Ecology andBiologica l Controlo f
Verticillium dahliae Promotor: prof, drM.J . Jeger hoogleraar in deecologisch e fytopathologie
Co-promotor: drA.J . Termorshuizen universitair docentbi j deleerstoelgroe p biologische bedrijfssystemen Propositions
1. With Arabidopsis thaliana, microsclerotia of Verticillium dahliae can be quantified down to 1microsclerotiu m g'1 soil. {This thesis).
2. Rather than direct detection methods such as plating techniques, bioassays integrate information on multiple soil factors leading to infection by Verticillium dahliae. {This thesis).
3. The dynamics of formation of microsclerotia of Verticillium dahliae in senescent host tissue is the result of a complex interaction between weather, host growth, number of root infections, and host senescence rate. {This thesis).
4. In quantifying microsclerotia of Verticillium dahliae in soil it should be taken into account that the amount of recoverable microsclerotia may change in time. {This thesis).
5. Consistency of biological control can be improved by applying compatible combinations of antagonists. {This thesis).
6. Biological control of plant pathogens will improve the quality of the environment.
7. Absence of above-ground symptoms of Verticillium dahliae does not exclude the presence of a pathogenic interaction below-ground.
8. Educating farmers in farmer's schools improves management of plant diseases and therefore leads to more sustainable agriculture.
9. Wisdom is knowing when to speak your mind and when to mind your speech. When we are filled with pride, we leave no room for wisdom {Our Daily Bread, RBC Ministries, Grand Rapids, Michigan, 1999).
10. We may not complain about so many thorns on roses but we should be thankful for the roses among thorns. 11. Proper education isdecisiv e for the quality of our future generations.
12. Understanding, paying attention, hospitality, and self control are thebasi s for building up good relationships among people having different cultural, religious, and social backgrounds.
Propositions attached toth e thesis: Ecology and Biological Control of Verticillium dahliae by L. Soesanto, defended on 29 ofMarc h 2000. L. Soesanto
Ecology and Biological Control of Verticillium dahliae
Proefschrift terverkrijgin g van degraa dva n doctor op gezagva n derecto r magnificus van Wageningen Universiteit, dr CM. Karssen, inhe topenbaa r teverdedige n opwoensda g 29maar t 2000 desnamiddag st e 16.00uu r ind eAul a van Wageningen Universiteit
J Bibliograpic data
Soesanto,L. ,200 0
Ecology and biological control of Verticillium dahliae PhDThesi s Wageningen University, Wageningen, the Netherlands With references -Wit h summary inEnglish , Dutch, and Indonesian ISBN 90-5808-192-3
Cover design: P.J. Kostense andL . Soesanto
The studies described in this thesis wereperforme d at theLaborator y of Phytopathology and Biological Farming Systems,th eDepartmen t of Crop Science, Wageningen University, Marijkeweg 22,670 9P GWageningen , theNetherlands .
Theresearc h was fully financed byth e Minister of Education and Culture,th e Republic of Indonesia, through the Higher Education Development Project (HEPII ) -AD BLoa n No. 1235- INO.
Printed byPonse n &Looije n B.V. Dedicated to mymothe r and my late father whopasse d away on 17tho fDecembe r 1998, tom ywif e andm ybrother s and sister and their family: Jones,Denny , Eli,Yenny , andAgu s ABSTRACT
Soesanto,L. , 2000.Ecolog y and biological control of Verticillium dahliae.Ph DThesis , Wageningen University, Wageningen, the Netherlands.
The dynamics of Verticillium dahliae, the causal agent of wilt disease in manycrop s including potato,cotton , and olive,wer einvestigated . Itsbiologica l control with Talaromycesflavus wit h or without additional Pseudomonasfluorescens wa s attempted.Arabidopsis thalianawa s selected as abioassa yplan t for studying aspects of ecology andbiologica l control of thepathoge n becauseo f its short life cycle andhig h sensitivity to thepathogen . The optimal temperature for production of microsclerotia,th e survival structures of thepathogen , both invitro an d onA. thalianawa sabou t 20°C. Microsclerotia incorporated in soil were exposed to arang e of conditions of temperature andmoistur e and quantified on several sampling occasions. One day after incorporation, densities were low, andwer eeve n lower overth e following 1-6 months,bu t subsequently densities increased. These changes were ascribed tochang e in the level of soil mycostasis rather than death andne w formation of microsclerotia. After application ofT. flavus to fresh organic debris containing microsclerotia followed bya 3-wee kincubatio n aboveground at 15o r25° C the population density ofT. flavus increased in soil,especiall y at 25°C.T. flavus significantly reduced the density of microsclerotia in soil,especiall y at 25°C,an d delayed thedevelopmen t of senescence ofA .thaliana a t 15an d 25°C.I ti sconclude d that above-ground application of T. flavus may lead to moreconsisten t effects. Theeffec t of P.fluorescens strain P60, originally isolated from atake-al l decline field continuously grown to wheat, onV. dahliaewa s also studied. Strain P60, andtw o otherisolate s of P.fluorescens, inhibited the invitro mycelia l growth of 20isolate s ofV. dahliae,reduce d formation of microsclerotia both invitro an d onA. thalianaan d theyretarde d senescence ofA .thaliana to arat elik e that of uninoculated plants.
Key words:Arabidopsis thaliana, biological control,bioassay , detection, eggplant, microsclerotia, Pseudomonasfluorescens, recovery, relative humidity, Solanum melongena, Talaromycesflavus, temperature, Verticillium dahliae. CONTENTS
Preface 11
Chapter 1. General introduction 13
Chapter 2. Arabidopsisthaliana a sa bioassa y plant for Verticillium dahliae 25
Chapter 3. Effect oftemperatur e on formation ofmicroscleroti a of Verticillium dahliae 37
Chapter 4. Recovery ofmicroscleroti a of Verticillium dahliaefro m soil as subjected to various treatments 49
Chapter 5. Above-ground application of Talaromycesflavus t o control Verticillium dahliae 61
Chapter 6. Control of Verticillium dahliaei nArabidopsis thaliana an d eggplant (Solatium melongena) by combination ofPseudomonas fluorescens and Talaromycesflavus Ti
Chapter 7. General discussion 89
References 95
Summary 107
Samenvatting 111
Ringkasan 115
Author's curriculum vitae 120 PREFACE
Thisthesi s isth eresul t of four yearso finvestigatin g theecolog y ofth eplan t pathogen Verticillium dahliaecarrie d out atth e Laboratory ofPhytopathology , section Ecological Phytopathology, Wageningen University, theNetherlands . First of allI deepl y would liket othan k God for Hismerc y to follow aPh Dprogram , for His guidance and strength duringthi shar dtime , and for Hisblessin g to finish theprogra m and defend mythesis .It' stru etha t "Ica nd o everything through Himwh o givesm e strength" (Philippians4:13). Thisresearc h was initiated in September 1995unde r the supervision ofD rA.J . Termorshuizen, recently moved to the Group ofBiologica l Farming Systems,Departmen t of Plant Science, Wageningen University. Ia mver y grateful for hispersisten t guidance,help , understanding, and support from thebeginnin g upt oth e end ofth eresearch . Aad,than k youver y muchfo r everything youhav edon et oencourag eme . Iwoul d liket othan kProf . DrM.J . Jeger for his efforts to attainth epromotion . Iacknowledg e my gratitude toD rJ.M . Raaijmakers for his encouragement, supervision, motivation, andreadin g the manuscript. Theprojec t was granted by theMinistr y ofEducatio n and Culture,th eRepubli c of Indonesia, through theHighe r Education Development Project (HEP II) -AD B LoanNo . 1235- INO,t owho m Ia m sincerely grateful for the financial support. Ials o extend my sincere gratitude toNuffi c both inJakarta , Indonesia, andi n TheHague ,th eNetherlands , for facilitating my stay inth eNetherlands . I deeply would liket o thank theRecto r ofth eUniversit y ofJendera l Soedirman (UNSOED) andth eDea n ofth eFacult y ofAgriculture ,UNSOED ,Purwokerto ,Indonesia , for theirpermissio n and support to improve my appreciation of sciencea tWageninge n University. I amdetermine d todistribut em y acquired skillst oUNSOED .M ythank s also got om y colleagues atth eDepartmen t ofPlan t Pests andDiseases , theFacult y of Agriculture,UNSOED .Th eDean' s Office for International Students,WAU , arranged andmanage d my accommodation, extension, andtravel .I a mver y grateful for their service. Iwoul d liket othan kProf .D rW . Gams (Centraalbureauvoo r Schimmelcultures,Baarn) , Dr W.J.Blo k (Laboratory ofPhytopathology) , DrJ .A .Hiemstr a (CPRO,Wageningen) , andD r M.P.M.Nagtzaa m (LNV, TheHague ) for useful comments on earlier versions ofth e manuscript. I further thank all themember s ofth e Laboratory of Phytopathology with special thanks toth e section of Ecological Phytopathology: DineVolker , Wout Hoogkamer,Dr s Gerrit Bollen, Dr Gerrie Tuitert, Dr Wim Blok, DrMari oNagtzaam , Jan-Kees Goud, Trudie Coenen, Dr Gitte Schober, Eelco Gilijamse, Herman Frinking,Jorg ed e Souza,Gerar d van Leeuwen, Angelique
11 Lamour,an d Corrie Geerds for theirhel p andnic eco-operation . For someo fth e fellow students, Hans Meerman,Pell aBrinkman , DioneBouchout , Gerben Messelink, Dereje Assefa Abera,an d other students for their kind co-operation during working in apleasan t atmosphere, Iexten d my thanks. Special thanks got o Willem Twijsel and his family for their kind support and companionship. Igiv em y thankst o Drs Gerrit Gort (section ofMathematics ) for his statistical advices.I als o thank themanagemen t ofth e greenhouse and climate chamber facilities, Unifarm, for assistance during my experiments. Wim,Bertus ,Pieter , Rick, Casper, and Rene,man ythanks ! I appreciate the Library ofFYT O (Heleen, Ans) andDuoton e for their service. Ireall y thank you. Iwis h to thank theIndonesia n Student Association ofWageninge n and thewhol e family ofIndonesia n communities, with special thanks got o Mrs.Widjorin i Eveleens and her family, theInternationa l Student Chaplaincy, theInternationa l Christian Fellowship, and my foreign friends, for their hospitality, spiritual support, andkin d co-operation during studying inth e Netherlands.Als o many thanks got o thoseI di d notmentio n here for their ideas and support. Finally, no list of acknowledgements wouldb ecomplete d without an acknowledgement ofver y special gratitude tom ymothe r andm y late father who invested in education but didno t live long enough to seeit sresult ,t o Mrs. SriRahay u andMrs .Ima m Soetorowit h their family, to mydea rwife , andm ybrother s and sister: Jones,Denny , Eli,Yenny , and Agus,wit hthei r family. Very special thanks got o you all for your support, encouragement, and understanding, duringm y study inth eNetherlands .Akhirnya, tiada senerai ucapan terima kasihyang lengkap tanpa ungkapan rasaterima kasih yang sangattulus danhormat kepada ibunda dan almarhum ayahndayang telah membekali kamidengan pendidikan tetapi tidak hidup cukup lama untuk menikmati hasilnya, kepadaibunda SriRahayu danibunda ImamSoetoro beserta keluarga, kepadaistriku tercinta, danadik-adikku: Jones, Denny,Eli, Yenny, danAgus beserta keluarga mereka masing-masing, atasdorongan, semangat, danpengertian yang telah diberikan khususnya selamamasa-masa sukar.
