaf disks taken 10day s after inoculation with B.cinerea sporesan dincubatio n at4° Ci nth edar k(magnificatio n isgive n on thephotograph ) whether dipping of whole cabbages into suspensions of these antagonists can prevent fungal spoilagedurin g coldstorag eo fcabbage si ntw ostorag etrials . References Leifert, C, Sigee,D .an dEpton ,H.A.S. ,1992a . Isolationo fbacteri aantagonisti ct opost - harvestfunga l diseases ofcol d storedB rassica spp.In Non-chemica l Approachest o CropProtectio ni nHorticulture .Ed .Crute ,I .HR IPublications ,Wellesbourn ep . 106- 109. Leifert, C, Sigee,D. , Stanley, R., Knight, C. and Epton, H.A.S., 1992b.Biocontro l of Botrytiscinerea and Alternaria brassicicola on Dutch white cabbage by bacterial antagonists atcol d storetemperatures .Plan tPathology ,i npress . Newhook,F.J. , 1951.Microbiologica l controlof Botrytis cinerea Pers. :Fr .I .Th erol eo f pHchange san dbacteria l antagonism.Annal so fMicrobiolog y 38:169-184. Wale, S.J., 1980. The post-harvest pathology of Dutch white cabbage in refrigerated storage.Ph.D .Thesis ,Mancheste rUniversity ,U K. Wilson,C.L .an dWisniewski ,M.W. , 1989.Biologica lcontro lo fpostharves tdisease so f fruits and vegetables. An emerging technology. Annual Review of Phytopathology 27:425-441. 205 Droughttoleranc e ofBotrytis squamosa, B. acladaan d potential antagonists J.Köhl, M.C. Krijgerand G.J.T. Kessel Summary Mycelial growth and conidial germination of Botrytis squamosa, B. aclada and anta gonistic species of Gliocladium and Trichoderma weredetermine d at water potentials between- 1 mPaan d- 7 mPao naga rwit hKC la sosmoticum . Gliocladium spp.wer ea t leasta sosmotoleran t asBotrytis spp.Trichoderma spp .wer edistinctl ymor esensitiv et o lowwate rpotentials .Th evariatio nbetwee nstrain swithi n thegener awa slow .I n abio - assayo ndea donio nleaves ,G. roseum andT. viride suppressed sporulation ofB. aclada at- 1 mPabu t at-6. 6 mPaonl yG. roseumwa s effective. Introduction Botrytissquamosa and B. aclada(B. allii)caus e leaf blight and neck rot in onions, respectively. During epidemics both pathogens produce inoculum on dead onion leaf tissue.T osuppres ssporulatio nof Botrytis spp. ,saprophyti cantagonist swer eintroduced . Peng and Sutton (1990) found Gliocladium roseuman d Trichoderma viride highly suppressive tosporulatio n ofB. cinereaafte r field applications ondea dleave so fstraw berry. In bioassays on dead onion leaves both antagonists suppressed sporulation of B.aclada almos tcompletel y(Köh let al., 1990). Oneo f themos tlimitin g abiotic factors for bothBotrytis spp .an dmicrobia l antagonists isth econtinuou s fluctuation in thewate rconten to fdea dtissu edu et oth e microclimate in the crop. In our study, the sensitivity to low water potentials of the pathogens and antagonistic Gliocladium spp.an dTrichoderma spp.wa scompared . Material and Methods Mycelial growthrate s Petridishe swit hmal textrac taga r( 1g maltextract.H , 15g agar.H )wer einoculate dwit h agardisk s( 5mm )fro m theedge so fcolonie so fth etes tfungi .Th ewate rpotentia lo fth e agar had been adjusted by adding KCl. A thermocouple psychrometer with multiple sampleholder s (Decagon Devices Inc.,Pullman ,WA ,USA )wa suse d todetermin e the water potential of the agar. Samples were left in the apparatus for 30 min before measuring to allow equilibration. Demineralised water, 0.5 M KCl, 0.5 M NaCl and saturated KClsolution s served asstandards .Plate swer eincubate da t 18°Can dth edail y mycelial growth rates werecalculate d from thecolon ydiameter s measured after 3t o7 days. To compare the susceptibility of the several fungi to low water potentials, the mycelialgrowt h wasexpresse d relativet oth egrowt hat- 1 mPa. 206 Spore germination Water agar (15 g agar.H) in sterile microtitre plates was inoculated with conidial suspensionsapplyin g 10|x lpe r6 m mwell .Conidi awer eobtaine dfro m cultureso nmal t agar after incubation at 18°Cfo r 5t o 10days .Th ecolonie s werefloode d with watert o remove the conidia. After filtration through adoubl e layer of cheesecloth, suspensions wereadjuste d to 1 x 104conidia.mH .Th ewate rpotential so fbot hth ewate raga ran dth e suspensions used for inoculations were adjusted by adding KCl. After an incubation period of 24h a t 18°C,plate swer eplace d for 60mi ni na chambe rcontainin g formalin vapour to stop further fungal growth. Fifty spores per treatment were counted in five replicates to determine the percentage germination; the length of ten germtubes of germinated conidia was measured in five replicates using a Minimop image analyzer (Kontron,Enching-Munich ,FRG) . Bioassays Antagonism of T. virideT00 4 and G. roseum 1813 against B. aclada was tested in a bioassay ondea d onionlea f segments.Therefore , autoclavedlea f segmentswer eplace d in Petri dishes containing water agar adjusted to- 1 or- 7 mPab y adding KCl.Leave s were separated from the agarb y sterile plastic grids to avoid diffusion of KCl into the leaves.Afte r 7day so f equilibration, themeasure d water potential of the leaves was -1 mPa or -6.6 mPa. Malt broth (10 g malt extract.H) was inoculated with conidia and incubatedfo r 14day sa t 18°Ci norde rt oproduc emycelium .Th emyceliu mwa s filtered, washed with sterile water and homogenised in a blender for 60 sec. Suspensions of mycelial fragments of B. aclada( 1 x lO^mH) were sprayed on the dead onion leaf segments infive replicate s pertreatment .Afte r 24h incubatio n at 18°C,th eantagonist s (1 x 105mycelia l fragments.ml-1) were applied. The water potentials of the mycelial suspensions wereadjuste d with KClt oth ewate rpotentia lo f thesubstrat et ob etreated . After 7 days at 18°C, the leaf surface covered with conidiophores of B. acladawa s estimated (class0 = 0% ; 1 = 1-5%; 2= 6-25% ; 3= 26-50% ;4 = 51-75% ;5 = 76-100% covered). Results Mycelial growth B. squamosawa s slightly more susceptible to low water potentials than B.aclada (Fig. la). Gliocladium spp. were at least as osmotolerant as Botrytisspp. , whereas distinctlyhighe rsusceptibilit y wasfoun d for Trichodermaspp .(Fig . lb and lc).Almos t novariatio nwa sfoun d betweenstrain swithi nth egener aTrichoderma an d Gliocladium. Spore germination At- 4mPa ,spor egerminatio n wasmoderat efo r B. acladaW ,an dhig h for Gliocladium roseum 1813, whereas spores of Trichoderma viride T004 failed togerminat e (Fig.2) . Sporeso fal lfung i failed togerminat ea t- 5 mPao rlowe rwate rpotentials .Th egermtub e growth in allfung i tested wasmor eaffecte d bylo wwate rpotential s than theirmycelia l growth (Fig.3 )an dT. viride wasth emos tsusceptibl especies . 207 (a) Botrytis spp. B. squamosa 100- ^^^ »•*. S,t " • B. aclada R cr ^^s. ^%""X-^ *"^ (3.3) 0 , , , •"--, *a*"tl -3 -5 -7 Water potential (MPa) (b) Gliocladium spp. 100- "^^~. G. catenulatum •"*•X. \\ "-»V NV 1814 (2.2) 0 ^S' G. nigrovirens ü 1815 (1.8) >. \S 50- \N. G. roseum > ^"^^J^ — 1813 (1.9) v J2 ^* ^-*l ^ *"*•••* ^*^**ö\^ ^ (c) Trichoderma spp. T. viride T004 (6.1) O 100 ö> T. viride T141 (4.7) o T. hamatum > T166 (6.6) E T. harzianum > 6 (5.7) jO -1 -3 -5 Water potential (MPa) Fig. 1. Relativemycelial growth (-1 mPa =100) of (a) Botrytis squamosa andB. aclada, (b)Gliocladium spp. and(c) Trichodermaspp. at different water potentials on malt agar. Growth ratesat-1 mPa inmm 24hr 1 in brackets. 208 100i Botrytis c aclada W .0 Gliocladium to roseum 1813 ç 'E Trichoderma ai viride T004 -3 -5 -7 Water potential (MPa) Fig. 2. Germination of conidia of Botrytisaclada W, Gliocladium roseum 1813 and Trichoderma viride T004 atdifferent water potentials onwater agar after 24 h at 18°C (meansof 50 conidia in five replicates). E *-> Botrytis e» aclada W Gliocladium roseum 1813 .O Trichoderma viride T004 (5 Water potentia Fig. 3. Germtube lengths of conidia of Botrytis aclada W., Gliocladium roseum 1813 andTrichoderma virideT004 atdifferent waterpotentials onwater agar after24 h at 18°C(means often germtubes infive replicates). Bioassay Onwe tleave swit h- 1 mPa,G. roseum 1813an dT. viride T004suppresse d sporulation ofB. aclada (Fig .4) .A t-6. 6m Pa ,onl yG. roseumwa s efficient. Discussion Droughttoleranc ei son eo fth eke yfactor s forth eapplicatio no fantagonist saime da tth e suppression of Botrytis spp. Potential antagonists in the genera Gliocladium and Trichoderma markedly differed from each other with respect to their growth on agars with decreasing water potential. Only Gliocladium spp. were at least as tolerant as Botrytisspp . The variation between strains within the genera was unexpectedly low. Consequently,selectio no fdrough ttoleran tstrain so fTrichoderma seem sles spromising . 209 -6.6 MPa 13 2 b Ä 1 water T.v. G.r. water T.v. G.r. Fig. 4. Sporulationof Botrytis aclada Won dead onion leavesat -1 mPaand -6.6 mPa afterapplication ofGliocladium roseum 1813or Trichoderma viride T004. Results obtained on agar with osmotica added are not necessarily representative of the conditions in the natural substrate wherematri c potential is more important. Alderman and Lacy (1984),foun d aclos e relationship between mycelial growth ofB. aclada ata range of water potentials on onion leaf tissue and on agar when KCl was used as osmoticum.I nou row nexperiments ,G. roseum 1813 wastoleran to flo wwate rpotential s on agar with low osmotic potential, as well as on dead leaf tissue with low matric potential,wherea sT. viride T004wa sles stoleran tunde rbot hconditions . References Alderman, S.C.an d Lacy,M.L. , 1984.Influenc e of temperature and water potential on growtho fBotrytis allii. Canadian Journal ofBotan y62:1567-1570 . Kohl, J., Molhoek, W.M.L. and Fokkema, N.J., 1990. Biological control of Botrytis aclada inonions .Phytopatholog y 80:1049(Abstract) . Peng,G .an dSutton ,J.C. , 1990. Biologicalmethod st ocontro lgre ymoul do fstrawberry . BritishCro pProtectio nConference s -Pest san dDisease sp .233-239 . 210 Efficacy ofa grapefruit seedextrac t BC-1000fo r controlo f Botrytiscinerea i n table grape in Chile MA, Esterio, J.G.Auger, E. Vazquez, M.Reyes andF. Scheelje Summary Theefficac y of BC-1000, asee d extract of grapefruit for control of B. cinerea in table grapes was tested in largefield experiment s in the 1990/1991 season. BC-1000 wasa s effective as benomyl or vinclozolin at concentrations of 1500 ppm. When applied in combination with fungicides the results were equivalent or superior, depending on the presenceo fbenomy lan dvinclozolin-resistan tstrains . Introduction InChile ,contro l ofBotrytis cinerea Pers.:Fr . in tablegrape s isbecomin g increasingly difficult becauseo f thedevelopmen t of strainso f thefungu s resistantt o benzimidazole anddicarboximid efungicides . Therefore, wehav edevelope d anatura l organicproduct , BC-1000, which has a different mode of action that could be useful for control of B.cinerea. BC-1000i sa nextrac to fgrapefrui t seedsknow nt ohav eantifunga l properties (Anonymus, 1990a,b). Its efficacy, either alone or in combination with chemical fungicides forcontro lo fB. cinerea i ntabl egrape swa steste di na numbe ro fexperiments . Someo fth eresult swer ealread ypublishe d (Esterio andAuger , 1990a,b). Material and Methods Aserie so fexperiment si nvineyard slocate d inth eCentra l valleyo fChil ewer ecarrie d outdurin g the 1990/1991season . In theexperiments ,al lwit h 15treatments ,BC-100 0(150 0ppm) ,benomyl ,benomy l +captan ,capta nan dvinclozolin ,(a tcommerciall yadvise drates )wer eapplie di nvariou s combinations at five growth stages during the development of berries of the cv. Thompson Seedless. Thetreatment san dgrowt hstage sar esummarise d inTabl e 1. Each treatmentha dfou r replicates,wit h2 0plant spe rtreatment . Toensur e aunifor m population of B. cinerea, aninoculatio n wasperforme d shortly before the pre-bloom stage by spraying two plants in the middle of each plot with a conidalsuspensio ncontainin g 9x 10*conidia.ml 1.Th esuspensio ncontaine dconidi ao f isolateso fB. cinereasensitiv ean dresistan tt o5 0pp mbenomyl ,i na 1: 1 ratio. Thefollowin g parameterswer eevaluated : 1) field infection levelthroughou tth efiv e growth stages; 2) final postharvest decay level (after storage of 21day s at0°C , and 11 days at 12°C); and 3) development of benomyl (50 ppm) and vinclozolin (20 ppm) resistantisolate susin g themetho d ofLerou x andGred t (1979).Concurrently ,a tes twa s carried out to evaluate any possible phytotoxic effects of BC-1000, on the vines and berrieso fcv sFlam eSeedless ,Thompso n Seedlessan dRibie ra t 1500an d 1800p p mi n comparison withstandar dcapta nsprays . 211 The infection level was determined from pre-bloom after harvest. Before the fungicide treatments were applied, 100 flowers or berries from each treatment were collected from thetw o central plants ineac h plot. These samples were incubated in Petri dishes containing water agar at20° C for2 0days , andperiodicall y assessed for infection. To determine post-harvest decay, two8. 2k g grape boxes were harvested from every plot; one box was subjected to the standard Botrytis postharvest control treatment (fumigation with S02), while the other remained untreated. Later, the boxes were kept at 0°C for a 21-day-period and thereafter left at room temperature for 2 to 13 days. The decay level per fruit box was determined after this period through visual inspection. Decay was also determined byweighin g the total berries which showed symptoms of the disease. Data were subjected to analysis of variance andDuncan' s multiple range test. The laboratory test were carried out at the Plant Pathology Laboratory of the Plant Health Department of the Faculty of Agricultural and Forest Sciences, University of Chile. Results and Discussion The data from one of the experiments aregive n in Table 2. Treatments with BC-1000 alone (Tl andT2 ) gave similar results to those with benomyl or vinclozolin alone (T12 Table 1. Overview of the different treatments inexperiments tocontrol B. cinerea in table grapes, cv. Thompson Seedless in the 1990/1991 season. Treatment Application Pre-bloom Full bloom Fruit set Véraison Preharvest (10 days before harvest) Tl BC1»> BC1 BC1 BC1 BC1 T2 BC22) BC2 BC2 BC2 BC2 T3 BC2 BC2 C C V T4 V3) B+C C C V T5 V B+C C C BC2 T6 V B+C BC2 BC2 V T7 B4>+C5> B+C C C V T8 B+C BC2 C C V T9 BC2 B+C C C V T10 B+C B+C C C BC2 Til V BC2 C C BC2 T12 B B B B B T13 V V V V V T14 Untreated control T15 Standard vineyard treatment i)BCl = BC-1000 1.200 ppm 2)BC 2 = BC-1000 1.500 ppm 3)B = benomyl 4)C = captan 5)V = vinclozolin 212 Table2. Infection of flowers or berries of cv. ThompsonSeedless by B. cinerea determined at the different growth stagesas treatments applied. (Eachfigure is the average offour replicates. Infectionsare given aspercentage of the untreatedcontrol). Treat Pre- Full Fruit Véraison Pre- Post- ment bloom bloom set harvest harvest Tl 11.75 2.75 1.0 4.25 5.75 1.45 T2 14.75 4.75 2.5 3.0 2.75 1.95 T3 18.5 6.5 1.25 3.75 3.5 1.05 T4 10.5 3.5 2.25 3.25 4.0 1.55 T5 16.5 6.0 2.0 4.25 4.3 0.80 T6 14.0 5.25 2.0 5.75 4.0 2.25 T7 14.0 5.25 2.25 3.25 5.25 0.50 T8 12.5 1.25 1.5 3.3 4.5 0.60 T9 14.75 5.25 1.25 2.05 3.5 1.25 T10 12.5 5.0 0.5 4.0 3.0 0.80 Til 12.5 6.75 1.75 5.0 3.0 1.35 T12 14.5 3.25 1.75 5.0 5.0 1.00 T13 11.25 5.75 2.0 5.8 4.5 2.75 T14 13.5 5.5 2.8 3.3 5.0 4.8 T15 16.5 5.25 1.5 4.0 5.0 1.25 Table 3. Percentage of isolates ofB. cinerea resistant to50 ppm benomyl obtained at various growth stages Treatment1) Growthstage s Pre- Full Fruit Véraison Pre- Harvest Post- bloom bloom set harvest harvest Tl 63.P ) 54.5 77.7 72.7 71.8 72.3 77.8 T2 42.4 74.7 72.2 71.5 73.9 66.5 70.8 T3 76.0 74.5 72.8 72.6 82.9 87.7 81.3 T4 87.6 67.6 89.1 84.8 77.1 85.6 88.0 T5 82.8 52.4 78.4 74.3 83.7 83.9 87.2 T6 63.2 73.9 80.6 80.8 89.4 91.3 88.3 T7 85.1 68.0 81.2 75.6 89.6 86.8 82.4 T8 46.4 37.2 77.9 84.2 75.0 88.4 74.7 T9 72.2 24.0 81.7 76.3 83.5 68.1 79.2 T10 48.6 42.3 86.8 77.5 72.9 72.0 70.7 Til 58.3 81.5 80.1 76.4 75.8 76.2 72.8 T12 38.1 55.1 91.7 84.3 83.2 84.2 88.8 T13 87.0 48.5 81.9 75.7 78.4 81.4 82.4 T14 53.4 13.6 82.6 83.4 82.5 79.4 79.0 T15 81.2 68.0 81.2 75.6 89.6 86.8 82.4 i)Se eTabl e1 2)Averag eo ffou r replicates,isolatin g threecolonie spe rtreatmen t 213 andTl3) . Somecombinations , such asT5 , T7 andT 8resulte d in alowe r percentageo f postharvest decay. The results of the other field experiments were not so successful, probably due to the presence of a larger number of fungicide-resistant strains in the population ofB. cinerea. Ahig h numbero fbenomyl-resistan t isolates were obtained in all field experiments, the number being highest in the benomyl-treated plots and the lowesti nth eBC-100 0treate dplot s(Tabl e 3). Therewa slittl edifferenc e in theincidenc e ofbenomy lresistan tisolate sbetwee n the treatmentsa tth een do fth eseason ,bu ta tearlie rdates ,ther ewa sa large rvariatio nwhic h couldno tb ecorrelate dwit htyp eo ftreatment .Mos to fth ebenomyl-resistan tisolate sals o showed somelimite d resistancet ovinclozolin . Novisibl edamag ewa ssee no nberrie so ro nth erachi si n thephytotoxicit ytests . The efficacy of BC-1000 in concentrations of 1500 ppm was similar to that of benomyl and vinclozolin and combinations of the three compounds increased their effectiveness, dependingo nth epresenc eo fresistan tisolate so fB. cinerea. Theseresult s wereobtaine d inexperiment scarrie d outi non eseason . References Anonymus, 1990a.Inhibitor yeffect s ofgrapefrui t seedextrac t(DF-100 )o ngrowt h and toxin production of Pénicilliumislandicum. Journal of the Korean Agricultural Chemical Society.Vol . 33(2) . Anonymus, 1990b.Antimicrobia l andantioxidan t activity ofgrapefrui t andsee dextrac t on fishery products. Bulletin of the Korean Fisheries Society, Vol.23 ,No .4 Pusan, Korea,1990 . Esterio,M .an dAuger ,Y. ,1990a . Determinaciónd el aefectivida d delproduct oBC-100 0 ene lcontro ld eBotrytis cinerea Pers .e nuv ad eexportació ncv .Thompso nSeedless . RegionMetropolitana .Temporad a 1989/1990.Santiago ,Faculda dC iencia sAgraria s yForestales ,Universida d deChile , Chile. Esterio,M .an dAuger ,Y. ,1990b . Determinaciónd el aefectivida dde lproduct oBC-100 0 ene lcontro ld eBotrytis cinereaPers .e nuv ad eexportació ncv .Thompso n Seedless. Regióon Metropolitana. Temporada 1990/1991. Santiago, Faculdad C iencias Agrariasy Forestales ,Universida d deChile ,Chile . Leroux,P . andGredt ,M. , 1979. Phénomènesd erésistanc ecroisé enégativ eche zBotrytis cinerea Pers. entre les fongicides benzimidazoles et les herbicides carbamates. Phytiatrie-Phytopharmacie 28:79-86. 214 CHEMICAL CONTROL -^fc- Chemical control ofBotrytis spp . M.L. Gullino Summary The main factors involved in designing effective chemical control strategies against Botrytisspp .o ndifferen t cropsar ereviewed .Som eo fth eproblem s relatedt oth eus eo f fungicides aresummarise d and theway s tocounterac t them arediscussed .Th enee d to integrateth eus eo fchemical swit h othercontro lmeasure s isstressed . Introduction Afe wBotrytis spp .caus esever elosse so nman yeconomicall yimportan tcrops .Althoug h alternativecontro l measures areofte n proposed andattempted ,especiall y inth ecas eo f protected crops,contro l of these fungi still relies mainly on extensive use of synthetic chemicals. However, in spite of the availability of several effective fungicides and improved application techniques, control of Botrytis spp. is often difficult and incomplete. This review focusses on some aspects of chemical control of Botrytisspp. , with specialemphasi s onth eproblem s thatca nb eencountere d underdifferen t situationsan d onth efactor s whichca ncaus ethes eproblems . Factorsaffectin g chemical controlo fBotrytis spp. Whenconsiderin g chemicalcontro lo fdisease scause db yBotrytis spp. ,i ti simportan tt o take into account the biology of the pathogen and crop diversity, cultural conditions, availability offungicide s andpossibl edevelopmen t ofresistanc etoward ssom eo fthes e fungicides. Pathogenbiolog y andcro pdiversit y Thecontro l ofBotrytis spp.i sextremel y difficult becauseo fth eabilit y ofthi spathoge n toattac kcrop s atalmos tan ystag eo fgrowth ,o rdurin g storagean dtransit ,an dt o affect allth eplan tparts ,includin g cotyledons,leaves ,stems ,flower s and fruits. Different species of Botrytisattac k different hosts. Among them, Botrytis cinerea Pers.: Fr. has the widest host spectrum and causes severe damage on several eco nomically important fruit, vegetable and ornamental crops. Other species for example B. acladaFresen. , B.fabae Sard., B, squamosa Walker, and B. tulipaeLin d show a restricted hostrang ean da sa consequenc egenerall ycaus eles ssever eeconomi closses . The use of fungicides to control the various species of Botrytis encounters different problems.Fo rinstance ,treatment sdevise d for protection ofcertai n parts ofa plan tma y notb esufficien t toprotec t othertissues .Fungicid eresistanc e spreads anddevelop s ata different ratei nvariou sspecies .Amon gthes especies , B. cinerea hasshow na particula r aptitudet odevelo pfungicid e resistance. 217 Crop diversity influences Botrytis control in terms of practical approaches, availabilityo fchemical san dimplementatio no fpossibl estrategies .I nfrui tcrop smos to f theavailabl efungicide s activeagains tthi spathoge nar eregistere dfo rus ei nfiel dsprays . A limited number of chemicals is registered for use as postharvest treatment with disparitiesbetwee ndifferen t countriesi nwhic hchemical sma yb eapplied .Toxicologica l and environmental considerations make the situation most complicated and rather diversified inth ecas eo fvegetable slik etomato ,strawberry ,lettuce ,cucumber ,onio nan d garlic, especially when these are grown in glasshouses or in polyethylene tunnels. In ornamentals,lik ecyclamen ,rose ,gerbera ,pelargonium ,tuli pan dUly ,aesthetic sstrongl y influences thechoic e of thefungicid e becausefungicid e residues mayb evisibl e onth e blossoms.Moreover , possible toxic effects on humans causes restrictions in the useo f somechemical s (Garibaldi andGullino ,1990) . Cultural conditions Different approachesar euse di ndesignin gchemica lcontro lstrategie si nope nfiel d ori n protected crops. Grey mould caused by B. cinerea is generally more serious in greenhouse crops, where conditions favour development and spread of the pathogen. However,mor einnovativ econtro lstrategie sca nb edesigne di ngreenhous ecrop s(Jarvis , 1989)an dfungicide s canb eapplie db ymean s ofenvironmentall y safer techniques.Fo r instance, formulations of dicarboximides which are active by sublimation have been developed, with the aim of reducing the amount of residues on fruit and atminimisin g farmer's exposure to chemicals during spraying. Fumigation with sulphur dioxide has been, and still is, widely used for table grape (Eckert and Ogawa, 1988). Postharvest treatments canb ecarrie dou tb yapplyin g chemicals inaqueou swashe san ddips ,spray s or as dust. Recently, sprays have been largely used in order to reduce environmental problems causedb ydischarg eo flarg equantitie s of fungicide. Available fungicides Two groups of chemicals, the benzimidazoles and dicarboximides which are highly activeagains tBotrytis spp .ar ecurrentl yavailable . Diethofencarb, aN-phenylcarbamate , has been developed over the past decade to control B. cinereastrain s resistant to benzimidazoles (Kato, 1988).Multisit efungicide s such aschlorothalonil , dichlofluanid andthira mar eals oavailable ,bu tsever erestriction s often limitthei rus eo nsom ecrops . Ethylene bisdithiocarbamate (EBDC) fungicides are still largely employed against B. squamosa, alone or in mixtures with dicarboximides (Lorbeer and Vincelli, 1990). Some ergosterol biosynthesis inhibiting fungicides (EBIs) have also shown useful activity againstBotrytis spp.