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Biology and Management of Box Blight Caused by Cylindrocladium Buxicola

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Biology and Management of Box Blight Caused by Cylindrocladium Buxicola Biology and management of box blight caused by Cylindrocladium buxicola By Sarah Elizabeth Healy A Thesis Presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Environmental Science Guelph, Ontario, Canada © Sarah Elizabeth Healy, 2014 ABSTRACT BIOLOGY AND MANAGEMENT OF BOX BLIGHT CAUSED BY CYLINDROCLADIUM BUXICOLA Sarah Elizabeth Healy Advisor: University of Guelph, 2014 Professor Tom Hsiang A new fungus, Cylindrocladium buxicola, which causes disease on boxwood was recently observed in Ontario. The fungus, isolated from samples from a nursery in Southern Ontario, was positively identified by DNA sequence comparisons as the cause of box blight. Whole boxwood plants required wounding for successful infection, however detached leaves did not. The ‘Green Series’ boxwood cultivar ‘Green Mountain’ was found to be the least susceptible to C. buxicola compared to other cultivars. Survival of C. buxicola in Southern Ontario was low after the winter, but was higher throughout the fall. Whole genome sequencing of two C. buxicola isolates revealed the presence of the MAT1-1 gene within a German isolate and MAT1-2 in an Ontario isolate, implying the possibility for sexual reproduction. All other 50 isolates tested were MAT1-2. The preventative use of fungicides to control box blight in nurseries, rather than curative use, is likely to provide a better management option for disease. ACKNOWLEDGEMENTS First and foremost I would like to thank Dr. Tom Hsiang for his guidance and support throughout my time as a graduate student in his lab. He provided me with the opportunity to conduct novel and interesting research in an area which I am very passionate about. With his encouragement and patience, I have acquired a vast skill set that I am sure will benefit me in future endeavors. I am also appreciative of the opportunities Dr. Hsiang has given me to share my scientific findings at international conferences and at industry meetings. I would also like to thank my advisory committee member, Dr. Goodwin, for his input and to my examination committee for taking the time to be part of my defense. Of course, this project would not have been possible without Mary Jane Ash and the other members of Sheridan Nurseries, thank you for your advice, never ending supply of boxwood plants, and friendly faces. To the members of the Hsiang lab: Linda you have given me constant support and advice during this project, not to mention a fantastic friendship. Thank you for your skilled script writing abilities, your vegan eating ways, and being my troubleshooting buddy. To Amy, you have been a support system for me since my undergrad and I thank you for your expertise in boxwood, fungicides, statistics and more. You were also the best tour guide of China I have ever had. To Miha, thank you for being my biggest believer and kindest friend. To Anne, you provided me with laughter and happiness every single day, thank you for giving me someone to lean on. To Craig, thank you for all of your help in the lab; and to Vince, thank you for your bioinformatics help. iii To my friends and family, thank you for being there for me and sticking with me during all my years of schooling. Especially to my parents, Sonia and Mike, you are the reason I am here today and your love and encouragement keep me going. To my office mates: Phil, Rochelle, Laura, Xin, and Jon, thank you for not only putting up with me all, but for providing me with friendships which I am sure will last a lifetime. Korri, thank you for being my motivation, best friend, and biggest source of joy. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ................................................................................................................ v LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES………………………………………………………………………….…xvii LIST OF APPENDICES .............................................................................................................. xix LIST OF ABBREVIATIONS AND ACRONYMS ................................................................... xxii LIST OF ABBREVIATIONS AND ACRONYMS CONTINUED .......................................... xxiii Chapter 1 Literature Review ........................................................................................................... 1 1.1 Introduction ........................................................................................................................... 1 1.1.1 Boxwood species and their uses .................................................................................... 1 1.2 Major fungal diseases on boxwood....................................................................................... 2 1.2.1 Macrophoma leaf spot.................................................................................................... 2 1.2.2 Phytophthora root rot ..................................................................................................... 3 1.2.3 Boxwood decline ........................................................................................................... 4 1.2.4 Volutella blight .............................................................................................................. 4 1.2.5 Box Blight ...................................................................................................................... 6 v 1.3 Box blight caused by Cylindrocladium buxicola .................................................................. 7 1.3.1 Signs and symptoms of Cylindrocladium buxicola ....................................................... 8 1.3.2 Life cycle of Cylindrocladium buxicola ........................................................................ 9 1.3.3 Host range and resistance of Cylindrocladium buxicola ............................................. 10 1.4 Genetic and molecular analysis of Cylindrocladium buxicola ........................................... 11 1.4.1 Genetic diversity within Cylindrocladium buxicola .................................................... 11 1.4.2 Mating type genes and sexual reproduction in Cylindrocladium buxicola.................. 12 1.4.3 Pathogen Identification ................................................................................................ 13 1.5 Cultural and chemical management methods ..................................................................... 14 1.6 Hypotheses .......................................................................................................................... 16 1.7 Objectives ........................................................................................................................... 17 Chapter 2 Pathogenicity, infection process, and survival of Cylindrocladium buxicola on Buxus and Pachysandra terminalis ......................................................................................................... 18 2.1 Introduction ......................................................................................................................... 18 2.1.1 Boxwood blight caused by Cylindrocladium buxicola ................................................ 18 2.1.2 Disease development and survival of Cylindrocladium buxicola ................................ 18 2.1.3 Host Range of Cylindrocladium buxicola .................................................................... 20 2.1.4 Causal agent identification ........................................................................................... 21 vi 2.1.5 Objectives .................................................................................................................... 22 2.2 Materials and methods ........................................................................................................ 23 2.2.1 Collection of Buxus specimens .................................................................................... 23 2.2.2 Media preparation and stock cultures .......................................................................... 24 2.2.3 Fungal isolation ............................................................................................................ 25 2.2.4 Temperature growth experiment for Cylindrocladium buxicola ................................. 26 2.2.5 Survival of Cylindrocladium buxicola under laboratory and field conditions ............ 27 2.2.6 Infection process of Cylindrocladium buxicola on detached ‘Green Mountain’ leaves ............................................................................................................................................... 29 2.2.7 Pathogenicity on detached leaves of Buxus and Pachysandra terminalis ................... 30 2.2.8 Pathogenicity on whole Buxus and Pachysandra terminalis plants ............................ 33 2.2.9 DNA extraction and PCR analysis ............................................................................... 34 2.3 Results and discussion ........................................................................................................ 36 2.3.1 Collection of Buxus species and fungal isolation ........................................................ 36 2.3.2 Koch’s postulates ......................................................................................................... 38 2.3.3 Temperature growth analysis
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