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Resistance to Fungicides in the Plant Pathogen Microdochium Nivale By

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Resistance to Fungicides in the Plant Pathogen Microdochium Nivale By Resistance to fungicides in the plant pathogen Microdochium nivale by Ryan Allan Russell Gourlie 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 © Ryan Allan Russell Gourlie, September, 2018 ABSTRACT RESISTANCE TO FUNGICIDES IN THE PLANT PATHOGEN MICRODOCHIUM NIVALE Ryan Allan Russell Gourlie Advisor: University of Guelph, 2018 Professor Tom Hsiang Samples of Microdochium nivale were collected from British Columbia and Ontario. The sampled isolates (161) and previously collected isolates (129) were tested for fungicide sensitivity by growing on amended media. Iprodione and propiconazole EC50 values ranged from 1 to 543 μg mL-1 and 0.001 to 0.87 μg mL-1 respectively. In field trials, resistance was significantly correlated with less disease. Draft genome assemblies of 13 isolates were searched for sequences associated with resistance. Alignments of the mnos-1 gene revealed a mutation (A3503G) and a deletion (Δ430-462) in iprodione-resistant isolates. Mutations in mnos-4 (G49A and C396T), and mutations in skn7 (214 SNPs, six deletions, two insertions) were associated with a subset of iprodione-resistant isolates. Mutations in the mrr1 gene (G2237A and G2392A) were associated with resistance to both fungicides in some isolates. No mutations in the propiconazole target site (cyp51) were found to be associated with propiconazole-resistant isolates of M. nivale. ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor Dr. Tom Hsiang for seeing my potential and giving me an opportunity to achieve my goals. His mentorship, unparalleled patience, and devotion to his students have helped me grow as a scientist, a writer, and a person. I would also like to thank my committee member Dr. Paul Goodwin for educating me, providing excellent feedback, and his unique insights into my project. To my partner, Kathryn Wilde, your unending love and support helped me through the hard times in my life and I wouldn't be here without you. Thanks to my parents and brother, Allan, Christine, and Kyle for their constant and unconditional encouragement through the years. To my sister Sarra for inspiring me to work harder, to be better, and for making me laugh. A special thanks to my lab and office mates Sara Stricker, Kate Stone, and Moez Valliani, for your friendship, coffee, and conversation. I would also like to thank the many undergraduate students and lab technicians who helped me in the lab (in no particular order): Hyo Choi, Karam Notay, Matthew Rudland, Jesse Pariselli, Joey Yang, Mina Nasr-Sharif, Jie Chen, Vivian Forte-Perri, and Shuhong Zhang. Thanks to the sample locations in British Columbia and Ontario that graciously allowed sample collection. A final sincere thanks to the Government of Ontario, Syngenta, and the turfgrass industry for funding my research. iii TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ....................................................................................................................... ix LIST OF APPENDICES .............................................................................................................. xiii LIST OF ABBREVIATIONS AND ACRONYMNS .................................................................. xv Chapter 1 General Introduction & Literature Review .................................................................... 1 1.1 Introduction ........................................................................................................................... 1 1.2 General Information about Microdochium nivale ................................................................. 2 1.2.1 Origin, distribution, and host range ................................................................................ 2 1.2.2 Disease cycle and disease descriptions ........................................................................... 2 1.3 Management of Diseases on Turfgrass ................................................................................. 4 1.3.1 Cultural control ............................................................................................................... 4 1.3.2 Chemical control............................................................................................................. 5 1.4 Fungicide Resistance ............................................................................................................. 8 1.4.1 Dicarboximide resistance ............................................................................................... 9 1.4.2 Demethylation inhibitor resistance ............................................................................... 11 1.4.3 Resistance-associated fitness costs ............................................................................... 12 1.5 High-throughput Sequencing and Analysis......................................................................... 14 1.6 Hypotheses and Objectives ................................................................................................. 16 1.6.1 Hypotheses.................................................................................................................... 16 1.6.2 Objectives ..................................................................................................................... 17 Chapter 2 Turfgrass sampling, fungal isolation, and fungicide sensitivity testing ....................... 25 2.1 Introduction ......................................................................................................................... 25 2.1.1 Fungicide resistance ..................................................................................................... 26 2.1.2 Fungicide resistance in Microdochium nivale .............................................................. 27 2.2 Methods and Materials ........................................................................................................ 28 2.2.1 Sample collection ......................................................................................................... 28 2.2.2 Fungal isolation and storage ......................................................................................... 28 2.2.3 Golf course fungicide application records .................................................................... 29 2.2.4 Fungicide sensitivity testing ......................................................................................... 30 iv 2.3 Results ................................................................................................................................. 33 2.3.1 Isolate collection ........................................................................................................... 33 2.3.2 Full concentration range EC50 testing ........................................................................... 33 2.3.3 Discriminatory concentration testing............................................................................ 34 2.3.4 Golf course fungicide applications ............................................................................... 36 2.4 Discussion ........................................................................................................................... 37 Chapter 3 Fungicide effects on insensitive isolates in the field .................................................... 62 3.1 Introduction ......................................................................................................................... 62 3.1.1 General overview of field resistance ............................................................................ 62 3.1.2 Field resistance to dicarboximide and DMI fungicides in other species ...................... 64 3.2 Methods ............................................................................................................................... 66 3.2.1 Isolate selection ............................................................................................................ 66 3.2.2 Inoculum production..................................................................................................... 67 3.2.3 Field trials to assess reduced level of control of lab insensitive isolates ...................... 68 3.2.4 Statistical analysis......................................................................................................... 70 3.3 Results ................................................................................................................................. 70 3.3.1 Isolate selection ............................................................................................................ 70 3.3.2 Fall 2016 ....................................................................................................................... 71 3.3.3 Fall 2017 ....................................................................................................................... 73 3.3.4 Spring 2018..................................................................................................................
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