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Athelia) Rolfsii and Related Species (Abstr. GENETIC DIVERSITY AND VARIATION IN SCLEROTIUM (ATHELIA) ROL FSII AND RELATED SPECIES COLLEEN E. HARLTON B. Sc., Simon Fraser University, 1991 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department Biological Sciences @ COLLEEN E. HARLTON 1995 SIMON FRASER UNIVERSITY April 1995 All rights resewed. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. APPROVAL Name: Colleen Esther Harlton Degree: Master of Science Title of Thesis: GENETIC DIVERSITY AND VARIATION IN SCLEROTIUM ROLFSII AND RELATED SPECIES Examining Committee: Chair: Dr. J. Borden, Professor Dr. Z. Punja, Associat$Profes , Senior Supervisor Department of Biological Sci Dr. M. Smith, Profe&or Department of Biological Sciences, SFU r. C.A. L6vesqu ReseaM Scientist ?griculture Cm& Research Station, Vancouver Dr. L. Druehl:Professor C Department of Biological Sciences, SFU Public Examiner Date Approved (&d /J. /PPi Y PARTIAL COPYRIGHT LICENSE I hereby grant to Simon Fraser Universi the right to lend my thesis, pro'ect or extended essay (the title o7' which is shown below) to users or' the Simon Fraser University Library, and to make partial or single copies only for such users or in response to a request from the library of any other university, or other educational institution, on its own behalf or for one of its users. I further agree that permission for multiple copying of this work for scholarly purposes may be granted by me or the Dean of Graduate Studies. It is understood that copying or publication of this work for financial gain shall not be allowed without my written permission. Title of Thesis/Project/Extended Essay Genetic Diversity and Variation in Sclerotium rolfsii and related species. Colleen E. Harlton (name) April 18, 1995 (date) ABSTRACT A worldwide collection of 119 isolates of Sclerotium (Athelia) rolfsii from 34 hosts and 22 different geographic areas, 11 isolates of S. delphinii from three hosts and three areas, and two isolates of S. coffeicola, were paired on potato dextrose agar (PDA) to determine mycelial compatibility groups (MCGs). A total of 49 MCGs were detected in S. rolfsii, three in S. delphinii, and two in S. coffeicola. Isolates originating from one host and one geographic area often belonged to the same MCG. Members of an MCG, however, were frequently comprised of isolates from widely different areas and hosts. There were 33 single-mem ber MCGs in S. rolfsii. The MCGs were then compared with variation in restriction fragment length polymorphisms (RFLPs) of amplified portions of mitochondria1 and nuclear rDNAs. Internal transcribed spacer (ITS)-RFLP analysis of 80 isolates of the three Sclerotium species, and three Athelia species (an outgroup), indicated that the Sclerotium species could be distinguished from each other and from the Athelia species. Using combined RFLP patterns generated by four restriction enzymes (Alul, Hpall, Rsal and Mbol), field isolates could be separated into ITS-RFLP Groups (ITS-RFLP-G). A total of 12 ITS-RFLP-Gs (I - XII) were observed in S. rolfsii, one in S. delphinii and two in S. coffeicola. MCGs were frequently found to be comprised of several ITS-RFLP-Gs, indicating that members were not clonally derived. Polymerae Chain Reaction (PCR)-amplified ITS regions for 29 single- basidiospore (SB) strains, which were obtained from four parental isolates of S. rolfsii (MCG 1 and ITS-RFLP-G Ill), were also digested with several different enzymes. Two SB ITS-RFLP patterns that segregated from the RFLP pattern observed in the parental isolates were observed using Mbol. The segregation of ITS-RFLP patterns among the SB strains (presumed homokaryons) suggests the occurrence of two distinct ribosomal DNA (rDNA) types in the parental isolates, each detected by its different restriction sites, which we refer to as rDNA subtypes (ITS-RFLP subtype patterns). Since the sum of restriction digest products in field isolates of S. rolfsii and S. delphinii frequently exceeded the amplified product (720 bp), this further confirms the presence of distinct rDNA subtypes which could reflect the heterokaryotic nuclear condition of field isolates. ITS-RFLP subtype patterns for field isolates of Sclerotium and Athelia spp. differing in endonuclease sites were represented in restriction maps for Alul, Hpall, Rsal and Mbol enzymes and phylogenetic analyses using parsimony were conducted. The similarity in banding patterns among S. rolfsii and S. delphinii isolates, the overlap in host range, and the results from phylogenetic analysis all suggest a close affinity between these two species. I suggest the designation of a subspecific variety to distinguish these isolates ie. S. rolfsii var. rolfsii, and var. delphinii. The results from this study illustrate the extent