(Reineck e etal., 1986 ) and recently developed fungicides with good anti-Botrytis activity are expected to appear on the market in the next few years. Benzimidazoles, originallyhighl yactive ,los tpar to fthei rimportanc edurin g thelas t decadeo nmos tcrop sdu et oth eappearanc ean dpersistenc eo fresistan tstrain s(se elater ) and to toxicological problems. At present, they have limited use on grapevine and vegetable crops (Gullino and Garibaldi, 1986a).I n the case of vegetable crops,besid e problems of resistance, theus eo f benzimidazoles hasbee n severely restricted in many areas. In Italy they can no longer be used on greenhouse-grown vegetables during the cultivation,althoug h theyca nb euse d aspostharves t treatment.I nth elatte rcas ether ei s 218 now a trend to replace them with more effective chemicals due to the presence of fungicide resistantstrain s(Ecker tan dOgawa ,1988) . Dicarboximides replaced benzimidazoles in most situations in the late 1970s and early 1980s. Theirinitia lhig h activity hasbeen ,a tleas ti npart ,los tdu et odevelopmen t ofresistanc eo nsom ecrop s(Lorenz , 1988),bu tthei rus eremain scrucia lo nmos tcrops . Diethofencarb, which showed remarkable activity against benzimidazole resistant B.cinerea, ha sbee nregistere dan duse di nsom ecountrie ssuc ha sFranc ean dIsrael ;how- ^ ever,it sus eagai nrapidl yle dt oth eoccurrenc eo fresistan tstrain so fB. cinerea (se elater) . Among ergosterol biosynthesis inhibiting fungicides (EBI), some conïpoundsjhow^ satisfactory activity againstBotrytis spp.Thei r fungicidal activity hasbee n particularly exploited against B. cinerea. For instance, tebuconazole, alone and mixed with toly- fluanid, showsa goo dactivit yagains tgre ymoul do nsevera lcrops .Prochlora zi sapplie d on tulip against B. tulipae (A. Koster, personal communication). However, the useo f EBIs against Botrytis spp. is uncommon and the selectivity of EBIs must be carefully evaluated.Repeate d application ingreenhouse sca ncaus ephytotoxicit y insom ecrops . Among new fungicides, the phenylpyrrole CGA 173506, the pyrimidine mepa- nipyrim (Maenoet al., 1990 ) and thetriazol e SSF-109(Murabayash i etal., 1990 )hav e been recently developed. CGA 173506 has a broad spectrum of activity, including Botrytis, Monilinia, Sclerotinia, Rhizoctonia, Alternaria, Fusarium, Septoria, Tilletia, Helinthosporium and Gibberella (Ghemann etal„ 1990) .Th e interest attracted by this chemicali srelate dt oth efac ttha ti tbelong st oa ne wchemica lgrou po ffungicide s with activity againstB. cinerea, Venturia spp.an dMonilinia fructicola (Maenoet al., 1990). Thetriazol eSSF-10 9seem st ob especificall y andhighl yactiv eagains tBotrytis spp. ,an d itsus e onbul bcrop sha sbee n reported (Anema etal., 1988) .I ti s highly desirable that fungicides like these with modes of action different from that of currently available chemicals,soo nbecom eavailabl et o growers. Multisitefungicides , althoughles seffectiv e thanspecifi c ones,ar einterestin gbecaus e ofthei rwide rspectru m of activitywhic hpermit scontro lo f secondary pathogens.The y are also good partners for specific fungicides in anti-resistance strategies. As a consequence, their use is still highly recommended, particularly on crops where the numbero f spraysi n aseaso ni shigh . Fungicideresistanc e Resistance tobenzimidazoles , dicarboximides and diethofencarb complicates chemical controlo fBotrytis spp.o nsevera lcrops .Th erelativ eimportanc eo ffungicid e resistance varieso ndifferen t cropsan di ndifferen t areas,accordin gt oth eselectio npressur eexerte d byth eus eo fthes egroup s ofchemical s (Staub,1991) . Atth etim eo fintroductio n ofth ebenzimidazoles ,ther ewer en oothe rhighl y effective fungicides that could havebee n used as companion materials. Accordingly, thebenzi midazoleswer eoverused ,an dresistanc edevelope dquickl y andlimite d their usefulness within 2t o4 year si nmos tareas ,wit hver yfe w exceptions (Delp,1988) . Dicarboximideresistanc edevelope di nth e 1980s,althoug hno ts orapidl yan di na les s dramatic way. This type of resistance now affects B. cinerea in many crops (Lorenz, 1988). Resistance to both benzimidazoles and dicarboximides is now widespread in B.cinerea o ngrapevine ,vegetabl ean dornamenta lcrop s(Gullin oan dGaribaldi ,1986b ; Leroux and Valentin, 1991). In the case of protected crops, resistance spreads quite rapidly through the entire greenhouse. A similar rapid spread of resistance has been 219 observed in commodities affected by postharvest attacks (Eckert and Ogawa, 1988). Resistancet obenzimidazole si sals opresen ti nB. squamosa (Presle yan dMaude ,1980) , B. tulipae (Duineveld andBeijersbergen , 1975)an dB. elliptica. Inth ecas eo fthi slatte r pathogen, dicarboximide resistance has also been reported (Migheli et al, 1989). In countries where diethofencarb is already registered, resistance to this chemical also developed rapidly(Lerou xan dValentin , 1991;Ela det al., 1992). Multisitefungicides , onth econtrary ,ar estil leffectiv e after yearso fuse .Onl yi nth e case of dichlofluanid, some loss of efficacy has been reported, probably due to the developmento fresistanc e(Malathrakis , 1989; Rewalet ai, 1991),bu ti nthes ecase sth e wide range in sensitivity of isolates within thewil d populations of B. cinerea mustb e considered (Garibaldi etal., unpublishedresults) . In crops which are frequently sprayed, monitoring for fungicide resistance is important because it permits the implementation of control strategies using chemicals whichar estil leffectiv e (Gullinoet al., 1989).Differen t monitoringtechnique shav ebee n described:som ear esimpl ean drapi dan dca nb edirectl yuse db ygrower so rtechnician s workingi nth eextensio n service(Lorenz , 1988). Mixtureso ffungicide s orrotatio no fthei rus ehelp st oreduc eth esprea do fresistance . The mixture of thiram with a dicarboximide showed good efficacy in the presence of fungicide resistancei nB. cinerea onsevera lcrop s(Boureau , 1988; Gullinoe tal., 1989; Creemers et al., 1990). It should be stressed, however, that the use of thiram is not recommended duet oit sactivit y againstsom ebenificia l organisms. Diethofencarb, a fungicide showing negative cross resistance to benzimidazoles, when used in mixtures withcarbendazi m controlsbenzimidazole-resistan t (RB)a swel l as benzimidazole and dicarboximide-resistant (RBD) populations of B. cinerea. This mixturerepresent s ashor tter manti-resistanc e strategy,abl et osolv eproblem s oflos s of efficacy due to fungicide resistance, but as described above, where its use has been intensive,doubl eresistanc et obenzimidazole s anddiethofencar b hasdevelope dalready . The mixture of carbendazim with diethofencarb cannot be used where benzimidazoles areno t permitted (i.e.greenhouse-grow n vegetables in Italy);thi s is onemor eexampl e of how the severe restrictions recently imposed in many countries complicate the implementation ofanti-resistanc estrategies . Strategiesi nchemica l control ofBotrytis spp. Although chemicals stillpla ya majo r rolei ncontro l ofBotrytis spp.o nmos tcrops ,i ti s clear that their use leads topractica l problems of various types in many cases.Fo rthi s reason, the exclusive use of fungicides can no longer be considered to be a workable approach toth econtro l ofBotrytis spp.o n mostcrops .Th echoic e of thechemica l still remainscrucial :ne wfungicide s having different modeso factio nar eurgentl yneeded . The integration of chemical treatments with cultural management systems, epide miologicalmodel san dbiologica lcontro lagents ,i sno wbecomin ga realisti can drationa l strategyfo r management ofdisease scause db yBotrytis spp. For B. cinerea, good results have been achieved on the greenhouse-grown crops tomato, cucumber and strawberry, by integrating the use of chemicals with careful control of environmental parameters. Reducing the persistence of water films on the plantb yincrease d ventilation andheatin g strongly reduced greymoul dincidenc eunde r severediseas e pressure on several crops (Elad andZimand , 1991;Gullin o etal., 1991; Nicotan dAllex ,1991) . 220 Acknowledgements Thisresearc h hasbee n supported by agran t from theMinister odelTUniversita ' edell a Ricercascientific a etechnologica . Manythank sar edu et oProfesso r A.Matt afo r criticalreadin g ofth emanuscript . References Anema,B.P. ,Bouwman , J.J. and deVlugt ,J. , 1988.Fluazinam : ane wbroa d spectrum fungicide for usei nbulbs .Mededelinge n FakulteitLandbouwwetenschappen , Rijks universiteitGen t53(2b):635-641 . Boureau, M., 1988. Intérêt de l'association de vinchlozoline et de thirame contre la pourrituregrise .Résultat sd edeu xannée sd'expérimentation . Phytoma399:39-41 . Creemers, P., Vanmechelem, A. and Bal, E., 1990. Ten years of control with dicar- boximide fungicides on strawberries. Mededelingen Fakulteit Landbouwweten schappen,Rijksuniversitei t Gent55:955-962 . Delp, C.J., 1988. Resistance management strategies for benzimidazoles. In Fungicide Resistancei nNort hAmerica .Ed .Delp ,C.J .AP SPress ,St .Paul ,MN ,US Ap .41-43 . Duineveld,T.L.J .an dBeijersbergen ,J.C.M. , 1975.O nth eresistanc et obenomy lo ffung i isolated from bulbsan dcorms .Act aHorticultura e47:143-149 . ' Eckert, J.W. and Ogawa, J.M., 1988. The chemical control of postharvest diseases: deciduous fruits, berries, vegetables and root/tuber crops. Annual Review of Phytopathology26:433-469 . Elad,Y. , Yunis,H .an dKatan ,T. ,1992 . Multiplefungicid e resistancet obenzimidazoles , dicarboximides anddiethofencar b infield isolate s ofBotrytis cinerea in Israel.Plan t Pathology41 , inpress . Elad,Y . andZimand ,G. , 1991.Experience si nintegrate d chemical-biological controlo f grey mould (Botrytis cinerea). West Paleartic Regional Section Bulletin 14(5):195 - 199. Garibaldi, A. and Gullino, M.L., 1990. Disease management of ornamental plants: a neverendin gchallenge .Mededelinge nFakultei tLandbouwwetenschappen , Rijksuni versiteitGen t55:189-201 . Gehmann, K., Nyfeler, R., Leadbeater, A.J., Nevill, D. and Sozzi, D., 1990. CGA 173506: a new phenylpyrrole fungicide for broad-spectrum disease control. ProceedingsBrighto n CropProtectio nConferenc e p.399-406 . Gullino, M.L., Aloi, C. and Garibaldi, A., 1989. Influence of spray schedules on fungicide resistant populations of Botrytis cinerea Pers. on grapevine. Netherlands Journalo fPlan tPatholog y 95(suppl. l):87-94. Gullino, M.L., Aloi, C. and Garibaldi, A., 1991.Integrate d control of grey mould of tomato.Wes tPalearti cRegiona l SectionBulleti n 14:211-215. Gullino, M.L. and Garibaldi, A., 1986a. Fungicide resistance monitoring as an aid to tomatogre y mould management. Proceedings Brighton CropProtectio n Conference p. 499-505. Gullino, M.L., and Garibaldi, A., 1986b.Resistanc e to fungicides in Botrytis cinerea: presentsituation .Notiziari oMalatti edell ePiant e 107:63-71. Jarvis,W.R. , 1989. Managingdisease si ngreenhous ecrops .Plan tDiseas e73:190-194 . Kato, R., 1988.Negativ e cross-resistance activity of MDPCan d diethofencarb against benzimidazole resistant fungi. InFungicid e resistance in North America. Ed.Delp , 221 C.J.AP SPress ,St .Paul ,MN ,US Ap . 1-40. Leroux, P. and Valentin, G., 1991. Evolution de la resistance de Botrytis cinerea aux fongicides dansl evignobl echampenoi s entrel e 1982e t 1990. AnnalesANPP , 3eme Conference Internationale surle sMaladie s desPlante sp . 845-851. Lorbeer, J.W. and Vincelli, P.C., 1990. Efficacy of dicarboximide fungicides and fungicidecombination sfo rcontro lof Botrytis lea fbligh to fonio ni nNe wYork . Plant Disease74:235-237 . Lorenz, G., 1988. Dicarboximide fungicides: history of resistance development and monitoring methods.In Fungicid e resistance in North America. Ed. Delp,C.J . APS Press,St .Paul ,MN ,US Ap .45-51 . Maeno,S. ,Miura ,I. ,Masuda ,K .an dNagata ,T. , 1990. Mepanipyrim (KJF-3535),a ne w pyrimidinederivative .Proceeding sBrighto nCro pProtectio nConferenc e p.415-422 . Malathrakis,N.E. , 1989.Resistanc e of Botrytis cinerea todichlofluani d in greenhouse vegetables.Plan tDiseas e73:138-141 . Maude,R.B. , 1980. Diseasecontrol .In Th ebiolog yo fBotrytis. Eds.Coley-Smith ,J.R. , Verhoeff, K.an dJarvis ,W.R .Academi c Press,Ne w York,Londo np .275-308 . Migheli, Q., Aloi, C. and Gullino, M.L., 1989. Presenza in Italia di ceppi di Botrytis elliptica resistenti aifung i cididicarb ossimidici.L aDifes a dellePiant e 12:3-10. Murabayashi, A.,Masuko ,M. , Shirane,N. ,Hayashi , Y.an d Makisumi,Y , 1990.SSF - 109, a novel triazole fungicide: synthesis and biological activity. Proceedings Brighton CropProtectio n Conference p.423-429 . Nicot,P .an dAllex ,D. , 1991.Gre ymoul do fgreenhous egrow ntomatoes : diseasecontro l byclimat emanagement ?Wes tPalearti c Regional SectionBulleti n 14:200-210. Presley,A.H .an dMaude ,R.B. , 1980. Controlof Botrytis cinerea an dBotrytis squamosa in overwintered salad onionsb yfungicid e spray.Annal s ofApplie d Biology94:197 - 204. Reinecke,P. ,Kaspers ,H. , Scheinpflug, H., Holmwood, G., 1986.BA YHW G 1608,a new fungicide for foliar spray and seed treatment against awid espectru m of fungal pathogens.Proceeding sBrighto n CropProtectio n Conference p. 41-46. Rewal, N., Coley-Smith, J.R. and Sealy-Lewis, H.M., 1991. Studies on resistance to dichlofluanid andothe rfungicide s inBotrytis cinerea. PlantPatholog y40:554-560 . Staub,T. , 1991.Fungicid eresistance :practica l experiencewit h antiresistance strategies andth erol eo fintegrate d use.Annua lRevie w ofPhytopatholog y 29:421-442. 222 Action ofstero l biosynthesis inhibitors against Botrytis cinerea MA. deWaard and C. Stehmann Summary Fungicides which inhibit sterol 14a-demethylation (DMIs) are registered to a limited extent for control of B. cinerea. Additional sterol biosynthesis inhibitors (SBIs) for control ofB. cinerea arerequired .Thi s led ust ostud yth eselectiv etoxicit y ofDMI st o B. cinerea. Acell-fre e sterol 14a-demethylaseassa yo fth efungu s ablet osynthesis eC4 - desmethyl sterols was developed. This opens the possibility to study the correlation between fungitoxicity of DMIs and the potency to inhibit the demethylase in an important target fungus. The assay also provides thepossibilit y to study quantitatively the structure-activity relationships of candidate fungicides and to screen for other potentialtarge tsite si nstero lbiosynthesis .Method st ostud yothe rparameter swhic hma y influence toxicity of DMIs against B. cinerea, such as accumulation in mycelium and resistance development, were alsodeveloped . Accumulation of fenarimol in mycelium wastransien tan dwa smediate d byenergy-dependen t efflux asdescribe d previously for other filamentous pathogens. Laboratory resistance was detected, but it was unstable upon subculturing of resistant isolates in the absence of selection pressure. This is probablycause db yth epoly -an dheterokaryoti c natureo fth e fungus. Introduction Inth elas ttw odecade schemica lcontro l ofgre ymoul dcause db yBotrytis cinerea Pers.: Fr.ha s been severely restricted by the development of resistance to benzimidazole and dicarboximide fungicides. Fungicides which inhibit sterol biosynthesis (SBIs) were developed during this period but only a few of them were registered for grey mould control.I ngeneral ,SBI sdi dno treplac eth ebenzimidazole s anddicarboximides .Thi si s of serious concern since SBIs have a number of advantageous features, such as a relatively low resistance risk. The unavailability of modern systemic fungicides for controlo fB. cinereama yresul ti na nincrease d re-useo fconventiona l fungicides which lack curative action.Grower swil l alsob eurge d torel y again muchmor eo n alternative methodso fdiseas econtrol .I nitself ,thi si sa goo ddevelopment ,bu t alternatives areno t alwaysavailabl eo rar edifficul t toachieve .I nth econtex to fthi ssituatio na stud yo nth e activity ofSBI sagains tB. cinereawa sstarted .Th estud ywil lb erestricte dt oth elarges t groupo f SBIs,th etriazole swhic hinhibi tstero lbiosythesi s byinhibitio n ofcytochrom e P450-dependent sterol 14a-demethylase activity (P45014DM). These are generally described asdemethylatio n inhibitors(DMIs) . Thegoa lo fth estud ywa st oinvestigat eparameter swhic hma yinfluenc e theselectiv e toxicityo ftriazol eDMI st oB. cinerea. Iti sexpecte dtha tfundamenta l knowledgegaine d about this topic may contribute to the development of DMIs which show a good field performance againstgre ymould . 223 Resultsan dDiscussio n Biological activity Thebiologica l activity oftriazol eDMI swa sinvestigate d inradia lgrowt htest so nPDA . EC50value s for radialgrowt h varied significantly among 17compound s tested.Amon g the compounds with the highest fungitoxicity found were itraconazole, an antifungal agent for control of mammalian pathogens (EC50 KH M), and tebuconazole (EC50 ICH M), the only triazole registered for control of B. cinerea. Triazoles with a low 6 -6 fungitoxicity weretriadimeno l(EC 5010" M) ,triadimefo n (EC505.10 M )an d flutriafol 6 (EC50 9.10" M). These results indicate a significant range in toxicity (900 fold) of triazoles.Th equestio n is whether thesefigure s correlate with differences in sensitivity of P45014DM or whether other factors are involved in selective toxicity, such as accumulation infunga l mycelium. Developmento fa cell-fre ebioassa yfo rstero l14a-demethylas e Theaffinit y ofDMI st oP450 14DMca nb eassesse d bymeasurin g spectrophotometrically the interaction between the DMIs and microsomal cytochrome P450 isozymes or by measuring the inhibitory potency on P45014DM activity. In studies with Pénicillium italicum measurement of the inhibitory potency on P45014DM activity gave the most relevantinfo r mation(Gua n etal, 1992a,b).Therefore , attempts weremad et odevelo p asimila rcell-fre e bioassayfo rth estero l 14ot-demethylaseo fB. cinerea. Th eprotoco lfo r preparationo fth eP. italicum assaywa sno tsuccessfu l forB. cinerea an di ttherefore ,ha d to be modified. Modifications were made in the mechanical disruption method of the mycelium and the nature of the incubation mixture. Details of the protocol will be published elsewhere. The final protocol yielded cell-free extracts which were able to incorporate20-30 %o f [14C] mevalonic acidi nnon-saponifiabl elipid so fwhic h 35-45% consisted C4-desmethyl sterols. Other sterols formed were lanosterol and eburicol. Incubation of themixtur ewit hth eimidazol eDM Iimazali l (10-7M )full y prevented the synthesiso fdesmethy lsterol san dinduce d theaccumulatio no feburicol ,th esubstrat eo f sterol 14a-demethylase. The IC50 value of imazalil (concentration which inhibits desmethyl sterol synthesis by 50%) was about 10"8M . Results of tests with various triazole DMIs will bepublishe d later.Th eresult s should elucidate whether compounds with a low toxicity towards B. cinerea have a relatively low inhibitory potency on P45014DM activity. If not, other factors influence negatively their toxicity such as low accumulation orfas t metabolicinactivation . Significance ofth estero l 14a-demethylaseassa y The development of the cell-free bioassay could provide a biochemical tool for agrochemical companies tod o quantitative studies of structure-activity relationships at theenzym e level and hence to optimise activity of DMIsspecificall y againstP450 14DM ofB. cinerea. Thisapproac hmigh tb eprofitabl e sinceB. cinereai sregarde d asa world wide target fungus in agriculture. Such an optimalisation has not been done before becauseo fth elac ko fa cell-fre e bioassayfo ran yfilamentou s plantpathogen ,excep tfo r therecentl y developed assayo fP. italicum. Asa substitute ,companie ssometime s usea 224 cell-free P45014DM assay ofSaccharomyces cerevisiae. In general, analogs of particular DMIsma y show acorrelatio nbetwee n toxicity towhol eorganism s such as Pyricularia oryzaean d Drechslerei sorokiniane and the activity in the P450,4DM assay of yeast (Fujimoto etal., 1988) . However,thi s mayno tb etru efo r B. cinerea sinceP450 14DMo f different organismsma yposses sa differentia l sensitivityt oDMIs .Hence ,optimalisatio n using the S. cerevisiae assay does not necessarily lead to compounds with maximum intrinsic activityt oB. cinerea. The cell-free bioassay developed here also opens the possibility to determine the inhibitory potency ofdifferen t stereo-isomers (diastereomers,enantiomers ) of thesam e compound onP450 14DMactivity . Such results aremor econclusiv efo r the identification of biologically active isomers than in vivo toxicity bioassays since in this instance metabolism mayinterfer e with aprope rinterpretatio n of theresults ,suc h asi n thecas e of triadimefon (Gasztonyi, 1981;Dea set al, 1984).A knowledg e of thetoxicit y ofth e various isomers of one DMI is important since it may be that only one isomer is responsiblefo r mosto f theactivity .Th eeffect s ofth eremainin g isomersshoul d alsob e takenint oacoun tsinc ethe yma yhav ebeneficia l ordeleteriou seffect s onnon-targe tsite s inth eplant .Hence ,i tma yb erelevan tt oalte rproductio no fthes eagrochemical si nsuc h a way that the content of the most active isomer in the product is as high as possible (Burdensa/. , 1987). Another spin off from using thecell-fre e bioassay istha tth etes tca n alsob euse d to identify compoundswhic hinterfer e instero lbiosynthesi sa tothe rsites .Thi sstatemen ti s confirmed by results of preliminary experiments with allylamines and morpholine fungicides. In addition, weidentifie d anexperimenta l sterol 14a-demethylaseinhibito r whichinduce d astero laccumulatio npatter ndifferen t from thosefoun d withcommercia l SBIs. This particular chemical inhibited both C4-desmethyl and C4,4-dimethyl synthesis.Suc ha chemica lma ylac kusua lcross-resistanc epattern s andb eo finteres tt o developne wcandidat e fungicides. Accumulation ofSBI si nfunga l mycelium Themechanis m oflaborator yresistanc ei n anumbe ro ffilamentou s fungi againstDMI s isbase d onincrease d energy-dependent efflux of thecompound s from mycelium. Such an increased secretion probably prevents accumulation of the compounds at the target sites to concentrations inhibitory to sterol 14a-demethylase (De Waard, 1980, 1988; Kalamarakis etal., 1991) . Accumulation inwild-typ eisolate swa salway shighe rtha ni n resistant mutants and was characterised by a transient accumulation of the fungicides. Theefflu x mechanism involvedha sneve rbee nelucidated . We demonstrated that energy-dependent efflux of fenarimol also operates in mycelium of B. cinerea. Incubation of mycelium of awild-typ e isolate with fenarimol (90 niM) gave the typical transient accumulation patterns. Accumulation after 1h of / «. incubation became stable and constant. Addition of inhibitors under such equilibrium conditions could induce an instantaneous increase in accumulation. The most active inhibitorsfoun d wereionophori cantibiotics .Thes ecompound sinterfer e withth eproto n motive force over membranes and hence may inhibit transport processes which are driven by this force. This suggests that energy-dependent efflux is also driven by membrane-bound enzymes.Furthe rresearc h should identify theseenzymes . 