Resistance to pear decline phytoplasma and its relationship to pathogen overwintering, host response and foliar symptoms by Mina Kaviani A Thesis Presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Plant Agriculture Guelph, Ontario, Canada © Mina Kaviani, August, 2014 Abstract Resistance to pear decline phytoplasma and its relationship to pathogen overwintering, host response and foliar symptoms Mina Kaviani Co-advisors: University of Guelph, 2014 Dr. D. M. Hunter Dr. P. H. Goodwin Pear Decline (PD) phytoplasma populations were measured using quantitative real-time PCR over a year in non-symptomatic pear trees (HW620 as scion) on the rootstocks, OH×F87 (PD resistant), OH×F69 (PD susceptible) and Bartlett (PD susceptible), growing in the Niagara region (Ontario, Canada). Seasonal patterns of PD populations were not greatly altered by the rootstock. Maximum populations occurred during late summer for leaves and shoots, and minimums during mid-winter for shoots and early spring for leaves, whereas maximum populations in roots were in mid-winter and minimums in mid-spring. PD populations for each month were typically less in leaves than in shoots, but both were much less than in roots. Regardless of tissue type, PD populations were lowest with OH×F87, then higher with OH×F69 and highest with Bartlett rootstock indicating that rootstock resistance affects PD populations in scion tissues. PD phytoplasma populations and expression patterns of six pear genes were also compared between leaves, shoots and roots of non- infected and PD-infected pear trees showing symptoms of leaf curling (selection 9328-1) or leaf reddening (selection 8824-1). The only significant differences in PD populations were higher numbers in roots of selection 9328-1 than 8824-1. More genes had significantly higher expression in PD-infected than non-infected tissues for selection 8824-1 than 9328-1 indicating stronger host responses. A comparison of PD-infected selection 8824-1 to 9328-1 showed greater up-regulation of genes in selection 8824-1 for sucrose synthase in leaves, acid invertase in leaves and roots, alcohol dehydrogenase in shoots, chitinase in all tissues, and phloem protein in roots, whereas greater up-regulation in PD-infected selection 9328-1 than 8824-1 was observed only in shoots for sucrose synthase, acid invertase and phenylalanine ammonia-lyase. Up-regulation of sucrose synthase and acid invertase could be associated to localized changes in sugars, while up-regulation of alcohol dehydrogenase, chitinase class III, phenylalanine ammonia-lyase and phloem protein2 could be part of triggered immunity. The results indicate that leaf reddening may be due to greater triggered immunity in selection 8824-1 resulting in an accumulation of anthocyanins, while leaf curling in selection 9328-1 may be due to water stress because of more dysfunctional phloem. Acknowledgments I would like to express my gratitude to my advisor, Dr. David Hunter, for providing me with the opportunity to pursue my research in plant agriculture. I benefited from his vast knowledge in pear breeding and horticulture, which enriched my experience in working on pears. I was fortunate to have Dr. Goodwin as my advisor, whose knowledge of plant pathology amazed me in several ways and his analytical knowledge helped me considerably in writing my thesis. I would like to thank the other members of my committee: Dr. Annette Nassuth for the assistance she provided in part of my lab work during my first part of study, sharing her expertise in molecular biology methods and teaching me the real meaning of organization in a lab; and Dr. Larry Erickson for serving on my advisory committee. I want to thank all the kind people I met at the Vineland Station campus of the University of Guelph and Agriculture and Agri-Food Canada (AAFC). My thanks go to Cheryl Collucci for welcoming me into the laboratory and teaching me many molecular techniques, and to Darlene Nesbitt whom I would never forget for her endless support and love during my lab work at Vineland Station in Dr. Hunter's lab. She has been a source of love and energy ever since. I must also acknowledge Dr. Peter Pauls for departmental support in covering my tuition fees. Appreciation also goes out to Jean Wolting who provided best advice to make my graduate program less stressful. Thanks also goes out to Dr. Chrystel Olivier and Dr. Istvan Rajcan, who kindly agreed to be my external and internal examiners, and their feedback in the final stages of my degree was invaluable. I would like to thank my parents and siblings for the support they provided me through my entire life and for their encouraging presence in my daily life throughout my graduate studies, despite the geographical distance. Last, but by no means least, I acknowledge my partner in life and my best friend, Mostafa, without whose love, and patience, I would not have finished this thesis. III Table of Contents Acknowledgments..........................................................................................................................III Table of Contents .......................................................................................................................... IV List of Tables ............................................................................................................................... VII List of Figures ............................................................................................................................. VIII List of Abbreviations ................................................................................................................... XII Chapter 1 ......................................................................................................................................... 1 Literature Review............................................................................................................................ 1 1.1. Introduction .......................................................................................................................... 1 1.2. Phytoplasma disease and their economic importance.......................................................... 2 1.3. Symptoms and spread of phytoplasma disease .................................................................... 3 1.4. 16SrX: Apple Proliferation group........................................................................................ 5 1.4.1. Pear Decline, Candidatus Phytoplasma pyri.................................................................... 6 1.4.2. PD resistance .................................................................................................................... 8 1.5. Phytoplasma detection and identification ............................................................................ 9 1.5.1. Electron microscopy and DAPI staining ........................................................................ 11 1.5.2. Dot-blot hybridization .................................................................................................... 12 1.5.3. Polymerase Chain Reaction ........................................................................................... 13 1.5.4. Apple proliferation (AP)-specific PCR .......................................................................... 14 1.5.5. Restriction Fragment Length Polymorphisms (RFLP) .................................................. 16 1.5.6. Real-Time PCR .............................................................................................................. 17 1.6. Colonization of woody plants by phytoplasmas ................................................................ 18 1.7. Phytoplasma genes and genomes ....................................................................................... 19 1.8. Phytoplasma pathogenicity and virulence ......................................................................... 22 1.9. Phytoplasma - plant interactions ........................................................................................ 24 1.9.1. Phytoplasma effects on plant development .................................................................... 24 1.9.2. Phytoplasma effects on photosynthesis and sugar metabolism...................................... 25 1.9.3. Phytoplasma effects on plant secondary metabolites ..................................................... 27 IV 1.9.4. Phytoplasma effects on plant gene expression ............................................................... 28 1.10. Research goals...................................................................................................................... 33 1.11. Hypothesis and objectives.................................................................................................... 34 1.11.1. Hypothesis......................................................................................................................... 34 1.11.2. Objectives.......................................................................................................................... 34 Chapter 2 ....................................................................................................................................... 41 Pear rootstock effects on seasonal colonization pattern of pear decline phytoplasma ................. 41 2.1. Introduction ............................................................................................................................ 41 2.2.