Wageningen, January 2000
12 CHAPTER1
GENERALINTRODUCTIO N
Verticillium dahliaeKleb . is a soilborne pathogen that causes wilt in awid erang e of hosts (Schnathorst, 1981).I t affects manycrops ,th e most economically significant being cotton,potato , and olive.I n addition many othercrop s can be affected seriously, including vegetables (artichoke, eggplant, cauliflower, pepper),fruit s (strawberry, grapevine), fruit trees (avocado,cocoa) ,ston e fruit, fibre and oil seed crops (apricot, kenaf {Hibiscus canabinus),linseed , sunflower), ornamentals (chrysanthemum, dahlia, rose),an d shadetree s (maple, ash).Som e reports on severe outbreaks ofV. dahliaear e listed in Table 1.1.
Table 1.1.Report s of severe yield lossescause db y Verticillium dahliae.
Major hosts Location Yield loss References Almond (Prunus dulcis) Italy I Luisi and Sicoli, 1993;Luis i etal., 1994 Artichoke (Cynara scolymus) Chile 22-70% Fernandez and Tobar, 1989 Italy - Cirulli et al., 1994 Ash (Fraxinus spp.) USA 20-30% Heffer and Regan, 1996 Avocado (Persea spp.) Chile - Latorre and Allende, 1983 Cauliflower (Brassica oleracea var. USA 95% Koike etal, 1994 botrytis) Cocoa (Theobroma cacao) Brazil 85% Resende et at, 1994 Cotton (Gossypium hirsutum) China - Shi etal., 1998 Spain - Bejarano-Alcazar et al., 1996 USA 100% Friebertshauser and DeVay, 1982 Eggplant (Solanum melongena) Japan - Hashimoto, 1989 Horseradish (Armoracia rusticana) USA 75% Eastburn and Chang, 1994 Linseed (Linum usitatissimum) UK >98% Fitter a/., 1991; 1992 Olive (Olea europaea) Greece - Thanassoulopoulos 1993 Spain 25% Bianco-Lopez et al., 1984 Paprika (Capsicum annuum) Israel 22% Tsror etal., 1998 Potato (Solanum tuberosum) Canada - Piatt etal., 1995 USA 50% Rowe etal, 1987 Israel 20-50% Susnoschi etal., 1974 Protea (Leucospermum cordifolium) USA - Koike etal., 1991 Pyramid tree (Lagunaria patersonii) Italy - Polizzi, 1996 Sunflower (Helianthus annuus) UK 25-90% Church and McCartney, 1995 Winter rape (Brassica napus) Germany 44% Daebeler etal., 1988 Precise yield loss not indicated.
13 Anextensiv e host list has been presented byPeg g (1974).Monocotyledonou s plantsar e generally considered non-hosts,wit h the notable exception ofbarle y (Mathre, 1986,1989).Th e pathogen occurs worldwide in the temperate and subtropical zones in all continents,bu t only sporadically in thetropics . Thepathoge n forms highlypersisten t survival structures, the microsclerotia,b y which it maysurviv e extended non-host periods.Give n the wide hostrang eo fV. dahliae,cro p rotation provides onlylimite d protection against thedisease ,a n alternation with monocotyledonous crops such ascereal s yielding thebes tresult s (Bollen etal., 1989).Contro l of verticilliumwil t has largelybee n based on chemical soil disinfestation, but because of the nonselective mode of action andth eenvironmenta l impact, suchtreatment s arebecomin gbanne d byth eauthorities . Currently, alternatives areno t available except for soil solarization in Mediterranean areas (Katan, 1981). Although microsclerotia are generally regarded asth etarge t structures tob econtrolle d through strategies of biological orcultura l control, theirecolog y and dynamics have onlyrarel y been studied in detail.Therefore , factors affecting formation, survival, anddeat h of microsclerotia of V. dahliaear eth e subject of thisthesis . BesidesV. dahliae,ther e are some other plant pathogenic species of Verticillium, i.e., V. albo-atrum Reinke &Berthold ,V. longisporum (C.Stark ) Karapapa, Bainbr. &Heale , andV. tricorpusIsaac .Thei r distinguishing morphological characters areliste d inTabl e 1.2.Verticillium species areknow n to degeneratequickl y after subculturing invitro (Domsc h etal., 1980),s oth e identification should be performed using fresh isolates. V. albo-atrum is known asth ecausa l agent of wilt in mainly hop,tomato , soybean, and alfalfa (Domsch etal., 1980;Atkinson , 1981).I ti sdistinguishe d from V. dahliaeb y the absence of microsclerotia and theformatio n of dark resting mycelium, which has less survival potential than the mycelium ofV. dahliae. Thebasa l cells of conidiophores are usually dark-coloured. In contrast toV. dahliae,V. albo-atrumma ybecom e air-borne through the formation of conidiao n infected shoottissu e (Jimenez-Diaz andMillar , 1988). V.tricorpus i sa wea k pathogen that is ablet oproduc e threetype s of resting structures, i.e., resting mycelium, microsclerotia, and chlamydospores (Heale and Isaac, 1965).Korole v and Katan (1999) mentioned that although V. tricorpuscolonize d theroot s and hypocotyls of seedlings, stems were only rarely infected. Paplomatas etal. (1997 )reporte d preliminary evidence thatV. tricorpusca n be used as abiocontro l agent of Rhizoctonia solanii n cotton seedlings. V.longisporum wasformerl y namedV. dahliaevar . longisporumStar k (Stark, 1961),bu t studies on the morphology, phylogeny, andecolog y revealed the necessity of recognition atth e level of species (Jackson andHeale , 1985; Karapapa etal., 1997). V.longisporum i s recognised
14 •8 -o
•Ii sIif ! S o S -a
o u
^ £ a 60 O jg
in ^ o g>5 w .„ c3 3 Q, -Q O •3! oo s Q *D *T •ff 2 ^ ro (N 23 « _hf_l o XI o o*. * .2 -~f 11 o a J3 *! i .s - « I) 3 Life cycleo f Verticillium dahliaean d disease symptoms The life cycle ofV. dahliaeca n be divided into adormant , parasitic, and saprotrophic stage (Figure 1.1). During the dormant stageth e fungus survives inroundis h toellipsoi d aggregateso f thick-walled, melanized cells called microsclerotia. Melanins aredark-pigment s found inanimals , plants,an d micro-organisms. They areno t essential for growth ordevelopmen t (Polak, 1989),bu t provide protection against infection and,consequently , play an important rolei n the survival. Microsclerotia ofV. dahliaehav e been shown toremai n viablei n soil for moretha n 10year s (Wilhelm, 1955; Powelson andRowe , 1993).Hawk e andLazarovit s (1995)foun d thatnon - melanized microsclerotia of amutan t hadn o persistence. In soil,V. dahliaelack s asaprotrophi c stage.Germinatio n of microsclerotia, being subject to mycostasis (Lockwood, 1977),take s place only under the influence of root exudates in the rhizosphere of host plants.Als o non-hosts havebee n reported toinduc e germination of microsclerotia (Schreiber and Green, 1963; Olsson and Nordbring-Hertz, 1985; Mol etal., 1995) . Mycostasis isprobabl y overcome byroo t exudates in therhizospher e where available carbon is not limited (Lynch andWhipps , 1990).Site s of infection of germinating microsclerotia areth e root tip,th eroo telongatio n zone,o rth epoint so f lateral root emergence. 16 Formation of microscierotia on dying tissue Microsclerotia in soil or on plant residues Germination of microsclerotia Colonization of shoot, stem and leaves Colonization of the cortex and stele Figure 1.1.Lif e cycleo f Verticillium dahliaei n potato (Courtesy DrM.P.M . Nagtzaami n Nagtzaam, 1998). Most of the root cortex infections seemt o affect the plant toa limite d extent andthe y remain superficial and small (Huisman, 1988;Geri k andHuisman , 1988).I n someplants ,roo t infections remain limited in sizeprobabl y as aresul t of aresistanc e reaction byth e host. However, in chrysanthemum considerable damage was observed in infected root cells (Hall and Busch, 1971).Th eendodermi s acts as anatura l barrier that apparently cannot bepenetrate d byV. dahliae, only very few root cortex infections seemt oreac h the vascular tissue.Huisma n (1982) estimated that only 5%o rles s of theroo tcorte x infections ultimately results in infection of the shoot. It has been postulated that vascular infections are successful only where the endodermis hasye tno tdevelope d (i.e.,a tth eroo ttip ) (Bowerset al., 1996),o rwher eth eendodermi s has been damaged, e.g.,b ynematode s (Pegg, 1974;Schnathorst , 1981;MacGuidwi n andRouse , 1990a). After penetration of the endodermis,th efungu s enters the vascular system where it forms conidia from inconspicuous intercalary phialides, alsoterme d budding (Gerik and Huisman, 17 1985;Ferrandino , 1995).Subsequently , theconidi a aretransporte d passively with the sap stream to end walls where theygerminat e andpenetrat e into thenex t vessel segment (Bell, 1992), followed again byproductio n of conidia.V. dahliaesecrete s pectinolytic enzymes that destroyth e middlelamella e of thexyle mparenchym a anddegrad e pectic compounds in the walls of vessels andtracheid s (Pierson etal, 1955;Heal ean dGupta , 1972).Parenchym a cells adjacent toth e infected xylem vessels become discoloured andfille d with gum-like material.I n most host species,xyle m discoloration can be seen byth enake d eye. Infected xylem vessels maybecom e clogged byth e secretion of material byth e parenchyma cells andb ymasse s of fungal hyphae. This,i n combination with production of wilt toxins (e.g., lipopolysaccharides) byth e pathogen, results in reduced respiration, reduced photosynthesis, and, finally, wilting (Orenstein et al, 1989;Mansoor i etal, 1995). Wilt symptoms appear initially atth e lower leaves,where ,typically , first half of an infected leaf shows disease symptoms,bu t ultimately the whole leaf wilts and drops off. In manycases , however, no one-sided wilting of leaves occurs,an dprematur e senescencei s the onlydiseas e symptom. In trees,onl y some branches maysho w wilting and they mayrecove r temporarily or completelyi n subsequent years (Hiemstra, 1998).Sinc e specific disease symptoms are often absent, thediseas e isonl y diagnosed reliably when the pathogen can be isolated from diseased plant material. Moreover, several Fusarium species cause similar disease symptoms. During and after plant senescence the saprotrophic stageo f thefungu s begins, resultingi n thecolonizatio n of the shoots androot s followed bymassiv e production of microsclerotia. Microsclerotia