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Cylindrocladium Buxicola Nom. Cons. Prop.(Syn. Calonectria

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Cylindrocladium Buxicola Nom. Cons. Prop.(Syn. Calonectria I Promotors: Prof. dr. ir. Monica Höfte Laboratory of Phytopathology, Department of Crop Protection Faculty of Bioscience Engineering Ghent University Dr. ir. Kurt Heungens Institute for Agricultural and Fisheries Research (ILVO) Plant Sciences Unit - Crop Protection Dean: Prof. dr. ir. Guido Van Huylenbroeck Rector: Prof. dr. Anne De Paepe II Bjorn Gehesquière Cylindrocladium buxicola nom. cons. prop. (syn. Calonectria pseudonaviculata) on Buxus: molecular characterization, epidemiology, host resistance and fungicide control Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences III Dutch translation of the title: Cylindrocladium buxicola nom. cons. prop. (syn. Calonectria pseudonaviculata) in Buxus: moleculaire karakterisering, epidemiologie, waardplantresistentie en chemische bestrijding. Please refer to this work as follows: Gehesquière B. (2014). Cylindrocladium buxicola nom. cons. prop. (syn. Calonectria pseudonaviculata) on Buxus: molecular characterization, epidemiology, host resistance and fungicide control. Phd Thesis. Ghent University, Belgium The author and the promotors give authorisation to consult and to copy parts of this work for personal use only. Any other use is limited by Laws of Copyright. Permission to reproduce any material contained in this work should be obtained from the author. The promotors, The author, Prof. dr. ir. M. Höfte Dr. ir. K. Heungens ir. B. Gehesquière IV Een woordje van dank…. Dit dankwoord schrijven is ongetwijfeld het leukste onderdeel van deze thesis, en een mooie afsluiting van een interessante periode. Terugblikkend op de voorbije vier jaren kan ik enkel maar beamen dat een doctoraat zoveel meer is dan een wetenschappelijke uitdaging. Het is een levensreis in al zijn facetten, waarbij ik mezelf heb leren kennen in al mijn goede en slechte kantjes. Gelukkig stond ik er niet alleen voor, en kon ik rekenen op de steun en hulp van vele fantastische mensen. Een oprecht woord van dank is daarom zeker op zijn plaats. Allereerst zou ik mijn promotoren en naaste begeleiders Monica Höfte (UGent), Kurt Heungens (ILVO P96), Filip Rys (PCS) en Johan Van Huylenbroeck (ILVO P21) willen bedanken voor het vertrouwen dat ze in mij en mijn project hebben gesteld. Bedankt voor jullie enthousiasme, ervaring en tijd. Onze momenten van overleg waren soms gezellig, soms intens, maar altijd verhelderend en waardevol. I sincerely want to thank the members of the examination committee for the thorough reading of my dissertation and the useful comments and suggestions. Thank you for improving this manuscript! Dit project was niet mogelijk zonder financiele steun van het IWT en de sierteeltssector. Ik wil iedereen van de buxusgroep en van het begeleidingscomité bedanken voor de jarenlange goede samenwerking. Robrecht, Jorien, Sam en Fien: we hebben samen veel tijd doorgebracht op dit project. Het werk zelf was niet altijd even leuk, maar jullie gezelschap heeft veel goed gemaakt! Allemaal hebben jullie in grote mate bijgedragen aan dit werk: bedankt. Ook alle ander collega’s van ILVO P96, ILVO P21 en PCS, teveel om hier op te noemen, verdienen een prominente plaats in dit dankwoord. Bedankt voor de goede werksfeer, voor de wetenschappelijk verantwoorde en niet-verantwoorde gesprekken tijdens de middagpauzes, voor de feestjes na de uren, voor allle steun, advies en technische ondersteuning als ik mijn experimenten weer eens te groot had ingeschat… Ik wil me bij iedereen oprecht excuseren voor mijn bovengemiddeld koffieverbruik en ondergemiddeld zangtalent. Ik hoop van harte dat we contact kunnen blijven houden, en dat we elkaar nog veel mogen zien tijdens de verschillende netwerkmomenten (congressen en seminaries, drinks, bowlings, kubb toernooien en instuiven etc.). V I also want to thank the people that made my trip to the US possible. Kelly and Miranda, Bennett and Lynn: thanks a lot for inviting me over and taking such good care of me. It was a highly inspiring experience and I’ll hope to ever meet you again. Uiteraard wil ik ook alle vrienden bedanken voor de nodige ontspanning. Of ik jullie nu ken van het lagere schooltje, secundaire of unief, uit de KLJ, via vrienden van vrienden, of gewoon van tegenkomen en blijven plakken: dank jullie voor etentjes, cafégesprekken, uitstapjes, BBQ’s, spelletjesavonden etc. Bedankt ook aan thuisfront. Ma en pa, broer en sœur, Nadine en Marc, Sophie en Timme, Julie en Myriam: jullie goede raad en steun waren uitermate belangrijk voor mij, ook al was ik niet altijd even enthousiast om over mijn thesis uit te wijden. Mijn laatste woord van dank gaat uit naar Valerie, mijn eeuwige steun en rots in de branding. Lieve schat, waar moet ik