225 Resistancedevelopmen t Selection for laboratory-resistance to DMIs in various monokaryotic fungi has been describedfrequently . Itca nb eachieve deasil yan dwa sshow nt ob epolygenicall ybase d (Van Tuyl, 1977). Resistance levels were relatively low. Recombination of different minorgene si nth esam estrai no rstepwis eselectio nyielde disolate swit hrelativel yhig h levelso fresistanc e(D eWaar dan dVa nNistelrooy , 1990;Kalamaraki s etal, 1991). Inth epresen t studiesw etrie d toselec t for resistance toDMI si nB. cinerea bymas s selection ofconidia ,whic hha dbee ntreate d oruntreate dwit hmutageni c agentso naga r with a minimal inhibitory concentration of different fungicides. Developing colonies were continuously subcultured on agarwit h sublethal concentrations of the fungicides. Resistancelevel swer edetermine di nradia lgrowt htest swit hinoculu mtake nfro mDMI - containing agaran dfro m thefirs t subcultures onaga rwithou t fungicides. Resistance levels of isolates tested with inoculum from fungicide-containing agar ranged from 6-19; resistance levels of the same isolates tested with inoculum from fungicide-free plates ranged from 2-4. This indicates that the decreased sensitivity observedi sno ta stabl egeneti ccharacter .Thi sma yrelat et oth epoly -an dheterokaryoti c natureo fB. cinerea. Attemptswil lb eundertake nt oacquir eisolate swit hstabl elevel s of resistance to DMIs. Only such isolates can be used to test whether the mechanism of resistancei sbase d onincrease defflu x from mycelium ordecrease d affinity ofP450 14DM toDMIs . Acknowledgement The authors thankMrs .MJ.C . Groenen andMr .J.H.N.M . Pijnenburg for carrying out the accumulation and fungicide resistance experiments and Mr. J.C. Kapteyn for participating inth edevelopmen to fth estero l 14a-bioassays. References Burden,R.S. ,Carter ,G.A. , Clark,T. ,Cooke ,D.T. ,Croker , S.J., Deas,A.H.B. ,Hedden , P., James, S.J. and Lenton, J.R., 1987. Comparative activity of the enantiomers of triadimenol and paclobutrazol as inhibitors of fungal growth and plant sterol and gibberellinbiosynthesis .Pesticid eScienc e21:253-267 . Deas, A.H.B., Clark, T. and Carter, G.A., 1984. The enatiomeric composition of triadimenolproduce ddurin gmetabolis m oftriadimefo n byfungi .Par tU : Differences betweenfunga l species.Pesticid eScienc e 15:71-77. DeWaard ,M.A . andVa nNistelrooy ,J.G.M. , 1980. Energy-dependentefflu x mechanism in a wild-type strain and fenarimol-resistant mutants of Aspergillus nidulans. PesticideBiochemistr y andPhysiolog y 13:255-266. DeWaard ,M.A . andVa nNistelrooy , J.G.M., 1988.Accumulatio n of SBIfungicide s in wild-type and fenarimol-resistant isolates of Pénicillium italicum. Pesticide Science 22:371-382. DeWaard ,M.A .an dVa nNistelrooy ,J.G.M. , 1990.Stepwis edevelopmen to flaborator y resistance to DMI-fungicides in Pénicillium italicum. Netherlands Journal of Plant Pathology96:321-329 . Fujimoto,T.T. , Quinn,J.A. ,Egan ,A.R. ,Shaber ,S.H . andRoss ,R.R. , 1988.Quantitativ e structure-activity relationship studieso fth efungitoxi c properties ofphenethy l 1,2,4- 226 triazoles.Pesticid eBiochemistr y andPhysiolog y30:199-213 . Gasztonyi, M., 1981.Th e diastereometric ratio in the triadimenol produced by fungal metabolism of triadimefon, and its role in fungicidal selectivity. Pesticide Science 12:433-438. Guan, J., Braks, H.J.M., Kerkenaar, A. and De Waard, M.A., 1992. Interaction of microsomalcytochrom eP45 0isozyme sisolate sfro m Pénicillium italicum withDM I fungicides. PesticideBiochemistr y andPhysiolog y42:24-34 . Guan,J. ,Stehmann ,C , Ellis,S.W. ,Kerkenaar ,A . andD eWaard ,M.A. ,1992 .Ergostero l biosynthesis in a cell-free preparation of Pénicillium italicum and its sensitivity to DMIfungicides . PesticideBiochemistr y andPhysiolog y42:262-270 . Kalamarakis, A.E., De Waard, M.A., Ziogas, B.N. and Georgopoulus, S.G., 1991. Resistance to Nectria haematococca var. Cucurbitae. Pesticide Biochemistry and Physiology40:212-220 . Van Tuyl, J.M., 1977. Genetics of fungal resistance to systemic fungicides. Thesis WageningenAgricultura l University,Th eNetherlands ,pp .137 . 227 Investigations on the dicarboximide resistance of Botrytis cinerea II.Incomplet e crossresistanc e inth e Rheingau J.F.R. Borge, N. Abolgassaniand E. Schlösser Summary All isolates of B. cinerea, representative for 2.600 ha of vineyards in the Rheingau, exhibited incomplete cross resistance to dicarboximide fungicides. As compared to sensitiveisolate s (ED505. 0 |xg.mH)the yha d alo wleve l of resistancet oiprodion ean d metomeclan (ED50 25 |xg.mH) and a high level of resistance to vinclozolin and procymidone(ED 5050 0|i,g.mH) . Introduction Resistance of Botrytis cinerea Pers.: Fr. to dicarboximide fungicides is widespread in fungal populations on various crop plants, including grapevine (Pommer and Lorenz, 1987). Theresistanc esituatio ni nth evineyard so fth eRheinga u hasneve rbee nassesse d before.Thus , B. cinereawa sisolate di n 1985an d 1986fro m representativevineyard so f theregion . Material and Methods B. cinerea was isolated from infected plants, as described by Borge (1988). Conidia produced werekep twithou tfurthe r subculturing onaga rmedi ab ystorag eo fconidi aa t 4°C. Agarplat eassa y The sensitivity to various fungicides was tested as described by Borge and Schlösser (1990). Detachedlea fassa y Detached leaves of Vitis vinifera cv.Miiller-Thurga u wereplace d onmois t filter paper disksi nPetr idishes ,wit hth eabaxia lsurfac euppermost .Th efungicide s wereapplie dt o leaves with a hand-sprayer to give afin e film. For inoculation, 50 |xldroplet s of a1 % maltextrac tsolution ,containin g750-110 0conidi awer eplace do nintercosta lareas .Afte r 10day sa t 18-22°Can da 12/1 2photoperio dwit hartificia l illuminationo f 10.000lux ,th e size of the necrotic lesions was measured and attributed to the following classes: 0= lesions absent; 1 = few minute lesions;2 = lesions of 1 x dropdiameter ; 3= 2x drop diameter; 4 = 3 x drop diameter; 5 = lesions > 3 x drop diameter. From the class distribution adiseas einde xwa scalculated : 228 _ rij+ 2n2+ 3n3+ 4n4 + 5n5 1 = n0 + tij + n2+ n3 + n4 + n5 Thevalue s for each variant of thelea f assayar eth eaverag e from threereplicate s with six inoculation points each. Results Agar plate assay The 20 isolates of B. cinerea collected in 1985 with 50 p,g a.i. vinclozolin.ml-1 as threshold level,differe d in their reaction to the four fungicides with regard to production of sclerotia (Table 1). Iprodione and metomeclan proved to be more effective than vinclozolin and procymidone. In 1986 resistant isolates were selected with vinclozolin resistance (V-R) and iprodione resistance (I-R), at 50 and 5 jxg a.i. as discriminatory levels, respectively. A similar reaction (Table 2) was observed with mycelial growth from fungal suspensions with six I-R andV- R isolates asshow n inTabl e 1,regardles s ofwhic h fungicide had been used to select the resistant isolate. ' Production of sclerotia on agar plates (Table 3) showed the same general pattern. Table1. Effect of dicarboximide fiingicides on sclerotia production in agar plates of 20 isolates ofB. cinerea collected in 1985 in the Rheingau. Fungicide number sclerotia/agarplate 1) 1 M-g a.i.ml" 0 5 10 25 50 Iprodione 41 44 42 0 0 Metomeclan 41 12 25 0 0 Vinclozolin 41 15 10 8 18 Procymidone 41 20 11 6 16 ') average of five replicates for each of the 20 isolates. Table2. Effect of dicarboximide fungicides on colony diameter from conidial suspensions on agar plates of six iprodione-resistant (I-R) and six vinclozolin-resistant (V-R) isolates ofB. cinerea collected in 1986 in the Rheingau. Fungicide colony diameter, as % of untreated check1) |xg a.i.ml"1 0 5 10 25 50 100 I-R V-R I-R V-R I-R V-R I-R V-R I-R V-R I-R V-R Iprodione 100 100 78 87 85 90 0 0 0 0 0 0 Vinclozolin 100 100 77 83 83 87 81 82 81 83 74 77 0 average of five replicates for each isolate. 229 *tfRis» Table 3. Effect of dicarboximide fungicides on sclerotiaproduction from conidial suspensions onagar plates of six iprodione-resistant (I-R) andsix vinclozolin-resistant (V-R) isolatesofB. cinerea collectedin 1986 in the Rheingau. Fungicide numbero fsclerotia/aga rplate 1) |xga.i.m H 0 5 10 25 50 100 I-R V-R I-R V-R I-R V-R I-R V-R I-R V-R I-R V-R Iprodione 48 72 119 25 59 23 0 0 0 0 0 0 Vinclozolin 68 39 106 19 21 21 12 49 10 16 9 14 0 averageo ffiv e replicates forever yisolate . Table 4. Reaction of three dicarboximide-sensitive isolates ofB. cinerea to iprodione andvinclozolin on detachedgrapevine leaves. Fungicide |xga.i.m H » 0 2.5 5.0 10.0 25.0 Iprodione 4.7» 3.7 2.7 0.4 0 Vinclozolin 4.8 3.9 2.5 0.9 0 ')lesio n indexi sth eaverag eo fthre ereplicate s ofsi xinoculatio nsite sfo rever yisolate . Table 5. Reaction of eightdicarboximide-resistant isolates of B. cinerea to iprodione andvinclozolin on detachedgrapevine leaves. Yearo fisolatio n (xg a.i.mH lesionindex 1) Iprod one 0 50 100 1985 4.8 1.3 0.4 1986 4.7 0.8 0.2 Vinclozolin 0 500 1000 1985 4.9 2.7 1.8 1986 4.8 2.5 1.7 ') averageo fthre ereplicate s ofsi xinoculatio n sitesfo r everyisolate . Detachedlea fassa y Agar plateassay s canb einformative , but often disagree with assays inplanta. Forthi s reason the reaction of resistant isolates was tested on detached grapevine leaves. Dicarboximide-sensitiveisolate sprove dequall ysusceptibl et ovinclozoli nan diprodion e (Table4) ,wit ha nED 50o f about5. 0 iiga.i.mH . With resistant isolates there was the same diverse reaction, as observed with the agarplate essays (Table 5).The y were more sensitive to iprodione than to vinclozolin, withED 50-valueso fabou t 25|x gan d50 0|x ga.i.mH ,respectively . 230 Discussion Onaga rplate sa swel la si ndetache d leaf assays,resistan tisolate so fB. cinerea showed arang eo freaction s toth efou r dicarboximides tested,bein g moresensitiv et oiprodion e andmetomecla ntha nt ovinclozoli nan dprocymidone .I nth elea fassay ,sensitiv eisolate s reactedequall yt oiprodion ean dvinclozolin ,wit ha nED 50o fabou t5. 0|x ga.i.mH ,whil e resistantisolate sha dED 50-valueso f2 5|x ga.i.m Hfo riprodion ean d50 0m ga.i.m H for vinclozolin, respectively. These differences were apparent, regardless whether the isolates had been selected for resistance towards iprodione or vinclozolin. This incompletecross-resistanc e wasdescribe d byVuli cet al. (1984) ,bu t theseauthor s also indicated that some isolates behaved this way, while others showed complete cross- resistance. Thevineyard s in the Rheingau consist of c. 2600h a in asingl e 35k m stretch along the Rhine, extending only 0.5 to 3.0 km from the river. As a result of the continuous grapevine cultivation for centuries a regional population of B. cinerea has probably evolved.Thi s mayb e thereaso n whyal l isolates from this region reacted similarlyan d withoutexceptio nt oth edicarboximid efungicides . Whetherpopulation si nothe rregion s behavesimilarl yremain st ob eestablished . The described phenomenon has two implications. First, complete cross resistance cannot automatically be assumed to occur in all areas in which resistance to a given fungicide has been detected. Second, in the case of incomplete cross resistance, some fungicides maystil lb eusefu l despiteth epresenc eo fresistan tisolates ,provide dthe yar e appliedwit hprudence . Acknowledgment This investigation was supported by a grant from the German Academic Exchange Service(DAAD )fo rJ.F.R . Borge. References Borge,J.F.R. , 1988. Untersuchungen zurResisten zvo nBotrytis cinerea Pers .gegenübe r Dicarboximid-Fungiziden imRheingau .Dissertatio nGiessen . Borge,J.F.R .an d Schlösser, E., 1990.Investigation s onth edicarboximid e resistanceo f Botrytiscinerea Pers .I .Distributio nan dqualit yo fvinclozolin-resistan tisolate si nth e Rheingau.Viticultura lEnologica l Science45:36-39 . Pommer, E.H. and Lorenz, G., 1987. Dicarboximide fungicides. In Modern Selective Fungicides.Ed .Lyr ,M .VE BGusta vFische rVerla gJen ap .91-106 . Vulic, M., Eberle, O. and Haberle, N., 1984. Metomeclan, ein neuer Dicarboximid- Wirkstoff mit breitem fungiziden Wirkunsspektrum. Mededelingen Fakulteit Land bouwwetenschappen, Rijksuniversiteit Gent49:293-301 . 231 Stability ofbenzimidazol e resistance and diethofencarb sensitivity ofBotrytis cinerea H. Ishii, M.Fukaya, F, Faretra, H. Takedaand Y. Tomita Summary Monosporeo rmonomycelia lisolate so fB. cinerea havin gth ephenotype s 'carbendazim- sensitive, diethofencarb-resistant' (S,R), 'highly carbendazim-resistant, diethofencarb- sensitive' (HR,S), 'intermediatelycarbendazim-resistant ,diethofencarb-resistant ' (IR,R), and 'highlycarbendazim-resistant , diethofencarb-resistant' (HR,R)wer eobtained .The y were phenotypically stable during subculture on media in the presence and absenceo f carbendazim.A mixtur eo fdiethofencar b andthiophanate-methy lwa shighl yeffectiv ei n reducinggrowt ho fth eHR, San dS, Risolates ,bu tno tagains tth eIR, Ran dHR, Risolates . Introduction In Japan, benzimidazole-resistant strains of Botrytis cinerea Pers.: Fr. appeared in the mid-70's and caused severe loss of disease control by benzimidazole fungicides (Takeuchi, 1987).Benzimidazol e resistance persisted in thefield , prompting theus eo f theN-phenylcarbamat efungicid e diethofencarb forcombattin gth eresistan tstrains . This fungicide, which is specifically active against benzimidazole-resistant strains, was synthesised inJapa n (Nakamura etal., 1986 )an dcommerciall y released inFranc e asa mixturewit hth ebenzimidazol e fungicide carbendazim.Thi swa s thefirs t example ofa practical application of the phenomenon of negatively correlated cross-resistance. In Japan,subsequently , diethofencarb wasregistere d as amixtur ewit heithe rthiophanate - methylo rprocymidone ,althoug hregistratio no fthes emixture sha sno tye tbee nobtaine d for grapes. The objectives of this work were: 1) to monitor field isolates for their resistancet obenzimidazole s andsensitivit yt odiethofencar b invineyards ;2 )t oexamin e thestabilit yo fresistanc eo rsensitivit yt ocarbendazi m and3 )diethofencar b invitro; and to test theeffect s of amixtur e of diethofencarb with thiophanate-methyl onB. cinerea isolatesi nth elaboratory . Material and Methods Monitoringo fbenzimidazol eresistanc ean ddiethofencar b sensitivity In 1990,isolate s ofB. cinerea were obtained from five commercial fields and 1 experi mental field in Akita Prefecture. In 1991, monitoring was repeated for the same experimentafield.l I ntha field,t a mixtur eo fdiethofencar b withthiophanate-methy lwa s appliedfive time s in total during the 2years .Mycelia l disks of each isolate previously culturedo npotat odextros eaga r(PDA )plate sa t24° Cfo r3 day swer etransferre d toPD A plates containing thiophanate-methyl (70% WP) or diethofencarb (25% WP). After incubation at 24°C for 2 days, the minimum inhibitory concentrations (MIC) of each fungicide weredetermined . 232 Stabilityo fresistanc eo rsensitivit yt ocarbendazi man d diethofencarb EightItalia nan dtw oJapanes eisolate swer eused .Eac hisolat ewa ssubculture do nPD A plates supplemented with carbendazim at 0,0.1, 10,an d 100ixg.mFa t 20°C to test for theirsensitivit y tocarbendazi m anddiethofencarb . After incubation at20° Cfo r 2 days, the inhibition of mycelial growth (%)wa s calculated for each fungicide. This test was repeatedever y 3day sove ra perio d of 18days ,an dthe nfou r timesever ymonth . Efficacy ofa mixtur eo fdiethofencar b with thiophanate-methyl Activity of thefungicid e wasdetermine d using the 'Cucumber FruitMethods ' reported by Tezuka and Kiso (1976).I n experiment 1,fou r isolates wereculture d separately on PDA plates. In experiment 2, conidial suspensions (ca. 1x 105conidia.mH ) prepared from four isolates were mixed in various ratios and dropped onto PDA plates. The cultures were incubated at 20°C for 4-5 days. Pieces of cucumber fruit dipped into a suspension of thiophanate-methyl (a.i.5 0mg.mH) ,diethofencar b (a.i. 83mg.mH ) ora mixtureo fthiophanate-methy lan ddiethofencar b atth e sameconcentration swer eplace d on the colonies grown on PDA plates. The cultures were kept at 20°C in a moist atmosphere.Th elengt h of decaycause db yB. cinereawa smeasure d4-5'day safte r the start ofincubation ,an dth eprotectiv evalu efo r thedeca ydevelopmen tb y the fungicide wascalculated . Results and Discussion Fukaya et al. (1979) reported that benzimidazole-resistant populations of B. cinerea declined in vineyards over the following 2 years when spray applications of benzimidazole fungicides were discontinued. In this work, the distribution of isolates 100 100 -A- .-A-- ^TTTT^ *> SS»o Untreated Treated with 80 1OO^g/m l 80 carbendazim X) (O 60 • 601 4-> +-> 3 01 40 Treated with • 40 2 S Untreated 100^g/ml ..-o. carbendazim .-O-. I...O-" V. 20 a 3 20 •«- -o-"/*— 4 5 6 10 Subculture Fig. 1. Isolate SAS405 (HR,S). Stability of carbendazim resistance and diethofencarb sensitivityin an isolate ofBotrytis cinerea. 233 showing different degrees of sensitivity to thiophanate-methyl and diethofencarb in fungal populations wasmonitore d inth esam evineyard s whereselectio n pressure from benzimidazole was removed after withdrawal of the chemical in 1975.O f 94 isolates testedi n 1990, seven(7.4% )wer ehighl yresistan tt othiophanate-methy l butmos to fth e remainingisolate swer esensitive .I na nexperimenta lvineyard ,o nth eothe rhand ,3 1 out of 36 isolates (86.1%) were highly resistant to thiophanate-methyl and sensitive to diethofencarb (HR.S) .I nth efollowin g year,12 5isolate sobtaine dfro m theexperimenta l vineyard contained 101 isolates (80.8%) which were highly resistant to thiophanate- methyl; one of them was resistant todiethofencarb . Isolates with the phenotype HR,R (highresistanc et obenzimidazole s andresistanc et odiethofencarb ) havebee ndescribe d previously (Faretra etal, 1989;Pallastr o and Faretra, 1992).I t was not clear however whetherappearanc eo fthes ene wvariant swa srelate dt oth eapplicatio no f diethofencarb inth evineyard .Fo rVenturia naschicola, causin gsca bo nJapanes epear ,th erecombinan t phenotypewit hhig hresistanc et obenzimidazole s andresistanc et oiV-phenylcarbamate s wasfoun d asa resul to fcrosse s ofth eisolate s(Ishi ian dVa nRaak ,1988) . Instability of dicarboximide resistance in B. cinerea has been explained by resensi- tisation in theresistan t strains (Lorenz and Eichhorn, 1982).I t has alsobee n suggested that heterokaryosis provides a mechanism for maintaining nuclei carrying genes for dicarboximide resistance (Summers et al, 1984). For benzimidazole resistance in B. cinerea,however ,circumstance smigh tb edifferent . Meyeret al. (1977)reporte dtha t a high level of resistance was retained for a long time and through many generations whenth efungu s wasgrow ni nth eabsenc eo fth efungicide .N oresistan tstrain sappeare d through the 'training culture'. In the present study, when monoascospore or mono- mycelial isolates were tested, the level of resistance or sensitivity to carbendazim or diethofencarb did not change markedly,irrespectiv e of theexistenc e of carbendazim in thecultur emedi a(Fig . 1).Th echange so fphenotyp efro m IR,R(intermediat eresistanc e to benzimidazole with resistance to diethofencarb) to HR,R, and from HR,R to IR,R TOO Treated with 100 100 jjg/ml carbendazim Untreated 60- X) N 20 -20 Untreated Treated with 100,ug/ml carbendazim i— a> -§ -60 ,4-> -100 •-100 4 5 6 10 Subculture Fig. 2. Isolate VB 73 (HR,R).Stability of carbendazim resistance and diethofencarb sensitivity inan isolate ofBotrytis cinerea. 234 Table 1. Effectsof a mixture of diethofencarb and thiophanate-methyl on grey mould developmenton cucumber fruit Plot Ratio(% ) ofisolate s Protectivevalu e ininoculu msource» ) S,R IR.R HR,S HR,R A 100 0 0 0 77.8 B 0 100 0 0 5.3 C 0 0 100 0 78.5 D 0 0 0 100 6.0 E 72 25 0 0 23.8 F 50 50 0 0 25.1 G 25 75 0 0 -56.5 H 75 0 25 0 75.0 I 50 0 50 0 81.2 J 25 0 75 0 100.0 K 0 75 25 0 -3.9 L 0 50 50 0 8.0 M 0 25 75 0 10.1 N 0 0 75 25 12!7 O 0 0 50 50 1.5 P 0 0 25 75 1.4 Q 50 25 25 0 -72.3 R 25 50 25 0 0.9 S 25 25 50 0 11.7 a) S,R: sensitivet ocarbendazi m andresistan tt odiethofencarb ; IR,R:intermediatel y resistantt ocarbendazi m andresistan tt o diethofencarb; HR,S:highl yresistan tt ocarbendazi m andsensitiv et o diethofencarb; HR,R:highl yresistan tt ocarbendazi m andresistan tt odiethofencarb . wereno tobserved .Th eHR, Rphenotyp eo fisolate sderive dfro m singleascospore swa s stable. On the other hand, the HR,R phenotype of isolates obtained by mass isolation from infected tissueswa sunstabl ei nsuccessiv esubculture s(Fig .2) .Th emass-isolatio n culturesseeme d tob eheterokaryon s ormixture so fdifferen t isolates. Recently, reduced performance of a mixture of diethofencarb and carbendazim has become apparent in French vineyards due to the increase in IR,R strains (Leroux, personal communication).Th eeffec t of suchdoubl e resistant strains onth edecreas eo f effectiveness of thefungicid e mixtureha s alsobee n reported invegetable s (Elad et al, 1992).Diethofencar b aloneo ri na mixtur ewit hthiophanate-methy lwa seffectiv e against the HR,S isolate in experiment 1 using the 'Cucumber Fruit Methods' (data not presented). However, these fungicides have no effect against the TR,R and the HR,R isolates. In experiment 2 involving mixed conidial suspensions of the isolates, the mixture of diethofencarb and thiophanate-methyl provided strong protection when the inoculum source comprised only the S,R (benzimidazole-sensitive and diethofencarb- resistant)and/o rth eHR, Sisolates .Whe nth eIR, Ro rHR, Risolate swer einclude di nth e inoculum, the fungicide efficacy decreased markedly (Table 1).Eve n when the double resistant isolates comprised 25 %o f the inoculum, the fungicide did not provide good 235 control,suggestin g thatth eIR, R and/orth eHR, R strainsca ncaus ea practica lproble m undersever ediseas epressure . Itha sbee ndemonstrate d thatth ebindin g ofcarbendazi m tocell-fre e proteini slowe r in highly carbendazim-resistant isolates and intermediately resistant isolates than in sensitiveisolate s (Ishii and Takeda, 1989).O n the other hand,biochemica l data on the binding of diethofencarb have not yet been reported. Methods for the double resistant strainsar eurgentl y required. Acknowledgements Theauthor swis ht othan kDr .T .Takeuchi ,Chiba-Ke n AgriculturalExperimen t Station, for providing fungal isolates and Sumitomo Chemical Co., Ltd. and Hoechst AG for providingfungicides .W ear eals ogreatfu l toDr .M . Grindle,Universit yo fSheffield , UK forcritica lreadin g ofthi smanuscript . References Elad,Y. ,Yunis ,H .an dKatan ,T. , 1992.Multipl efungicid e resistancet obenzimidazoles , dicarboximides anddiethofencar b infiel d isolates ofBotrytis cinerea inIsrael .Plan t Pathology41:41-46 . Faretra, F.,Pollastro , S. and DiTonno ,A.P. , 1989.Ne w natural variants ofBotryotinia fuckeliana {Botrytis cinerea) coupling benzimidazole-resistance to insensitivity toward theAT-phenylcarbamat ediethofencarb . Phytopathologia Mediterranea 28:98- 104. Fukaya, M., Kato, S. and Sato, H., 1979. Studies on the tolerance of grey mould of grapes. I.Toleranc eo fgre ymoul dan dsom eo fi tcharacteristics . Bulletino fth eAkit a Fruit-TreeExperimen t Station 11:33-38. Ishii, H. and Takeda, H., 1989. Differential binding of a iV-phenylformamidoxime compound in cell-free extracts ofbenzimidazol e -resistant and -sensitive isolateso f Venturia nashicola, Botrytis cinerea andGibberellafujikuroi. NetherlandsJourna lo f PlantPatholog y9 5(supplemen t 1):99-108. Ishii, H. and Van Raak, M., 1988. Inheritance of increased sensitivity to Af-phenyl- carbamates in benzimidazole-resistant Venturia nashicola. Phytopathology 78:695- 698. Lorenz, D.H. and Eichhorn, K.W., 1982. Botrytis cinerea and its resistance to dicarboximidefungicides .Europea nan dMediterranea nPlan tProtectio nOrganisatio n Bulletin 12:125-129. Meyer,J.A. ,Pourtois ,A. ,Lumande ,K .an dMaraite ,H. , 1977. Tolerancet obenomy li n Botrytiscinerea an dFusarium oxysporum. Netherland sJourna lo fPlan tPatholog y8 3 (supplement l):215-227. Nakamura,S. , Kato,T. ,Noguchi ,H. ,Takahashi ,J .an dKamoshita ,K. , 1986.Biologica l activityan dmod eo factio no fa resistanc ebreaker ,diethofencarb .Abstract so fth e6t h International Congress ofPesticid eChemistry :3E-01 . Pollastro, S. and Faretra, F., 1992. Genetic characterization of Botryotiniafuckeliana (Botrytis cinerea) field isolates with high resistance tobenzimidazole s coupled with insensitivity toth eJV-phenylcarbamat ediethofencarb . PhytopathologiaMediterranea : (inpress) . Summers,R.W. ,Heaney ,S.P . andGrindle ,M. , 1984.Studie so fa dicarboximid eresistan t 236 T heterokaryon of Botrytis cinerea. Proceedings of the 1984 British Crop Protection Conference -Pest san dDiseases:453-458 . Takeuchi,T. ,1987 .Studie so fth eepidemiolog y offungicid e resistantgra ymol dstrains . SpecialBulleti no fth eChiba-Ke nAgricultura lExperimen t Station14:1-75 . Tezuka, N. and Kiso, A., 1976. A test for chemical effect using cucumber fruit to multiple-hostfungi . Journalo fPesticid e Science 1:321-324. 