Understanding the Biology and Epidemiology of Dickeya solani: a Recently Emerged Bacterial Pathogen of Potato Rachel Maria Kelly PhD Thesis Heriot-Watt University & Science and Advice for Scottish Agriculture (SASA) August 2018 The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any of the information contained in it must acknowledge this thesis as the source of the quotation or information." i Abstract Dickeya solani has emerged as a major threat to potato production in Europe and Israel. Its potential impact on the Scottish potato industry was studied with the goal to develop better control strategies. Molecular techniques were employed to understand the diversity of the D. solani and its comparison to other Dickeya spp. and to map the origin and spread of infection. Using nine housekeeping genes, a MLSA typing scheme was created and published online to facilitate its future tracking. MLSA established genetic variation between D. solani isolates and it was determined that D. solani is likely to be clonal. Due to the similarities between D. solani isolates, which was further aided with the sequencing of three full D. solani genomes, single nucleotide polymorphisms were discovered. Eight SNPs were identified and further investigated; allowing for the development of pyrosequencing assays. Two real-time PCR assays were developed to improve the diagnostics of D. solani by detecting and identifying D. solani specifically. These assays were further evaluated as part of the Euphresco II project on blackleg and soft rot disease. The spread of D. solani is likely to be through latently infected seed potatoes; therefore, the transmission of D. solani from infected seed potato to daughter tubers was studied. It has also been suggested that D. solani can survive and spread through stored tubers and the potential for D. solani to survive in plant material was assessed. The effectiveness of disinfectants commonly used in agriculture was also investigated and it was determined when used at the manufacturer’s recommended concentration, they were effective at controlling the pathogen on surfaces. ii Dedicated to Kelly family: Mary, Shaun, Natalie, Lauren and Timothy. iii Acknowledgments There are no adequate words to express the gratitude I have for my supervisor, Gerry Saddler, who has shown tremendous patience and support throughout this whole process. His guidance has been invaluable and he went above and beyond what I ever expected. Having him as my supervisor was the key factor in my completion of this thesis and the reason I loved this project so much. I would like to thank Wilf Mitchell from Heriot-Watt University who also provided guidance and support and took the time to read this thesis in his retirement, and Euan Brown who stepped in at the end of my PhD studies to represent me as a supervisor. Without Euan’s advice and support it would have been impossible for me to complete and submit this work and I am so grateful for his patience and for the time he has sacrificed to guide me. Completing this research as part of a larger project afforded me the opportunity to work with a number of collaborators and I would like to thank Ian Toth, Sonia Humphris and Leighton Pritchard from JHI and John Elphinstone from Fera for sharing their knowledge and time and in return, for allowing me to share my own work. I am grateful to the entire Diagnostic and Molecular Biology department at SASA, who were crucial in the research process, especially Greig Cahill, Andrew Jeffries, Karen Fraser, Alex Reid, Vince Mulholland and David Kenyon, who not only put up with me in the lab but also provided guidance and advice. With their support I was never able to get discouraged and from them I learned so much. I am appreciative of all my friends and colleagues at SASA who provided support (especially during my rants and mini- breakdowns!) and a wonderful environment in which to complete my PhD. Finally, I want to thank my family, especially my parents, who have supported me unconditionally through this process. My family have always inspired me and driven me to be the best I could be and without them I never would have undertaken this PhD, let alone complete it. I am eternally grateful for all of you and love you all. iv ACADEMIC REGISTRY Research Thesis Submission Please note this form should be bound into the submitted thesis. Name: Rachel Maria Kelly School: Engineering and Physical Science Version: (i.e. First, Final Degree Sought: PhD Resubmission, Final) Declaration In accordance with the appropriate regulations I hereby submit my thesis and I declare that: 1. The thesis embodies the results of my own work and has been composed by myself 2. Where appropriate, I have made acknowledgement of the work of others 3. Where the thesis contains published outputs under Regulation 6 (9.1.2) these are accompanied by a critical review which accurately describes my contribution to the research and, for multi-author outputs, a signed declaration indicating the contribution of each author (complete Inclusion of Published Works Form – see below) 4. The thesis is the correct version for submission and is the same version as any electronic versions submitted*. 