areforme d mostprominentl y in thecorte x of stems, and morerarel y in the leaves (cotton) of herbaceous plants.The y are alsocommonl y formed in petioles of woodyhost s (Rijkers etal, 1992). Epidemiology of verticilliumwilt s Verticillium wilt is considered to bea monocycli c disease, because a single propagule (i.e., a microsclerotium) causes only oneinfectio n within one growing season and new microsclerotia mayb eforme d in the season of infection, but they will not leadt one w infection in the same season. Alinea r relationship between thedensit y of microsclerotia in soil andth enumbe r ofroo t infections per unit of root length has been reported frequently (Huisman and Ashworth, 1976; Ashworth etal, 1979;Nagtzaa m etal, 1997).V. dahliaema yb econsidere d an ecologically obligate vascular pathogen. Ashor t growth through soil to reach aroo t ispossibl y at the expense of the energy contents of themicrosclerotia . Itha s been shown that microsclerotia maygerminat e independently of anexterna l nutrient supply (Ben-Yephet andPinkas , 1977).However , asdescribe d inth e previous paragraph, ina n unsterile soil,V. dahliaei s subject toth ephenomeno n of mycostasis.Lockwoo d (1977) hypothesized that propagules can germinate byconsumin g exudates accumulated on their surface. In an unsterile soil,thes e exudates areremove d byothe r organisms,thu s preventing germination. Mycostasis has been interpreted asecologicall y advantageous for any soil-borneplan t pathogen because in the absence of ahos t their germination hyphae would be lysedreadily . Inth e rhizosphere, however, wherethroug h theexudatio n of nutrients byth eroo t carbon limitation is counteracted (Lynch andWhipps , 1990),microscleroti a ofV. dahliaegerminat e readily (Molan d Van Riesen, 1995).Th eproductio n of new microsclerotia from germinated microsclerotia has alsobee n reported (Farley etal., 1971),bu t this new microsclerotium receives less energy than the original one,a s the uptake of nutrients from the surrounding soil is considered tob e highly unlikely. The energy contents of amicrosclerotiu m are likely tob e sufficient to support hyphal growth over aver y short distance towards aroot . Each singlecel l of amicrosclerotiu m is able to germinate once.Thus ,microscleroti a are able to germinate repeatedly. Alinea rrelationshi p hasbee n observed between densityo f microsclerotia in soil androo t infection ordiseas e incidence (Ashworth etal., 1979;Nagtzaa met al., 1997).However , in several other studiesn o significant correlation was found between microsclerotial density in soil andinfectio n oryiel d loss (Ashworth etal, 1972;DeVa y et al, 1974;Davi s andEverson , 1986;Bejarano-Alcaza r etal., 1995) .Som edispersa l ofV. dahliae through the soili spossibl eb y mesofauna orabioti cfactors . Uneven distribution of the microsclerotia may alsopla y arole .Bejarano-Alcaza r etal. (1999 ) observed infection in all parts of the root systems of eggplants that were inoculated byplacin g about 100 microsclerotia adhering to a9-1 8 mm2-sized tape against theroo t for threedays .The y suggested that production of conidia on microsclerotia and subsequent transport of theseconidi a bywate r flow orth e mesofauna mayexplai n the distribution of infection onth e eggplant root system.Transmissio n of conidia of Coniothyrium minitansi n soil byth emesofaun a hasbee n shown byWilliam s and Whipps (1995).However , for time-spans longertha n afe w weeks,microscleroti a areth eonl y survival structures ofV. dahliaei n soil (Green, 1969). The main mechanisms of dispersal ofV. dahliaear e probably through transport of infested soil byagricultura l tools orb yth e wind,b yusin g infected plant material orb yinfeste d soil adhering to plant material.Row e etal. (1997 ) showed ahig h incidence of infection inpotat o seed tubers. Seed transmission seems tob e animportan t mechanism of dispersal of V. dahliaewhic h 19 has been demonstrated for safflower (Klisiewicz, 1975),sunflowe r (Sackston and Martens, 1959; Richardson, 1990),cotton , eggplant, linseed, soybean, and tomato (Richardson, 1990),an d spinach and beet (Van der Spek, 1972). Genetic variation ofV. dahliae,presenc e of root-infecting nematodes and environmental conditions mayexplai n differences in therelatio n between soil inoculum density and disease incidence among fields. Although V. dahliaeha s long been regarded as afairl y homogeneous species,severa l pathotypes and,mor erecently , four orfiv e vegetative compatibility groups have been recognised (Jeger etal, 1996).Thes e vegetative compatibility groups exhibit some specialization with respect tohos t range,aggressiveness , and distribution. Forexample , vegetative compatibility group4 A is most commonly encountered in solanaceous crops inEurop e andAmeric a (Joaquim andRowe , 1991).O f courseth e virulence of particular isolates of V. dahliaedepend s also on the susceptibility of thehos t cultivar (Zilberstein etal., 1983). Plant-pathogenic nematodes areknow n toenhanc e verticillium wilt severity{e.g., Harrison, 1971;MacGuidwi n and Rouse, 1990ab;Bower s etal, 1996).Th emechanis m of this interaction is not well-understood. Both direct (provision of infection courts forV. dahliaeb y root wounding caused bynematodes ;Ma i and Abawi, 1987) and indirect interactions (increased susceptibility; Faulkner etal, 1970)hav e been suggested. The interaction between verticillium wilt androot-infectin g nematodes is likely to beo f considerable importance, given the wide host ranges and strong survival capabilities of both nematodes andV. dahliae. Moderately high temperatures favour yield loss in potato (Francl etal., 1990).However , supra-optimal temperatures curtail further development ofV. dahliae.I n potato-growing areas disease development ofV. dahliaei s limited toth e springan d autumn, because temperatures become too high in summer (Tsror etal., 1990).Germinatio n of conidia and mycelial growth at temperatures above 31-33°C (Pullman etal., 1981 )an d the development ofV. dahliaei n infected plants stop attemperature s above 27°C (Roweet al, 1987;Koik e etal, 1994).Thi s mayb eth e major reason thatV. dahliaei so f less importance in tropical regions.Excessiv e soil moisturei s known toenhanc e potato early dying in irrigated areas with an aridclimate .Thi s mayb ecause d by afas t transpiration stream (Cook, 1973),althoug h several other factors such aseffect s onroo t growth androo t exudation haveals obee n proposed (Pullman andDeVay , 1982;Gaudreaul t et al, 1995).I n more temperate areas theeffec t of soil moisture is,however , less evident (Bollenet al, 1989;Haverkorte f al, 1990). Yield reduction is mainly theresul t of blockage of the xylem vessels,closur e of the stomata, reduced photosynthesis, andearl y senescence (Haverkort etal, 1990;Bowde n and Rouse, 1991; Schnathorst, 1981).Th e production of toxins byV. dahliaepossibl y also 20 contributes to yield reduction (Healean dGupta , 1972;Orenstei n etal, 1989). New microsclerotia areforme d when infected plant parts senesce. V.dahliae leave sth e xylem,colonize s theparenchymatou s tissues and forms microsclerotia. When plant parts bearing microsclerotia are incorporated into the soil, aggregated microsclerotia areinitiall y held together byth e shoot tissue,bu t gradually, asth e shoot decomposes,th emicroscleroti a fall apart.Thi s is reflected in an apparent increase in soil inoculum density oneo rtw o years after the incorporation of infected plant material (Mol etal, 1996a).Wilhel m (1955)reporte dtha t microsclerotia may survive in soil for 12-14years .Population s of microsclerotia gradually decrease with timedow n to levels where damage to the host isinsignificant . Bollen etal. (1989 )foun d that growing non- hosts for four years was sufficient toeliminat e damage toV. dahliaei npotato .Th eeffect s of environmental conditions on the survival of microsclerotia havebee n studied only rarely. Artificially produced microsclerotia mayhav edifferen t survival patterns from those formed naturally. In unsterile soil,the y survived onlyu p to6 0 days (Lazarovits etal, 1991). Moist conditions resulting from heavyrain s andfloodin g of potato fields were found to induce production of microsclerotia ofV. dahliaeo nth e surface of moribund stemsan d leaves after plant senescence (McKeen andThorpe , 1981; Stapleton etal, 1993).V. dahliae,however , alsoca n grow, sporulate, and form microsclerotia under drycondition s of -100 to -120bar s (approx.p F 5) (Ioannou etal, 1977a).Furthe rLazarovit s etal. (1991 )reporte d that microsclerotia in water-saturated soils lost viability morequickl y (less than 10hours )tha n in soilstha t were air-dryo ra t 50%o f field capacity. Mol and Scholte (1995b) found that covering theplan t remains with soil did not reduce theproductio n of microsclerotia. Root colonization by V. dahliaewa s suppressed consistently bya soil water tension of -0.01MP a (pF2 ) (Gaudreault et al, 1995).Th efinding s of Ioannou etal. (1977a ) andPowelso n andRow e (1993)tha tearly - season irrigation can be an effective component of integrated disease management support this. Management of verticillium wilt disease Manynon-hosts , including various monocotyledonous crops,ca n be symptomless carriers ofV . dahliae.Attempt s have been made toreduc e inoculum densities ofV. dahliaeb y inducing germination of microsclerotia bynon-hosts .However , new microsclerotia wereproduce d inth e roots of non-hosts,resultin g in insufficient control ofV. dahliae(Mo l etal, 1996a). Breeding for resistance ortoleranc e hasbee n successful for only afe w crops. Commercial resistant or tolerant crops areonl y available in tomato and cotton production (Allen, 1994) andi n 21 sunflower (Miller andGulya , 1995).Toleranc ei s availablei n other crops such aspotato ,bu