EPIDEMIOLOGY AND MOLECULAR CHARACTERIZATION OF PSEUDOMONAS SPECIES ON BLUEBERRY PLANTS by Anna DiCarlo B.Sc., University of Manitoba, 2002 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Biological Sciences Faculty of Science Anna DiCarlo 2012 SIMON FRASER UNIVERSITY Summer 2012 All rights reserved. However, in accordance with the Copyright Act of Canada, this work may be reproduced, without authorization, under the conditions for “Fair Dealing.” Therefore, limited reproduction of this work for the purposes of private study, research, criticism, review and news reporting is likely to be in accordance with the law, particularly if cited appropriately. Approval Name: Anna DiCarlo Degree: Master of Science Title of Thesis: Epidemiology and molecular characterization of Pseudomonas species on blueberry plants Examining Committee: Chair: Gerhard Gries, Professor Zamir K. Punja Senior Supervisor Professor Margo M. Moore Supervisor Professor Julian Guttman External Examiner Assistant Professor, Department of Biological Sciences Simon Fraser University Date Defended/Approved: August 20, 2012 ii Partial Copyright Licence iii Abstract Severe bacterial blight occurs periodically in British Columbia’s blueberry fields for unknown reasons. Pseudomonas syringae pv. syringae (Pss) - the putative causal organism, P. viridiflava, P. fluorescens, P. tolaasii, and P.s. pathovars aceris, tagetis, apii, and antirrhini were isolated from historically blighted fields. Three Pss isolates screened on lilac plantlets were pathogenic. A two-year field study of Pss-inoculated blueberry plants showed that Pseudomonas strains are a predominant part of blueberry stem microflora. Internal populations overwintered at low levels (101-102 CFU/g), but when wounds were present during inoculation 106 CFU/g survived. Maximum epiphytic populations reached 105 CFU/g in March-April and internal populations reached 106-107 CFU/g in Feb-May. With wounding, maximum internal populations reached 107-108 CFU/g as early as Jan-Feb. A colony blot method was developed to identify Pss from diseased tissues. Combined factors including weather, inoculum load and host phenology may contribute to the sporadic nature of bacterial blight. Keywords: Pseudomonas syringae; blueberry; epiphytic survival; overwintering; molecular detection iv Dedication For my father, who loves his land and all the things that grow there. v Acknowledgements I would like to thank my advisor, Dr. Zamir Punja, for allowing me to pursue my research interests, for his advice and humour and, above all, for countering my protracted delays with his usual professional alacrity. It has been a pleasure to work in his lab. I also thank Dr. Margo Moore for serving on my advisory committee and for her encouragement and insight. She initiated my renewed and much deeper appreciation of bacteriology. Funding for this research was provided by Agriculture and Agri-Food Canada’s Advancing Canadian Agriculture and Agri-Food program through the Investment Agriculture Foundation of British Columbia. Matching funding was provided by the British Columbia Blueberry Council. I would like to express my gratitude to the blueberry growers of British Columbia for their continued interest and support of research and especially the growers of Delta, Cloverdale and Abbotsford who allowed repeated sampling in their fields. BC blueberry growers continue to produce extremely high quality fruit under increasingly difficult circumstances. Jimmy Gaskin (Gaskin Farms) and Ray Biln (Silver Valley) generously donated potted plants for field experiments and JRT Nurseries donated tissue cultured plantlets. I would like to thank Dr. Janice Elmhirst (Elmhirst Diagnostics) and Grant McMillan (ICMS) for providing space for the field trials. Dr. Diane Cuppels (AAFC Southern Crop Protection and Food Research Centre), Dr. Peter Sholberg (AAFC Pacific Agri-Food Research Centre, Summerland) and Dr. Steven Lindow (UC Berkeley) kindly shared bacterial strains and Lynn Lashuk (Engage Agro) donated BlightBan® A506. Dr. Chris Wendt (NuFarm Agricultural Inc.) donated a sample of Blightban C9-1. Vippen Joshi and Maria Jeffries of the BC Ministry of Agriculture’s plant diagnostic lab provided guidance and use of the Biolog microplate reader and software and also shared bacterial strains. Thanks to Grigory Khaskin for assistance with the rotovap and Dr. Dipankar Sen’s lab for loan of equipment. SFU Biological Sciences technicians Fan Sozzi-Guo, Cheryl Leonard, Alex Fraser and Leslie Dodd kindly helped with innumerable items in the lab and greenhouse. Thanks to Karen Bedford (AAFC Pacific Agri-Food Research Centre, Summerland) for her suggestions and sharing reprints. SFU (graduate fellowship) provided financial assistance. This research would not have proceeded without the input and guidance of Dr. Siva Sabaratnam and Mark Sweeney of the BC Ministry of Agriculture. Thank you for vi the hours of discussion, suggestions and direction. Mark also obtained a sample of Serenade® on my behalf. I would like to thank Karina Sakalauskas of the BC Blueberry Council for her friendship and her great efforts in being an effective liaison between growers and researchers. Members of the Punja lab past and present, especially Dr. Syama Chatterton, Dr. Owen Wally and Sarah Chen provided invaluable advice, assistance and laughs. I would also like to express my deep appreciation to my partner Ron for his support over the years. vii Table of Contents Approval .......................................................................................................................... ii Partial Copyright Licence ............................................................................................... iii Abstract .......................................................................................................................... iv Dedication ....................................................................................................................... v Acknowledgements ........................................................................................................ vi Table of Contents .......................................................................................................... viii List of Tables ................................................................................................................... x List of Figures................................................................................................................. xi List of Acronyms ............................................................................................................ xiii 1. Introduction .......................................................................................................... 1 1.1. Highbush blueberry ................................................................................................ 1 1.1.1. Economic importance in the Fraser Valley .................................................. 2 1.1.2. Botany......................................................................................................... 2 1.1.3. Horticulture ................................................................................................. 4 1.1.4. Breeding ..................................................................................................... 5 1.1.5. Pests and management .............................................................................. 5 1.2. Pseudomonas syringae pv. syringae van Hall (Pss) ............................................... 7 1.2.1. Description .................................................................................................. 7 1.2.2. Optimal growth conditions ........................................................................... 7 1.2.3. Taxonomy ................................................................................................... 9 1.2.4. Methods of identification ............................................................................. 9 1.2.5. Habitat ...................................................................................................... 12 1.2.6. Host plant species ..................................................................................... 14 1.2.6.1. Woody host plants ...................................................................... 14 1.2.6.2. Infection process ........................................................................ 16 1.2.7. Virulence factors ....................................................................................... 17 1.2.7.1. Toxins ......................................................................................... 18 1.2.7.2. Extracellular polysaccharides ..................................................... 21 1.2.7.3. Indole-3-acetic acid synthesis ..................................................... 23 1.2.7.4. Type three secrection system and effectors ................................ 24 1.2.7.5. Ice nucleation activity .................................................................. 26 1.2.7.6. Cell wall degrading enzymes ...................................................... 27 1.2.8. Environmental fitness ................................................................................ 28 1.3. Bacterial blight: Blueberry-Pseudomonas pathology ............................................. 31 1.3.1. Symptomology .........................................................................................