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Increasing Accuracy of Powdery Mildew (Ascomycota, Erysiphales) Identification Using Previously Untapped DNA Regions

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Increasing Accuracy of Powdery Mildew (Ascomycota, Erysiphales) Identification Using Previously Untapped DNA Regions Increasing accuracy of Powdery Mildew (Ascomycota, Erysiphales) identification using previously untapped DNA regions Thesis submitted for the degree of DOCTOR OF PHILOSOPHY Oliver Ellingham School of Biological Sciences Plant and Fungal Systematics Research April, 2017 Supervisor: Dr Alastair Culham Declaration: I confirm that this is my own work and the use of all material from other sources has been properly and fully acknowledged. Oliver Ellingham i Abstract The powdery mildews (Ascomycota, Erysiphales) are a group of obligate biotrophic fungi found on nearly 10,000 angiosperm plant hosts globally including many that are important horticultural and agricultural plants. Infection can greatly reduce the appearance and vigour of the host therefore reducing attractiveness and yields significantly. A reliable and efficient method is required for unambiguous identification of these often cryptic species such that spread to new areas and/or new hosts can be detected rapidly and controlled early. This research aims to combine currently accepted techniques – host identification, fungal morphological analysis, DNA sequencing of the fungal rDNA ITS region – with sequencing of additional nuclear DNA regions in order to increase the reliability of the identification process via BLAST, DNA Barcoding, and phylogenetic reconstruction. Samples were collected through the Powdery Mildew Survey (a citizen science scheme), begun in 2014 and concluded in 2016. Generic fungal DNA primers were found to amplify non-powdery mildew species, some of which were mycoparasites, as well as powdery mildews, and were therefore not a useful technique for accurate identification of powdery mildews. Consequently specific primers were developed for the amplification of the Actin, β-tubulin, Chitin synthase, Mcm7, Translation elongation factor 1-α, and Tsr1 regions. Results indicate that several of these regions could be used alongside ITS to increase identification power (reliability and accuracy), with regions Mcm7 and β- tubulin performing particularly well. These rapid diagnostic techniques could provide a valuable tool for plant quarantine, and plant breeding, particularly for greater security in the movement of plants and plant products in trade. ii Acknowledgements It is my great pleasure to reach this stage and be able to thank everyone involved in the powdery mildew PhD. I offer sincere thanks to the funders who made the research possible; the Biotechnology and Biological Sciences Research Council and the Royal Horticultural Society. As well as my initial supervisor, Béatrice Henricot, and principal supervisor Alastair Culham for the inception of the project and seeing a fungal systematist in me. I am also indebted to John David who stepped in for the lion’s share as my secondary supervisor. Thanks also must go to my lab mates, particularly Maria Christodoulou for her time and Masters student Waheed Arshad for his help in 2014. I am grateful to Roger Cook, whose early masterclasses set me on my powdery way and to Levente Kiss for the inception and organisation of the Powdery mildew summer school. Special thanks must go to the powdery mildews themselves whose lifestyle and beautiful forms are always varied and rarely boring. Thanks for providing me with such a fascinating study organism and introducing me to the wonders and utility of fantastic Fungi. I wish to show my gratitude to the science outreach staff of the British Mycological Society, Botanical Society of Britain & Ireland, Plant Heritage (NCCPG), and the British Society of Plant Pathology for help promoting the Powdery Mildew Survey. I am also grateful to the outreach staff of the Royal Horticultural Society as well as their pathology and media teams who hosted me on numerous occasions. I would like to use this opportunity to thank my friends and family and the welcoming staff at C.U.P. for offering me refuge from academia and fellow researchers who I am honoured to call friends: Lionel Smith, Matthew McFrederick, Gedimimas Baltulionis, and Irene Teixidor. Finally, I am truly indebted and thankful to Halala Hewazy for her unwavering supply of great food, friendship, smiles, and understanding. iii Contents CHAPTER 1: INTRODUCTION ........................................................................................................................... 1 1.1: POWDERY MILDEWS ......................................................................................................................................... 1 1.2: THE IMPORTANCE OF FUNGAL DISEASES ................................................................................................................ 2 1.3: IMPORTANCE OF POWDERY MILDEW .................................................................................................................... 3 1.4: PM BIOLOGY ................................................................................................................................................... 5 1.5: ESTABLISHED SPECIES IDENTIFICATION .................................................................................................................. 6 1.5.1: Established relationships, phylogeny, and taxonomy ........................................................................ 14 1.6: DNA SEQUENCE ANALYSES ............................................................................................................................... 17 1.6.1: NCBI GenBank Nucleotide BLAST ....................................................................................................... 17 1.6.2: Phylogenetics ..................................................................................................................................... 17 1.6.3: DNA barcoding ................................................................................................................................... 18 1.7: UK PM BASELINE REVIEW ................................................................................................................................ 19 1.8: AIMS ........................................................................................................................................................... 20 CHAPTER 2: SAMPLING – THE POWDERY MILDEW CITIZEN SCIENCE SCHEME ............................................... 21 2.1: INTRODUCTION .............................................................................................................................................. 21 2.1.1: Citizen science .................................................................................................................................... 21 2.1.1.1 Value, usage, and caution ...................................................................................................................... 21 2.1.1.2 Use in the project .................................................................................................................................. 22 2.2: MATERIALS AND METHODS .............................................................................................................................. 25 2.2.1: Citizen science launch & engagement ............................................................................................... 25 2.2.2: Sample collection ............................................................................................................................... 25 2.2.3: Sample handling ................................................................................................................................ 26 2.2.4: Culturing of PM samples .................................................................................................................... 30 2.3: RESULTS ....................................................................................................................................................... 31 2.3.1: Samples received and social engagement ......................................................................................... 31 2.3.2: Sample distribution ............................................................................................................................ 32 iv 2.3.3: Host profiling ..................................................................................................................................... 32 2.3.4: Culturing of PM samples .................................................................................................................... 36 2.4: DISCUSSION .................................................................................................................................................. 38 2.4.1: Samples received and social engagement ......................................................................................... 38 2.4.2: Sample distribution ............................................................................................................................ 40 2.4.3: Host profiling ..................................................................................................................................... 41 2.4.4: Culturing of PM samples .................................................................................................................... 41 2.5: CONCLUSIONS ............................................................................................................................................... 42 CHAPTER 3: SPECIES IDENTIFICATION USING ESTABLISHED TECHNIQUES ..................................................... 43 3.1: INTRODUCTION .............................................................................................................................................
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