t presently the choiceo f potatocultivar s isdominate d byth erequirement s of the processing industry andpreference s ofth econsumers . Croprotatio n is commonly recommended asa metho d to control V. dahliae.Croppin g non-hosts for four years ormor e markedly reduces populations ofV. dahliae(Bolle n etal, 1989). However, given the widehos t rangeo fV. dahliae,non-host s mayno t be aneconomi c alternative to the farmer. Moreover, crops such ascotto n andpotat o aregrow n continuously in variousareas . Under these circumstances soil steaming orfumigatio n has been applied.Thes e methods, however, become more and morerestricte d due tohig h costs,healt h risk for workers, and adverse effects onth e environment. Soil solarization provides control in areas with aMediterranea n climate (Katan, 1980). Soil inundation mayals ob eeffectiv e provided that low oxygen levels develop and temperatures areno t too low (Ioannou etal., 1977b).I nth e Netherlands, Blok etal. (2000 ) obtained atleas t 90%reductio n of inoculum density ofV. dahliaeb yinducin g anoxic conditions through the incorporation of fresh organic matter and overnight irrigation followed byth eplacemen t ofa plastic tarp.I n addition toth e direct effect on the fungus, nematodes stimulating verticillium wilt wereals ocontrolled . Conn andLazarovit s (1998)wer eabl et oreduc everticilliu m byth e application of chicken manure. Other organic amendments that showed an effect onV. dahliae arealfalf a and oat residues (Green andPapavizas , 1968),chiti n (Jordan etal, 1972)o rbarle y (Harrison, 1976).Davi s etal. (1996 )conclude d that green manure treatments with sudangrass or maize gavebes t control of verticillium wilt. Koike etal. (1997 ) and Subbarao etal. (1999 ) demonstrated theeffectivenes s of broccoli residues tocontro l verticillium wilt in cauliflower. The removal of debriscontainin g microsclerotia seems alogica l approach tocontro l V. dahliaebut , accordingt oMo l etal. (1995) ,thi streatmen t wasinsufficien t to allow narrow rotations ofpotato . Soils that are suppressive toV. dahliaear ebarel y reported. Ashwort h etal. (1976 ) showed that Cu2+ wasinvolve d in the suppression ofV. dahliae. Thiseffec t was noticed when no microsclerotia wererecovere d from soils which previously had contained high densities of them. Further data on this phenomenon haveno t been reported. Keinath and Fravel (1992) were ablet o induce suppressiveness in thegreenhous e byrepeatedl y cropping potato on the same soil.Th e induction of this suppressiveness varied with soil type,bu t itsnatur e was not further investigated. Thephenomeno n ofdiseas e suppression, which is understood in several other soilborne pathosystems (e.g., Baker and Cook, 1974;Oyarzu n etal., 1997, 1998)i s still not explained for verticillium. 22 Thepotentia l of the application of biocontrol agents tocontro l verticillium wilt isno w being studied intensively. About 25potentia l antagonists have been tested. In most, if not all, cases,thes e antagonists havebee n selected from studies on other sclerotial parasites.Th e studyo f parasites of microsclerotiai shampere d bythei r small size.Recentl y investigated antagonists include Trichoderma(Gliocladium) virens(Johnsto n etal, 1994),Trichoderma koningii (Georgieva, 1992)an dothe r Trichoderma species,Bacillus subtilis, Pseudomonas fluorescens, and Streptomycesflavofungini (Ber gan dBallin , 1994),Pythium oligandrum (Al-Rawah i and Hancock, 1998)and ,mos t notably, Talaromycesflavus (Maroi s etal, 1982;Fravel , 1996).T. flavus isabl et o parasitize microsclerotia ofV. dahliae(Fahim a etah, 1992)an d sclerotiao f several otherpathogen s such asSclerotinia sclerotiorum (McLaren etal, 1986), Rhizoctonia solani(Boosalis , 1956),an dSclerotium rolfsii(Mad iet al, 1992).Furthermore ,T. flavus i sabl e toproduc e enzymes such asglucos e oxidase that generatestoxi c peroxide,chitinase ,glucanase , andcellulase .Mad i etal. (1997 ) showed that culture filtrates ofT. flavus affecte d germination andmelani n formation of microsclerotia. McLaren etal. (1986 )reporte dpartia ldisintegratio no f melanized material of sclerotia of S. sclerotiorum that wereparasitise d byT. flavus. Althoug h T. flavus isregarde d as aroo t colonizer, the factors affecting thepopulatio n dynamics in the rhizosphere areinsufficientl y understood (Fahima andHenis , 1995; Nagtzaam andBollen , 1997). RecentlyT. flavus ha sbee n registered as abiocontro l agent byProphyt a (Rostock, Germany) for the control in oil-seed rape and tomato.Bot h root dipping of young plants ormixin g of the suspended product is advised (Zeise, 1997;Koch , 1999).S ofar , application ofT. flavus t oth e soil has yielded inconsistent results,probabl y because of competition with the resident microflora. About this thesis The main goal of the present study was togathe r fundamental ecological knowledge on the formation and survival of microsclerotia. Detailed quantitative information about factors influencing theseprocesse s may leadt one w prospects for controllingV. dahliae.A secondar y goal wast ofurthe r improve biological control of verticillium wilt.W ehypothesize d that the above-ground application of thebiocontro l agent Talaromycesflavus ont o microsclerotia- containing debris would lead toreductio n of inoculum density ofV. dahliae.I n addition, the effects of adiacetylphloroglucinol-producin g strain of Pseudomonasfluorescens o n verticillium wilt was studied. 23 InChapte r 2a bioassa y ispresente d for thequantitativ e studyo f the formation of microsclerotia andfo r evaluating theeffect s of biocontrol agents usingArabidopsis thalianaa sa test plant.A. thalianawa schose n because of its short life cycle and its sensitivity toman y plant pathogens.Man y other bioassay plantsrequir e either labour-intensive methods,involvin g the plating of root or shoot fragments todetec t orquantif y V. dahliae,o rthe ynee d alon g period before disease symptoms develop, ord ono t allow to detect low population levels of, e.g.,1 microsclerotiumpe rgramm e soil,tha t may still damage asusceptibl ecrop . In Chapter 3th eeffect s of constant and variable temperatures on the formation of microsclerotia onA. thalianaar e assessed. In thefiel d alarg evariatio n in densityo f microsclerotia iscommonl y observed in senescent host tissue.W e studied whether temperature affects theproces s of microsclerotium formation, either directly"o rindirectl y through plant senescence. In Chapter4 factors affecting thedensit y of microsclerotia distributed in unsterile soil are investigated. Factors studied include temperature, moisture, anddifferen t methodso f incorporation of organic debris. Chapter 5studie s effects of various methods of application of the antagonist T.flavus on survival of thepathoge n on potato stems covered with microsclerotia ofV. dahliae. In Chapter 6th eeffect s of a2,4-diacetylphloroglucinol-producin g strain of P.fluorescens o n verticillium wilti nA. thalianaan d eggplant, with or without in combination withT. flavus are reported. Finally, in Chapter 7topic s of bioassays, formation and survival of microsclerotia, and biocontrol are discussed in amor e general context. 24 CHAPTER 2 ARABIDOPSISTHAUANA ASA BIOASSA Y PLANT FOR VERTICILLIUMDAHUAE Summary Arabidopsisthaliana wa s used asa bioassa yplan t todetec t low inoculum levelso f Verticillium dahliaei n soil. Although inth efiel d crucifers arepredominantl y infected byV. longisporum, underth etes tcondition sA. thalianabecam ereadil yinfecte d byfou r isolates ofV. dahliae. Disease symptoms included early senescence,bu tn o typical verticillium wilt. Increasing amounts of infection ofbot h root and shoot werenote d atdensitie s of 1,3,10 , 30,an d 100 microsclerotia g"1soil .A log-linea rrelatio n between inoculum density andth e area-under-the-disease-progress curve wasestablished . Even atth elowes t inoculum density of 1 microsclerotium g"1soil , 5% of theroo t length wasinfecte d withV. dahliae,an d30 %o f thestems .Th emos t sensitive parameter was thedensit y of new microsclerotia formed in the shoot.Th ehighe r theinoculu m densityth e greaterth e density of microsclerotia in the shoot, indicating that multiple root infections leadt oa more intense colonization ofV. dahliae. Disease progress was fastest at 20°C, and slower at 10, 15,an d 25°C.A bioassa y withA. thalianafo r assessing infestation of fields with V.dahliae can be recommended. Introduction Verticillium dahliaeKleb .cause sconsiderabl e economic losses through wilting and early senescence in diverse crops such aspotato ,cotton , rose, strawberry, and maple (Schnathorst, 1981).Thi sroot-infectin g pathogen forms abundant survival structures, microsclerotia, in diseased, senescent tissue.Give n the widehos t range ofV. dahliae, crop rotation providesonl y limited protection against thedisease , an alternation with monocotyledonous crops such as cereals giving thebes t results (Bollen etal., 1989).Contro l ofV. dahliaeha s largely been based on the application of chemical soil disinfestants, but because of their impact on the soil 25 ecosystem, thegroundwate r quality, andth econtributio n toth edepletio n of theozonospher eb y methyl bromide,thes emethod s arebecomin g unacceptable. For the development andevaluatio n of new management strategies againstV. dahliae, assays areneede d that, rapidly andwit h limited labour input, provide information about the density of V. dahliaemicroscleroti a in soil toevaluat e thenee d for control orcontro l efficacy. Changes in the soil inoculum density ofV. dahliaear eofte n assessed byplatin g asoi l suspension onto asemi-selectiv e agarmedium , but these determinations are not always reliable (Termorshuizen etal., 1998).Moreover , theyexclud e effects of competition in the rhizosphere ando f induced resistance.Therefore , bioassays havebee n recommended to study theecolog yo f V. dahliae(e.g., Evanset al., 1974),fo r selection of resistance toth epathoge n (Palloix etal., 1990)an d for theevaluatio n of biocontrol agents {e.g., Nagtzaam etal., 1998).However , bioassays sofa r available areeithe r labour-intensive, involving plating of root or shoot fragments to detect orquantif y V. dahliae,o rdiseas e symptoms take alon gtim et o develop. Moreover, presently available bioassays areno t able to detect low population levels of e.g. 1 microsclerotium pergramm e soil that may stilldamag e asusceptibl e crop (Nicot andRouse , 1987). Arabidopsisthaliana (L. )Heyhn .havin g anunusuall y small genome among greenplant s hasbecom e aguine ypi gfo r studies on geneexpression . In preliminary experiments, atthi s institute,A. thalianawa sfoun d tob e sensitive toV. dahliae.W etherefor e decided totr yit s use asa tes t plant inbioassay s forV. dahliae.A. thalianai suse d in many studies, with fungi (Koch and Slusarenko, 1990),plant-pathogeni c bacteria (Whalen etal., 1991;Tsuj i and Somerville, 1992),nematode s (Sijmons etal., 1991),an d viruses (Sosnova and Polak, 1975),i n part because of itsshor t life cycle.Cruciferou s crops such asoi l seed rape are long known tob e preferentially infected byheterodiploi d strainso fV. dahliaeformerl y known asV. dahliaevar .longisporum, but recently this taxon was elevated to species level (Karapapa etal., 1997).Tabret t etal. (1995) , however, showed that the haploidV. dahliaeals o was able toinfec t the crucifer A. thaliana. Material and methods Fungalcollections, maintenance, andinoculum preparation Fourcollection s ofV. dahliae,K 3 and VI, V3,an d V4,wer e obtained in summer 1996 and 1997, respectively, from diseased potato stemscontainin g microsclerotia in the municipalities of Veenhuizen, Nieuweroord, Nieuw-Balinge, andBruntinge , respectively, in theProvinc eo f 26 Drenthe,th e Netherlands.Th epotat o stems werecu t into 1-cmpieces ,allowe d to airdr y for about ten days atroo mtemperature , and stored in plastic boxes until further use. To separate the microsclerotiafro m potato stem tissue the material was ground and sieved over screens with mesh sizes of 106an d2 0 um.Th eresidu eremainin g on the 20u m sieve was suspended in water containing 0.08% agar.Th e agarwa s added toobtai n ahomogeneou s suspension of microsclerotia. Thenumbe r of microsclerotia in the suspension was determined in small subsamples using adissectin g microscope at 25x magnification. To determine the germinabilityo f microsclerotia, 25microscleroti a wereplate d individually in Petri dishes containing Modified SoilExtrac t Agar (MSEA)mediu m (Harris etal., 1993)b yusin g an insect pin00 0 (Emil Arlt,Australia) .Th ePetr i dishes were incubated upside down in thedar k for 14 daysa t 23°C,an dth e number of germinated microsclerotia was counted. Microsclerotia from collections VI, V3,an dV 4wer e surface-disinfested in 1% NaCIO for 1 min,washe d three times in sterile water each for 1 min,blotte d dryo n sterile filter paper, andplate d onto Potato-Dextrose Agar (Oxoid) amended with 50pp moxytetracyclin . From this culture monoconidial isolates wereprepared .Thi s culture waspropagate d bygrowin g in Czapek- Dox liquid medium (Oxoid)fo r 7day s at23° C in ashakin g incubator (Gallenkamp Orbital Incubator). After incubation, conidia were filtered through cheese cloth and suspended in sterile distilled water to obtain 1.0 x 106conidi a ml"1 suspension. Plantmaterial andgrowth conditions A. thalianaecotyp eColumbi a wasuse di n this study. Apreliminar yexperimen t didno t indicate significant differences in colonization with three otherecotype s ofA .thaliana, i.e., Cap e Verde Islands,Landsber g erecta,an d Wassilewskaija. Seeds ofA .thaliana wer eprovide d byProf .D r M.Koornnee f (Laboratory of Genetics, Wageningen University). Seeds were spread over sieved and wetted potting soil in aplasti c box covered with aca p in ordert omaintai n high humidity. Theboxe swer eincubate d in aclimat echambe r with 16h ligh t (PhilipsTD32W/8 4HF ) and 8 h dark at20°C . Todetermin e infection byV. dahliae, A. thalianawa sharveste d after itsrosett e had completely died. Roots were washed on asiev e by spraying tap water toremov e organic matter. Stem segments were surface-sterilized in 1%NaCI Ofo r 30 sec,roo t segments in 96%ethano l for 1 sec,the n washed twicei n sterile water,drie do n sterilefilte r paper, plated separately inPetr i dishes containing MSEA and incubated at25° C inth e dark for 14days . 27 Disease assessment Senescence of rosette leaves and stems (including stems,ste m leaves,flowers an d siliquae)wa s evaluated separately byassessin g senescence indices (SI)rangin g from 0 to 5i n weeklyintervals . Forth erosett e leaves:0 =0-5 %o f therosett e leaves showing somenecrosis ; 1 =6-15% ;2 = 16- 25%; 3= 26-50% ;4 = 51-75%;an d 5= 76-100% .Fo r the stems:0 =n o necrosis; 1 = stems still green but 1-5% of leaves showing necrosis; 2= 1-5% of the stembrownis h or yellowish, but 6- 25% of theleave s showing necrosis, and 1-5% of pods and/orflower s yellowish orbrownish ; 3= 6-25% of the stems brownish oryellowish , 26-75%o f the leaves showing necrosis, and 6-25%o f the podsbrownish ; 4 =26-50 %o f the stems brown, 26-100% of the leaves showing necrosis,26 - 50%o f thepod s brown and 1-75% of theflower s yellow orbrown ; and 5= mos t of all stems, leaves,flowers , andpod s show necrosis.Th e disease index (DI)o f verticillium-inoculated plants wasdefine d as SIafte r subtraction of the values of SIdetermine d for thecontro l plants.Usin g theseindices ,th e area-under-the-senescence-progress curve (AUSPC) and the area-under-the- disease-progress curve (AUDPC) werecalculate d (Campbell andMadden , 1990). Production of microsclerotia ofV. dahliaewa s quantified after A.thaliana ha d completely died. Shoots androot swer eseparate d andth eroot swer ewashe db ygentl ysoakin g in standing tapwater . All parts of the plant were then dried atroo m temperature for aweek . Both dried shoots androot s ofA . thalianawer e weighed and ground using amorta r andpestl e and suspended in 0.08% water agar.Th e suspension was weighed and homogenized. The densityo f microsclerotia in the suspension was determined bydirec t counting of at least three small subsamples under the dissecting microscope at 16x magnification measuring approximately 20 ul Experiments Effect of inoculumdensit y ondiseas edevelopment .Th eexperimen t wascarrie d out asa completely randomized block design using densities of 0,1, 3,10, 30,an d 100microscleroti ag" 1 dry soil and was performed with 10replication s pertreatment . Plastic pots (w/w/h, 7/7/8 cm) wereuse d containing mixed sieved potting soil and sand (2/1, v/v).Four-week-ol d seedlings ofA . thalianawit h the first rosette leaf emerging wereplante d andplace d in aclimat e chamber at2 5° C under a 16/8h light/dark regime.A repeate d experiment showed similar results, and onlythos eo f thefirs t experiment arepresente d here. Effects of temperature. The optimal bioassay temperature and the pathogenicity of three isolates ofV. dahliaewer e determined in aroot-di p experiment with isolates VI, V3,an d V4.Four-week - 28 old seedlings ofA .thaliana wer euproote d carefully andth eroot swer ewashe d in steriledistille d water, dipped for 20mi n in aconidia l suspension ofV. dahliae(10 6conidi a ml"1),an d plantedi n pots with mixed potting soil and sand asdescribe d above.Th epot s were placed at 10,15,20, and 25°C, under the same light regime. Results Disease symptoms became visible asearl y as4 weeks after planting the seedlings ofA . thaliana in infested soil or dipping the seedlings in aconidia l suspension. Symptoms consistedo f chlorosis followed bynecrosis , sometimes accompanied by some stunting.Th etypica l unilateral wilting of leaves orplants ,ofte n described for verticilliumwil t was not observed. Nine weeks after planting,mos t rosette leaves werecompletel y dead and 11week s after planting the stems hadnearl y orcompletel y died.Durin gth e final stage of senescence,microscleroti awer e formed abundantly, especially onth e stems,bu t many were also observed on allothe r plant parts, including theroo t system.Th edr yweigh t of the shoot was not significantly affected byV. dahliae. Natural senescenceprogresse d slowlya tlo wtemperatures . Senescence duet oV. dahliae wasmos t stronglyenhance d at 20°Cbu t less at 15an d25° C (Table 2.1). Senescence began earlier andprogresse d faster atincreasin g densities of microsclerotia in soil (Figure 2.1). Since senescence isa rapi d natural process inA .thaliana al l plants eventually became senescent; thus differences in senescence between treatments tend to converge atth een d Table 2.1. The time (days)i t takes for the plants to die of seedlings that were root-dip infected with three isolates of Verticillium dahliae(10 6cf u ml"1suspension ) to uninfected control plants. Isolate of Verticillium dahliae VI V3 V4 I Temperature (°C) Life span ofArabidopsis thaliana relative toth econtro l 10 0.81 0.86 0.82 15 0.82 0.80 0.79 20 0.66 0.59 0.66 25 0.76 0.71 0.71 'Control plants had life spans of 293, 247, 178,an d 99day sfo r incubation temperatures of 10, 15,20 ,an d 25°C respectively. 29 a2 MICROSCLEROTIA MICROSCLEROTIA z 9 G''SOI L G' SOIL sin in 5 -*-10 -*-10 -*-30 -*-30 -•-100 -•-100 4 5 6 7 8 9 10 11 4 5 6 7 8 9 10 11 WEEKSAFTE RTRANSPLANTIN G WEEKSAFTE RTRANSPLANTIN G Figure 2.1. Senescence progress for rosette leaves and stems ofArabidopsis thaliana a t different inoculum densities of Verticillium dahliae.(A ) Senescence index, (B)Diseas e index = senescence index minus the senescence index estimated for thecontro l plants. MICROSCLEROTIA MICROSCLEROTIA G'1 SOIL G"' SOIL -*-10 -X-10 -*-30 -•-100 -*-30 -•-100 5 6 7 8 9 10 11 5 6 7 8 9 10 11 WEEKSAFTE RTRANSPLANTIN G WEEKSAFTE RTRANSPLANTIN G Figure 2.2.Senescenc e progress for rosette leaves and stems ofArabidopsis thalianaa t different inoculum densities of Verticillium dahliae.