237 -L Effectiveness ofsom efungicide s and fungicide combinations onBotrytis cinereaisolate s N.Delen andT. Ozbek Summary B. cinereai s oneo f theimportan tpathogen s ofgreenhous ecrop si nTurkey .Becaus eo f thecontinuou s applications of fungicides, ahig hproportio n of thepathoge n population isresistan tt obenzimid a zoles,dicarboximide s andcaptan .In vitro testsan dglasshous e experiments suggest that mixed formulations of tebucanazole + tolylfluanid, tebuca- nazole + dichlofluanid, diethofencarb + carbendazim and dichlofluanid alone are the mosteffectiv e fungicides. Phosetyl-Alstimulate d theresistanc erespons ei n thehost . Introduction Botrytis cinerea Pers.: Fr. is one of the most important pathogens of greenhouse vegetables and ornamental crops in Turkey, but because of the continuous fungicide application,B. cinerea hasbecom e resistant toth ebenzimidazole s (Delen etal., 1984) . Nowsevera lisolate s ofth epathoge n havebee n isolated from greenhouseplants ,whic h aremoderatel y resistant todicarboximide s andmultisit efungicide s (Delen etal, 1984, 1985).Becaus echemica l control of thepathoge n isbecomin g moredifficul t inTurkey , the sensitivity of B. cinerea to the recommended chemicals was studied. Some of the more recent fungicides and fungicide combinations were also evaluated for control of B. cinereai nTurkey . Material and Methods Infected tissueswer ecollecte di nTurke yfro m thevegetabl ean dornamenta lgreenhouse s located in the coastal regions of the Aegean and the Mediterranean in 1991. The sensitivity of 37 isolates to fungicides was tested on MM medium, consisting of 20g glucose, 1.5 g asparagine 1.0 g K2HP04, 0.5 g MgS04.7H20, 0.1 g FeCl3, 1g yeast extract, 20 g difco bacto agar per 1000 ml distilled water (Delen et al., 1984). Five selectedisolate swer estudie din vitro. Isolate s90/5-CA Pan d91/12- Kwer esensitiv ean d isolates 91/13-K and 91/23-1 wereles s sensitive tobenzimidazole s and dicarboximide, whereas isolate 91/26-1 was sensitive to dicarboximides but less sensitive to benzimidazoles.Th eeffectivenes s ofth echemical sliste d inTabl e4 o nth eisolate swa s determined using seedlings of broadbean (Viciafabae) (Meunier etal., 1985).Isolate s 90/5-Cap,91/13- Kan d91/23- 1wer euse di ntw osteps ;i nth efirs tnewe rchemical swer e tested; the second step was used triggering a resistance response in the host, combinations of phosetyl-Al with iprodione or carbendazim were used (Cohen and Coffey, 1986). 238 Results Thesensitivit y ofcollecte d isolatest ochemical s recommended inTurke yfo r controlo f B. cinerea aregive ni nTabl e1 . The data in Table 1 show, that several isolates were relatively insensitive to the recommended fungicides. Someo fth eisolate swer eespeciall yinsensitiv et ocoppe roxychlorid e+ iprodion e+ phosetyl-Al (Table 2). Tebucanazole + tolylfluanid, tebucanazole + dichlofluanid, dichlofluanid alone and diethofencarb + carbendazim were the most effective for the controlo fisolate sresistan to rsensitiv et obenzimidazol e andiprodione . The efficacy of phosetyl-Al combinations with carbendazim or iprodione are summarised inTabl e4 .Th eiprodion e+ phosetyl-A lcombinatio nwa sth emos t effective treatmentfo rcontro lo fgre ymould .Carbendazi m+ phosetyl-A lwa smor eeffectiv e than carbendazimalone . Table 1. SensitivityofB. cinereaisolates to recommendedfungicides inTurkey in 1991. Fungicide Numbero f distributiono f isolates ED50value si nisolat epopulatio n <0.3 0.3-1 1-3 3-10 10-30 30-100 100-300 >300 Iprodione 37 37.1 18.9 24.3 21.6 0.0 0.0 0.0 0.0 Benomyl 37 29.7 2.7 0.0 0.0 0.0 0.0 0.0 67.6 Carbendazim 17 29.4 0.0 0.0 0.0 0.0 5.9 5.9 58.8 Dichlofluanid 23 26.1 17.4 13.0 30.4 13.0 0.0 0.0 0.0 Thiram 23 8.1 0.0 8.7 52.2 30.4 0.0 0.0 0.0 Mancozeb 24 8.3 0.0 0.0 0.0 4.2 54.2 12.5 20.8 Captan 23 4.3 4.3 4.3 34.8 30.4 13.0 8.7 0.0 Table 2. Effectof fungicides on germination ofconidiaof five isolates ofB. cinerea. Fungicideso r doses Germination ofconidia (%o fcontrol ) 1 i combinations ^ (|i,g.ml-ij Isolates 90/5-Cap 91/12-K 91/13-K 91/23-1 91/26-1 Anilazine 0.3 90.6 100.0 100.0 96.3 66.2 1.0 46.3 81.2 100.0 71.5 37.5 3.0 32.2 100.0 100.0 65.2 47.7 10.0 14.8 27.8 38.0 22.7 11.1 Copper 0.3 66.5 100.0 100.0 100.0 88.5 Oxychloride+ 1.0 - 31.0 - - - Iprodione+ 3.0 82.7 59.8 42.0 100.0 76.7 Phosetyl-Al 10.0 94.5 100.0 82.0 50.2 72.6 0 Imazalil, prochloraz, flusilazole, myclobutanil, tebucanazole, tebucanazole + tolyl fluanid, tebucanazole + dichlofluanid, diethofencarb + carbendazim completely inhibitedconidia lgermination . 239 Table 3. Statisticalgrouping of the charactersaccording totheir effectiveness, as tested on broadbeans. Fungicide diseaseseverit y(% ) Tebucanazole+ tolylfluani d 0.00 a') Tebucanazole+ dichlofluani d 0.00a Dichlofluanid 4.31a b Diethofencarb +Carbendazi m 4.31 ab Anilazine 22.79b e Tebucanazole 25.31 be Imazalil 26.45b e Prochloraz 26.57b e Myclobutanil 27.71 c Flusilazole 35.22 cd TBZ 36.14 cd Iprodione 43.24c d Carbendazim 51.14 de Control 67.71 e •)Figure sfollowe d byth esam elette rd ono tdiffe r significantly, p= 0.05. Table4. Statisticalgrouping ofthe charactersaccording totheir effectiveness, as tested on broadbeans. Fungicide diseaseseverit y(% ) Phosetyl-Al+ iprodion e 15.61 a1) Iprodione 28.46a b Phosetyl-Al +cabendazi m 33.25a b Carbendazim 38.85 be Phosetyl-Al 45.50b e Control 55.57c 0 Figuresfollowe d byth esam elette rd ono tdiffe r significantly, p= 0.05. Discussion Reducedsensitivit yo fB. cinereat oth erecommende dfungicide s isseriousl y influencing thechemica lcontro lo fth epathoge ni nTurkey .Th emos timportan taspec to fth eproble m is,tha tth erati oo f theisolate swit h reduced sensitivity isincreasin g eachyear .Becaus e ofheterokaryosi s(Cate nan dJinks , 1966),fungicid e resistancei sexpecte di nB. cinerea. Forthi sreaso nne wan deffectiv e productswil lb ea continuin g need.Thi swor kshowe d thatmixture so ftebucanazol e+ tolylfluanid , tebucanazole+ dichlofluanid , diethofencarb + carbendazim and dichlofluanid alone were effective against grey mould. These chemicals may have some resistance risks (Katan etal, 1989;Malathrakis , 1989),an d this should be taken into consideration in designing control strategies. The results obtained with phosetyl-Al perhaps suggest that this chemical may act indirectly by stimulating aresistanc erespons ei nth ehos t(Cohe nan dCoffey , 1986). 240 References Caten, CE. and Jinks, J.L., 1966.Heterokaryosis : its significance in wild homothallic ascomycetes and fungi imperfecti. Transactions of the British Mycological Society 49:81-93. Cohen, Y.an d Coffey, M.D., 1986.Systemi c fungicides and thecontro l of oomycetes. AnnualRevie wo fPhytopatholog y24:311-338 . Delen, N., Yildiz, M. and Maraite, H., 1984. Benzimidazole and dithiocarbamate resistanceo fBotrytis cinerea o ngreenhous ecrop si nTurkey .Mededelinge nFakultei t Landbouwwetenschappen, Rijksuniversiteit Gent49:153-161 . Delen, N., Maraite, H. and Yildiz, M., 1985. Sensitivity of Botrytis cinerea to dicarboximides in Turkey.Quadern i Delia Scuola di Specializzazione in Viticultura edEnologi aUniversit éd iTorin o9:278-279 . Katan,T. ,Elad ,Y an dYuniz ,H. , 1989.Resistanc et odiethofencar b (NPC)i nbenomyl - resistantfield isolate so fBotrytis cinerea. PlantPatholog y38:86-92 . Malathrakis,N.E. , 1989.Resistanc e of Botrytis cinerea todichlofluani d in greenhouse vegetables.Plan tDiseas e73:138-141 . Meunier, S., Maraite, H. and Meyer, J.A., 1985.Charactéristique s culturales et patho- géniques de souches de Botrytis cinerea résistantes aux imides cycliques de fraisiersan Belgiques. European and Mediterranean Plant Protection Oganisation Bulletin15:395-401 . 241 The chemical control ofBotrytis cinereai n fruit culture P. Creemers Summary B. cinerea is themos t important fruit rotdiseas e onpom e and small fruits in Belgium. Theappearanc ei n 1979o fstrain so fB. cinerearesistan tt odicarboximide si nstrawberr y fields led to different control strategies. Combinations of dicarboximides with the fungicides thiraman dtolylfluani d wereth emos teffective , eveni nstrawberr yfields wit h a relative high population of resistant strains. The development of new fungicides belonging to different chemical groups are in progress with positive perspectives for controlo fB. cinerea inth enea r future. Thermonebulisation is a new development in the control of postharvest diseases of pome fruits. This method has important advantages in comparison with preharvest treatments orothe rpostharves t applications such asdippin g ordrenching . Introduction In Belgium, Botrytis cinerea Pers.: Fr. is an important pathogen responsible for severe yield losses of ripe and near-ripe strawberries. To control B. cinerea, repeated applications of different fungicides are applied during the blossom period. The development of resistant strainst obenzimidazole s anddicarboximide s has necessitated thesearc hfo rne wstrategie st opreven to rt odela yth ebuild-u po fresistanc etha tdiminis h theefficac y of thesecompounds . B.cinerea als ocause spostharves tgre ymoul do fpom efruits .I nBelgium ,gre ymoul d is the major disease of pear, and on apple the second most severe after Gloeosporium (Creemers,1989) . The same fungicides used in strawberry culture, can be used to control postharvest fruit rot of pome fruits. In Belgium, two to three treatments are usually applied before harvest. Recently a new method called thermonebulisation has been tested (Chapon et al, 1988),whic hi sbase d onth etherma l fogging offungicide s inth estorag eroom .Th e methodi sbase do ntherma lfoggin g ofchemical st oproduc edroplet so fc .1 JJL.Compare d withdippin g ordrenching ,thi smetho d offers anumbe ro fadvantages ;th eapplicatio ni s done after fruits are stored without further handling; 200 ton fruits can be treated per hour;n oproblem s with residualchemica lsolution s occur aswit hdippin g ordrenching ; nosprea do fspore st ohealth yfruit s occursan dth etreatmen tca nb erepeate dfo rproduct s with ashor tlif edurin gstorage . Results and Discussion ResistanceotB. cinerea tobenzimidazole san ddicarboximide si nstrawberrie s In 1969,th eResearc h Station atGorse mdi dth efirs t trialswit hbenzimidazole s(MBC ) 242 for thecontro lo fB. cinerea onstrawberrie s (Balet al, 1989). In 1973,afte r tenweekl y sprayapplication s toth eeverbearin g cultivarOstara ,th eefficac y ofth ebenzimidazole s decreased sharply. In June-bearing strawberries where only four to five treatments against B. cinerea were applied, the efficacy of benzimidazoles also decreased, albeit slower.Sinc e 1983 theMB Cfungicide s haven olonge rcontrolle d thispathogen . Atpresent ,eve no nplot swhic hha dno tbee ntreate dwit hMB Cfungicide s duringth e lastfe w years,application s ofthes ecompound s hadn oeffec t onB. cinerea. Thisshow s that resistance to MBC is no longer limited to plots regularly subjected to MBC treatments. Dicarboximide fungicides, such as vinclozolin, iprodione and procymidone were introduced later. Here also, dicarboximide-resistant strains were soon isolated from everbearing strawberries. The resistance level was much lower and resistant strains showed alsoa lowe rfitnes s comparedt owil dstrains .Thi sle dt oa decreas ei n frequency ofdicarboximide-resistan t strains whenn odicarboximide s hadbee napplied . The selection pressure for resistance is higher on everbearing than on June-bearing strawberries asles s than half of thenumbe r of treatments areapplie d within oneseaso n toth elatter .Th enumbe ro fdicarboximide-resistan t strainsincrease ddurin gth elas tyear s onJune-bearin g strawberries. With better control strategies it was possible to increase the efficacy of the dicarboximides in strawberry fields wheredicarboximide-resistan t strains werepresent . Thebes t resultswer eobtaine d when thedicarboximide s werecombine d with thiram or tolylfluanid (Table 1 and2) . Theefficac y of thedicarboximide s on June-bearing strawberries wasbette r than on everbearingstrawberries ,th emos tpronounce ddifferenc e beingfoun d withprocymidon e (84,1%agains t8,2%) . Dicarboximideresistanc eoccurre dmor eofte n onth eeverbearin gstrawberrie sa swa s expected. Recentlyw eals oobserve da tendenc yfo ra decreas ei nth eactivit y ofdicarboximide s in June-bearing types; the efficacy was comparable to the older fungicides thiram and tolylfluanid. The combination of dicarboximides with these compounds considerably improved controlo fB. cinerea (Creemerset al, 1990; Gochaan dCreemers ,1991) . Efficacy ofdiethofencar b againstB. cinerea infrui t culture Diethofencarb, afungicid e withnegativ ecros sresistanc et obenzimidazoles ,offer s more possibilitiesfo ralternatin gfungicide s ofdifferen t chemicalgroup si nth econtro lschem e (Gullinoetal, 1989; Knightsetal, 1991). For the control of natural populations of B. cinereai t is necessary to combine diethofencarb with benzimidazoles. Sumico, a coformulation of diethofencarb with carbendazim has been registered in Belgium for commercial use on strawberries since 1990. It is possible to apply Sumico until 3day s before harvest. In 1992 a request for registration of Sumico as an agent for control of B. cinerea and Gloeosporium sp. on stored apples andpear sha sbee n submitted. Sumico showed a good activity against B. cinerea in June-bearing and everbearing strawberries.Th eadditio n ofthira mt oSumic ogav ea nimportan tincreas ei nth eleve lo f control (Table 3). On everbearing strawberries where three sprays of Sumico were followed with three sprays of tolylfluanid there was also an improvement in control compared withth etw ocompound s alone(Tabl e4) . 243 Table 1. Control ofB. cinerea onJune-bearing strawberry, cv. Elsanta infour trials in 1990and 1991. Fungicide Doserat e Efficacy (%) g alare-') Iprodione 10 80.3 c» Procymidone 5 84.1 c Thiram 16 79.5 c Tolylfluanid 12.5 78.6 c Procymidone+ thiram 5+16 94.3 ab Procymidone+ tolylfluanid 5+ 12. 5 95.2 ab Vinclozolin+ thira m 5+ 1 6 97.4 a Iprodione+ thira m 10+16 90.7 b Averagenumbe ro f treatments:6 Averagedegre eo finfectio n inth euntreate d plots:13,8 % ')Value sfollowe d byth esam elette rar eno tsignificantl y different atp = 0.0 5 Table2. ControlofB. cinereaon everbearing strawberry, cv. Selvain threetrials in 1990 and1991. Fungicide Doserat e Efficacy (%) g a.i.are') Procymidone 5 8.2 e1) Thiram 16 62.7 d Tolylfluanid 12.5 76.0 c Procymidone+ thira m 5+16 88.7 ab Procymidone+ tolylfluanid 5+12.5 80.5 bc Vinclozolin+ thira m 5+16 90.2 a Iprodione+ thira m 10+16 79.2 c Averagenumbe ro ftreatments :1 4 Averagedegre eo finfectio n inth euntreate d plots:13.6 % ') Seefoo tnot ei nTabl e1 . Because of the mode of action of Sumico, we recommended that the number of applications within one season should be limited to avoid the selection of strains of B. cinerea which areresistan t tobot h components i.e. diethofencarb and carbendazim. Wehav eisolate dsuc hstrain sfro meverbearin gstrawberr yfield swhic hwer etreate dwit h only Sumico. Besides the possibility of reducing resistance build-up against MBC fungicides, alternationo rcombinatio nwit hthira mo rtolylfluani d hasa positiv eeffec t on the control of other fruit rot fungi, like leather rot (Phytophthora cactorum)an d anthracnose{Colletotrichum gloeosporioides) (Tables3 an d4) . Sumicowa sals oeffectiv e againstB. cinerea i npom efruit s wherebenzimidazole sar e no longer effective. Normally three sprays are applied in the last 5 to 6 weeks before harvest,th elas t applied 14day s before harvest. Thecontro l ofB. cinerea with Sumico wassuperio rt ospra yprogramme sbase do nvinclozoli n (Table 5). Postharvest dipping treatments were more effective for control of B. cinerea than preharvest treatments. In Belgium, some fungicides (imazalil, thiabendazole) are 244 Table3. ControlofB. cinereaon June-bearing strawberry cvs Gorella, Berluta, Sivetta, Vicodaand Elsanta in nine trialsduring 1987-1991. Fungicide Doserat e Efficacy (%) Fungicide gai.are- 1 Diethofencarb +carbendazi m 2.5+ 2. 5 88.3 b») Diethofencarb +carbendazi m 2.5+ 2. 5 97.4 a +thira m + 16.0 Tolylfluanid 12.5 83.3 b Vinclozolin 5 77.0 be Vinclozolin+ thira m 5+16 95.9 a Averagenumbe ro ftreatments :5. 7 Averagedegre eo finfectio n inth euntreate d plots:18.8 % ') Seefoo t notei nTabl e1 . Table4. Control ofB. cinerea ineverbearing strawberry cvs Rapella andSelva in eight trialsduring 1987-1991. Fungicide Doserat e Efficacy (%) g ai.are • Diethofencarb +carbendazi m 2.5+ 2. 5 73.8 b» Diethofencarb +carbendazi m 2.5+ 2. 5 88.0 a alternated with tolylfluanid 12.5 Tolylfluanid 12.5 67.8 b Vinclozolin 5 39.6 c Vinclozolin+ thira m 5+16 89.2 a Averagenumbe ro f treatments:1 5 Averagedegre eo finfectio n inth euntreate d plots:16.8 % ') Seefoo t notei nTabl e1 . permitted for postharvest use,bu t in practice are rarely applied. These treatments have some advantages biologically and ecologically compared to preharvest applications, such as reduced useo f chemicals peruni to f fruits, noeffec t on benificial organismsi n theorchard ,bette rcontro lo ffrui t rotb ya bette rcoverag eo fth efruit s by theprotectio n ofwound smad eb ypicking ,separatio nbetwee n fruits fordirec tsal eo rfo r long storage anda reduce drisk fo rth eselectio no fresistan tstrain so fwhic hth einfectio n sourcei si n theorchard . Thermonebulisation Experimentswer edon et ocompar ethermonebulisatio n anddipping .Th ebes tcontro lo f B. cinerea wasobtaine db ydippin g (Table6) .A tth efirs t assessment in February,ther e was a difference in efficacy between the preharvest treatments and the thermone bulisation,bu tb yth esecon d assessmenti nJun en odifferenc e wasdetected . 245 Table 5. ControlofB. cinereaon pear cv. Conférence inthree trialsduring 1988-1990. Fungicide Doserat e (a.i)Efficac y (%) Pre-harvest treatments (3treatments ) Diethofencarb +carbendazi m 375+ 37 5 g.ha-1 86.5 Vinclozolin 750g.ha- i 73.0 Post-harvest treatmentb ydippin g Diethofencarb +carbendazi m 250+ 250mg. H 98.2 Vinclozolin 500mg. H 92.8 Averagedegre eo finfectio n inth euntreate dplots :14.8 % Table 6. Control of B. cinerea onpear, cv.Conférence duringstorage using different methods. Method Doserat e(a.i. ) Efficacy (%) February '91 June '91 Preharvest Vinclozolin 3x 75 0g.ha- i 86.5ab » 82.2 a +Carbendazi m 3x 37 5 g.ha-1 Dipping Vinclozolin 500mg. H 94.7 a 86.4a + Thiabendazole 500mg. H +Imazali l 250mg. H Thermonebulisation Vinclozolin 550gr/5 0to n 72.9b 80.0a Thiabendazole 480gr/5 0to n 26.7c 0.0b Untreated %infectio n 22.4 18.0 l) Seefoo t notei nTabl e1 . Theperio do f 14day sbetwee nharves tan dthermonebulisatio n mayhav eaffecte d this resultsan dbette rcontro lma yhav ebee nachieve di fthi streatmen twa scarrie dou trapidl y after harvest. Because of MBC resistant strains the efficacy of thiabendazole was inadequate. Although the efficacy of thermonebulisation is lower than that of dipping, withmodificatio n itma yreplac eth enee dfo r pre-harvestsprays . Newfungicide s againstBotrytis cinerea In 1991 some new fungicides from different chemical groups were evaluated for the control of B. cinerea on strawberries.On eo f these compounds, CGA 173506,i sbase d onth enatura lsubstanc epyrrolnitrin ,a metabolit eo fPseudomonas cepacia andbelong s to the chemical class of the phenylpyrroles. The chemical structure for the other compoundscanno tye tb egiven .The yar epresente db yth eRS Gnumber s40 4an d184 . 246 Table7. Fungicidalcontrol ofB. cinereaon three strawberry cultivarsin 1991. Fungicide June-bearing Everbearing Doserat e Elsanta Elsanta Vicoda Selva Selva g ai.are-1 Trial1 Trial2 2year s Trial1 Trial2 CGA 173506 5.0 94.6 a1) 99.0a 91.2a 95.9a 89.9 a RSG404 8.0 96.9a 98.1a 62.3 b 91.7a b 66.4b RSG18 4 5.0 87.6 abc 93.3a b 89.0 a 90.0a b 71.0 ab RGS 184+ thira m 3.75+ 16.0 95.3 a 96.2a b 78.8 a 90.9a b 81.3 ab Tolyfluanid 12.5 68.2 de 89.4 ab 64.1b 75.2b 73.4 ab Thiram 16.0 80.6be d 86.5b 55.7b 44.6c 66.4b Procymidone 5.0 62.0e 69.2c 11.8 c 42.1 d 6.3c Vinclozolin 5.0 +Thira m 16.0 93.0 ab 99.0 a 62.7b 87.6a b 85.2 ab Untreated %infectio n 12.8 13.0 33.3 15.1 16.0 J) Seefoo t notei nTabl e1 These new fungicides showed a good activity against B. cinereao n June- and everbearing strawberries (Table 7). The efficacy was comparable, or belter than the standard vinclozolin+ thira mcombination . The release of several fungicides with a different mode of action offers more possibilitiest oalternat eth efungicide s andt opreven to rt odela yth eselectio no fresistan t strainsfo r aspecifi c fungicide groupi nth enea r future. References Bal,E. ,Gilles ,G .an dCreemers ,P. ,1989 . Problemso fresistanc ei nBelgia n Strawberry culture.Act aHorticultura e265:473-482 . Chapon, J.E, N'Guyen T.C., Seng, J.M. and Bompeix, G., 1988. Traitements de postrécolte des maladies des fruits avec des antioxydants et des fongicides par thermonebulisation.Deuxièm econférenc e internationale surle smaladie sde splantes , Bordeauxp .735-742 . Creemers, P., 1989.Chemica l control of parasitic storage diseases on apple and pear. ActaHorticultura e258:648-653 . Creemers, P., Vanmechelen, A. and Bal, E., 1990. Ten years of Botrytis cinerea Pers. control with dicarboximides fungicides on strawberries. Mededelingen Fakulteit Landbouwwetenschappen, Rijksuniversiteit Gent55:955-962 . Gocha, N. and Creemers, P., 1991.Us e of thiram in strawberries, apples and pears. Mededelingen Fakulteit Landbouwwetenschappen, Rijksuniversiteit Gent 56:501- 515. Gullino,M.L. ,Aloi ,C .an dGaribaldi ,A. , 1989.Experience so fchemica lan d biological controlo fgre ymoul d ofstrawberry .Act aHorticultura e265:507-512 . Knights, I.K., Neumann, G.L. and Creemers, P., 1991.Diethofencarb : a new fungicide for the control of Botrytis cinerea in strawberries. Mededelingen Fakulteit Landbouwwetenschappen, Rijksuniversiteit Gent52:491-500 . 247 Effect offungicid e application techniques on the control of Botrytiscinerea an d development offunga l resistance M. Besriand F. Diatta Summary The fungicides used to control tomato grey mould caused by B. cinerea are usually sprayed,bu tsom efarmer s applythe mals oa sa past et oth este mlesions .Applicatio no f benomyl and thiophanate-methyl to plants previously inoculated, did not control the fungus. Procymidone, applied as spray or paste, reduced lesion length by 25.6% and 42.5% respectively and reduced the percentage of wilted plants, particularly when applied asa paste . Painting theB, cinerea lesions with benomyl, thiophanate-methyl and procymidone increasedth efrequenc y ofresistan tspores .N oresistan tspore swer edetecte d onspraye d oruntreate dlesions . Introduction Chemical control of tomato pathogens, particularly Botrytis cinerea Pers.: Fr., is intensive along the Moroccan atlantic coast. Despite the large number of fungicide applications(20-2 5pe rcrop) ,gre ymoul dremain sth emos timportan tdiseas eo fthi scro p (Diatta, 1984; Hormatallah, 1984).Diatt a(1984 )reporte dtha tB. cinerea occursi n96 % of the plastic greenhouses surveyed. Farmers use several fungicides to control the disease, but particularly thiophanate-methyl (Pelt 44), benomyl (Benlate) and procymidone (Sumisclex). These are either sprayed or applied as a paste on the stem lesions.Abundan tsporulatio no fB. cinerea i softe n observedeithe ro nth espraye dplant s oro nth epainte dlesions . Under Moroccan conditions (Besri and Diatta, 1985) and elsewere (Dekker, 1976, 1982; Delp, 1980; Lerouxet al, 1982;Loren zan dEichhorn , 1982),fungicid e resistance tobenomyl , procymidone and thiophanate-methyl is widely distributed. However, data relatedt omethod so ffungicid e applicationo nresistan tstrai ndevelopmen ti nB. cinerea ando nefficienc y ofcontro lar eno tavailable . Material and Methods Frequency ofsporulatio n ofB. cinerea on treated stem lesions Plastictunnel si nth eimportan ttomat ogrowin garea so fDa rBouazza ,Chtouka ,Oualidi a and Abda where benomyl, procymidone and thiophanate-methyl are used to treat the diseased plants were chosen for our study. In each region, one hundred lesions per treatment for each fungicide were observed 4 months after planting to examine sporulation ofth e fungus. 248 Efficacy ofth efungicid e application techniques Stemso ftomat oplant s(cv .Daru s 149)wer ewound-inoculate d withB. cinerea, usinga tomatolea f discpreviousl ysteam-heate d andinoculate d withth efungu s (Diatta, 1984). The fungicides were applied one week after inoculation when lesions developed. Application wasdon eeithe r aspast e(0. 