5. My thesis for the award referred to, deposited in the Heriot-Watt University Library, should be made available for loan or photocopying and be available via the Institutional Repository, subject to such conditions as the Librarian may require 6. I understand that as a student of the University I am required to abide by the Regulations of the University and to conform to its discipline. 7. Inclusion of published outputs under Regulation 6 (9.1.2) shall not constitute plagiarism. 8. I confirm that the thesis has been verified against plagiarism via an approved plagiarism detection application e.g. Turnitin. * Please note that it is the responsibility of the candidate to ensure that the correct version of the thesis is submitted. Signature of Date: Candidate: Submission Submitted By (name in capitals): Signature of Individual Submitting: Date Submitted: For Completion in the Student Service Centre (SSC) Received in the SSC by (name in capitals): Method of Submission (Handed in to SSC; posted through internal/external mail): E-thesis Submitted (mandatory for final theses) Signature: Date: Page 1 of 2 RDC Clerk/Nov 2018 ACADEMIC REGISTRY Inclusion of Published Works Declaration This thesis contains one or more multi-author published works. In accordance with Regulation 6 (9.1.2) I hereby declare that the contributions of each author to these publications is as follows: Citation details e. g. Author 1 and Author 2, Title of paper, Title of Journal, X, XX-XX (20XX) Author 1 Contribution…. Author 2 Contribution…. Signature: Date: Citation details e. g. Author 1 and Author 2, Title of paper, Title of Journal, X, XX-XX (20XX) Author 1 Contribution…. Author 2 Contribution…. Signature: Date: Citation details e. g. Author 1 and Author 2, Title of paper, Title of Journal, X, XX-XX (20XX) Author 1 Contribution…. Author 2 Contribution…. Signature: Date: Page 2 of 2 RDC Clerk/Nov 2018 Contents Abstract………………………………………………………………………………….i Dedication.......................................................................................................................iii Acknowledgments………………………………………………………………...........iv Declaration Statement...………………………………………………………………..v List of Tables….…...…………………………………………………………………...xi List of Figures...............................................................................................................xiii List of Publications...….……………………………………………………………...xiii Chapter 1. Introduction….…………………………………...………………………..1 1.1. The History of Potato....……………………………………………...…………..1 1.2. The Development of the Cultivated Potato....…………………….……………...2 1.3. The Significance of Potato as a Crop...…………..................................................3 1.4. Modern Potato Breeding and Advancements.....………………………………...4 1.5. Potato Genomics...….……………………………………………………………6 1.6. Seed Classification Schemes……………………………………………….….....7 1.7. Scottish Potato Production…………………………………………………….....7 1.8. Threats to Potato Production……………………………………………….…...10 1.9. History of Dickeya spp…………………………………………………….……13 1.10. Dickeya spp. on Potato………………………………………………………...16 1.11. Economic losses due to Dickeya spp.…………………………………...…….16 1.12. Factors influencing Dickeya Disease Development…………………………..17 1.13 . Transmission and Survival of Dickeya spp.…………………………………..18 1.14 .The importance of Diagnostics of Dickeya spp.…………………………….....19 1.15 . Aims and Objectives of PhD project……………………………………….....20 Chapter 2. The Development of a Real-Time PCR Diagnostic Assay specific for Dickeya solani………………………………………………………………………….22 2.1. Introduction…………………………….………………………...……………..22 2.1.1 Potato Industry of Great Britain…………………………………………...22 2.1.2 Diagnostics and Detection of Potato Pathogens…………………...……...22 2.1.3 Molecular Advancements in Techniques for Diagnostics and Detection….23 2.1.4 Polymerase Chain Reaction………………………………………………..24 2.1.5 Detection Methods for Dickeya sp.………………………………………...25 2.1.6 Specific Aims of Developing PCR Assay……………………………….......26 2.2. Materials and Methods………………………….……………………………....28 2.2.1 Cultures…………………………………………………………………….28 vii 2.2.2 Real time assay……………………………………………………………..28 2.2.3 Validation of Real-Time Assays…………………………………………....29 2.2.4 Further validation of PCR assays: “Ring Test.”…………………………..30 2.3. Results from Real-time Diagnostic Assays…………………….…..…………...34 2.3.1