(A )Area-under-the-senescence-progres s curve (AUSPC),(B )Area-under-the-disease-progres s curve (AUDPC) =AUSP C minusAUSP C estimated for the control plants. 30 • rosette,repetitio n1 AUSPC= 5.7(±0.7)log(ID+1)+6.5(±0.6) • rosette,repetitio n2 AUSPC= 6.0(±0.5)log(ID+1)+7.2(±0.6) • stem,repetitio n1 AUSPC= 6.3(±0.6)log(ID+1)+8.5(±0.6) xstem ,repetitio n2 AUSPC= 7.4(±0.6)log(ID+1)+8.3(±0.7) 100 LOG (MICROSCLEROTIA G'1 SOIL) Figure 2.3. Regression lines for the area-under-the-senescence-progress curve (AUSPC) asa function of the transformed (10log)soi l inoculum density. of the life cycle ofA .thaliana (Figur e 2.1).Th e disease index {i.e., the senescence index scored for the inoculated seedlings minus the senescence index scored for the non-inoculated seedlings) was maximal 6-8 weeks after transplanting the seedlings into infested soil for therosett e leaves, and6- 9 weeks for the stems (Figure 2.1).Diseas e indices for therosett e leaves generally gaveles s variable results than those for the stems.I n several cases disease indices decreased temporarily for the estimates based on the stems,perhap s indicating that compensation growth had occurred. Thearea-under-the-senescence-progres s curves steadilyincrease d and showed the largest differences at the final observation date (Figure 2.2).Th e area-under-the-disease-progress curves showed few differences with the area-under-the-senescence-progress curves (Figure 2.2). At the latest observation date (9an d 11wee k after transplanting for the rosette leaves and stems respectively), alog-linea r relationship existed between inoculum density and the area-under-the- senescence-progress curve (Figure 2.3),wit h adjusted-R2 values closet o0.70 . Atharvest , 11 weeks after transplanting, plating of the stem bases of seedlings that hadbee n inoculated at1 microsclerotium g"1soi l resulted in 30%incidenc e ofV. dahliae(Figur e 2.4).Individua l colonies growing out from the roots could not becounte d because most root infections appeared tob e systemic.Therefore , the percentage of root length covered withV. dahliaeafte r 14day so f incubation on MSEA wasdetermined , resulting in 5%roo t infection atth e lowest inoculum level. Infection gradually increased with increasing inoculum levels,leadin g up to 100fo r stem and 70% for root infection, at 100microscleroti a g"1soi l (Figure 2.4). 31 100 w tit i o LU uoj z O UJ O o o 0 1 3 10 30 100 MICROSCLEROTIA /G DRY SOIL STEM ROOT Figure2.4 .Incidenc e of Verticillium dahliaei nth este mbas e andpercentag e ofroo t length infected ofcompletel y senescentArabidopsis thalianaa sreveale d byplatin g in relation to soil inoculum density. Thenumber s ofmicroscleroti a g"1 dry shoot tissuewer ecorrelate d with the inoculum density ofV. dahliae.Whil e at 1 microsclerotia g"1 soil 1.6 xlO3± 9.0 xlO2microscleroti a g"1 dry shoottissu ewa s found, at 100microscleroti a g"1 soil an 18-fold increasewa s observed (Figure 2.5).Irrespectiv e ofth e inoculum level,incidenc e ofmicroscleroti a inth e shootswa s 100%. However, not all the stemsproduce d by asingl etes tplan t contained externally visible microsclerotia (Figure 2.5);th eproportio n of stemswit h externally visible microsclerotia was strongly correlated with inoculum density. Discussion The four isolatesteste d hadconidia l lengths upt o 6.0 um, indicating that theybelon g toV. dahliae,an d not toV. longisporum, whichha sconidi a upt o 8.8 um (Karapapa etal., 1997) . Problemswit hverticilliu m wilt in oil seed rape(Brassica napusvar . oleifera)an d other crucifers havebee n associated withV. longisporum, andmostl y notV. dahliae,whils t on non-crucifers theyappea rt ob ealway sassociate d withV. dahliae,an dno tV. longisporum(Okol iet al., 1994 ; Subbarao etal., 1995).However , when inoculating V. dahliaeo rV. longisporum on crucifer or non-crucifer hosts,diseas e regularly occurred (Subbarao etal., 1995,Tabret t etal., 1995 ; 32 yd C en UJ O : PS w > 1 3 10 30 100 MICROSCLEROTIA G"1SOI L Figure 2.5.Microscleroti a in shoot tissue after complete senescence ofArabidopsis thaliana asa function of soil inoculum density. (A)Th e amount of microsclerotia g"1dr y shoot tissue,(B )Th e percentage of stems perplan t exhibiting externally visible microsclerotia. Karapapa etah, 1997).Apparently , in thefiel d othermechanism s areoperativ e that suppress disease development or dispersal of thepathoge n inV. dahliaeI crucife r andV. longisporumI non-crucifer combinations. A. thalianawa s susceptible toal l four isolates ofV. dahliaeuse di n thisstudy .Eac h isolate caused similar disease symptoms and formed microsclerotia in all parts of the senescent plants.However , typical disease symptoms of verticilliumwilt , such aspartia l wilting of the leaves,wer eno tobserved . Allparameter s measured,excep t shootdr yweight , showed strong positive correlations with soil inoculum density ofV. dahliae. Even at 1 microsclerotium g"1soil , stem and root infection was detected andth e area-under-the-senescence-progress curve differed significantly from that of the control plants.Th e sensitivity of detectingV. dahliaeb yplatin g stem base fragments appear tob e lower than that of assessing microsclerotia in the shoot, 33 senescence index,o rroo t plating.Fo r example, microsclerotia were detected in all stems of plants exposed to 1 microsclerotium g"1soil ,althoug h only 30%o f them showed presence ofV. dahliae on plated stembas efragments . Nagtzaam etal. (1997 )foun d that plating of 5-mm pieces of stem bases of eggplant orpotat o wasmuc h less sensitive than plating sap squeezed from 5-cm stem pieces.Thi s indicates either that low populations ofV. dahliaear e unable to grow out from the stempiece s ontoth e agar medium ortha t the pathogen maysimpl yb e absent from the small stem pieces.Usin g ELISA and plating, Van derKoppe l and Schots (1995) mentioned uneven distributions ofV. dahliaei n stems of various hosts.Likewise ,i n ourplating s of the5-cm-lon g pieces of the stem base ofA . thaliana,V. dahliaema yhav e been absent, while being present in otherpart s of the stem. The association ofV. dahliaewit h thecrucife r A. thalianama yb e rare under field conditions, if extant at all,bu t for bioassay purposes it appears tob e well-suited. Thebioassa y is relatively fast as already 6 weeks after planting final disease scores can be made.Th e bioassay appears to be suited to detect low levels of soil infestation withV. dahliae.Assessmen t of disease 6-9 weeks after planting is optimal when the differences with thecontro l are most prominent. The disease index isth eparamete r most easilydetermined . More sharply defined criteria includeth e number of isolations ofV. dahliaeobtaine d from the shoot orroot , and the amounto f microsclerotia formed onth e shoot after complete senescence of the plants.Instea do f determining the amount of microsclerotia peruni t of dry weight of shoot material, asemi quantitative index for microsclerotial density mayals ob e used (data not presented).However , we prefer the useo f disease indices because theydiscriminat e sufficiently between the different inoculum densities.A. thalianaresponde d more sensitively to low inoculum densities than other bioassay plants such aseggplan t (Nagtzaam etah, 1997)o rthor n apple (Evans etal., 1974).Fo r example, at several (butno t all)observatio n dates 5-7 weeks after planting, disease indices differed significantly (P< 0.05) between inoculum densities 0,1, and 3microscleroti a per grammedr y soil.Diseas e progressed most quicklyi n plants grown at25°C ,bu t the life spano f theV. dahliae-infected plants was most influenced at 20°C (Table 2.1).Therefore , we recommend atemperatur e of 20°C for the bioassay. In all test plants,th eproportio n of stems exhibiting externally visible microsclerotia was strongly positively correlated with inoculum density. This mayindicat e that a single infection on aroo t isno t ablet o infect all stems of aplant .Th e amount of microsclerotia g"1dr y shoot tissue reaches rather similar values for inoculum densities of 1,3 ,an d 10microscleroti a g'1 soil, i.e., 1.6 x 103± 9. 0 x 102,2. 2 x 103± 6.4 x 102,an d 3.8 x 103± 1.1 x 103,respectively , if only the stems showing externally visiblemicroscleroti a are taken into account. However, at 30 andespeciall y at 34 100microscleroti a g"1soil ,highe rdensitie s of microsclerotia, viz.,8. 5 x 103± 4. 3x 103an d2. 9 x 104± 6. 