5g.m H ofwater )b ypaintin g thelesions ,o ra s sprays at 250, 750 and 300 ppm for benomyl, thiophanate-methyl and procymidone respectively.Fiv ereplicate softe n plantswer euse dfo reac hfungicid e andtreatment .Th e lesionlength swer emeasure d 14day safte rfungicid e applicationan dth eefficienc y ofth e fungicide determinedb yth eform ula : A-B x10 0 Efficiency (%)= A Where A= length increase of thecontro l lesion and B= length increase of the treated lesion. Thepercentag eo fwilte d plantswa scalculate d 40day safte r inoculation ofplants . Frequencyo fresistan tspore so fB. cinerea producedo ntreate d tomatoplant s Sporesfro m stemlesion sspraye do rpainte dwit hth ethre efungicide s weresuspende di n wateran dth econcentratio n adjusted to 103spores.mH .PD APetr idishes ,amende dwit h benomyl (Benlate 50%), thiophanate-methyl (Pelt 44), procymidone (Sumisclex) at concentrations 20, 20 and 2 ppm respectively were inoculated with 0.5 ml spore suspension and the dishes were incub ated at 25°C for 6 days. The frequency of the resistant spores (F %) was then calculated by the same formula as above where A represents thenumbe r of colonies onPD A andB th enumbe r ofcolonie s on fungicide- amended PDA. Fifteen isolates per treatment in five replicates were tested for each fungicide. Results Frequencyo fsporulatin g lesions B. cinereasporulate d on tomato plants sprayed or painted by the three fungicides (Table 1), but the percentages of the sporulating lesions treated with benomyl and thiophanate-methyl were higher than those treated with procymidone. The sporulation frequency on the sprayed plants is also higher than on the painted ones for the three fungicides used, but neither spraying nor painting the lesions completely inhibited sporulation. Effect ofapplicatio n techniqueo nlesio nlengt han dpercentag eo fwilte dplants . Theapplicatio n ofbenomy lan dthiophanate-methy l asspray so ra spast edi dno tinhibi t thegrowt h oflesions .Th etreatmen tefficienc y varied from 1.2% to 5.4% 14 days after the inoculation. Procymidone applied as a spray or as paste reduced lesion length by 25.6% and42.5 % respectively (Table2) . 249 Table 1. Frequencyof sporulating B. cinerea lesionsalong theMoroccan atlantic coast. Region %sporulatin g lesions Sprayed Painted BD TM P B TM P DarBouazz a 80 92 4 48 55 0 Chtouka 86 85 3 38 62 2 Oualidia 73 95 6 49 52 5 Abda 59 88 7 53 49 3 Mean 74.5 90 5 39.5 54.5 2.5 ')B = benomyl, TM = thiophanate-methyl, P = procymidone. The fungicides were appliedb yth egrower s asspray so ra spaste . Table 2. Effect of fungicide application technique on B. cinereastem lesions after 14 days. Fungicide Sprayed Painted lesionlengt h (mm) % control" lesionlengt h(mm ) % control Benomyl 95.0 5.4 89.2 3.2 Thiophanate-methyl 91.5 1.2 99.0 2.2 Procymidone 81.5 25.6 53.2 42.4 Control 96.8 96.8 ')control ,compare d tolesio nlengt hbefor e fungicide applied. There weren o significant differences between plant parts treated with benomyl and thiophanate-methyl, either sprayed orpainted , and the control plants.I n procymidone- treated plants sprays and painting reduced wilting from 95% incontrol s to4 9 and28 % respectively. Frequencyo fresistan tspore so fB. cinerea Table3 show stha tpaintin gth este mlesion swit hth efungicide s increases the frequency of the resistant spores ofB. cinerea tobenomyl ,thiophanate-methy l andprocymidone . Noresistan tspore st oprocymidon ewer edetecte d onspraye d oro ncontro llesions . Discussion Toou rknowledge ,n oresearc h onth eeffec t of thefungicid e application techniqueha s beendon ewit hB. cinerea control oro nth edevelopmen t of resistance. Sporulationwa s higher on tomato plants sprayed with benomyl and thiophanate-methyl than on those painted withth esam efungicides . Similarresult swer efoun d withprocymidone ,bu tth e percentageo fsporulatin g lesionswa slower . The curative treatments through spraying or painting the lesions did not stop their development,bu tprocymidon eapplie da sspra yo rpast edecrease dlesio nlengt han dth e 250 Table 3. Frequency (%) of resistant spores ofB. cinerea growing in theagar medium amendedwith thefungicide. Origino f Benomyl Thiophanate-methyl Procymidone isolates (20ppm ) (20ppm ) (2 ppm) Paintedlesio n 80a » 93 a 2a Sprayedlesio n 30b 42b Ob Control 15c 18 c Ob 0 Figuresfollowe d bydifferen t letters aresignificantl y different, p = 0.01 numbero fwilte dplants .Therefor e fungicides mustb eapplie d preventively. Thefungicid e applicationtechnique suse db yth egrower shav elimite deffec t ongre y mould,bu tincreas eth epopulatio no fresistan tconidia ,particularl ywhe nth este mlesion s were painted. Theresult s indicate that thelatte r practice isprobabl y responsible for an importantincreas eo fth eresistan tB. cinerea isolate scompare dt oth esprayin gtechnique . We therefore advise painting of lesions should be avoided and an integrated disease management strategy to control the pathogen introduced (Ogawa etal, 1977,Dekker , 1982). References Besri,M .an dDiatta ,F. , 1985. Résistanced eBotrytis cinereaagen td el apourritur egris e de la tomate aux benzimidazoles, dicarboximides et sulfamides. European and Mediterranean PlantProtectio n OrganisationBulleti n 15:379-386. Dekker, J., 1976.Acquire d resistance tofungicides . Annual Review of Phytopathology 14:405-428. Dekker, J., 1982. Countermeasures for avoiding fungicide resistance. In Fungicide Resistance in Crop Protection. Eds. Dekker, J. and Georgopoulos, S.G., PUDOC, Wageningen p. 177-186. Delp, C.J., 1980.Copin g with resistance to plant disease control agents.Plan t Disease 64:652-657. Diatta,F. , 1984.Efficacit é del alutt echimiqu econtr eDidymell a lycopersici etBotrytis cinerea et problème de résistance aux fongicides. Thèse, Institut Agronomique et VétérinaireHassa nII ,Rabat ,Morocco . Hormatallah,A. , 1984.L'oïdiu md el atomat ed ûà Oïdiopsi staurica .Gamm ed'hôte se t méthodes de lutte. Thèse, Institut Agronomique et Vétérinaire Hassan n, Rabat, Morocco. Leroux, P., Lafon, R. and Gredt, M., 1982. La résistance de Botrytis cinereaau x benzimidazoles et aux imides cycliques. Situation dans les vignobles alsaciens, bordelais etchampenois .Europea n andMediterranea n PlantProtectio n Organisation Bulletin 12 :137-143. Lorenz, D.H. and Eichhorn, K.W., 1982. Botrytis cinerea and its resistance to dicarboximidefungicides .Europea nan dMediterranea nPlan tProtectio nOrganisatio n Bulletin 12:125-129. Ogawa, J.M., Gilpatrick, J.D. and Chiarappa, L„ 1977. Review of plant pathogens resistantt ofungicide s andbacteriocides .FA OPlan tProtectio n Bulletin 25:97-111. 251 Effect on grey mould ofpresenc e ofBotrytis cinerea strains showing reduced sensitivity to dicholofluanid A.C. Pappasand K. Elena Summary In the winter of 1987-88 during monitoring of tomato and rose crops infected by B. cinereai nprotecte d cropping,i twa sfoun d thata hig hpercentag eo fisolate sshowe d reduced sensitivityt odichlofluani d invitro. Thefield efficac y ofthi sfungicid e wasno t decreasedthroughou ta 3-yea rtria lo ntomato .Spray scontainin gdichlofluani d provided thebes tprotectio n againstB. cinerea causingpeta lspottin g onroses . Introduction Thewidesprea ddistributio no fstrain sof Botrytis cinerea Pers. : Fr. showingresistanc et o benzimidazoles and dicarboximides has restricted the number of compounds available forgre ymoul dcontro li ngreenhous ecrops .Th erecentl yintroduce dfungicid e diethofen- carb offered apossibl e alternative for use in places wherehig h levels of carbendazim- resistant strains predominate (Pappas and Elena, 1988).Nevertheless , insensitive bio- tyr^st obot hcarbendazi m anddiethofencar b havebee nalread ydetecte di nIsrae l(Kata n et'al., 1989). Thelong-standin g protectant fungicide dichlofluanid isstil l usedextensivel y against greymould ,bu tdoubt sabou tit sefficac y wereclaime dfollowin g theisolatio no fstrain s exhibiting awid erang eo fsensitivit yt othi sfungicid e whenteste din vitro (Malathrakis, 1989). Moreover,suc hdichlofluani d 'resistant' isolates alsoshowe d variablesensitivit y totw oothe rmultisit efungicides , chlorothalonilan dcapta nbot huse dagains tgre ymoul d (Elena and Pappas, 1989). A re-evaluation of the performance of dichlofluanid in the field in places where strains of B. cinerea with reduced sensitivity weredetected , was therefore necessary. Material and Methods Todetermin e the frequency of infections caused by strains of B. cinerea with reduced sensitivity to dichlofluanid, the 'point inoculation method' was applied. Infected plant tissueswer ecollecte di npolyethylen ebag san dincubate dovernigh ta troo mtemperature . Conidiafro m freshly sporulatinglesion so feac hsampl ewer ethe nasepticall ytransferre d with a needle onto malt extract agar (Oxoid) containing 0.1 or 3 jig a.i.mH of the fungicide dissolved inacetone .Th efinal concentratio n ofth esolven ti nth emediu mwa s always 1%.Whe n necessary the sensitivity to dicarboximides andbenzimidazole s was detected on media amended with 3an d 10iig.m H iprodione or 100jJLg.m Hbenomyl , respectively. In some cases, the sensitivity to N-phenylcarbamates was also tested in media containing 100fJLg.ml- 1diethofencarb . Solventsi nthes etest swer eaceton efo r iprodione 252 and dimethyl sulfoxide for benomyl and diethofencarb. Following 24 h incubation at 21°Ci ndarkness ,spor egerminatio no feac hisolat ewa sexamined . Inthre esuccessiv eyear s(1985-88) ,dichlofluanid , aloneo ri n alternating spraysan d combined programmes with specific fungicides, wasevaluate d againstgre y mould ina protected commercial tomatocro p inTymbaki .I n all trials plants of tomatocv .Tomb o weregrow na ssingl estem so nvertica lstring san dplante d5 0c mapar ti nmiddl eOctobe r as six rows spaced at 1m . Each treatment was applied to a 50-plant randomised plot surrounded byunspraye d guardplant san dreplicate d threetimes . The fungicides used were: dichlofluanid; iprodione; metomeclan; chlorothalonil; carbendazim + diethofencarb (Sumico). These fungicides were applied until run-off (112 0Lha -1)usin ga hand-operate dknapsac kspraye ra ta pressur eo f6 kg/cm 2.Fungicid e applications were made on 8-11 occasions each growing season at 15-day intervals commencing the first fortnight of December and terminating in April. Prior to each application ripe fruit was picked and weighed and the number of infected tomatoes in eachplo trecorded . Fungicidetreatment sdurin g the 1987-88perio d wereals ocompare d onrose sfo r the control of petal spotting on flower buds caused by B. cinerea. In this experiment, 4- month oldplant so fros ecv .Soni awer eplante d inJul y 19872 0c mapar ti nseve nrows . The experiment was done in a commercial glasshouse in the Marathon region. Each treatmentwa sapplie d onthre e9 m lon g randomised plots.Fungicide s werespraye d on six occasions at 10-day intervals between October-December 1987. Before each application five flowers pertreatment/replicat e werecollecte d atrando m andexamine d for petalspottin g after storagefo r 3day sa t5°C . Results The monitoring of tomato fruit rots on two occasions during the winter of 1987-88 in Tymbaki region revealed that 80 out of 150 infections were due to B. cinerea strains snowingreduce d sensitivityt odichlofluanid . Thespore so fthes eisolate sgerminate do n media containing 1|xg.m H of the fungicide. However, germtube length was less than doubleth espor ediameter ,an dth egerminatio nwa scompletel yarreste da tconcentration s of 3 jjLg.ml-1. Isolations of such strains were few in previous years where spore germination of most isolates was inhibited by dichlofluanid at concentrations of 0.2 |xg.mF. Ahig hincidenc e of strains with reduced sensitivity todichlofluani d wasdetecte d in 4-month-oldcommercia l roses in theregio n ofMarathon .I n asurve y madei nOctobe r 1987,th espore so f 23ou to f7 0isolate swit horiginate d from infected rosesgerminate d onmedi acontainin g 1 ixg.mH dichlofluanid. The predominance of B. cinereastrains , showing moderate resistance to dicar- boximides and high resistance to benzimidazoles, was confirmed in Tymbaki and Marathon. All benzimidazole-resistant isolates tested however, were found to be sensitivet o diethofencarb. In the 1985-1986 disease control trial, all fungicide applications, except iprodione, significantly reduced fruit rot and in creased the yield of tomato. Better control was providedb yth etan k mixtureo fdichlofluani d plushal f strength metomeclan (Table 1 ). Iprodione alone failed to control the disease, presumably due to infection by strains moderatelyinsensitiv et odicarboximides . In the 1986-87 trial, dichlofluanid alone and in combined spray programmes gave 253 Table 1. Evaluationof fungicides for controlof fruit rotin tomato causedby B. cinerea inthe 1985-86season. Treatment/concentration (a.i.) Yield No.rotte d (kg) fruits Dichlofluanid(0.1%) 85 10.7 a Iprodione(0.05% ) 84 18.0b Metomeclan (0.05%) 95 8.0a Dichlofluanid (0.1%+ iprodion e (0.025%) 96 9.3a Dichlofluanid (0.1%)+ metomecla n (0.025%) 92 6.7a Untreated 81 18.0b S.E.D. - 1.44 Datafollowe d byth esam elette rd ono tdiffe r significantly atp = 0.05 . Table 2. Evaluation ofvarious spray-programmesfor control ofB. cinereafruit rot in tomato inthe 1986-87season. Treatment/concentration(a.i. ) Yield No.rotte d (kg) fruits Dichlofluanid (0.1% ) 104 53.7 be Dichlofluanid (0.1% )+ iprodion e(0.025% ) 113 36.7a Dichlofluanid (0.1%)*)followe d by dichlofluanid (0.1%)+ iprodion e(0.025% ) 97 50.0ab c Dichlofluanid (0.1%)*)followe d by iprodione (0.05%) 97 57.0 c Chlorothalonil (0.1%)*)followe d by chlorothalonil (0.1% )+ iprodion e(0.025% ) 109 39.7a b Untreated 80 95.7 d S.E.D. - 4.63 Datafollowe d byth esam elette rd ono tdiffe r significantly atp = 0.05. *)Thi sfungicid e wasapplie d inth eth efirst fou r spraysonly . satisfactory control andincrease d theyiel do ftomato .Th ebes tcontro lwa sachieve db y repeated applications of the tank mixture of dichlofluanid with half-strength iprodione (Table2) . During 1987-88 trials, applications based on dichlofluanid significantly controlled grey mould in both crops tested. In tomato, the use of carbendazim + diethofencarb (Sumico)eithe ralon eo ri nhalf-strengt h tankmixe swit hdichlofluani d further decreased the number of infected fruit and increased yield. By contrast, Sumico alone was less effective in rose for control of the number of flowers affected by lesions caused by B. cinerea. For this type of infection, applications containing dichlofluanid weremor e effective (Table3) .I nbot hcrops ,sporulatin g lesionswer estrongl y suppressed onplot s whichreceive d Sumicosprays . No apparent differences in sensitivity to dichlofluanid were found among isolates originating from plantswhic hreceive dvariou streatments .O nrose ,afte r thecompletio n of six sprays in the overall treatment with dichlofluanid, inoculum with reduced sensitivityt othi schemica lwa sincrease db y5% . 254 Table 3. Evaluation ofvarious fungicide applications againstB. cinerea intomato and rose culture inthe 1987-1988season. Treatment/concentration(a.i. ) Tomato Rose Yield No.rotte d petal spotting (kg) fruits sin-'p° % Dichlofluanid (0.1%) 170 40.7b » 43.2 47.0a Dichlofluanid (0.1%)+iprodione(0.025% ) 171 47.3b 47.1 53.7a b 'Sumico' (0.025%+ 0.025% ) 178 16.3 a 54.8 66.7 bc Dichlofluanid (0.1%)+ 'Sumico' (0.0125% +0.0125% ) 190 23.3 a 45.6 51.0a b Iprodione(0.025% )+ 'Sumico' (0.0125%+ 0.0125% ) 171 22.0 a 62.4 78.0c d Iprodione(0.05% )alternate dwit h 'Sumico' (0.025% + 0.025%) NT2) NT 58.3 72.3 cd Untreated 160 89.0c 66.4 83.3 d S.E.D. 4.12 3.5 0 Datafollowe d byth esam elette rd ono tdiffe r significantly atp = : 0.05. 2)N T= no tteste d Discussion A wide variation in sensitivity to dichlofluanid, chlorothalonil and captan had been demonstrated among field isolates ofB. cinerea inth eearl y years of their commercial introduction. All these wide spectrum compounds mainly inhibit spore germination whilemycelia lgrowt h ismuc hles s affected (Pappas, 1978). Thepresen t findings show thatB. cinerea population sshif tt obecom ele sssensitiv et odichlofluani d incrop sgrow n under cover. This was probably due to high selection pressure among the wild strains followed by the continuous use of this fungicide. The increased frequency of less sensitive strains, however, should not be considered as resistance development in the field. Dichlofluanid efficacy inlocalitie s of high inoculum pressurewa s alwaysinferio r to that provided by the site specific compounds (Hunter et al, 1979).Th e results of the present work show thatdichlofluani d maintains its moderate effectiveness against grey mould in the field, although the frequency of strains exhibiting reduced sensitivity in vitrowa smuc hincreased .Thes efinding s arei nagreemen twit hthos eobtaine db yothe r workers. Inoculation experiments have shown that conidia of B. cinerea from isolates withreduce d sensitivity failed toinfec t plants sprayed with field-strength dichlofluanid (Elena andPappas , 1989 ; Rewalet ai, 1991). Afurthe r improvementi ndichlofluani d efficacy wasachieve ddurin gthi swork ,whe n it was applied in mixes with compounds having specific modes of action such as iprodione or Sumico used at half recommended strength. Moreover, in cases of non- aggressive infections, the continuation of inclusion of multisite inhibitors in control programmes appeared morejustifiable . Thus,dichlofluani d and its mixtures effectively restrictedth espottin go nros epetal swhe nth emor especifi c compoundsfaile d tod othis . 255 However,th epossibilit y ofa gradua ldeclin ei nth eeffectivenes s ofconventiona lbroad - spectrumfungicide s mustb econsidere d inplannin g theirmor erationa lus ei nprotecte d crops. References Elena, K. and Pappas, A.C., 1989. Sensitivity of Botrytis cinereaPers . isolates to dichlofluanid,chlorothaloni l andcaptan .Proceeding s5t hNationa lPhytopathologica l Congress,Helleni cPhytopathologica l Society pp. 47. Hunter, T., Jordan, V.W.L. and Pappas, A.C., 1979. Control of strawberry fruit rots caused by Botrytiscinerea and Phytophthora cactorum. Proceedings British Crop ProtectionConference .Pest san dDisease sp .177-183 . Katan,T. ,Elad ,Y . andYunis ,H. , 1989.Resistanc et odiethofencar b (NPC)i nbenomyl - resistantfiel d isolates ofBotrytis cinerea. PlantPatholog y38:86-92 . Malathrakis, N.E., 1989.Resistanc e ofBotrytis cinerea to dichlofluanid in greenhouse vegetables.Plan tDiseas e73:138-141 . Pappas,A.C. , 1978. Botrytis controlo nsof t fruit. PhDThesis ,Universit yo fBristol . Pappas,A.C . and Elena, K., 1988.Sensitivit y to diethofencarb and control ofBotrytis cinerea Pers. in greenhouse grown plants. Proceedings of the 5th International Congress ofPlan tPathology ,Kyot op .336 . Rewal, N., Coley-Smith, J.R. and Sealy-Lewis, H.M., 1991. Studies on resistance to dichlofluanid andothe rfungicide s inBotrytis cinerea. PlantPatholog y40:554-560 . 256 Control ofBotrytis cinerea i n cut roseflowers b y gibberellic acid,ethylen e inhibitors and calcium O. Shaul, Y. Elad, B.Kirshner, H.Volpin andN. Zieslin Summary Botrytis blight, caused by B. cinerea is a widespread problem in rose {Rosa hybrida) flower production in greenhouses. Despite efforts to reduce damage caused by the diseaseb ymodificatio n ofenvironmenta lcondition san dus eo ffungicides ,man y flowers carry latent infections and these become visible during shipment or storage. The physiological status of the flower probably inf luences the change from latency to developmento fspreadin glesions .Th eeffec t ofethylen einhibitor san dcalciu mnutritio n on susceptibility of rose flowers to the disease was studied and both factors were effective in controlling the disease. Exogenously applied gibberellic acid also showed potentialfo rdiseas econtrol . / Introduction Botrytisflower blight ,cause db yBotrytis cinereaPers. :Fr. ,i sa widesprea d problemo f roses (Rosa hybrida) grown in greenhouses. The pathogen attacks mainlyflowers, bu t also causes necrotic lesions on young canes. Latent infection occurs and lesions are invisible at harvest, but develop under humid conditions or asflowers matur e (Elad, 1988b).Th emos tsever eeconomi c damageoccur swhe ndiseas edevelop s onflowers i n storage or in transit (Elad, 1988a; Hammer and Marois, 1988). Fungicides are recommended for controlo fth ediseas eo nrose s (Hammer andMarois , 1988), butthei r efficacy islimited .Rose sar eharveste d generally in thebu d stage,bu tinfection s occur between sprays on flowers that have opened slightly. Populations of the pathogen resistant to dicarboximide, benzimidazole and diethofencarb have developed (Katan, 1982;Kata net al., 1989) .On emetho d ofpartiall y controlling thediseas e onrose s ist o lowergreenhous ehumidit yb yincrease dventilatio n andheatin g(Maroi set al., 1988). Development offlower bligh t on petals occurs in two stages. Initially, infection is followed by restricted colonisation, which is not visible to the naked eye, and then cessationo fdevelopment .I nth esecon dstag esmal lo rspreadin gnecroti clesion sdevelo p whenenvironmenta lcondition san dhos ttissue sar econduciv e(Elad , 1988b).Th efactor s affecting the transition of tissues from resistant to susceptible are not understood, but senescenceo fpeta ltissue si sprobabl yinvolved . Ethylenean dCalciu m Therol eo fethylen ei nth edevelopmen t ofBotrytis bligh tha sbee ndemonstrate d (Elad, 1988a)an dthi si ssupporte db yth efac t thatinhibitor so fethylen eproductio n oractivit y can reduce disease severity (Elad, 1988a; Elad and Volpin, 1988). Cut roseflowers fertilised with nutrient solutions containing elevated concentrations of calcium ions, 257 «rt» produced less ethylene and wereles s affected byBotrytis blight than those with lower levelso fCa 2+(Ela dan dVolpin , 1988; Volpinan dElad ,1991) . Physiologicalstresse stha tincreas eth epermeabilit yo fplan tcel lmembrane senhanc e diseasedevelopment , withleake d exudates serving asa nutrien t source of thepathoge n (Sol, 1965, 1967) and it is known that addition of Ca2+ ions to plant tissue retards senescence (Ferguson, 1984; Pooviah and Leopold, 1973) and limits membrane permeability (Simon, 1978).Severa lothe rplan tdisease sar einhibite db yth eadditio no f Ca2+ions ,th einhibitio nbein gassociate dwit hth eabilit yo fth eio nt ostrengthe nth ecel l wall and inhibit degradation by pectinolytic enzymes (Corden, 1965; Liptay and Van Dierendock, 1987). Volpin and Elad (1991), found that the addition of calcium nitrate to the nutrient solutions used to fertilise rose plants of cvs Golden Times and Mercedes reduced the severity of postharvest Botrytis blight. The susceptibility of cut flowers of cv. Golden Times was increased by treatment at 4° and 10"C for 2 days, but this effect was suppressed by addition of calcium,mor e so at 10°tha n at4°C .Col d storage of rosesa t 8°C shortens vase life, and storage at 3°C results in severe damage to the plasma- membrane. The induced susceptibility of flowers to Botrytisbligh t caused by cold storagecoul db edu et oincrease dleakag efro m plasmamembranes(Faraghe ran dMayak , 1984). At 10°C, Ca2+ may limit the membrane damage caused by low temperature exposure, and thereby inhibit leaching. At4°C ,Ca 2+ was probably unable to overcome thedamag ecause d toth emembrane . In amediu m containing Na-pectate asth ecarbo n sourcean d 3m Mcalcium ,growt h of B. cinerea and polygalacturonase activity were inhibited as compared with a Na- pectatemediu m withoutcalcium .Pectat ei s animportan tcompoun d inplan t cellwalls , and more than 60%o f the calcium in the plant is found in cell walls (Rossignal et al., 1977).B. cinerea produces atleas tfou r endo-polygalacturonases(Marcu s and Schejter, 1983). Theinabilit y ofB. cinerea toutilis epectat ei nth epresenc eo fcalcium , therefore, issuggeste d asa mod eo f actiono fth eCa-ions . Gibberellicaci d Onth ebasi so fthes ereport sth ephysiologica l statuso fth ecu tflower s appearst opla ya major role in the change from latent infection to the appearance of spreading lesions. Goszczynska etal. (1990 ) showed that the vase-life of rose flowers was improved and senescenceo fpetal sdelaye d after pre-treatmentwit hgibberelli caci d(GA 3). Theobjectiv eo fou rstud ywa st oexamin eth eeffect s ofth eexogenousl yapplie dG A 3 ongre ymoul di nros eflowers , andth egrowt h ofB. cinerea invitro. Material and Methods Rose flowers (Rosa x hybrida cv. Mercedes) grown in an experimental greenhouse or supplied by commercial growers were used in the experiments. The flowers were harvested whensepal san dpetal sstarte d to unfold. Detached petals,o rflower s budslef t intacto nth estem ,wer espraye d torun-of f with 1 mMsolution so fgibberelli caci d(GA 3Sigma ,USA) .Followin gth evariou streatments , the intact flowers were placed with their stem base in water and the buds enclosed in plastic bags.Th e detached petals wereinserte d through a sliti n aplasti c netwit h their basei nwater ,an dcovere dwit ha transparan tpolyethylen esheet . Bothintac tflower san d 258 detachedpetal swer ekep ti n anilluminate d growthchambe ra t20±2° Can d90-95 %r.h . (Eladan dVolpin ,1988) . Experimentswer erepeate d 3 to6 time san deac htreatmen twa s replicated 10times . Disease severity was assessed on a scale 0 to 5, where 0=healthy petals and 5=completely affected petals.I nal l theexperiment s (moretha n 20),te nflower bud s or petals were used asreplicate s ineac h treatment. The results were analysed statistically andmean swer ecompare db yDuncan' s multiplerang etest . Disks from the growing margins of mycelium from cultures or drops of conidial suspension of B. cinereawer e inoculated onto plates of PDA containing different concentrations of GA3 and incubated at 20±2°C. The linear growth of mycelium was measured daily and the germination of the conidia was examined under a light microscopeafte r 24h . Results and Discussion Thedevelopmen to fgre ymoul do ndetache dpetal san dintac tbuds ,eithe rwit ho rwithou t inoculation withconidia ,wa sinhibite d by application of a 1 mMsolutio n of GA3.Thi s Table 1. The effect of gibberellic acid on grey mould causedby B. cinereain rose flowers. Cultivar Days of incubation Disease index (0-5) Control GA3D Detached petals Mercedes 16 2.8a 0.2b Ilseta 9 2.8a 0.9b Celica 9 3.8a 0.2b Madelon 9 1.4a 0.8a Madam Delbar 9 2.5a 0.5b Cadillac 5 4.6a 2.0b Sonia 6 2.4a 0.5b Gabriela 11 3.1a 0.6b Golden Times 8 3.1a 4.6b Whole flowers Mercedes 14 4.7a 3.2b Fresco 7 3.1a 3.8a Golden Times 6 2.7a 2.8a Sonia 6 1.9a 0.9a Gabriela 10 4.7a 2.2b Pasadena 9 2.1a 0.3b Jaguar 8 1.3a 1.7a ')Whol eflower s werespraye d with 1 mMgibberelli c acid;detache d petals weredippe di n0.05 7m Mgibberelli c acid. Statistical significance (p = 0.05) between control and treatment is indicated by a different letter. 