GENETIC DIVERSITY AND POTENTIAL BIOLOGICAL CONTROL OF RUBUS SPECTABILIS Grace Sumampong B.Sc., Malaspina University-College, 1999 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Biological Sciences O Grace Sumampong 2005 SIMON FRASER UNIVERSITY Summer 2005 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. APPROVAL Name: Grace Sumampong Degree: Master of Science Title of Thesis: Genetic diversity and potential biological control of Rubus spectabilis Examining committee: Chair: Dr. C.K. Kennedy, Associate Professor Dr. Z.K. Punja, Professor Department of Biological Sciences, S.F.U. Dr. S.F. Shamoun, Research Scientist Canadian Forest Service Dr. M.M. Moore, Associate Professor Department of Biological Sciences, S.F.U. Dr. G. Allen, Associate Professor Department of Biology, University of Victoria Dr. Collette Breuil, Professor Department of Wood Science, U.B.C. Public Examiner rSk( 7A7oJ Date Approved I ABSTRACT A mycobiota survey was conducted on Vancouver Island and mainland BC to identify potential biocontrol agents of salmonberry. The species, which included 17 isolates of Bot~tiscinerea, 3 Phoma argillacea and 1 Septoria rubi, were pathogenic on detached leaves. Greenhouse trials showed P. argillacea induced foliar and stem necrosis on intact plants with an 18-hr leaf wetness period and mycelial inoculum of 2.6~10~CFUImL. In a host range test, three conifer species showed seedling tolerance and red raspberry showed initial damage but no cane blight when inoculated with P. argillacea. Salmonbeny DNA was isolated from leaves collected from five widespread populations. RAPD-PCR was used to fingerprint individuals, generating 35 loci with a 75.5% average variability. Diversity within populations was high, with an average heterozygosity of 0.38. Among population differentiation was poor, with an FSTof 0.0454; therefore, an average 5.3 migrants per generation is required to maintain this variation. ACKNOWLEDGEMENTS A big "thank you" to friends and colleagues at PFC (Shamoun Lab) and at SFU (Punja Lab) fbr their encouragement, support and help. A special thank you to Jennifer (Jen) Wilkin and Cheryl Konoff for their help with field collections, Jen for technical advice, suggestions and discussions, and Nitin Verma for being a great fellow graduate student and a dear friend. I would like to thank my funding agencies, including Natural Resource Canada, Weyerhaeuser Canada Inc., Science Council of British Columbia - GREAT Award, and Western Forest Products. I am very thankful to my supervising committee, Drs. Simon Shamoun, Zamir Punja, Margo Moore and Geraldine Allen, and all those who assisted through advice and discussions, technical support and reviews of my thesis. I wish to dedicate this work to my beloved "Lola", Eustacia Sumampong. Finally, I would like to thank my beautiful daughter, Rochelle, and Stan Khan for their patience, understanding, support and unconditional love. Rochelle, you have been my strength and inspiration. TABLE OF CONTENTS Approval ............................................................................................. ii ... Abstract ............................................................................................. 111 Acknowledgements ................................................................................. iv Table of Contents .................................................................................. v ... List of Tables ...................................................................................... VUI List of Figures ........................................................................................ x Chapter 1. Literature review ................................................................... 1 1.1. Rubus spectabilis .............................................................................. 1 Introduction ......................................................................... 1 Origin and distribution ............................................................ 1 Life history and biology ............................................................ 3 Taxonomy. phylogeny and diversity ............................................. 5 Ecological and other uses ......................................................... 8 Rubus spectabilis as a weedy species in conifer regeneration sites ......... 9 Management ....................................................................... 11 Fungal pathogens that infect R . spectabilis .................................... 13 Biological control of forest weeds with plant pathogens ............................... 15 1.2.1. Introduction ....................................................................... 15 1.2.2. Classical strategy ................................................................. 16 1.2.3. Inundative strategy .............................................................. -16 1.2.3.1. Discovery ................................................................... -17 1.2.3.2. Development ................................................................ 18 1.2.3.3. Deployment ................................................................. 18 1.2.3.4. Examples of inundative biological control agents for control of R . spectabilis and other forest weeds ................................... 19 1.2.4. Regulation ....................................................................... -21 1.2.5. Host range ........................................................................ 22 1.3. Evaluation of Didymella applanata (anamorph Phoma argillacea) as a potential biological control agent for R . spectabilis ................................... 24 1.3.1. Life cycle and biology ........................................................... 24 1.3.2. Taxonomy of P . argillacea ...................................................... 26 1.3.3. Phoma argillacea host range .................................................. -30 1.3.4. Fungal mode of infection ........................................................ 31 1.4. Objectives of research ..................................................................... 33 Chapter 2 . Occurrence of Phoma argillacea on Rubus spectabilis in British Columbia and an evaluation of its potential as a forest weed biological control agent ..................................................................................... 34 2.1. Introduction ................................................................................... -34 2.2. Materials and Methods ....................................................................... 35 2.2.1. Pathogen survey ..................................................................... -35 2.2.2. In vitro pathogenicity tests ........................................................ -37 2.2.3. Colony growth and spore germination ............................................ 37 2.2.4. Greenhouse virulence tests ......................................................... 38 2.2.4.1. Propagation of plants ........................................................... 38 2.2.4.2. Inoculum preparation ........................................................... 38 2.2.4.3. Effect of post-inoculation conditions ......................................... 39 2.2.5. Effect of P. argillacea on non-target hosts ....................................... 40 2.2.6. Data analysis ......................................................................... 41 2.3. Results and Discussion ...................................................................... 41 Pathogen survey ..................................................................... 41 Colony growth and spore germination ............................................ 43 Greenhouse virulence tests ......................................................... 44 Effect of different leaf wetness periods on infection of salmonbeny by P. argillacea ..................................................................... 50 Effect of inoculum concentration on infection of salmonbeny by P . argillacea ..................................................................... 51 Effect of filtrate on infection of salmonbeny by P. argillacea ...............51 Effect of P . argillacea on non-target hosts ....................................... 51 Chapter 3 . Genetic Diversity of Rubus spectabilis in Coastal British Columbia .....59 3.1. Introduction .................................................................................... 59 3.1.1. Life history and vegetative growth ...............................................-59 3.1.2. Plant population genetics ............................................................. 60 3.1.3. Population genetics of clonal plants .............................................. 62 3.1.4. Genetic markers ..................................................................... 65 3.1.5. Objectives ........................................................................... -69 3.2. Materials and Methods ....................................................................... 70 3.2.1. Site description ...................................................................... -70 3.2.2. Sample collection .................................................................... 70 3.2.3. DNA isolation ........................................................................ 70 3.2.4. PCR reaction .......................................................................