8 x 103respectivel y wereobserve d inth e stems.Th emuc h higher densityo f microsclerotia at 100microscleroti a g"1soi l indicates that repeated infection of the shoot leadst o a more thorough colonization and subsequently tohighe r amounts of microsclerotia g"1shoot . Therefore wehypothesiz e that asingl e shoot infection is not ablet o colonize the shoot completely. The most sensitive bioassays reported sofa r recommend the determination of thenumbe r of rootinfection s peruni t of root length onthor n apple (Daturastramonium) (Evan set ah, 1974). This method allowes adetectio n limit of about 2-3 microsclerotia g"1soil .Th edetectio n limit reported here of atleas t 1 microsclerotiumg" 1soi l is alsolowe rtha n with otherplant s tested such aspotat o and eggplant (Nagtzaam etal., 1997).Thi s mayb e duet o thehig h sensitivity ofA . thaliana to systemic root infection. Whilei n many host plants the great majority of root infections remain localized (Huisman and Gerik, 1989),i nA .thaliana w eobserve d mainly systemic infections, with microsclerotia present inside thexyle mtissu e of roots after plating onto Ethanol Agar. Itwas ,therefore , impossible tocoun t the number ofroo t infections in our bioassay. Nevertheless,th e percentage of root length occupied byV. dahliae 14day s after plating onto MSEA was strongly correlated with soil inoculum density (Figure 2.4).Give n thelo w standard errors, wetherefor e recommend, in linewit h Evans etal. (1974) ,t o useth e amount of root infection as the best estimate for soil inoculum density ofV. dahliae.I f however, the aim is solely to determine whether agive n soil is infested withV. dahliae,th e less labour-intensive methodo f observing the senescence severity 6-9 weeks after planting 4-week-old seedlings ofA .thaliana in infested soil mayb e used. 35 CHAPTER 3 EFFECT OFTEMPERATUR E ONTH EFORMATIO N OFMICROSCLEROTI A OF VERTICILLIUMDAHLIAE Summary Microsclerotium formation by six isolates of Verticillium dahliaewa s studied at different temperatures both invitro an d inArabidopsis thaliana. Mycelial growth appeared to beoptima l at 25°C,bu t microsclerotium formation wasgreates t at20° C (two isolates) or 15-20°C (oneisolate) . Seedlings ofA .thaliana wer e root-dipped in aconidia l suspension, planted, andeithe r placeda t 5,10, 15,o r25°C ,o r left at 20°C until the onset of senescence, after which some of the plants wereplace d at5 , 10, 15,o r 25°C.Th e amount of microsclerotia peruni t of shoot weight was assessed in relation to isolate and temperature. Generally, theoptima l temperature for production of microsclerotia was 20°C.Tw oisolate s each produced about ten times moremicroscleroti a than 2 each of the other four isolates.Fo rthes e isolates,hig h R adj.-values of 0.77 and0.6 6 were obtained in multiple regressions, with temperature andit s square ashighl y significant (P< 0.001) 2 independent variables.R adj.-values for the otherisolate s varied between 0.28 and 0.39. Moving plants todifferen t temperatures atth e onset of senescence led to microsclerotial densities intermediate between densities on plants that had grown atconstantl y 20°C and plants grown at other temperatures.Thi s suggests that vascular colonization rate and rateo f microsclerotium formation are similarly affected bytemperature . The senescence rateo f plants appeared unimportant except for plants grown at 25°C, which showed highest amounts of microsclerotia peruni t of plant weight in themos trapidl y senescingplants .Temperatur e andisolat e are regarded asprimar y factors in themicrosclerotiu m formation in host tissue. Introduction Verticillium dahliaeKleb .i s asoil-born e wilt pathogen of awid erang eo f crops such as vegetables (e.g.potato ,tomato ,eggplant) ,ornamental s (e.g. chrysanthemum), fruit trees(e.g., cacao,olive) ,an d shade trees (e.g.ash , maple) (Pegg, 1974;Schnathorst , 1981; Hiemstra, 1998). The pathogen can survive for 14year so r more (Wilhelm, 1955)i n soil asmicrosclerotia , which 37 are small, multi-celled and melanized structures. Germination of microsclerotia in the rhizosphere isinduce d byroo t exudates (Schreiber andGreen , 1963;Schnathorst , 1981).Afte r penetration of the vascular system,th e pathogen istransporte d passively upwards through the xylem vessels. Disease symptoms develop asth eresul t of physical blockage of the xylem vessels and the production of toxins.Finally , verticillium wilt results inearl y senescence of the infected plant. When infected plant parts aredyin g ordead ,th epathoge n grows intoth e parenchymatous tissue where itform s abundant microsclerotia upt o 2 x 105g' 1 dryshoo t tissue ormor e (Molan d Scholte, 1995a).However , among and within fields theformatio n of microsclerotia in shoot tissueca n varyconsiderabl y (Mol and Scholte, 1995b).Althoug h it seems likelytha t spatial pattern in inoculum densityexplain s most of the spatial variation in formation of microsclerotia (Smith andRowe , 1984),i t maya tth e sametim eb eth econsequenc e of variation in formation of microsclerotia. The choiceo f host cultivar hasbee n shown to strongly influence densityo f microsclerotia inhos t tissue (Slattery, 1981;Davi s etal., 1983).I n addition, environmental factors maypla y an important role. Temperature may affect formation of microsclerotia either directly or indirectly through plant senescence.Heal ean d Isaac (1965) reported that the optimum temperature for microsclerotiumformatio n byV. dahliaeproduce d on Czapek-Dox Agar was24°C . Brinkerhoff (1969)reporte d on PDA optimum temperatures for mycelial growth and formation of microsclerotia of 22-25°C.I n cotton leaves,som e formation of microsclerotia continued even at5 and 30°C,thoug h with low densities,bu t it was stopped at 32°C (Brinkerhoff, 1969).Formatio n of microsclerotia in infected tomato tissue in unsterile soil amended with Potato-Dextrose Broth occurred at all temperatures tested (12-33°C) and wasoptima l at 24°C (Ioannou, 1977a). The goal of the present study was toinvestigat e theeffec t of temperature on microsclerotium formation invitro an d inplanta. Arabidopsis thaliana wa s used as ates t plant because of its high susceptibility toV. dahliaean d short life cycle (Chapter 2).T o avoid variation due todifference s in inoculum density, seedlings were root-dipped in ahig h density conidial suspension.T o study temperature effects on events prior to microsclerotium formation, plants grown atconstan t temperatures werecompare d toplant s left at 20°C until the first symptomso f senescence were observed, after which theywer eplace d at the test temperatures of 5, 10,15, and 25°C. 38 Materials and methods Inoculumof V. dahliae Six monospore cultures ofV. dahliaewer e obtained from afla x stem (isolate CI) orpotat o stems (isolates C2,C3 ,VI , V3,V4 )collecte d from fields in the province of Gelderland (isolates Cl- C3)o rDrenth e (isolates V1-V4),th eNetherlands .Conidia l suspensions wereprepare db y growing theculture s in Czapek-Dox liquid medium (Oxoid) for 7day s at 23°C in ashakin g incubator. After incubation, conidia werefiltere d through cheeseclot h and suspended in sterile distilled water toobtai n 106conidi aml" 1. Effectof temperature invitro Cultures of isolates VI-V4 were grown onEthano l Agarmediu m (Nadakavukaren and Horner, 1959) at 10, 15,20 ,25 ,o r 30°C in the darkfo r 25days .Colon y size was measured every 2-3day s andnumber s of microsclerotiape rcolon ywer ecounte d atth een d of theincubatio n time.T o determine thenumber s of microsclerotia, ameasure d sectoro f thecolon y wastaken , ground using mortar andpestle , suspended in 0.08%wate r agar, andth e microsclerotia were directly counted in three small subsamples (each 15ul )o f the suspension using adissectin g microscope (magnification 25x) . Effectof temperature inplanta Seedlings ofArabidopsis thaliana ecotyp e Columbia were germinated in steamed potting soil. After 40 daysa t22° Cth efirs t rosette leaves wereforme d andth eplant s uprooted.Th eroot swer e washed in sterile distilled water, dipped for 20mi n in aconidia l suspension, planted inblac k plastic pots (w/w/h, 7/7/6 cm)containin g sievedpottin g soil mixed with sand (2/1, v/v),an d placed at 5, 10, 15,20 ,o r 25°C for experiment 1(isolate s C1-C3) or 10, 15,20 ,o r 25°C for experiment 2 (isolates VI-V4) respectively. Another set of plants wasplace d first at20° C anda t the onset of senescence transferred toth e temperatures of 5(experimen t 1 only), 10, 15,o r25°C . The onset of senescence was defined asth e stage when rosette leaves and lower stem leavesha d started toyello w and first seeds werebein gproduced . Atthi s stage,n o microsclerotia could be observed within the plant tissue.Ever y treatment wasreplicate d ten (experiment 1)o rsi x (experiment 2) times.Plant s werewatere d asnecessar y and placed in a 16h-lighte d (Philips TD 32W/8 4 HF)climat e chamber. 39 90 EC UJ 60 < Q >- 30 o u III III 10 10 10 15 15 15 20 20 20 25 25 25 30 30 30 V1 V3 V4 iiuiUlVI V3 V4 V1 V3 V4 V1 V3 lV4 V1 V3 V4 TEMPERATURE (°C), ISOLATE 10 10 10 15 15 15 25 25 25 30 30 30 V1 V3 V4 V1 V3 V4 V1 V3 V4 VI V3 V4 TEMPERATURE (°C), ISOLATE 20 20 20 25 25 25 30 30 30 V1 V3 V4 V1 V3 V4 V1 V3 V4 TEMPERATURE (°C), ISOLATE Figure 3.1. Effect oftemperatur e onthre eisolate s of Verticillium dahliaegrowin g on Ethanol Agar. (A)Colon y diameter, (B)Numbe r ofmicroscleroti a per colony, (C)Numbe r of microsclerotia mm'2 colony. 40 Plants were harvested after when dead. After cutting off the shoot, pots were soaked in standing tapwate r for 30mi n andth e soil wasremove d from theroots . Shoots and roots were dried, weighed, and stored separately inpape r bags until further analysis.Bot h dried shoots androot s were ground separately using amorta r andpestl e and suspended in 0.08% water agar.Th e density of microsclerotia in the suspension wasdetermine d bydirec t counting of at least three dropletso f known volume (approximately 20ul ) under adissectin g microscope. Numbers of microsclerotia weretransforme d logarithmically (logio(x+l))prio r todat a analysis.Th eexperimenta l design was asplit-plo t design with temperature asmain-plo t factor and constant versus different temperature andV. dahliaeisolat e as sub-plot factors. Results Effectof temperature on mycelial growth andmicrosclerotium formation invitro Maximum growth occurred at 25°C for all isolates,wit h growth at 15an d 20°Conl y slightly less (Figure 3.1A). Growth at 10an d 30°C was about half the growth at 25°C (P< 0.001). Growth rate of three isolates (CI -C3 ) wasbroadl y similar. Formation of microsclerotia percolon yan d permm 2colon y varied considerably (P< 0.001), with anobviou s maximum at 20°Cfo r isolates V3 andV4 ,an d amaximu m at 15-20°C for isolateV I (Figure 3.1B.C). Effectsof V. dahliaeand temperature onArabidopsis thaliana Theeffec t ofV. dahliaeo n growth ofA .thaliana wa sdetermine d only in experiment 2.Plant s grown atlowe r temperatures generally grew more slowly and yielded higher dryweights .O n average, uninoculated