259 effect ofGA 3wa sobserve di nsevera lros ecultivar s(Tabl e 1).Th eexten to finhibitio no f grey mould varied in flowers obtained from different production regions in these experiments. Neither growth of the mycelium nor germination of the conidia was affected by addition of GA3.Onl y at 10m MGA 3,th elinea rgrowt h of themyceliu m wasinhibite d by 20%. Sinceth egrowt han dgerminatio no fB. cinerea wer eunaffecte d byadditio no fGA 3t o the growth medium, the suppression of grey mould development in the rose flowers sprayedwit hGA 3ma yresul tfro m adecrease d sensitivity oftissue st oth epathoge ndu e to the inhibition of senescence of petals by GA3 (Goszczynska et al., 1990). These authorsals oshowe d thatsenescenc eo fth eGA 3-treatedros eflower s wasinhibited ,eve n inth epresenc eo fhighe rlevel so fethylen eevolve db yth eGA 3-treatedtha nth euntreate d flowers. Aged rose flowers are more sensitive to ethylene than the younger flowers (Lukaszewska et al, 1990). Therefore, we cannot exclude the possibility that GA3- inducedinhibitio no fgre ymoul di nou rwor kwa sth eresul to fa GA-induce ddecreas ei n petalsensitivit yt oethylene . Allth eflower s from thenumerou ssource sinvestigate d inthi sstud ywer einfecte d to varying degrees by B. cinerea. Large quantities of fungicides are used to control this disease,wit ha concomitan tincreas ei nth eresistanc eo fthi sfungu s tofungicide s (Katan etal., 1989) . Replacement offungicide s withgibberelli n and/orothe rgrowt h regulators may therefore be advantageous for control of plant diseases and reduce the ecological hazardscause db yexcessiv epesticid euse . References Corden, M.E., 1965. Influence of calcium nutrition on Fusarium wilt of tomato and polygalacturonase activity.Phytopatholog y 55:22-24 . Elad, Y., 1988a. Involvement of ethylene in the disease caused by Botrytis cinerea on rosean dcarnatio n flowers and thepossibilit y ofcontrol .Annal s ofApplie d Biology 113:589-598. Elad, Y., 1988b. Latent infection of Botrytiscinerea in rose flowers and combined chemicalan dphysiologica l controlo fth edisease .Cro pProtectio n 7:361-366. Elad,Y . andVolpin ,H. , 1988. Theinvolvemen t ofethylen ean dcalciu m ingre ymol do f Pelargonium, Ruscus,an dros eplants .Phytoparasitic a 16:119-131. Faragher, J.D. and Mayak, S., 1984. Physiological responses of cut rose flowers to exposure to low temperature: Changes in membrane permeability and ethylene production.Journa lo fExperimenta lBotan y35:965-974 . Ferguson, C.H.R., 1984. Calcium in plant senescence and fruit ripening. Plant Cell Environment7:477-489 . Goszczynska, D.M., Zieslin, N., Mor, Y. and Halevy, A.H., 1990. Improvement of postharvest keeping quality of 'Mercedes' roses by gibberellin. Plant Growth Regulators. 9:293-303. Hammer,P.E . andMarois ,J.J. , 1988.Postharves tcontro lo fBotrytis cinerea o ncu trose s withpicro-cupric-ammoniu mformate .Plan tDiseas e72:347-350 . Katan, T., 1982. Resistance to 3,5-dichlorophenyl-N-cyclicimide (dicarboximide) fungicides in the grey mould pathogen Botrytiscinerea on protected crops. Plant Pathology31:133-141 . Katan,T. ,Elad ,Y andYunis ,H. ,1989 . Insensitivityt odiethofencar b(NPC )i nbenomyl - 260 resistanfieldt isolate so fBotrytis cinerea. PlantPatholog y38:86-92 . Liptay, A. and van Dierendock, P., 1987.Calciu m retards physiological collapse and subsequentmicrobia l degradation ofmun gbea n (Vigna radiata L.Wilczek )sprouts . CanadianJourna lo fPlan t Science67:537-548 . Lukaszewska,A J. , Tonecki,J. ,Woltering ,E.J . andGorin ,N. , 1990.Effec t of ethylene and silver thiosulfate on vase life of 'Sonia' roses. Gartenbauwissenschaft 55:118- 121. Marcus, L. and Schejter, A., 1983. Single step chromatographic purification and characterization on the endopolygalacturonases and pectinesterases of the fungus Botrytiscinerea. PlantPatholog y23:1-13 . Marois, J.J., Redmond, J.C. and MacDonald, J., 1988.Quantificatio n of the impacto f environment on the susceptibility of Rosa hybrida flowers to Botrytis cinerea. Proceedingso fth eAmerica n Societyo fHorticultura l Science 113:842-845. Pooviah,B.W . andLeopold ,A.C. , 1973.Deferra l oflea f senescencewit hcalcium .Plan t Physiology52:236-239 . Rossignol,M. ,Lamant ,D. ,Salsac ,L .an dHeller ,R. , 1977. Calciumfixatio n byth eroot s of calcicole and calcifuge plants: The importance of membrane systems and their liquid composition. In Transmembrane Ionic Exchange in Plants. Thellier, M., Monnier,A. ,Demarty ,M .an dDainty ,J. ,eds .Edition sd uCNRS ,Pari se tEdition sd e l'Université,Rouen ,Franc ep .483-490 . Simon, E.W., 1978. The symptoms of calcium deficiency in plants. New Phytologist 80:1-15. Sol, H.H., 1965. Alternation in the susceptibility of Vicia faba to Botrytisfabae by various pretreatments of theleaves .Netherland s Journal of Plant Pathology 71:196- 202. Sol,H.H. , 1967. Theinfluenc e ofdifferen t nitrogensource so n(1 )th esugar san damin o acidsleache dfro mleaves ,an d(2 )th esusceptibilit yo f Viciafaba t oattac kb yBotrytis fabae. Mededelingen Fakulteit Landbouwwetenschappen, Rijksuniversiteit Gent 32:768-775. Volpin, H. and Elad, Y., 1991. Influence of calcium nutrition on susceptibility of rose flowers toBotrytis blight .Phytopatholog y 81:1390-1394. 261 Mannans from Botrytis cinerea strains resistant to dicarboximide fungicides O. Kamoen, H.Geeraert and G. vander Cruyssen Summary Isolateso fB. cinerea resistantt odicarboximid efungicide s areusuall y moresensitiv et o high osmotic strength than non-resistant isolates. Osmo-sensitivity of dicarboximide- resistantisolate swa scorrelate dwit hhighe rconcentration so frhamno-galacto-mannans , butno twit hsolubl eglucans ,i nth ecultur emedium . Introduction Dicarboximide-sensitive isolatesof Botrytis cinerea Pers. : Fr. (Bc-S)ar emor etoleran to f high osmotic strengths than dicarboximide-resistant ones(Bc-R )(Albert , 1979;Lerou x andGredt ,1979) . Thehighe rsensitivit yo fBc- Ri sprobabl ydu et odefectiv ecel lwal lsynthesi swit hth e resulttha tth ewal lma ygiv einsufficien t supportt oth eplasmalemma .Her ew eteste dth e hypothesis that more soluble fungal cell wall fragments could be released in liquid cultureso fBc- Risolates . Material and Methods ThreeBc- R and threeBc- S isolates (Table 1)wer egrow n in 300m lErlenmeye r flasks with 150 ml Czapek-Dox medium. The medium was inoculated with a 1 ml spore Table 1. Geneticstatus andorigin ofisolates ofB. cinerea, used inthe experiments Isolatesan dhos tplant s Statusan dsourc e Dicarboximide-sensitive (Bc-S) Bc-L69(apple ) homokaryotic,Dr .Laubert , 1971, Switzerland (SchönenbergTG ) Bc-L5 (raspberry)homokaryotic ,Dr .Laubert , 1971, Switzerland (Meerlischachen SZ) Bc-Sl (from grape) field isolatefro m Angers(F )- Coteaud uLayo n(1988 ) Dicarboximide-resistant(Bc-R) Bc-Rl VGlf (strawberry) field isolatefro m Meerle(Belgium ) selected onvinclozolin-containin g medium Bc-R2C Gl a (strawberry) field isolatefro m Meerle(Belgium ) selected onvinclozolin-containin g medium Bc-RF16(fro m grape) homokaryotic,Dr .Faretra , 1989, Bari,Ital y 262 Table 2. Precipitation ofpolysaccharides from culture media of isolates ofB. cinerea resistantand non-resistant to dicarboximides. p Isolate ED50 Mycelialdr y P05 itoP4 weight(g ) mg/gmyc. mg/gmyc. Bc-L69 <1 1.31 150.7 7.7 Bc-L5 <1 2.87 19.8 6.7 Bc-Sl <1 12.28 30.9 31.2 Bc-RlV Gif 5 2.97 147.7 307.2 Bc-R2V Gla >10 5.88 61.2 92.8 BC-FR16 5 0.63 14.8 55.9 Table 3. Sugar analysis of the culturemedium of B. cinerea,after DEAE column separation. Fractions Sugar(% ) 8-13 14-19 20-22 50-60 62-72 glucose 66 38 15 21 / 12 mannose 21 21 67 64 66 galactose 12 39 16 12 16 rhamnose 12 2 2 6 suspsensioncontainin g 106spore san dincubate do na rotar yshake r(10 0rpm )fo r6 days . Culturefiltrate s wereobtaine db yvacuu mfiltratio n throughglassfibe r filters (SSG F92 , 50m mi na millipor efiltration syste mX X10.047 )an dmycelia lweight swer edetermine d after dryingunde rvacuu ma t90°C .T oobtai npolysaccharides ,th emediu mwa s acidified (pH +3) and ahal f volume ofethano l added togiv eimmediat eprecipitatio n of glucans 6 6 (MWbetwee n 10 an d0. 5x 10 Da).Thi sprecipitate ,calle dP 05 appeare da sa slurr ytha t wasremove dimmediately .Furthe raddition so fethanol ,on etil lfou rtime sth evolum eo f the medium, precipitated polysaccharides of lower molecular weight (between 50an d 3 5x 10 Da) .Thes eprecipitate scalle dP p P2andP 4consiste do f rhamno-galacto-mannans (Dubourdieu, 1978; Kamoen and Dubourdieu, 1990). The different precipitates were washedwit hethanol ,redissolve di ndistille dwater ,dialyse dfo r2 4h ,lyophilyse dan dth e dryweight sdetermined . The amount of polysaccharides in the precipitates was determined with anthrone- sulphuricaci dreagen tan dth eprotein swer emeasure daccordin gt oBradfor d (1976).Th e sugarcompositio n ofsevera lprecipitate swa sexamine db yga schromatograph y(Zanett a etal, 1972).Fro m someprecipitate s thepolysaccharide s were separated from proteins by anion exchange chromatography on DEAE-Sepharose CL-6B (Pharmacia), firstly eluted with 10m Mtri sbuffe r atp H9 an dthe nwit h0 t o0. 5M NaC li nth esam e buffer. Thebes tseparatio n wasobtaine d bypre-treatmen t of thesampl eb y ultrasonication and byelutio n witha stepwis eincreas eusin gNaCl . Results and Discussion The growth of the isolates differed substantially, but growth rates had no relationship 263 Fig. 1. DEAE-columnfor analysisof a rhamno-galacto-mannanfrom B. cinereaisolate R1VGif resistantto dicarboximid.es. 264 with resistance of isolates todicarboximide s (Table 2).Th egluca n obtained in the P05 fraction alsoshowe dgrea tvariatio nbu tthes edifference s wereunrelate d toresistanc eo f theisolates .Th eamoun t of rhamno-galacto-mannans obtained in the P, to P4 fractions washighe rfo r theBc- Risolate stha nfo rth eBc- Sisolates . Further analysis showed that the sugar content of the P2 ethanol precipitate of the sensitive isolate Bc-L69 and the resistant isolate Bc-RlV Gif contained mainly mannose,som egalactos e andonl ytrace so frhamnose . Glucosewa salway spresen tan d itwa sno tclea ri fthi swa sa resul to fgluca ncontaminatio n ori fglucos ewa sa nessentia l parto fth erhamno-galacto-mannan .Smal lamount so fprotein swer eals oco-precipitate d with the polysaccharides (Fig. 1). Table 3 shows the sugar composition of several fractions from thesam eseparation .Mos tpolysaccharide s eluted from theunboun d part of theDEA Ecolumn . Fractions 18t o 28containe d the rhamno-galacto-mannans, with some glucose. Proteins present in mg amounts were almost completely separated from thepolysaccharides ;onl ya trac eo fresidua lprotei nco-elute di nth efraction s 55t o80 . A higher amount of rhamno-galacto-mannans was found in shake cultures from isolates of B. cinerea resistant to dicarboximides than from susceptible isolates. The higher release of rhamno-galacto-mannans may have an effect on the behaviour and survivalo fBc- Risolate si nnature .I ngenera lBc- Risolate sar eles scompetitiv ei nnatur e thanBc- Sisolate san dthe ydisappea ri nth efiel di nth eabsenc eo fth efungicide s (Beever etal., 1991) .Marait eet al. (1980)examine dth ecompetitivit y ofBc- Sisolate san dBc- R isolates invitro; two Bc-R isolates of six were competitive with the Bc-S isolates but competitivityin vitro wa sprobabl ydu et osporulatio nan dgrowt hrat ean dcanno texplai n completely thedecreas eo fBc- Risolate si nth efiel d inth eabsenc eo f fungicides. Dicarboximide-resistant isolates are highly sensitive to high osmotic pressure.Thi s feature tooca nonl ypartl yexplai nth elo wfitne s of Bc-Risolate si nnatur efo r inman y infected tissuesth efungu s doesno tgro w athig hosmoti cpressure . Thehig hsecretio no frhamno-galacto-mannan sb yBc- Risolate si nvitr oma yprovid e an additional explanation for these polysaccharides are known as elicitors of plant defence (Dixon and Fuller, 1977; Kamoen et al, 1980) and might give elicitation of defence reactions.A sa consequenc ethe yma yb eles sabl et osurviv ea sa parasite . References Albert, 1979. Wirkungsmechanismus und Wirksamkeit von Vinclozolin beiBotrytis cinerea (Pers). Ph.D. Thesis. Hohen Landwirtschaftlichen Facultät Rheinischen Friedrich-WilhemsUniversität , Bonn,German ypp . 96. Beever, R.E., Pak, H.A. and Laracy, E.P., 1991. An hypothesis to account for the behaviour of dicarboximide-resistant strains of Botrytis cinerea in vineyards. Plant Pathology.40:342-346 . Bradford, M., 1976.A rapi d and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein dye binding. Analytical Biochemistry72:248 . Dixon, R.A. and Fuller, K.W., 1977. Characterisation of components from culture filtrates of Botrytiscinerea whic h stimulate phaseolin biosynthesis inPhaseolus vulgaris cell suspensioncultures .Physiologica lPlan tPatholog y 11:287-296. Dubourdieu, D., 1978.Etud ede spolysaccharide s sécrétées parBotrytis cinerea dansl a baied eraisin ,Ph.D .Thesis .Universit éBordeau xH , France,pp .160 . Kamoen O. and Dubourdieu, D., 1990.Phytotoxi c secretions from Botrytis cinerea in 265 grapes. Mededelingen Fakulteit Landbouwwetenschappen, Rijksuniversiteit Gent 53:799-804. Kamoen O., Jamart, G., Declercq, H. and Dubourdieu, D., 1980. Des éliciteurs des phytoalexines chezl eBotrytis cinerea. AnnalesPhytopathologi e 12:365-376. Leroux,P . andGredt ,M. , 1979. Phénomènesd erésistanc ecroisé enégativ eche zBotrytis cinerea Pers. entre les fongicides benzimidazoles et les herbicides carbamates. Phytiatrie-Phytopharmacie 28:79-86. Maraite, H., Meunier, S., Pourtois, A. and Meyer, J.A., 1980. Emergence invitro an d fitness of strains of Botrytis cinerea resistant to dicarboximide fungicides. Mededelingen Fakulteit Landbouwwetenschappen, Rijksuniversiteit Gent 45:159- 167. Zanetta,L.P. ,Beckenbridge ,W.C . andVincendon ,G. , 1972.Ga sliqui dchromatograph y oftrifluoracetateso f O-methylglucosides.Journa lo fChromatograph y69:291-304 . 266 Bacillusbrevis as a biocontrol agent against Botrytiscinerea o nprotecte d chinese cabbage S.G. Edwards andB. Seddon Summary Bacillus brevis produces theantibiotic ,gramicidi n S whichi sfungicida l toB. cinerea in vitro. Testsin vitro usin gB. brevis spore sshowe dtha tgramicidi nS associatedwit hthes e spores was also active against B. cinerea. Testsusin g leaf disks and seedlings failed to confirm theseobservation s inplanta andi twa sfoun d thatgramicidi n S bindst oth elea f surfacean di sinactiv ei nthi sform .Fiel dtrial susin gChines ecabbag ea sa protecte dcro p showed that biocontrol of B. cinerea with B. brevis was achievable with protection as gooda sth estandar d fungicide treatment.B. cinerea i sdependen tupo nsurfac e waterfo r germination andinfectio n andi ti sthough ttha tB. brevis controlledB. cinerea infection byreducin g theperio do flea fwetness . Introduction Bacillusbrevis Nagan o is one of three strains of B. breviswhic h produces a single antibiotic, gramicidin Swhic h since its discovery (Gause and Brazhnikove, 1944) has beenknow nt ob ebactericidal ;onl yrecentl yha si tbee nshow nt ohav efungicida l activity also (Murray et al., 1986). Gramicidin S is active towards Botrytiscinerea Pers.: Fr. (Davis et ai, 1987) which causes grey mould and is a major pathogen of numerous importantcrop s(Jarvis , 1977). Thediseas ei sdifficul t tocontro lbecaus eo fit sabilit yt o attackcrop sa talmos tan ygrowt hstag ean ddevelo pfungicid e resistance(Maude ,1980) . The research described in this paper aimed to test the potential of B. brevis to control B. cinerea. Thiswor kwa sfacilitate d byth eavailabilit yo fpurifie d gramicidin S andth e acquisition ofa nantibiotic-negativ e mutant,B. brevis E-l (Iwakiet al., 1972). Results Antagonism in vitro Tests of antagonism of B. brevis toB. cinerea werecarrie d outi n liquid culture.Flask s (100ml )containin g2 0m lglucos epepton ebrot h(Blakeman , 1975)wer einoculate dwit h conidia or mycelal plugs of B. cinerea and incubated for 5day s in an orbital shaker at 25°C,10 0rp mi nth epresenc eo fgramicidi n S (Sigma)o rspore so fB. brevis (containin g 4.1m Mgra micidi n S/105spores )o rth emutan tE-l . After incubation themyceliu mwa s dried, weighed and theincreas e inbiomas sdetermined . Gramicidin Swa sshow n tob e fungicidal to B. cinerea (Fig. 1). The antibiotic-negative mutant E-l failed to inhibit B.cinerea, suggesting thatth eantagonisti cactivit yo fB. cinerea wa sdu et oth epresenc e of gramicidin S.Germinatio n ofB. cinerea wascompletel y inhibitedb ygramicidi n Sa t 5(xM. 267 Gramicidin S B. b Nagano B.bE-J Germination Growth • --o --O i i i 1 i i i i i i"1 i r—i r—i 1 30 40 50 60 70 80 90 100 Gramicidin S cone. (fjM) or equivalentB. brevis spore cone. (10 6 spores/ml = 4.i/M GS) Fig. 1. Inhibition of germination andmycelial growth ofB. cinerea bygramicidin S and B. brevis in liquid culture. Antagonism in planta Infection droplets containing conidia ofB. cinerealabelle d with Calcofluor M2 were pla^d on Chinese cabbage (Brassica campestris subsp.pekinensis) leaf sections (5x3 cm)fro m leavespreviousl y sprayed untilrun-of f withgramicidi n S,o rspore so fB. brevis or the mutant E-l. The sections were incubated for 5 days at 15°C and 100% r.h. in the dark. After such time the infection droplets were dried at 60"C and observed by fluorescence microscopy (Polyvar, Reichert-Jung; Excitation filter Band Pass 330-380 nm, Emission filter Long Pass 418 nm). The concentrations of gramicidin S and spores required to achieve inhibition of B. cinerea germination (500 \xMgramicidi n S and 2 x 107B. brevis spores.mF) werehighe r on thelea f sections than invitro, with only 65 and 48% inhibition of B. cinerea germination achieved, respectively. Chinesecabbag eseedling s wereals ospraye d withgramicidi n S,o rspore s ofB. brevis orth emutan t E-1,drie d and sprayed thefollowin g daywit hB. cinerea( 10 3conidia.mF) . The seedlings were incubated for 20 days and assessed. The gramicidin S and B. brevis did not control damping-off on Chinese cabbage seedlings caused by B. cinerea. The highest concentrations, 500 jxMgramicidi n San d 2.1 x 107spores.ml-1,wer e phytotoxic to the seedlings. The B. brevis E-l treatment was not phytotoxic, implying that phytotoxicity was due to gramicidin S. Gramicidin Swa s strongly adsorbed to the leaf surface and in this form was inactive. Antagonism in situ A polythene tunnel planted with Chinese cabbage in 24 plots of 24 plants was used in trials during 1990 and 1991.Ther e were six replicates of three treatments positioned in two 3x3 Latin squares plus a row of border plots at either end as guard plants. The 268 Table 1. Controlof grey mould on Chinese cabbage in polythene tunnels by B. brevis and iprodione. Treatment Diseaseinde x Trialno .2 Trialno .3 Untreated 40.3 28.4 Iprodione 13.6(66 ) 6.7 (76) B. brevis 14.2 (65 8.2(71) LSD 9.8 14.7 Thepercentag econtro lo fgre ymoul dwit hrespec tt oth euntreate dcontro lplot si sgive n inparentheses . treatments were as follows: 1) B. brevis; 2) iprodione (1.5 kg/1000 1/haRovra l WP, Embetec) and 3)untreate d control.Th eplant swer espraye d in treatments 2an d 3unti l run-off at2- 3 weekinterval susin ga hand-hel dapplicato rwit ha hollo wcon enozzl ewit h an output of 1.1 l.mùr1 at 3 bar pressure. During the first trial in 1990 there was a prolonged period of warm,dr y weather which resulted in aver y low incidence of grey mould infection and no differences in disease levels between treatments. However, no phytotoxicitywit hth eB. brevis treatmen toccurred .I nth esecon dan dthir dtrial si n1991 , greymoul ddevelope dan da tharves teac hplan twa sassesse dusin g adiseas escal e0-5 , whichdenote d0 = healt yan d5 =sever einfectio n ofth ecabbag eheart .Th etota ldiseas e index for each plot was calculated and used in an analysis of variance. Differences between the treatments were highly significant (p = 0.001). Table 1show s thediseas e indexfo rtreatment sfo rth etw ofield trials .Th eB. brevis treatmen treduce dth eincidenc e ofgre ymoul d toa leve lequivalen tt otha to fIprodione . Leaf wetnessstudie s Duringth etrial si twa snote dtha tB. brevis-ttcatod plot sdrie dquicke rafte rirrigatio ntha n untreated ones. This phenomenon was further investigated in greenhouse studies.Th e periods oflea f wetness onChines ecabbag eleave sspraye d withB. brevis anduntreate d leaves were recorded using automated sensors (Envirolog, Envirolog Measurement Ltd.). The electrical resistance (Ohm) across the leaf surface was measured and correlated toth ewate rpresen to nth elea fsurface ; themor ewate ro nth elea f surface,th e lower the electrical resistance. Fig. 2 shows the duration of leaf wetness of B. brevis- treated and untreated plants, as indicated by the electrical resistance across the leaf surface.Th eB. brevis treatmentreduce dth eperio do flea f wetnessb y7 9% . Discussion Initialdat afro m in vitro work suggested thatantibiosi s wasth emod eo fantagonis mb y B.brevis agains tB. cinerea. However,th eantibioti cwa sles seffectiv e inplanta becaus e itwa sadsorbe dt oth elea f surfacean dwa sinactiv ei nthi sstate .Nevertheless ,biocontro l of B. cinerea with B. brevis was achieved in twotrial s with protected Chinesecabbag e indicating anothermod eo f antagonism. B. cinerea needs high humidity for germination and infection, and epidemics are related to periods of wet, humid weather (Yunis et al, 1990). Practical methods of 269 3- aJ Dry s-yx L"AW _ 101 d f cT ü ö 3- te « 100 - Wet '-'/ L 0 lp Time(h) 20 30 * * T - Control leaf Arrows indicate times of irrigation Sprayed leaf Fig. 2. Leafwetness ofcontrol and Bacillus brevis sprayed leaves. controllingB. cinerea includ eavoidanc eo fhig hhumidit yan dminimisin gperiod so flea f wetness. B. cinereaha s been controlled successfully in environmentally controlled greenhouses (Jarvis,1989) . Iti spostulate d thatB. brevis controlledB. cinerea infection ofth eprotecte d Chinese cabbage by reducing the period of leaf wetness. If confirmed, this treatment may be effective whencombine dwit ha nintegrate dcontro lsystem ,bu twoul dno tb eeffectiv ei f prolonged periodso fhig hhumidit yprevail . Acknowledgment Thiswor kwa ssupporte d byAFR CGran tno .PG1/506 . References Blakeman, J.P., 1975. Germination of Botrytis cinerea conidia in vitroi n relation to conditions on the leaf surface. Transactions of the British Mycological Society 66:305-311. Davis,S.J. , McKay,T. ,Campbell ,D.P .an d Seddon,B. , 1987.Antifunga l activity ofth e peptideantibioti cgramicidi n S(abstract) .Journa lo fApplie dBacteriolog y63:xxiv . Gause,G.F .an dBrazhnikova , M.G., 1944.Gramicidi n San dit s usei n thetreatmen to f infected wounds.Natur e154:703 . Iwaki, M., Shimura, K., Kanda, M., Kaji, E. and Saito, Y., 1972. Some mutants of Bacillusbrevis deficien t in gramicidin S formation. Biochemical and Biophysical Research Communications48:113-118 . Jarvis, W.R., 1977. Botryotinia and Botrytis species: taxonomy physiology and pathogenicity.Monograp hNo . 