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Dogwood Anthracnose Caused by Discula Destructiva on Cornus Spp

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Dogwood Anthracnose Caused by Discula Destructiva on Cornus Spp Dogwood Anthracnose Caused by Discula destructiva on Cornus spp. in Canada by Mihaela Stanescu A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Environmental Biology Guelph, Ontario, Canada © Mihaela Stanescu, December, 2013 ABSTRACT DOGWOOD ANTHRACNOSE CAUSED BY DISCULA DESTRUCTIVA ON CORNUS SPP. IN CANADA Mihaela Stanescu, Advisor: University of Guelph, 2013 Professor T. Hsiang The most important fungal disease of dogwoods in North America is anthracnose caused by Discula destructiva. This disease affects Cornus florida (flowering dogwood), C. nuttallii (Pacific dogwood), and C. kousa (kousa dogwood). It has not been well studied in Ontario nor anywhere in Canada. In this study, over 2,500 fungal isolates were obtained from symptomatic samples of C. alba, C. alternifolia, C. amomum, C. kousa, C. florida, C. nuttallii, C. racemosa and C. sericea. To help distinguish between foliar symptoms of different etiologies, a “Dogwood Disease Symptom Guide” was produced. Isolates were divided into 13 fungal morphotypes, of which D. destructiva accounted for 39% of all isolates. Pathogenicity testing of Discula destructiva on C. florida satisfied Koch’s postulates, and this fungus was confirmed as the causal agent of dogwood anthracnose in southwestern Ontario (C. florida) and southwestern British Columbia (C. nuttallii). Wounds and leaf trichomes may provide a point of entry and help the pathogen survive endophytically without producing symptoms on “non-host” plants such as oak, maple and pear. The pathogen was found to survive for over 12 weeks at -20 °C, and the optimal growth temperature was found to be between 20-25 °C, but temperatures as high as 30 °C inhibited the growth, and the fungus died after one week incubation at 40 °C. The finding of only one mating type within D. destructiva populations (122 isolates) explains the lack of sexual reproduction of this fungus in North America, and along with the SSR results, reconfirms the low genetic variability within its populations. ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Tom Hsiang for giving me the opportunity to obtain my MSc. He helped me not only with his knowledge, guidance and expertise, but also with his encouragements and his constant trust in my ability to complete this project, although at times I wavered. I am also thankful to the other members in the Advisory Committee, Dr. John McLaughlin and Dr. Richard Wilson, for their knowledge and competent advice. I am thankful to the Ontario Ministry of Natural Resources who funded this research. The input of collaborators who helped with sample collection, and the support of nurseries which donated plant material, are also much appreciated. I am also thankful to the lab technician Angie Darbison, who started this project and helped with the DNA extraction of the first Discula destructiva isolate obtained in our lab, for the purpose of genome sequencing. I am very grateful to my lab mates, who with their advice and friendly attitude made my life at the lab a lot easier. Especially I would like to thank Amy Shi and Linda Jewell, for helping me get through the hard times at the beginning of my graduate studies, and also for their constant advice. I am also grateful to my family and friends for their encouragements. Finally, I would like to thank my husband, Adrian, for his unconditional love and priceless support which helped me bring this project to an end. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS...................................................................................................... iii TABLE OF CONTENTS.......................................................................................................... iv LIST OF TABLES.................................................................................................................. viii LIST OF FIGURES.................................................................................................................. x LIST OF APPENDICES.......................................................................................................... xiii LIST OF ABBREVIATIONS AND ACRONYMS.................................................................. xiv Chapter One: Literature Review................................................................................................ 1 1.1 Introduction..................................................................................................................... 1 1.2 Dogwood (Cornus) species............................................................................................. 2 1.3 Major fungal diseases of dogwoods................................................................................ 8 1.3.1 Spot anthracnose caused by Elsinoe corni.............................................................. 8 1.3.2 Septoria leaf spot caused by Septoria cornicola..................................................... 8 1.3.3 Limb dieback caused by Colletotrichum gloeosporioides and C. acutatum........... 9 1.3.4 Powdery mildew caused by Microsphaera penicillata and Phillactinia guttata ... 10 1.3.5 Dogwood anthracnose caused by Discula destructiva............................................. 10 1.3.5.1 Disease history and distribution........................................................................ 10 1.3.5.2 Symptoms......................................................................................................... 12 1.3.5.3 Morphology and reproduction of Discula destructiva ..................................... 14 1.3.5.4 Population structure and origin of Discula destructiva.................................... 15 1.3.5.5 Infection process.............................................................................................. 17 1.4 Hypothesis and objectives............................................................................................... 19 Chapter Two: The Causal Agent of Dogwood Anthracnose...................................................... 33 2.1 Introduction ...................................................................................................................... 33 2.1.1 Morphological identification of fungal isolates ...................................................... 33 2.1.2 Molecular identification of fungal isolates.............................................................. 34 2.1.3 Dogwood diseases .................................................................................................... 35 2.1.4 Objectives................................................................................................................. 36 2.2 Materials and methods..................................................................................................... 37 2.2.1 Sample collection..................................................................................................... 37 2.2.2 Media preparation...................................................................................................... 37 iv 2.2.3 Fungal isolation from symptomatic dogwood tissues............................................. 38 2.2.4 Identification of fungal isolates with morphological techniques............................ 38 2.2.5 DNA extraction...................................................................................................... 39 2.2.6 Sequencing the genome of Discula destructiva...................................................... 40 2.2.7 Specific primer design and selection...................................................................... 40 2.2.8 PCR amplification................................................................................................... 43 2.2.9 Sequencing of PCR products and result analysis ................................................... 43 2.2.10 Pathogenicity testing ........................................................................................... 44 2.3 Results............................................................................................................................ 45 2.3.1 Symptomatic samples of dogwood......................................................................... 45 2.3.2 Morphological characteristics of fungal isolates.................................................... 47 2.3.3 Molecular identification of morphotypes............................................................... 49 2.3.4 Genome sequencing and assembly......................................................................... 50 2.3.5 Primer amplification................................................................................................ 50 2.3.5.1 Testing specific primers.................................................................................. 51 2.3.5.2 Amplification results with the specific primers.............................................. 53 2.3.6 Testing the pathogenicity of Discula destructiva ................................................... 53 2.4 Discussion........................................................................................................................ 54 Chapter Three: Biological Characteristics, Pathogenicity and Genetic Variation in Discula destructiva.................................................................................................................................. 73 3.1 Introduction...................................................................................................................... 73 3.1.1 Growth and survival
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