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Analysis of Three Biological Control Agents and Naturally-Occurring Fungal Colonizers

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Analysis of Three Biological Control Agents and Naturally-Occurring Fungal Colonizers Analysis of three biological control agents and naturally-occurring fungal colonizers on the survival of sclerotia of Botrytis squamosa, Sclerotinia sclerotiorum and Sclerotium cepivorum by Laura C. Barbison A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Environmental Sciences Guelph, Ontario, Canada © Laura C. Barbison, 2014 ABSTRACT ANALYSIS OF THREE BIOLOGICAL CONTROL AGENTS AND NATURALLY-OCCURRING FUNGAL COLONIZERS ON THE SURVIVAL OF SCLEROTIA OF BOTRYTIS SQUAMOSA, SCLEROTINIA SCLEROTIORUM AND SCLEROTIUM CEPIVORUM Laura Barbison Advisors: University of Guelph, 2014 Professor G. J. Boland Professor M. R. McDonald Management of sclerotia-forming phytopathogenic fungi is difficult due to the resilience of their sclerotia within soil. This thesis assessed the efficacy of three biological control agents (BCAs), Coniothyrium minitans, Microsphaeropsis ochracea, and Trichoderma atroviride, on laboratory-produced (LP) sclerotia of Botrytis squamosa, Sclerotinia sclerotiorum and Sclerotium cepivorum under field conditions for 20 months. Trichoderma atroviride was most consistent, reducing survival of B. squamosa and S. sclerotiorum to 19 and 23%. S. cepivorum did not vary. Further investigations compared the survivability of LP and field-produced (FP) sclerotia of S. sclerotiorum. Survival of LP sclerotia of S. sclerotiorum was greater than that of FP, which were heavily colonized with other fungi. Colonizers were isolated, identified, and evaluated for antagonistic and pathogenic abilities. 80% of isolates demonstrated antagonistism, and two isolates demonstrated pathogenicity. It is evident that sclerotia are not sterile propagules, and that antagonistic soil-dwelling microorganisms are important influential factors on sclerotial survival. ACKNOWLEDGEMENTS I would like to take this opportunity to express my sincere gratitude to the individuals and groups that have supported me throughout my continued education and completion of this degree at the University of Guelph. Firstly, my deepest appreciation to my co-advisors Dr. Greg Boland and Dr. Mary Ruth McDonald for providing me with this opportunity, and for their continuous support, assistance, and guidance. Your insightful encouragement has allowed me to develop my analytical and communication skills which will forever be an asset as I move forward in life. I would also like to extend my gratitude to the final member of my committee, Dr. Katerina Serlemitsos Jordan. Your insightful support throughout the program has broadened my view and further developed my understanding of plant epidemiology. The assistance of several colleagues was invaluable for the completion of this thesis research. Much appreciation to the team at the Muck Crops Research Station for their field assistance, especially Michael Tesfaendrias for his technical guidance and expertise. To Dr. Monica Parker for her assistance with molecular laboratory techniques. Your innate ability to relay concepts and information has been of great benefit to me, and your continuous offer of guidance has been greatly appreciated. Thanks to Sara Stricker for her assistance with field work preparations. To Melody Melzer, although I did not have the joy of working with you during my graduate degree, your guidance during my time as a graduate student was essential for my success throughout this degree - thank you. Furthermore, much appreciation to Dr. Brian Allen and Michelle Edwards for their expert assistance with the statistical analyses. iii I am very thankful for the generous funding agencies that provided financial assistance both for this research and for my academic studies. Specifically, the Ontario Ministry of Agriculture, Food & Rural Affairs, University of Guelph, and the Ontario Processing Vegetable Growers. The contributions from both Plant Products Co. Ltd. and ProPhyta GmbH are also much appreciated. I must also recognize my family and the invaluable support that they provide me with each and every day. To my parents, your unwavering encouragement ever since I was young has brought me to challenge myself, and has allowed me to achieve many things that would not have been possible without you. Finally, these acknowledgements are far from complete without recognizing my close friends, both in and outside of the graduate program. Every ounce of support that I have received throughout this process has been greatly valued. Thanks to every one of you! iv CONTENTS ACKNOWLEDGEMENTS ........................................................................................................... iii LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix LIST OF ABBREVIATIONS ......................................................................................................... x CHAPTER 1 - LITERATURE REVIEW ....................................................................................... 1 1.1 Introduction .......................................................................................................................... 1 1.2 Sclerotia-Forming Fungi ...................................................................................................... 1 1.3 Sclerotia Survival ................................................................................................................. 3 1.3.1 Structural Aspects ............................................................................................................. 5 1.3.2 Environmental Conditions ................................................................................................ 6 1.3.3 Evaluating Survival .......................................................................................................... 7 1.4 Pathogens of Interest ............................................................................................................ 8 1.4.1 Botrytis squamosa ............................................................................................................. 8 1.4.1.1 Biology of B. squamosa ............................................................................................ 9 1.4.1.2 Management of B. squamosa .................................................................................. 12 1.4.2 Sclerotinia sclerotiorum ................................................................................................. 14 1.4.2.1 Biology of S. sclerotiorum ...................................................................................... 15 1.4.2.2 Management of S. sclerotiorum .............................................................................. 18 1.4.3 Sclerotium cepivorum ..................................................................................................... 21 1.4.3.1 Biology of S. cepivorum ......................................................................................... 22 1.4.3.2 Management of S. cepivorum ................................................................................. 25 1.5 Biocontrol Agents .............................................................................................................. 27 1.5.1 Biocontrol Agents of Interest .......................................................................................... 28 1.5.1.1 Coniothyrium minitans............................................................................................ 28 1.5.1.2 Microsphaeropsis ochracea .................................................................................... 30 1.5.1.3 Trichoderma atroviride ........................................................................................... 31 1.5.2 Factors Affecting BCA Efficacy .................................................................................... 34 1.6 Conclusions ........................................................................................................................ 34 1.7 Thesis Objectives ............................................................................................................... 36 v CHAPTER 2 - LONG-TERM SURVIVAL OF SCLEROTIA OF BOTRYTIS SQUAMOSA, SCLEROTINIA SCLEROTIORUM, AND SCLEROTIUM CEPIVORUM TREATED WITH THREE BIOLOGICAL CONTROLS UNDER FIELD CONDITIONS ....... 38 2.1 Abstract .............................................................................................................................. 38 2.2 Introduction ........................................................................................................................ 39 2.3 Materials and Methods ....................................................................................................... 42 2.3.1 Production and collection of sclerotia ............................................................................ 42 2.3.2 Long-term survival of sclerotia untreated and treated with three biocontrols in a natural setting .................................................................................................................. 44 2.3.3 Statistical analysis ........................................................................................................... 45 2.4 Results ................................................................................................................................ 47 2.4.1 Survival of untreated sclerotia in a natural
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