15,Canadia nDepartmen to fAgriculture ,Ottaw ap .9 - 10. 270 Jarvis,W.R. , 1989.Managin gdisease si ngreenhous ecrops .Plan tDiseas e73:190-194 . Maude,R.B. , 1980. DiseaseControl . InTh ebiolog yo fBotrytis. Eds.Coley-Smith ,J.R. , Verhoeff, K.an dJarvis ,W.R .Academi cPress ,Londo np .275-308 . Murray,T. ,Leighton ,F.C .an d Seddon,B. ,1986 .Inhibitio n offunga l sporegerminatio n by gramicidin San d its potential usea s abiocontro l againstfunga l plantpathogens . Letterso fApplie dMicrobiolog y 3:5-7. Yunis, H.,Elad ,Y andMahrer ,Y , 1990. Effects ofai rtemperatur erelativ ehumidit yan d canopywetnes so ngre ymol do fcucumber si nunheate dgreenhouses .Phytoparasitic a 18:203-215. 271 Integration ofbiologica l andchemica l control for greymoul d Y. Eladand G. Zimand Summary The biocontrol agent Trichoderma harzianum (T39) prepared as a wettable powder (Trichodex) was sprayed in field experiments for the control of grey mould (Botrytis cinerea) was compared with the conventional fungicides vinclozolin, iprodione, diethofencarb + carbendazim and tebuconazole + dichlofluanid in greenhouse-grown cucumbers, tomatoes and strawberry and in vineyards. Effective control was usually achievedb yth ebiocontro lpreparatio nwhe napplie d alonebu ti nstrawberry ,ther ewer e often failures in control. Tank mixes of the biocontrol agent with dicarboximide fungicides were effective for controlling grey mould in all crops. Alternation of Trichodexwit hdicarboximid efungicide s orwit hdiethofencar b +carbendazi m generally resulted in a level of control similar totha t obtained with the standard fungicides. The alternating programme achieved areductio n in fungicides residues in thefruit s andth e surrounding ecosystem, whilst the population of B. cinerea was exposed less to the fungicides andtherefor e lesslikel yt odevelo presistanc et othem . Introduction Moredevelopmen t andcommercia lexploitatio n ofbiologica l control systemsfo r insect pests has occurred than for plant diseases.On eo f thereason s for this isth e availability of cheap and efficient chemical fungicides and their ease of application to foliage. Another problem is that the biotic and abiotic factors in the phylloplane vary and are generallyunfavourabl e for anintroduce d microflora (Elad, 1990). However, a number of foliar diseases, including several caused byBotrytis cinerea Pers.: Fr., can be reduced by pre-inoculation of the phylloplane with epiphytic filamentous fungi, yeasts or bacteria (Blakeman and Fokkema, 1982) and several bacteria and actinomycetes controlled grey mould of lettuce (Wood, 1950). Biological control of grey mould was suggested by Newhook in 1957. He sprayed tomato plants with Cladosporium herbarium and Pénicillium spp. and reduced the incidence of grey mould of fruits. Later, bacteria and Streptomyces spp.wer e reported to be effective in suppressingB. cinerea o nleave so fbeetroot ,onio nan dlettuc e(Clar kan dLorbeer ,1977 ; Pennock-Vos et al., 1990; White et al., 1990). C. herbarium effectively controlled blossom blight and fruit rot of underripe strawberries (Bhatt and Vaughan, 1962). Redmond etal. (1987)controlle d rosegre ymoul dwit hExophialajeanselmei. Satisfactory controlo fB. cinerea usin gisolate so fTrichoderma an dGliocladium spp . on grapes (Dubos and Bulit, 1981;Bisiac h etal, 1985;Gullin o and Garibaldi, 1988), strawberry(Tronsm oan dDennis ,1974 ; Pengan dSutton , 1990)an dappl e(Tronsm oan d Raa, 1977) has been achieved. Combinations of Trichodermaharzianum with 272 vinclozolin at reduced rates were tested for the control of the disease on cyclamen (McCaine tal. , 1985). Material and Methods Wehav e tested apreparatio n ofT. harzianum in greenhouses on tomato (Lycopersicon esculentum), cucumber (Cucumis sativus) and strawberry (Fragaria ananasa) and in vineyards( Vitis viniferea) fo rcontro lo fgre ymould .Th eexperiment swer eperforme d at several sitesi n Israel.Th egreenhouse s werecovere d with0.1 5 mm-thick polyethylene (Infrasol 266,ultraviole t absorbing -UV A- + infrare d repellent- I R- ;Ginnegar ,Israel ) and the soil (sandy loam) was fumigated with methyl bromide (Bromine Compounds, Be'erSheva ,Israel )prio rt oplanting . Eachplo tcontaine d 10t o1 5plant san dguar drow s were used between treated rows. The experiments were laid out in randomised block designwit hfiv ereplicates .Treatment swer egive naccordin gt ocommercia lpractic ean d the disease was evaluated on the fruits or stem nodes of the greenhouse crops and on grapeberries . An isolate of T. harzianum (T-39) previously isolated from a cucumber fruit was grown and formulated byMakhteshi m Chemical WorksLtd . (Be'er Sheva,Israel) .Th e preparation -Trichode x 25W P- wa s applied ata rat eo f 0.2-0.4% (0.5g.b 1 a.i.) unless otherwisespecified .Th efollowin g fungicides wereuse di nth eexperiments :vinclozoli n (Ronilan 50 WP, BASF AG, Ludwigshafen, Germany) 0.5g.H; iprodione (Rovral 50 WP,Rhone-Poulenc ,Lyon ,France )0. 5g.H ;a factory-prepare d mixtureo f diethofencarb (250 g-kg-1) and carbendazim (250 g.kg-1) (Resec WP,Aga n Chemicals Co., Ashdod, Israel),0.2 5 g.Heach; and afactory-prepare d mixture of tebuconazole (100g.kg-> )an d dichlofluanid (400g-kg- 1)(Silvacu r WP,Baye r Ltd., Ludwigshafen, Germany),0.1 5+ 0.6g.H , respectively. Thefungicide s andth ebiocontro lagen twer espraye dt oru nof fwit ha knapsac kmoto r sprayer(Ech oSH R200E ,Kioriz oCorp. ,Japan )a ta volum eo f 1000l.ha- 'usin ga Conje t DX6spra ynozzle .Population s ofT. harzianum werepresen t ata leve l of 3x 105t o8 x 105CF Upe rleaf ,an d 10%o ftha to nfruits , butremaine d highafte r thesecon d andthir d sprays. Calculations were made for average disease reduction compared with non-treated controls.Th e percentage of cases giving positive results wascalculate d three times:i n the first, second and third calculation a reduction of 40, 50 and 60%repesctivel y was regarded aspositive . Results and Discussion Table 1 summarisesth eresult so f7 0experiments . T. harzianum isolateT-3 9prove d tob ea reliabl ebiocontro l agent of cucumbergre y mould.I nmos to fth eexperiment sth epreparatio nsignificantl y decreased theseverit yo f the disease. In some experiments, when the biocontrol preparation was applied along withth edicarboximid efungicide s iprodioneo rvinclozolin ,a slightl ybette refficac y was observed, compared with either agent alone. This may be due to the presence of resistancet othes efungicide s inpopulation s ofB. cinereai nth egreenhouse . The ultimate aim of our work was to reduce the usage of fungicides during the growing season without loss of control; for this reason we alternated T. harzianum applicationwit hfungicides . Aleve l ofcontro lsimila rt otha tobtaine d withth estandar d 273 Table1. Efficacy ofTrichoderma harzianum T39 (formulated as Trichodex)for control ofgrey mould in seventy experiments. Method of control Minimum % for Grape Cucumber Tomato Strawberry seccessful disease reduction 35') 13» 11D 11» Average grey mould 37.0 35.3 26.7 41.8 severity (%) Standard fungicide: 69 35 69 80 % control % Expts. with 402> 94 62 100 100 effective control 50 84 54 90 91 60 66 38 70 91 T. harzianum: - 50 47 61 42 % control % Expts. with 402> 67 69 100 45 effective control 50 54 69 82 36 60 36 31 45 27 T. harzianum + fungicide - 68 45 X» 73 tank mix, % control % Expts. with 402> 88 50 X 100 effective control 50 87 50 X 100 60 62 50 X 100 Alternate sprays of 86 52 70 74 T. harzianum/fungicide: (%) control % Expts. with 402> 100 100 100 100 effective control 50 78 100 100 100 60 67 33 100 100 ') Number of experiments 2) See Material and Methods for calculation of wthresholds considered as positive control.Threshold s were chosen atth e rang e of 40 to 60%diseas e reduction, according to common control achievements obtained in greenhouses under commercial conditions 3) X = not tested fungicide treatments recommended forgre ymoul dcontro lwa s achieved,wit hn o failures in control as occurred with Trichoderma alone. The alternating use of biocontrol agent and fungicides had two beneficial effects; first, there was a reduction in fungicide residues in fruits and the surrounding agro-system. Second, the population of the pathogen was exposed less to the fungicides and were then less likely to develop resistance. Resistance to common fungicides has become widespread in greenhouse populations of B. cinerea (Elad et al, 1992) and consequently the range of chemicals 274 availablefo rcontro lo fgre ymoul di slimited .Simila rresult shav ebee nobtaine di nItal y bysprayin gdicarboximid e+ thira malternatel ywit h Trichodermat ocontro lgre ymoul d intomatoe s andstrawberrie s(Gullin oet ai, 1991). McKenzie et al. (1991) concluded that poor activity often shown by Trichoderma against B. cinerea under severe disease pressure can be explained in part by its poor survival on the phylloplane in the field. The preparation tested in the present study ensured satisfactory survival of an efficient population of the antagonist for 1mont h under commercial greenhouse conditions and a moderate population also became established in the non-treated control plots. This was probably due to secondary distributiono fth efunga l propagulesi nth eai ro fth egreenhouses . Secondarydistributio n mayb eimportan tfo restablishmen to fth ebiocontro lagent ,bu ti nthes etes tcondition si t wasinsufficientl y effective toreduc egre ymoul di nth econtrols . An examination of the relationships between microclimatic conditions and disease control achieved by Trichoderma revealed a negative effect of surface wetness and a positiveeffec t oftemperatur e above20°C . Thisma yexplai nth efailur e ofT. harzianum to control grey mould in Experiment 3. These results showed that it is possible to incorporatebiocontro l ofplan tpathogen s intointegrate dpes tmanagemen tprogrammes . Moreisolate so f Trichoderma whicheffectivel y control thediseas eunde ra wide rrang e of conditions are needed, as the pressure to develop biological controlstrategie s is increasing (Elad, 1990). Meanwhile,contro lca nb eachieve d byus eo fcombination so f thebiocontro l agentan d fungicides. References Bhatt,D.D .an dVaughan ,E.K. , 1962. Preliminary investigation onbiologica lcontro lo f graymol d (Botrytis cinerea) ofstrawberries .Plan tDiseas eReporte r46:342-345 . Bisiach,M. ,Minervini ,G. ,Vercesi ,A .an dZerbetto ,F. , 1985.Si xyear so fexperimenta l trialsi nbiologica lcontro lagains tgrap evin egre ymould . ProceedingsEight hBotryti s Symposium (Alba,Torino ,Italy )Quadem id iViticoltur ae dEcologi adell ' Universita diTorin o9:285-297 . Blakeman,J.P . andFokkema ,N.J. ,1982 .Potentia lfo rbiologica lcontro lo fplan tdisease s onth ephylloplane .Annua lRevie wo fPhytopatholog y20:167-192 . Clark,CA . and Lorbeer, J.W., 1977.Th erol eo f phyllosphere bacteria in pathogenesis byBotrytis squamosa andB. cinerea ononio nleaves .Phytopatholog y67:96-100 . Dubos, B. and Bulit, J., 1981.Filamentou s fungi as biocontrol agents on aerial plant surfaces. InMicrobia l Ecology of the Phylloplane, Blakeman, J.P. (Ed.). Academic Press,London ,p .353-367 . Elad, Y., 1990. Reasons for the delay in development of biological control of foliar pathogens.Phytoparasitic a 18:99-105. Elad, Y., Yunis, H.J. and Katan, T, 1992. Multiple fungicide resistance to benzi- midazoles, dicarboximides and diethofencarb infield isolate s of Botrytis cinerea in Israel.Plan tPatholog y41:(i npress) . Gullino, M.L., Aloi, C, Benzi, D. and Garibaldi, A., 1991.Biologica l and integrated controlo fgre ymoul di nvegetabl ecrops .Petri a 1:149-150. Gullino,M.L .an dGaribaldi ,A. , 1988. Biological andintegrate d control of greymoul d of grapevine: Results in Italy, European and Mediterranean Plant Protection Oganisation Bulletin 18:9-12. McCain,A.H. ,Sciaroni ,R.H. ,Welch ,K.E . andPierce ,L.E. , 1985. Greymol dcontro lo f 275 cyclamen using Trichoderma harzianum Rifai and reduced rates of vinclozolin. University of California, Berkeley,CA .Flowe r andNurser y Reportfo r Commercial Growers. Fallan dWinte rp . 1. McKenzie, L.L., Benzi, D., Dellavalle, D. and Gullino, M.L., 1991. Survival on the phylloplane of strains of Trichoderma spp. antagonistic to Botrytiscinerea. Petria 1:133-134. Newhook,F.J. , 1957.Th e relationship of saprophytic antagonism tocontro l ofBotrytis cinereaPers . on tomatoes. New Zealand Journal of Science and Technology B, 38:473-481. Peng,G . andSutton ,J.C. , 1990. Biologicalmethod st ocontro lgre ymoul do fstrawberry . BrightonCro pProtectio n Conference -Pest s andDisease s3C233-240 . Pennock-Vos,M.G. , Roebroeck,E.J.A .an d Skrzypczak, Cz., 1990.Preliminar y results onbiologica lcontro lof Botrytis cinereai nforce d tulips.Act aHorticultura e226:425 - 428. Redmond, J.C.,Marois , J.J. andMacDonald , J.C., 1987.Biologica l control ofBotrytis cinereao nrose swit hepiphyti cmicroorganisms .Plan tDiseas e71:799-802 . Tronsmo,A . andDennis ,C. , 1974.Th eus eo fTrichoderma specie st ocontro lstrawberr y fruit rots.Netherland s Journalo fPlan tPatholog y83:449-455 . Tronsmo, A. and Raa, J., 1977. Antagonistic action of Trichoderma pseudokoningii against the apple pathogen Botrytis cinerea. Phytopathologische Zeitschrift 89:216- 220. White, J.G., Linfield, CA., Lahdenpera, M.L. and Uoti, J., 1990.Mycosto p - a novel biofungicide based onStreptomyces griseovirides. Brighton CropProtectio n Confer ence- Pes tan dDisease s3C221-226 . Wood, R.K.S., 1950. The control of diseases of lettuce by the use of antagonistic organisms.I .Th econtro lo fBotrytis cinerea Pers .Annal so fApplie dBiolog y38:203 - 216. 276 Control ofBotrytis spp .i n tulip with reduced input of chemical crop protection A.Th.J. Kosterand L.J. van derMeer Summary Foliagean dflowers o fman yflowerbul b speciesca nb einfecte d byBotrytis spp .causin g the so-called 'fire' disease. At present, these diseases arecontrolle d mainly by weekly sprays of the crops with dithiocarbamate compounds, supplemented with a dicar- boximideo rbenzimidazol ecompoun di nthre espray saroun d flowering time. To reduce the input of chemical crop protectants and to avoid the use of ethylene thiourea (ETU)-forming dithiocarbamate compounds (e.g. zineb, maneb, mancozeb), which are expected to be banned in the future, field experiments were Carried out in among others tulips with a number of potentially effective alternative chemicals.W e tested chlorothalonil, chlorothalonil plus prochloraz, fluazinam, and CGA 173506 sprayedi nlo wdosage sonc eever yfortnight . Thesecompound s wereapplie dalon eo ri n acombinatio n withdicarboximid e orbenzimidazol e fungicides. Data obtained indicate thatlow-dosag e applications of chlorothalonil,chlorothaloni l plus prochloraz, or fluazinam in combination with dicarboximide or benzimidazole fungicides atfortnigh t intervals,resulte d ina 70-82 %reductio n of inputs andi ncontro l ofBotrytis spp .a tleas tequa lt otha to fth epresentl yapplie dsprayin gscheme . Introduction Many flowerbulb crops, including tulips, lily and gladiolus, grown in thefield ca n be infected by Botrytis causing a disease called 'fire'. The specialised Botrytis spp., B.tulipae i ntuli p(Beaumon tet ai, 1936;Price , 1970), B.elliptica i nlilie s(Ward ,1888 ; Doss et al, 1988) and B. gladiolorum in gladiolus (Timmermans, 1941) are mainly responsible for the disease. Depending on the conditions, B. cinerea may also infect bulbousplant s(Schönbeck , 1967). In tulip, infected leaves and stems initially show characteristic small, round, dark greenlesions ;thes etur nwhitis hwit ho rwithou tperiphera lwater-soakin go r'aggressive ' spreading lesions develop which cause early senescence with subsequent loss of bulb yield.Petal sar eextremel y susceptiblet oBotrytis infection andmassiv esporulatio nca n generallyb eobserve d onnecroti ctissues . Atpresent , 'fire' disease is controlled bycultivatio n techniques,suc h as removalo f flowers and other plant debris,b y crop rotation and by spraying with fungicides. Over severalyears ,weekl yspray swit hdithiocarbamate s supplemented witha dicarboximid e ora benzimidazol ecompoun di nthre espray saroun dflowerin g timehav ebee n effective (Anonymous, 1980). Dithiocarbamate compounds are expected to be banned in the future, particularly because of the persistent and mobile properties of the metabolite ethylene thiourea (ETU) in sandy soils used for flowerbulb cultivation (Lagas et al, 277 1990), but for the more general need to reduce the input of agrochemicals in current agrosystems. Aresearc h programme was started to find alternative chemicals,t o reduceth e input of chemical crop protectants, to improve spraying techniques and to develop non- chemical control methods and apredictio n system for timing of fungicide application. This paper describes experiments designed to test alternative chemicals that can be appliedeffectivel y inlo wquantities . Material and methods Plantmateria lan dspra yschedule s Bulbs of tulip cv. Renown (circumference 9-10 cm) were harvested, stored at 17°C, dipped before planting in awate r suspension of captan andcarbendazi m (a.i.0.5 %an d 0.2%v/v ,respectively ) andplante d inth efiel d inNovember . Cropswer espraye d with awate rsuspensio n 500l.ha- 1o f mancozeb,chlorothalonil , chlorothalonil/prochloraz,fluazina m orC GA 173506(Ciba-Geig y AG)wit ho rwithou t procymidoneand/o rcarbendazi m inamount sgive ni nTabl e 1. Cropswer espraye dwit h a portable field-plot sprayer (AZO, Ede,Th e Netherlands; 1.2 F nozzle) weekly or at fortnightly intervals from thetim etha tleave so fneighbourin g plantsreache deac hothe r untilplant sbega nt osenesce . Experimental design The experiments were performed in 1989, 1990 and 1991 on various locations in The Netherlandsusin g plotso f 1.5 m2wit h 150plante dbulb seac hi nthre ereplicates ,an da n adjacent sprayedguar drow ,suc htha tth esprayin gare afo reac hplo twa s2. 5m 2.Th etes t plots wereno t inoculated. The percentage of diseased foliage was recorded c. 1 month before harvesting the bulbs, and the weight of harvested bulbs was determined. Bulb yields were expressed relative to yields from untreated control plots which were set at 100.Dat awer eanalyse d using analysis ofvarianc e( p= 0.05) . Results The effects of sprays with various combinations of chemical crop protectants on the infection of tulip plants by B. tulipae in 1990 and 1991 are presented in Table 2.Th e results obtained in 1989 were similar (data now shown). All treatments were effective against B. tulipae. Compared with current weekly sprays of dithiocarbamates around flowering time, applications at fortnightly intervals of chlorothalonil alone were significantly lesseffective . Allothe rtreatment swer eas ,o rmore ,effective . Asexpected , data on bulb yield and on percentage of diseased foliage showed a strong inverse relationship. Noharmfu l effects ofth evariou scombination s ofchemical swa sobserve d intulips . Discussion Combinations of chemicals with different modes of action were more effective than chemicalsapplie dalone .I npractice ,apar tfro m theefficac y ofth efungicides , therisk o f 278 Table 1. Thevarious treatments carriedout to controlB. tulipae infield experiments in 1989,1990and 1991. Treatment Codeuse di n Table2 Untreated A Mancozeb 1.88 kgai.ha- 1,wit hth e B addition oftwic e0.2 5k ga.i.ha- 1 procymidone andonc e0.1 5k ga.i.ha 1 carbendazim, atweekl yinterval s Chlorothalonil,0.7 5k g ai.ha-1, atfortnightl y intervals C Chlorothalonil,0.7 5k gai.ha- 1, with D theadditio n of0.12 5 kga.i.ha- 1 procyimidonean d0.07 5 kg ai.ha-1 carbendazim atfortnightl y intervals Chlorothalonil/prochloraz,0.7 5 and0.2 3 kgai.ha- 1respectivel y E Chlorothalonil/prochloraz,0.7 5an d0.2 3 F kgai.ha^respectively ,wit h thesam e additionsa smentione d underD Fluazinam,0. 5k ga.i-ha- 1 G Fluazinam,0. 5k ga.i.ha- !,wit h thesam e H additionsa smentione d underD CGA 173506,0.5k ga.i.ha- 1 J Botrytis speciesbecomin g resistant andth eimplication s for theenvironmen t and safety ofworkers ,ar eals oimportant . Unlike otherBotrytis species (Pommer and Lorenz, 1983, Presly and Maude, 1986; Lörcher et al., 1987), strains of B. tulipae resistant to dicarboximide and/or benomyl compounds have not been found in the field. To avoid the development of specific fungicide resistance, applications of compounds with various modes of action in alternation or in combination are generally preferred to consecutive applications of a singlecompound . Application of fluazinam orCG A 173506alon e also would be unwise, sincerecen t resultsobtaine d withCG A 173506sho wtha tBotrytis spp.ca nacquir eresistanc et othi s phenylpyrrole compound under laboratory conditions (Hilber, personal communi cations);relevan tdat ao nfluazina m arelackin ga tpresence .Consequently ,chlorothaloni l orfluazina m with additions of chlorothalonil/prochloraz with or without additionsma y 279 Table 2. Effects of combinations offungicides on thecontrol ofB. tulipae in tulip cv. Renown. Treatment 1990 1991 %disease d bulb %disease d bulb foliage yield foliage yield A 80 100 70 100 B 33 118 23 142 C 40 108 33 134 D 27 114 13 150 E — - 23 137 F - — 13 151 G 37 Ill 20 153 H 23 120 13 164 J 23 123 10 150 LSD 15.60 10.96 7.60 7.41 be usedeffectivel y and without seriousrisks o fresistanc e inB. tulipae. Acombinatio n ofCG A 173506an daddition s hasno tye tbee ninclude d inou rexperiments . In comparison with the presently applied standard treatment with dithiocarbamate, considerablereductio ni n thetota lvolum eo fspraye dchemical seac hyea rwer erealise d usingth ealternativ ecombination so ffungicides .Thi sreductio namounte dt o7 0an d82 % for chlorothalonil/prochloraz plus additions and for fluazinam plus additions, respect ively.Moreover ,furthe r release ofET Ufro m tulipcro pspray swa sprevented . Thereductio n of theamoun t offungicide s used andparticularl y thereductio n of the frequency of applicationhel pt oreduc eexposur eo fworker st ofungicide s inth e field. References Anonymus, 1980. Bloembollencultuur36:976-977 . Beaumont, A., Dillon Weston, W.A.R. and Wallace, E.R., 1936.Tuli p fire. Annals of Applied Biology23:57-88 . Doss, R.P., Christian, J.K. and Chastagner, G.A., 1988. Infection of lily leaves from conidia ofBotrytis elliptica. Canadian Journalo fBotan y66:1204-1208 . Lagas,P. ,Va nMaaren , H.L.J., VanZoonen , P.,Baumann , R.A., Heusinkveld, H.A.G., Van der Heeden, W.N. and Koeleman, M., 1990. Report nr. 725803001, RIVM, Bilthoven,Th eNetherlands ,pp . 