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Fungicide-Resistance Management Tactics: Impacts on Zymoseptoria Tritici Populations

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Fungicide-Resistance Management Tactics: Impacts on Zymoseptoria Tritici Populations View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Central Archive at the University of Reading Fungicide-resistance management tactics: impacts on Zymoseptoria tritici populations Thesis submitted in fulfillment of the Degree of Doctor of Philosophy School of Agriculture, Policy and Development Hilda Dooley January 2015 Abstract Azoles and Succinate Dehydrogenase Inhibitors (SDHIs) are the main fungicides available for septoria tritici blotch control, causal agent Zymoseptoria tritici. Decline in azole sensitivity, in combination with European legislation, poses a threat to wheat production in Ireland. Azole fungicides select CYP51 mutations differentially; it was hypothesised that using combinations of azoles could be an effective anti-resistance tool. Naturally inoculated field experiments were carried out in order to understand the impacts of using combinations of azoles, epoxiconazole and metconazole, on azole sensitivity. Approximately 3700 isolates were isolated and their sensitivity to both azoles analysed. Findings showed that limiting the number of applications, by alternating each fungicide, slowed selection for reduced azole sensitivity. Limiting azole use by reducing doses did not reduce selection for decreased azole sensitivity. Although not complete, cross-resistance was observed between the two azoles, which will lead to general reduction in azole sensitivity. A sub-selection of isolates from each treatment at each location were analysed for changes in the CYP51 gene. Sequence analysis identified 49 combinations of mutations in the CYP51 gene, and three different inserts in the CYP51 promoter. Intragenic recombination also featured in these populations. Baseline studies of five new SDHIs were carried out on 209 naturally infected, non- SDHI-treated isolates. With the exception of fluopyram, cross-resistance was apparent between the SDHIs. Analysis of 2300 isolates found that when compared to the solo products, mixing the SDHI isopyrazam and the azole epoxiconazole increased epoxiconazole sensitivity, but had no apparent effect on isopyrazam sensitivity. SDHI resistance-conferring mutations were absent in the baseline and experimental isolates. As long as azoles are used, Z. tritici populations will continue to evolve towards resistance. Combining different modes-of-action, SDHIs and multi-sites, with azoles will relieve some of that selective pressure. To get the best out of available fungicides, they should be used in combination with host resistance and good crop management practices. i Declaration I confirm that this is my own work and the use of all material from other sources has been properly and fully acknowledged. Signed ………………………………. Date………………………………….. ii Acknowledgements This PhD was funded by Teagasc under its Walsh fellow scheme. The Walsh fellow scheme also funded an invaluable three month study trip to the phytopathology lab in ETH, Zurich. I would like to take this opportunity to thank a few people who supported me throughout my PhD studies. To all Teagasc research officers, technicians and farm staff who helped me whenever I needed it. Special thanks to Jim Grace and Liz Glynn for all the help with field experiments and for helping to find data which went rogue at times. Thanks Jeanne Mehenni-Ciz for the help in the lab. To the undergraduate students who helped me through the long day’s disease assessing and isolating, thank you Aaron Mullins, Gillian Darby, Clair Jouan and Aurelien Lepenettier. To all of the other PhD students at Teagasc, for keeping me sane during stressful times, and for supplying the gossip that kept me smiling, thank you. To my supervisors: for furnishing me with the skills needed to complete such a mammoth task as a PhD; for allowing me to develop as a researcher at my own pace; for such graceful reactions to my tears of frustration; and especially for almost never disagreeing with each other, thank you Steven Kildea, Mike Shaw and John Spink. At this time I must also thank Bruce McDonald (and everyone at ETH) for welcoming me into the phytopathology lab at ETH, and for facilitating the collaboration with Patrick Brunner who advised and assisted with analysis and discussion points for Chapter 3. Bruce and Patrick, thank you. To all of my family and friends who have accepted without question my virtual absence for the last two years. To my parents, who instilled in me the work ethic that got me through this PhD, and who are still working hard to keep all of their children, grandchildren and friends happy, thank you. Last, but by no means least, to Mike, for putting up with me over the last three years, I may have cracked by now if it weren’t for you. Thank you. iii Table of Contents Abstract......................................................................................................................................... Declaration................................................................................................................................. ii Acknowledgements................................................................................................................... iii Table of Contents...................................................................................................................... iv List of Figures......................................................................................................................... viii List of Tables ............................................................................................................................ xi Abbreviations.......................................................................................................................... xiv Chapter 1: Introduction...............................................................................................................1 1.1 Preface ..............................................................................................................................1 1.2 Biology of Zymoseptoria tritici ........................................................................................2 1.3 Controlling Zymoseptoria tritici .......................................................................................3 1.3.1 Crop management ......................................................................................................3 1.3.2 Host resistance ...........................................................................................................4 1.3.3 Fungicides..................................................................................................................5 1.4 Resistance evolution .........................................................................................................8 1.4.1 Fungicide resistance...................................................................................................8 1.4.2 History of fungicide resistance in Zymoseptoria tritici isolates ................................9 1.4.3 The development and spread of fungicide resistance in Zymoseptoria tritici .........11 1.5 Resistance management..................................................................................................12 iv 1.6 Aims and objectives of this Ph.D....................................................................................16 Chapter 2: Effect of azole fungicide mixtures, alternations and reduced dose rates on azole sensitivity in the wheat pathogen Zymoseptoria tritici.............................................................18 2.1 Introduction.....................................................................................................................18 2.2 Materials and methods....................................................................................................20 2.2.1 Disease and yield assessments.................................................................................21 2.2.2 Sampling Zymoseptoria tritici .................................................................................21 2.2.3 Isolating Zymoseptoria tritici...................................................................................22 2.2.4 In vitro sensitivity testing ........................................................................................25 2.2.5 Data analysis ............................................................................................................25 2.3 Results.............................................................................................................................27 2.3.1 Variability before fungicide applications ................................................................27 2.3.2 Main contrasts..........................................................................................................31 2.3.3 Principal components analysis.................................................................................34 2.3.4 Disease severity and its relationship with selection.................................................38 2.3.5 Effects if fungicides on yield ...................................................................................41 2.4 Discussion.......................................................................................................................42 Chapter 3: Molecular mechanisms associated with reduced azole sensitivity and the genetic structure of azole treated populations of Zymoseptoria tritici in Ireland .................................49 3.1 Introduction.....................................................................................................................49
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