beginnen? Bedankt, voor het geduld en begrip gedurende de laatste maanden, ik beseft dat het niet altijd gemakkelijk was. Bedankt, omdat je er altijd bent geweest als ik steun kon gebruiken. Bedankt, omdat jij als geen ander me aan het lachen kan maken. Bedankt, omdat je me elke dag opnieuw verwondert en sprakeloos achterlaat met je fascinerende, totaal onlogische gedachtegang. Kortom: bedankt, omdat je me elke dag opnieuw gelukkig maakt! Bjorn April 2014 VI Table of Contents Table of Contents .................................................................................................................................. VII List of Acronyms and Abbreviations ....................................................................................................... XI Problem statement and thesis outline .................................................................................................... 5 Chapter 1: General introduction ............................................................................................................. 7 1.1 Buxus ....................................................................................................................................... 9 1.2 Cylindrocladium buxicola ....................................................................................................... 16 1.3 Molecular detection: Real-time PCR ..................................................................................... 22 1.4 Molecular fingerprinting techniques ..................................................................................... 24 Part I: Genetic and phenotypic variation in C. buxicola .................................................................. 29 Chapter 2: Identification and characterization of two genetic lineages in Cylindrocladium buxicola .. 31 2.1 Introduction ........................................................................................................................... 33 2.2 Materials and methods ......................................................................................................... 34 2.3 Results ................................................................................................................................... 41 2.4 Discussion and conclusions ................................................................................................... 54 Chapter 3: Population dynamics in Cylindrocladium buxicola based on newly developed SSR markers ............................................................................................................................................................... 61 3.1 Introduction ........................................................................................................................... 63 3.2 Material and methods ........................................................................................................... 64 3.3 Results ................................................................................................................................... 66 3.4 Discussion and conclusions ................................................................................................... 73 Part II: Detection, spread and introduction pathways of Cylindrocladium buxicola ............................. 77 Chapter 4: real-time PCR assays for the detection of Cylindrocladium buxicola in water, air and plant samples .................................................................................................................................................. 79 4.1 Introduction ........................................................................................................................... 81 4.2 Materials and methods ......................................................................................................... 81 4.3 Results ................................................................................................................................... 89 4.4 Discussion and conclusions ................................................................................................... 97 VII Chapter 5: Distances and pathways of Cylindrocladium buxicola dispersal ....................................... 103 5.1 Introduction ......................................................................................................................... 105 5.2 Materials and methods ....................................................................................................... 106 5.3 Results ................................................................................................................................. 114 5.4 Discussion and conclusions ................................................................................................. 125 Part III: Host susceptibility, plant-pathogen
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