41. Löcher, F.J., Lorenz, G. and Beetz, K.J., 1987.Resistanc e management strategies for dicarboximide fungicides in grapes:result s of six years' trial work. Crop Protection 6(3):139-147 . Pommer, E.H. and Lorenz, G., 1983. Resistance of B. cinerea to dicarboximide fungicides: aliteratur ereview .Cro pProtectio n 1 :221-230. Presley,A.H .an dMaude ,R.B. ,1986 . Fungicidetoleranc ei nBotrytis squamosa. Report ofth eNationa lVegetabl eResearc h Station,Wellesbourne ,U K 36:62-63. Price,D. , 1970.Tuli pfir ecause db yBotrytis tulipae (lib.)Lind. ;th elea fspottin gphase . Journalo fHorticultura l Science45:233-238 . 280 Schönbeck, F., 1967. Untersuchungen über Blüteninfektionen. V. Untersuchungen an Tulpen.Phytopathologische s Zeitschrift 59:205-224. Timmermans, A.S., 1941. Het Botrytis-totde r gladiolen, veroorzaakt door Botrytis gladiolorum. Laboratorium voor Bloembollenonderzoek, Lisse, The Netherlands. Mededeling nr.6 7pp . 32. Ward,H.M. , 1888. Alil ydisease .Annal so fBotan y2:319-382 . 281 Control ofgre y mould oftomatoe s in greenhouses with fungicides and antagonists N.E. Malathrakis andEJ. Klironomou Summary Tomatoesgrow n in greenhouses in Greecesuffe r severedamag e caused byB. cinerea. Controlo fthi sfungu s isbase d entirely onfungicid e application anddurin g recentyear s strains of the pathogen resistant to fungicides have predominated. To find alternative control methods, the fungi Trichoderma harzianum, Pénicillium sp. and Acremonium alternatumhav e been assessed as potential biocontrol agents in tomatoes. In one experiment where strains of the pathogen resistant to fungicides predominated, the antagonists were applied as suspensions containing 106 spores.mH at 4 and 7 day intervals,an dcompare dwit hthre efungicide s currentlyuse dagains tB. cinerea. Neither the antagonists nor the fungicides were effective. In the second, the antagonists were applied atconcentration s of 106 and 5x 106spores.m H at7 da yintervals ,o r at5 x 10 6 spores.mH alternated with iprodione every third week. In this experiment, the antagonists alternated with iprodione or iprodione alone, were equally effective. The Pénicillium isolate established more readibly on the petals of tomato flowers than T. harzianum, whereas A. alternatum was not recovered from flowers. B. cinerea was isolated more frequently from tomato flowers treated with Pénicillium sp. and A. alternatum atconcentration s concerning 106spores.mH , than incontrols .B. cinerea also was recovered in lower frequency from flowers treated with antagonists plus iprodione,o riprodion ealone . Introduction Duet ofungicid e resistancei nBotrytis cinerea Pers. :Fr .contro lo fgre ymoul di nsevera l greenhouse-grown crops has been difficult. To overcome this several workers have studied the effectiveness of microbial antagonists, combinations of conventional fungicides, and the integration of antagonists with fungicides (Gullino and Garibaldi, 1988; Creemers, 1992). Preliminary experiments which evaluated Pénicillium sp., Trichoderma harzianum andAcremonium alternatum aspotentia l biocontrol agents for B.cinerea in vitro o ro nyoun gbea nplant shav egive npromisin gresult s(Malathraki san d Kritsotaki, 1992). This paper examines the effectiveness of three antagonists, several fungicides andantagonist si nmixe dprogramme swit hiprodione . Material and Methods Themethod san d materials aredescribe d elsewhere(Malathraki s and Kritsotaki, 1992). Twoexperiment swer eperforme d ina plasti cgreenhous efro mearl ySeptembe runti llat e April.Th etreatment s were arranged in arandomize d block design with four replicates. Thetreatment s were applied commencing in midNovembe r when thetemperatur e and 282 Table 1. Effectiveness ofAcremonium alternatum andTrichoderma sp.against tomato greymould. Treatments No. infection sites/plot 15 Feb 13 March T. harzianum lOV-W 47a 75a T. harzianum \Wlà 42 a 60a b A. alternatum PWd 46a 62a b A. alternatum lOWd 34 a 48b Iprodione0.05 %+ dichlofluani d 0.12% 48 a 62a b Iprodione0.05 %+ thira m0.20 % 52 a 60a b Iprodione0.05 % 51a 64a b Dichlofluanid 0.12% 47a 70a b Sumico0.05 % 40 a 62 ab Control 41a 51b Figures followed by the same letter do not differ significantly according to Duncan's MultipleRang eTes t( p= 0.05) . Table 2. Effectivenessof antagonists integratedwith iprodione againstgrey mould %Infecte d fruits No.Infecte d sites per plant perplo t 26Marc h 18 April 18 April Acremonium sp.10 6 8.5 8.0a 44a Acremonium sp.5 x 10 6 9.4 5.2 ab 32b c Acremonium sp.5 x lO6^-iprodion e0.05 % i 1.9 1.6c lid Pénicillium sp.10 6 10.2 5.9 ab 35 abc Pénicillium sp.5 x 10 6 9.5 5.9 ab 33 bc Pénicillium sp.5x1 ^ +iprodion e0.05 % 2.3 1.6c lid Trichodex0.2 % 8.6 3.1 bc 27 c Trichodex0.2 %+ iprodion e0.05 % 2.9 1.7c lid Iprodione0.05 %0. 8 1.0c 6 d Control 10.0 6.2 a 41a b Figures followed by the same letter do not differ significantly according to Duncan's MultipleRang eTes t( p= 0.05) . humidity became conducive to grey mould. In thefirst experimen t A. alternatum and T.harzianum were applied as a suspension containing 106 spores.mH at 4 and 7 day intervals and compared to several fungicides (Table 1). In the second experiment A. alternatum, Pénicillium sp. and Trichodex (a commercial formulation of T. harzianum;Makthesi mCo ,Israel) ,alon eo rintegrate dwit hiprodione ,wer eevaluated . Thefirst tw ofunga l antagonists wereapplie d assuspension scontainin g 106an d5 x 10 6 spores, ml-1. Trichodex was applied at the rate of 2g. H of water.Th e antagonists and iprodione were applied at 7 and 15 day intervals respectively. Treatments in which A. alternatum, Pénicillium sp. and T harzianum were applied at the same rates and 283 alternatedever ythir dwee kwit hiprodione ,wer eals ocompared .Iprodion eapplie dalon e at 15 dayinterval swa suse da sa standar d treatment. The number of infection sites on plants in each plot and percentage infected fruits were recorded. In the first experiment, the severity of theghos t spot symptoms on the fruits was also recorded on a0 t o5 scale .I n thesecon d experiment, thefunga l flora of tomatoflower s wasals omonitored . Senescing flowers werecollecte d from allth eplot s andte nsmal lpiece so feac hwer eplate dseparatel yo npatot odextros eaga r(PDA) ,i nth e presence of antibiotics and incubated at 22°C for 3 days. The fungal colonies were counted and the percentage colonies of each antagonist recovered was calculated. The sensitivity of the population of B. cinereat o iprodione and dichlofluanid was also monitored. Results Inth efirs texperimen tneithe rth efungicide s north eantagonist swer eeffectiv e (Table1) . TheED 50value s for iprodione for sporegerminatio n inB. cinerearange d from 5t o1 1 jjLg.ml"1 throughoutth ework .N osignifican tdifference s occurredbetwee ntreatment sfo r percentageinfecte d fruits orghos tspot ,bu tsom edifference s wereinitiall yfoun d forth e numbero finfectio n sites/plot treated withiprodion e andplot streate dotherwise . In thesecon d experiment iprodione alonea t 15 day intervals,o rintegrate d withan y of the antagonists every third week controlled grey mould (Table 2)effectively . When antagonistswer euse dalone ,T. harzianumwa sslightl yeffective , butPénicillium sp .an d A. alternatum, irrespective of the inoculum concentration, were ineffective (Table 2). Isolates showed that T. harzianum and Pénicillium sp. were established on senescent tomato flowers but A. alternatum was not (Table 3).B, cinerea was isolated regularly from senescenttomat oflowers ,bu tles sfro m plotstreate dwit hiprodion etha nfro m other plots.Th eED 50valu efo r iprodionefo rB. cinerea throughoutth ecroppin g seasonwa s < 2ixg.m H andfo r dichlofluanid <0. 1|xg.mH . Discussion Neither the fungicides nor the antagonists applied alone were consistently effective against grey mould in this study.Th e inadequate performance of thefungicide s can be explained on the basis of the data obtained by resistance monitoring. In the first experiment,th eED 50value so fiprodion et oB. cinerea range dfro m 5 to 11 mg.mHwit h nosignifican t difference between thetreatment s andshowe dclearl y thatB. cinereawa s resistant toiprodione , asdescribe d by Katan (1982) , andn oeffectiv e control could be expected. Similarly, thefailur e ofdichlofluani d inthes eexperiment s mayb e thati nth e pathogenpopulatio nstrain swit hreduce dsensitivit y(ED 50value srangin gfro m0. 7t o2. 7 ixg.mH) prevailed throughout thegrowin g season as described by Malathrakis (1989). Thiswor kprovide sfurthe r evidencetha tdichlofluani d undergreenhous econdition sma y fail to control grey mould when strains with reduced sensitivity are present from the beginning of theseason . Pappas (1992) reported effective control of grey mould under the same conditions, but in this case the resistant isolates represent c. 50% of the population atabou tth emiddl eo fth egrowin gseason . Inth esecon d experiment,a populatio n ofB. cinerea sensitive toiprodion e prevailed 1 throughoutth egrowin g season(ED 50< 0. 1ixg-ml- )an di tcontrolle d greymould . Iprodione integrated with Pénicillium sp.,T. harzianum and A. alternatum applied 284 Table 3. Fungalflora of tomato flowers treated by fungicides andantagonists I n m IV V VI') A. alternatum 106 29 0 19 3 11 1 A. alternatum 5x 10 6 14 0 63 1 4 3 A.alternatum 5 x 10 6 +iprodion e0.05 % 1 0 59 5 37 3 Pénicillium sp.10 6 27 0 97 0 0 1 Pénicillium sp.5 x 10 6 1 0 97 0 0 6 Pénicillium sp.5 x 10 6 +iprodion e0.05 % 3 0 98 3 14 1 Trichodex0.2 % 12 0 10 43 0 7 Trichodex0.2 % +iprodion e0.05 % 16 0 55 35 3 1 Iprodione0.05 %3 0 12 7 1 1 Control(water )1 2 0 16 0 41 4 ') I =Botrytis II =Acremonium sp. III =Pénicillium sp. IV =Trichode x V = Cladosporum VI =Other s every third week was equally effective as iprodione alone at 15 day intervals. The contribution tothi sresul tdu et oth eantagonists ,i sdifficul t to assessbecaus ether ewa s no treatment with iprodione alone applied at 3 week intervals. In our experience iprodioneapplie d at3 wee kinterval si sno tusuall y so effective. Except T. harzianum which was slightly effective, the antagonists alone were ineffective, and this contrasts markedly with results from thelaboratory . Atleas t in the second experiment this could be partly explained by the data obtained from the monitoring of the fungal flora of the flowers. A. alternatum was not established on flowers andthi stissu einitiate sth einfectio n ofth efruits ; henceth efailur e ofthi s fungus tocontro lgre ymould .I nth elaborator yA . alternatumestablishe dsatisfactorel y onyoun g beanplant s andcompete d againstB. cinerea attemperature s aslo w as 10°C.It s failure toestablis h on tomato flowers mayb e due to its low competitiveness to the microbial flora onthi stissue . T. harzianum and Pénicillium sp.wel l established atbot h spore concentrations, but only the former species was effective. T.harzianum applied as Trichodex has been specifically selected andcommerciall y formulated for control ofB. cinerea. Itha sbee n testedan dfoun dt ob eeffectiv e againstgre ymoul db yothers .Pénicillium specie sar epar t of the natural flora of tomato flowers and have been monitored on the plots of all the treatments and regularly from several greenhouses (Malathrakis, unpublished). It was probably present in sufficient densities aspar t of the natural flora in allth etreatments , including the controls. On this basis there was no check without Pénicillium sp. for comparison. In these cases, the role of the natural flora against grey mould can be evaluated onlyi ffungide s effective againstthem ,bu tno tagains tB. cinerea, areuse di n theexperiment . 285 Whatever theprotectio n of thenatura l population ofPénicillium sp.is ,however ,i ti s probable, as Fokkema (1991) claimed, that the application of large amounts of highly effective and competitive strains would deprive the remains of flowers of nutrients, which are considered to be the main food base of B. cinerea and might reduce fruit infection. Hence more research is needed to select such strains from the natural population. References Creemers,P. ,1992 .Th echemica lcontro lof Botrytis cinerea i nfrui t culture.Proceeding s ofth eXt hBotrytis symposium ,Crete ,Greece . Pudoc,Wageningen ,Th eNetherland s p.242-247 . Fokkema, N.J., 1991. The phyllosphere as an ecological neglected milieu: A plant pathologistspoin t ofview . InMicrobia lecolog y ofleaves .Andrew ,J.H .an dHirano , S.S.(Eds) .Springe rVerla gp .3-18 . Gullino,L .an dGaribaldi ,A. , 1988.Biologica l andintegrate d controlo fgre ymoul do f grapevine:Result si nItaly .Europea nan dM editerranea nPlan tProtectio nOganisatio n Bulletin 18:9-12. Katan, R., 1982. Resistance to 3,5-dichlorophenyl-N-cyclic imide ('dicarboximide') fungicides in the grey mould pathogen Botrytiscinerea on protected crops. Plant Pathology 31:133-141. Malathrakis,N.E. , 1989.Resistanc e ofBotrytis cinerea todichlofluani d in greenhouse vegetables.Plan tDiseas e73:138-141 . Malathrakis,N.E .an d Kritsotaki,O. , 1992.Effec t ofsubstrate ,temperatur e and timeo f application on theeffectivenes s of three antagonistic fungi againstBotrytis cinerea. Proceedingso fth eXt hBotrytis symposium,Crete ,Greece .Pudoc ,Wageningen ,Th e Netherlands p. 187-191. Pappas, A. and Elena, K., 1992. Grey mould control where dichlofluanid reduced sensitivity strains of Botrytiscinerea Pers. occur. Proceedings of the Xth Botrytis symposium,Crete ,Greece .Pudoc ,Wageningen ,Th eNetherland s p.252-256 . 286 NAMESAN DADDRESSE S OF PARTICIPANTS AND AUTHORS ABOLGASSANI, A.,Institut eo f Phytopathology and Applied Zoology,Justus-Liebig - University,Justu sLudwigstraß e 23,D-630 0Giessen ,Germany . ALOI,C , Department of Plant Protection, University of Torino,Vi aGiuri a 15,1012 6 Torino,Italy . ANDERS OHMAN, Novo Nordisk A/S, Plant Protection Division, Plant Pathology, NovoAlle ,280 0Bagsvaerd ,Denmark . ANTONAKOU,M. ,Hellafar m Inc.,6 0Agio uKostantino u Str., 10437Athens , Greece. ARTJNOS,J. ,Cib aGeigy ,Hellas ,Amthousa ,Attik i 10010, Greece. AUGER, J.G., Plant Health Department, Faculty of Agricultural and Forest Sciences, University ofChile ,P.O .Bo x 1004,Santiago ,Chile . BAICU,T. ,Researc h Institute for Plant Protection, Bulv.Io n Ionescu del aBra d no.8 , 71592Bucharest ,Romania . BALIS, F.J., Department of Plant Pathology, Cornell University, Ithaca, New York 14853,U.S.A . BARBIER, M., Laboratory of Grape Sciences, Faculty of Science, University of Burgundy,P.O .Bo x 138,21004 ,Dijon ,France . BAR-NUN,N. ,Departmen t ofBotany ,Th eHebre wUniversit y ofJerusalem ,Jerusale m 91904,Israel . BENLIOGLU,S. ,Plan tProtectio nResearc h Institute,P.O . Box49 ,Bagda tCadess i250 , 06172Yenimahalle ,Ankara ,Turkey . ' BENZI,D. ,Departmen t ofPlan tProtection ,Universit y ofTorino ,Vi aGiuri a 15,1012 6 Torino, Italy. BERGMANS, G.J.B., Wageningen Agricultural University, Department of Phyto pathology,P.O .Bo x8025 , 6700A EWageningen ,Th eNetherlands . BESRI, M., HASSAN II Institute of Agronomy and Veterinary Medicine, Rabat, Morocco. BESSIS,R. ,Laborator yo fGrap eSciences ,Facult yo fScience ,Universit y ofBurgundy , P.O.Bo x 138,21004 ,Dijon ,France . BODMER,M. , Swiss Federal Station for Fruit-Growing,Viticultur e and Horticulture, CH-8820Wädenswill ,Switzerland . BORGE, J.F.R., Institute of Phytopathology and Applied Zoology, Justus-Liebig- University,Justu sLudwigstraß e23 ,D-630 0Giessen ,Germany . BOULAY,J. ,LVM HResearch ,Grou pLVMH , LouisVuiton ,Moé tHennes y 48-50Ru e deSeine ,9270 3Colombes ,France . BOURBOS,V.A. ,Subtropica lPlant san dOlive sTree sInstitut eo fChania ,Laborator yo f PlantPathology ,7310 0Chania ,Greece . BRANDES, W., Bayer AG, PF-Zentrum Monheim, PF-E/F, 5900 Leverkussen- Bayerwerk,German y BRINKMANN, R., Agrochemicals Division Development, Fungicides Product Devel opment,D-509 0Leverkusse n Bayerwerk,Germany . BUSCHHAUS, H„ Schering AG, Agrochemical Division, P.O. Box 650311, D-1000 Berlin 65,Germany . CHUDEK, J.A., Department of Chemistry, University of Dundee, Dundee DD14HN , Scotland,Unite d Kingdom. CREEMERS, P., Research Station of Gorsem, Brede Akker 3, 3800 Sint Truiden, Belgium. DELEN, N„ Department of Plant Protection, Agricultural University of Ege, 35100 BornovaIsmir , Turkey 289 DE WAARD, M.A., Wageningen Agricultural University, Department of Phyto pathology,P.O .Bo x8025,670 0A EWageningen ,Th eNetherland s. DIATTA, F., HASSAN II Institute of Agronomy and Veterinary Medicine, Rabat, Morocco. DUBOS,B. ,Nationa l Agricultural Research Institute,Plan tPatholog y Station,B.P . 81, 33883Villenav ed'Orno nCedex ,France . EDWARDS,S.G. , Departmento fAgriculture ,Universit yo fAberdeen ,58 1Kin gStreet , Aberdeen AB9 1UD,Unite dKingdom . ELAD,Y. ,Departmen to fPlan tPathology ,ARO ,Th eVolcan iCenter ,Bet-Daga n50250 , Israel. ELENA,K, .Benak iPhytopathologica l Institute, 14561Kiphissia ,Athens ,Greece . EPTON,H.A.S. ,Departmen t of Cell and Structural Biology,Universit y of Manchester, Schoolo fBiologica lSciences ,Oxfor dRoad ,Mancheste rM1 39PT ,Unite dKingdom . ESTERIO,M.A. ,Plan tHealt hDepartment ,Facult yo fAgricultura lan dFores tSciences , Universityo fChile ,P.O .Bo x 1004,Santiago ,Chile . FARETRA,F. ,Departmen to fPlan tProtection ,Universit yo fBari ,Vi aAmendol a 165/a, 70126Bari ,Italy . FISCHER,B. ,Institut eo f PlantProtection ,Nussalle e9 ,D-530 0Bon nI ,Germany . FOKKEMA, N.J., Research Institute for Plant Protection (IPO-DLO),P.O .Bo x 9060, 6700G WWageningen ,Th eNetherlands . FONTARA,A. ,Departmen t ofAgriculture ,Plan tProtectio n Service,Hippokratou s3-5 , 10164Athens , Greece. FUKAYA,M. ,Tenn oBranch ,Akit aFrui tTre eExperimenta l Station,Tenno , Akita010 - 02,Japan . GARIBALDI, A.,Departmen t of PlantProtection , University of Torino,Vi aGiuri a15 , 10126Torino ,Italy . GEERAERT, H., Ministry of Agriculture, Agricultural Research Center Ghent, Burg. vanGansberghelaa n 96,B-982 0Merelbeke ,Belgium . GEORGOPOULOS,G. ,Lapaphar mInc. ,7 3Menandro u Str., 10437Athens , Greece. GOODAY,W.G. ,Departmen t of Molecular andCel lBiology ,Universit y of Aberdeen, Marischal College,Aberdee nAB 9 IAS,Unite dKingdom . GOODMAN,B.A. , ScottischCro pResearc h Institute,Invergowrie ,Dunde eDD 25DA , Scotland,Unite dKingdom . GORRIS,L. ,Agrotechnologica l ResearchInstitut e(ATO-DLO) ,P.O . Box 17,6700A A Wageningen,Th eNetherlands . COTTER, V.T., American Cyanamid Fungicide Discovery, P.O. Box 400, Princeton, NewYerse y08543-0400 , U.S.A. GRAMMATICAKIS,N. , Intrachem Hellas Ltd., 31 Kiffissias Avenue, 11523Athens , Greece. GRINDLE, M., University of Sheffield, Department of Molecular Biology and Biotechnology,P.O . Box594 ,Firt hCourt ,Wester nBank ,S heffiel d S102UH ,Unite d Kingdom. GULLINO,M.L. ,Departmen t of PlantProtection , University ofTorino ,Vi aGiuri a 15, 10126Torino ,Italy . GÜRER,M. ,Plan tProtectio n ResearchInstitute ,Ankara ,Turkey . HANSEN, H.J., Danish Government Institute of Seed Pathology for Developing Countries,Ryvang s Alle78 ,D K290 0Hellerup ,Denmark . HEALE,J.B. , PlantCel l& Molecula r ScienceResearc hGroup ,Lif e SciencesDivision , 290 King's College (University of London) Kensington Campus,Campde n Hill,Londo n W87AH ,Unite dKingdom . HERBERT,B. , Schering AG,PF-FD ,Mullerstraß e 170-178,D-10 0Berlin ,Germany . HICKS, B.R., The Britisch Sulphur Cooperation Limited, CPM, Editorial Office 6, TorcrossGrov eCalcot ,Reading ,Berk sRG 37AT , United Kingdom. HILBER,U.W. , SwissFedera l Station for Fruit-Growing, Viticulture and Horticulture, CH-8820Wädenswill ,Switzerland . HOANG-VAN,K. ,Universit yo fGeneva ,Laborator yo fGenera lMicrobiology ,Science s in, CH-1211,Genev a4 , Switzerland. ISHn, H.,Frui tTre eResearc h Station,Ministr y of Agriculture, Forestry andFisheries , 2-1Fujimoto ,Tsukuda ,Ibarak i305 ,Japan . JEANDET, Ph., Laboratory of Grape Sciences, Faculty of Science, University of Burgundy,P.O .Bo x 138,21004,Dijon ,France . JDAKLI, H., Faculty of Agriculture, Plant Pathology Department, Bâtiment 47, 13 AvenueMarécha lJuin ,B-503 0Gembloux ,Belgium . JOHNSTON,D.J. , Scottish Crop Research Institute,Invergowrie , Dundee DD2 5DA, United Kingdom. JOUSSAUME,C , Rohman dHaa sEspan aS.A. ,Spai n KALAMARAKI,A. ,Departmen to fAgriculture ,Plan tProtectio nService ,Hippokratou s 3-5,10164Athens ,Greece . KALTSIKES,P. ,Agricultura l University of Athens,Departmen t of Plant Breeding and Biometry,Ier aOdo s75,1185 5Athens ,Greece . KAMOEN,O. ,Ministr y of Agriculture,Agricultura lResearc hCente rGhent ,Burg ,va n Gansberghelaan 96,B-982 0Merelbeke ,Belgium . KAMPP, J., Biotechnolocical Institute, Holbergsvej 10, P.O. Box 818, DK-6000, Kolding,Denmark . KERSSIES,A. ,Researc h Station for Floriculture, Linnaeuslaan 2a, 1431J VAalsmeer , TheNetherlands . KESSEL, G.J.T., Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700G WWageningen ,Th eNetherlands . KETTERER,N. ,Institut eo fPlan tProtection ,Nussalle e9 ,D-530 0Bon nI ,Germany . KIRSHNER, B.,Departmen t ofPlan tPathology , ARO,Th eVolcan iCenter , BetDaga n 50250,Israel . KLIRONOMOU,E.J. ,Plan tProtectio n Institute,Heraklio ,Crete , Greece. KÖHL,J. ,Researc hInstitut efo rPlan tProtectio n (IPO-DLO),P.O . Box9060,670 0G W Wageningen,Th eNetherlands . KOSTER,A.ThJ. ,Bul bResearc hCenter ,P.O .Bo x85,216 0A BLisse ,Th eNetherlands . KOUKOUTOS, D.,Technologica l Education Institute, P.O. Box 140,7111 0Heraklio , Crete,Greece . KOULIZAKIS, M., Hellapharm Inc., 60 Agiou Konstantinou Str., 10437 Athens, Greece. KRDGER, M.C., Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700G WWageningen ,Th eNetherlands . KRITSOTAKI,O. ,Plan tProtectio nInstitute ,Heraklio ,Crete , Greece. KUSTERS-VAN SOMEREN,M. ,Wageninge n Agricultural University, Department of Genetics,Sectio no fMolecula rGenetics ,Dreijenlaa n 2a,670 3H AWageningen ,Th e Netherlands. LARRAIN,P.M. ,Chil eChemicals ,P.O . Box239-9 ,Santiago ,Chile . 291 LATORSE, M.P., Rhone Poulenc Agro, Fungicide Service, 14 Rue P. Baizet, 69009, Lyon,France . LEIFFERT, C, Department of Cell and Structural Biology, University of Manchester, Schoolo fBiologica l Sciences,Mancheste rM1 3 9PT, UnitedKingdom . LE MENN, R., Department of Cell Biology & Electron Microscopy, University Bordeaux1,35 1 Coursd el alibération ,3340 5Talence , France. LEONE,G. , Research Institute for Plant Protection (IPO-DLO), P.O.Bo x 9060,670 0 GWWageningen ,Th eNetherlands . LORBEER,J.W. ,Departmen t ofPlan tPathology ,Cornel lUniversity ,Ithaca ,Ne wYor k 14853,U.S.A. . LORENZ, D., Department for Research and Education in Agriculture, Viticulture and Horticulture, Institute of Phytopathology, LLFA Neustadt, Breitenweg 71, 6730 Neustadta nde rWeinstraße ,Germany . LORENZ,G. ,BAS FAG ,Agricultura lResearc hCente rLimburgerhof ,Car lBoschstraß e 64,W-670 3Limburgerhof ,Germany . LOUWERSE, A., Research Institute for Plant Protection (IPO-DLO), P.O.Bo x 9060, 6700G WWageningen ,Th eNetherlands . MAHRER, Y., Department of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel. MALATHRAKIS, N.E., Technological Education Institute, P.O. Box 140, 71110 Heraklio,Crete , Greece. MANDERS, B.J.C., Wageningen Agricultural University, Department of Genetics, Section of Molecular Genetics, Dreijenlaan 2a, 6703 HA Wageningen, The Netherlands. MARAZAKI, M., Technological Education Institute, P.O. Box 140, 71110 Heraklio, Crete, Greece. MARKELLOU, A., Technological Education Institute, P.O.Bo x 140,7111 0Heraklio , Crete, Greece. MAYER, A.M., The Hebrew Univeristy of Jerusalem, The Institute of Life Science, Department ofBotany ,Giva tRam ,9190 4Jerusalem ,Israel . MOLHOEK, W.M.L., Research Institute for Plant Protection (IPO-DLO), P.O. Box 9060,6700G WWageningen ,Th eNetherlands . MOLLER,I. ,Nov oNordis k A/S,Plan tProtectio nDivision ,Plan tPathology ,Nov oAlle , 2800Bagsvaerd ,Denmark . NGUYEN-THE CH, National Agronomique Research Institute (INRA), Avignon Research Center, Plant Pathology Laboratory, Domaine St. Maurice, P.O. Box91 , 84143 Montfavet Cedex,France . NIELSEN,P.V. , Centerfo r FoodResearc h atth eTechnica lUniversit yo fDenmark ,Th e MycologyGroup ,Departmen to fBiology ,Buildin g 221,Th eTechnica lUniversit yo f Denmark,DK-2800 ,Lyngby ,Denmark . NIELSEN,R. ,Nov oNordis k A/S, 1 Ks33 ,Nov oAlle ,280 0Bagsvaerd , Denmark. NIKLIS, N.D., Aristotle University, Laboratory of Pomology, Thessaloniki, 54006, Greece. 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