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. , 2000 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, theDepartmen 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