plants grown continuously at 10,15, 20,an d 25°C were completely dead 293,246,178, and9 9day s after inoculation respectively (Figure 3.2).Inoculatio n withV. dahliae accelerated senescence by26 ,36,45 , and 34%respectively , ascompare d toth e uninoculated plants (Figure 3.2).Inoculate d plants weighed 7-67% less than control plants (Figure 3.3).Th e strongest growth reduction occurred for allthre eisolate s at25°C .Th enegativ e effect of isolate VI onplan t dry weight was more apparent athighe r temperatures: a9 %reductio n at 5°Cu pt o 67% at25°C .Fo risolate s V3an dV4 , growthreductio n was alsoconsiderabl e at 10an d 15°C, with arang e of 20-28% and 21-25%respectively . Iti sremarkabl e that the growth reduction observed with isolate V4 at 20°C wasonl y 7%.Roo t dryweight s werequit e low andhighl y variable, probably because theyha d alreadybee n partly decomposed. Shoot dryweigh t and life span of individual plants were significantly (P< 0.001) andpositivel y correlated (Pearson R= 41 0.72). However, thereductio n of shoot dry weight due toV. dahliaewa s not correlated with the reduction of life span duet oV. dahliae. Plants grown at 20°C and moved to 10,15, or 25°C at the onset of senescence showed more similar life spans and dryweight s tothos eo f plants grown continuously at 20°C than those of plants grown continuously at 10,15, or 25°C (Figures 3.2, 3.3).Thus ,plant s moved to 10 or 15°Cha d shorter life spans,an dthos e moved to 25°C had longer life spanstha n plants thatha d been grown atthes e temperatures continuously. Except for isolate VI at 25°C,al l plants thatha d been moved had greater reductions in dryweigh t and life spans duet oV. dahliaetha n plants that grew continuously atth e sametemperatur e (Table 3.1). Analysis of variance indicated ahighl y significant interaction (P< 0.001)betwee n isolate, temperature, and the act of moving plants to different temperatures atth e onset of senescence,o n thedensit yo f microsclerotiai n shoot tissue.Th e average number of microsclerotiaforme d per gramme dryweigh t was almost identical for shoot androot , 1.4 x 105an d 1.3 x 105respectivel y (averages from untransformed data).Isolate s CI, VI, V3,an d V4 formed on average 8.8 x 103 - 1.8 x 104microscleroti a g"1shoo t dryweight , whilst isolates C2an d C3 formed on average 2.9 x Table 3.1. Average percentage of reduction of shoot dryweigh t and life span of the test plants due to Verticillium dahliae.Constan t =plant sgrow n continuously attemperature s indicated; Changed = plants grown at20° C and moved toth e temperatures indicated at the onset of senescence. Temperature treatment Constant Changed Constant Changed Isolate Temperature (°C) Plantdr y weight Life span VI 10 9.4 51 19 21 15 10 58 18 45 20 38 - 39 - 25 67 47 31 31 V3 10 20 25 13 33 15 21 35 21 48 20 11 - 43 - 25 30 53 30 35 V4 10 28 45 19 21 15 25 41 21 46 20 6.8 - 39 - 25 66 76 21 26 42 CONSTANTTEMPERATUR E CHANGEDTEMPERATUR E ISOLATEV 1 ISOLATEV 3 ISOLATEV 4 25 10 TEMPERATURE(°C ) --m--Contro l Inoculated Figure 3.2. Effect ofthre e isolates of Verticillium dahliaean d oftemperatur e on life spano f Arabidopsisthaliana. Plantswer emaintaine d either atconstan t temperatures, orthe ywer e grown up at 20°C andplace d atth eindicate d temperatures atth eonse t of senescence ('changed temperature'). 43 CONSTANTTEMPERATUR E CHANGEDTEMPERATUR E ISOLATEV 1 x o m a H o o OTX 1 •• X e> !! 'kv ui ^ i Q I- O o X (0 10 15 20 25 ISOLATEV 4 10 15 20 25 10 TEMPERATURE(°C ) • Control A- Inoculated Figure 3.3.Effec t ofthre e isolates of Verticillium dahliaean d oftemperatur e on shoot dry weight ofArabidopsis thaliana. Plants weremaintaine d either atconstan t temperatures, orthe ywer e grown up at 20°C andplace d at the indicated temperatures atth e onset of senescence ('changed temperature'). 44 105- 3.0 x 105microscleroti a g"1shoo t dryweigh t (averages from untransformed data).Mos t isolates showed anoptimu m microsclerotium production peruni t shoot dryweigh t at 15-20°C (Table 3.2).Isolate s V3 and V4 showed almost identical production of microsclerotia at 15an d 20°C. The numbers of microsclerotia g"1shoo t dryweigh t forplant s grown atconstan t temperatures andfo r plants moved from 20°Ct odifferen t temperatures atth eonse t senescence was 1.6 x 105an d 1.5 x 106(average s from untransformed data)respectively . The effect of movingplant s todifferen t temperatures atth eonse t of senescence generally ledt omicrosclerotia l densities in plant tissueclose r tothos eo f plants grown atconstan t 20°C.I n most casesth e optimum temperature for microsclerotium formation of moved plantsremaine d 15-20°C.Fo r isolates CI and C3, the effect of movingplant s atth e onset of senescence to 5°Cresulte d ina significant (P< 0.01) increase in the density of microsclerotia g"1shoo t dry weight ando f number microsclerotia perplan t (data not shown)compare d toplant s that hadgrow n continuously at5° C (Table 3.2).Fo r isolates CI, C2,C3 ,an d VI, asimilarl y significant effect was found for plants thatha dbee n moved to 10°C.However , the effect of moving plants to different temperatures was largely insignificant for isolates V3 and V4.Movemen t of plants to 25°C led to a significant increase in the number of microsclerotia per shoot dryweigh t for two isolates and a significant decreasefo r oneisolate . Similareffect s werefoun d for theproductio n of microsclerotia on the root, although these optima were less prominent for isolates C2an d C3 (data not shown). Attempts to describe the density of microsclerotia in plant material for all isolates and treatments in one (multiple) regression as afunctio n of temperature, the square of thetemperature ,plant , shoot, orroo t dry weight, and/orpathogenicit y of theisolat e (defined asth eeffec t of the isolate 2 on growth reduction) using multipleregressio n ledt oa maximu m R adj. of 0.28 with the squareo f thetemperatur e and dryshoo t and root weight as significant terms (P< 0.01) . When onlyth e 2 separate isolates treated atconstan t temperatures wereconsidered , high R adj. values of 0.77 and 0.66 wereobtaine d onlyfo r isolates C2an d C3, with temperature andit s square ashighl y 2 significant (P< 0.001) terms.R adj. values for theothe r isolates varied between 0.28 and 0.39, with the temperature and its square (isolate CI) oreffec t of the isolate on growth reduction (isolates V1-V4) as significant (P< 0.01 )terms . No significant correlations between life span and density of microsclerotia in host tissue werefoun d when allth edat awer econsidered . However, significant correlations between life span anddensit y of microsclerotia in host tissue were found for plants grown at25°C ,wher ea significant negative correlation was found (Pearson R = -0.62).Plant s atthi s temperature containingth e highest microsclerotia densities hadth eshortes t life spans. 45 Discussion Thetemperatur e optimum for formation of microsclerotia invitro (15-20°C )i s lower than that for hyphal growth ofV. dahliae(25°C) .Wit hA. thaliana,a n optimum of 20°C wasfoun d for the formation of microsclerotia bymos t isolates.W e wereunabl e todescrib e microsclerotial density asa functio n of other variables measured, such asplan t dryweight , amount of microsclerotia in theroot , orth eeffec t ofV. dahliaeo nplant , shooto rroo t dryweight .Isolate s C2 and C3 produced aboutte n times moremicroscleroti a than the other isolates.Whe n the individual 2 isolates wereconsidered , regression resulted in high R adj-values for isolates C2 and C3only , wheretemperatur e and its squarewer e highly significant. Isolates C2 and C3produce d c.te n times more microsclerotia peruni t plant weight than the other isolates. Microsclerotium production in these otherisolate s (CI, VI, V3,V4 )ma yb elimite d byfactor s that aredifferen t to those in isolates C2an dC3 . Mol etal. (1996b ) observed that soil inoculum density ofV. dahliaeafte r incorporation of debriso f arang eo fhost san dnon-host s interacted strongly with theV. dahliaeisolat e useda s inoculum. An isolate ofV. dahliaeobtaine d from asoi l cropped continuously to potato for 14 yearsle d to higher inoculum densities when potato was grown than when beans were grown; and an isolate obtained from asoi l cropped continuously to beans for 10year s led to higher inoculum densities when beans weregrow n than when potato wasgrow n (Mol etal., 1996a).W e found that different isolates originating from potato (except isolate CI from flax), can lead to widely different numbers of microsclerotia in host tissue.I tca n be expected that in thefiel d these isolates would quicklyoutcompet e the otherisolates . However, other conditions, such assoi l moisture andit spersistenc e mayals oinfluenc e therat eo f success of particularisolates . Soesanto andTermorshuize n (Chapter 2)foun d that thedensit y of microsclerotia formed in shoot tissuedepend s on the soilinoculu m density, andconclude d that multiple root and vascular infections are needed toobtai n maximum quantities of microsclerotia in the shoot. Inth e present experiments, ahig h level of root infections was ensured byapplyin g aroo t dip inoculum of 106conidi aml" 1,an dconsequentl y thenumbe ro froo t infections wasno t alimitin g factor. An effect of temperature on vascularcolonizatio n ratema yexplai n the difference in microsclerotial densities between moved and unmoved plants at low temperatures. It isprobabl e thatV. dahliae grew and colonized more tissue at20° C than at 5o r 10°C.Th e higher biomass ofV. dahliaei n the vascular system resulted in more microsclerotia produced for the moved plants.Whe n plants were moved todifferen t temperatures,th eformatio n of microsclerotia had yett obegin ; consequently the fewer microsclerotia produced in plants moved to low temperatures compared 46 o o o o