of intraspecific variability within S. rolfsii and represent a molecular approach to studying the genetic composition of populations of this widespread plant pathogen. The combination of MCG and ITS-RFLP types provided a unique characterization of many of the isolates, which could tk used to monitor the spread of an isolate or to detect subsequent changes in the population structure within a localized area or over a larger scale. ACKNOWLEDGEMENTS I would like to express my appreciation to my committee members, and to Dr. L. D. Druehl, my public examiner. The encouragement, advice, and continued enthusiasm of my senior supervisor, Dr. Z. K. Punja is gratefully acknowledged. A special thanks to Dr. C. A. Levesque for his contribution of molecular expertise, time-saving computer skills, and support of the project, and to Dr. M. J. Smith for valuable discussions, 'molecular' suggestions and encouragement. For the numerous discussions, constructive editorial comments, and friendship, I wish to express my sincere gratitude to Charlene Mayes. I am also indebted to John Boom for sharing his molecular knowledge, Margarita Gilbert for her assistance with DNA isolations, Heidi Zimmerling for technical assistance, and Gerard C. Adams, Jr. for sharing unpublished ITS sequence data. To my lab colleagues at Simon Fraser University and at Pacific Agriculture Research Center, and to my family and friends, thanks for your invaluable support and companionship. Finally, Carlos Neves whose ever-present moral support, computer knowledge and cooking skills made the completion of this thesis possible; Evelyn Neves, whose contagious energy and life discussions were always welcomed during the last three years. TABLE OF CONTENTS APPROVAL ....................................................................................................... ii ... ABSTRACT ........................................................................................................ III ACKNOWLEDGEMENTS ................................................................................... v TABLE OF CONTENTS ..................................................................................... vi ... LIST OF TABLES .............................................................................................. VIII LIST OF FIGURES ............................................................................................ ix CHAPTER I........................................................................................................ 1 INTRODUCTION .................................................................................... 2 General ........................................................................................ 2 Diseases caused by Sclerotium rolfsii .......................................... 3 Taxonomy .................................................................................... 4 Sclerotium rolfsii and related species ........................................... 5 Sclerotium rolfsii Nuclear condition and sexual cycle ................................... 6 Sources of genetic variation in higher fungi ................................. 7 Heterokaryosis .............................................................................9 Parasexuality .................................................................. ............. 10 Sexual versus somatic (vegetative) incompatibility ...................... 12 Vegetative compatibility and mycelial compatibility groups .......... 13 Molecular approaches to studying fungal diversity ...................... 18 Restriction Fragment Length Polymorphisms (RFLP) and Polymerase Chain Reaction (PCR) ............................ 18 Mitochondria1 DNA Variation in fungi ................................. 19 Nuclear ribosomal DNA (rDNA) and the Internal Transcribed Spacer (ITS) .................................................. 21 Phylogenetic inferrence from restriction site data ............. 26 CHAPTER 11 ....................................................................................................... 29 INTRODUCTION .................................................................................... 30 MATERIALS AND METHODS ................................................................ 30 Fungal isolates and culture maintenance..................................... 30 Mycelial compatibility groups ...................................................... 31 Culture conditions and DNA isolation .......................................... 31 DNA amplification and enzymatic digests .................................... 35 Data analysis ................................................................................ 36 Phylogenet ic analysis ..................................................................
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