Epidemiology and Population Biology of Inonotus Tomentosus As a Pathogen of Forests of British Columbia

AN ABSTRACT OF THE THESIS OF Katherine JoAnn Lewis for the degree of Doctor of Philosophy in Botany and Plant Pathologypresented on April 10. 1990. Title: Epidemiology and Population Biology of Inonotus tomentosus as a Pathogen of Young Forests in British C9Ilumbia. A Abstract approved: < Redacted for Privacy Dr. Everett M. Hansen The root disease fungus Inonotus tomentosus, common in the old growth boreal forests of British Columbia, poses a threat to the health of second growth forests established on sites with a previous history of root disease. Colonized stumps occur in groups of 1 to 6; the groups are clumped within a clearcut. Therefore, surveys for disease incidence need to employ wide (10 m) transects and cover at least 10% of the stand area. The fungus remains viable in stumps for at least 30 years and can infect roots of regeneration trees in contact with stump roots. Spruce stump roots cause twice as many infections as pine stump roots because more spruce roots are colonized, they are longer, and they are horizontally oriented. Trees growing within 200 cm of diseased spruce stumps have a 25% chance of infection. At 350 cm - 400 cm the chance of infection is 10%. Trees growing within 50 cm and 250 cm 300 cm of diseased pine stumps have a 25% and 10% chance of infection respectively. Within a stand, disease centres are usually small (less than 5 trees) and composed of a single genotype. Larger centres consist of several genotypes. Electrophoretic protein profiles and vegetative compatibility tests were co-supportive methods used to determine genotype similarities. The clumped distribution of disease centres and the frequency of unique genotypes suggest that spores may have an important role in disease spread. Infection of roots by I. tomentosus occurs through direct penetration of the bark of small roots, or by infection of a feeder root. In roots approximately 5 cm diameter or less, the fungus grew in or on the bark and often preceded decay in the wood. In larger roots fungal growth is in the heartwood; radial growth to the barkis limited until root death. Inoculations of mature spruce and pine trees suggest that spruce is more readily infected than pine, due in part to greater resin production and phenol accumulation by challenged pine roots. Infection of root wood by I. tomentosus caused host cell death at the hyphal front, increased phenolic deposition, peroxidase activity, and starch depletion beyond the hyphal front. Inonotus circinatus (a pathogen of pine in eastern North America) and I. tomentosus show consistent differences at the protein level as indicated by protein electrophoresis and southern hybridization of a homologous probe (random genomic clone) to total genomic DNA. Protein patterns of I. tomentosus isolates are more variable than patterns of I. circinatus which may be due to greater diversity of I. tomentosus hosts and climate. Epidemiology and Population Biology of Inonotus tomentosus as a Pathogen of Forests of British Columbia by Katherine JoAnn Lewis A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed April 10, 1990 Commencement June 1990 APPROVED: Redacted for Privacy Professor of Botany and Plant Pathology in charge of major Redacted for Privacy Chairman of the DepartInt of Botany and P1Xt Pathology Redacted for Privacy to School Date thesis is presented April 10, 1990 To my parents, Alan and Carolyn Lewis, and to "Wimp Tours" Acknowledgements Financial support for this thesis was provided by the Forest Resource Development Agreement (Provincial Direct Delivery) administrated by the Victoria, B.C. office of the Ministry of Forests, Protection Branch. Funding for the molecular genetics work was provided by a Grant-in-Aid of research from Sigma Xi. To these agencies I am grateful. Additional financial and moral support was provided by my parents, and my two grandmothers, who have seen me through yet another degree with as much enthusiasm as when I began as an undergraduate. I feel very lucky to have had the opportunity to work with Everett Hansen, my major professor. If I had to do it all over again, he would still be my first choice, for many reasons. I also feel lucky to have had the diverse committee that I did. To Duncan Morrison in particular, I extend my appreciation for his help in the field, and my admiration of his knowledge of forest pathology. There are many people who helped me out and kept me laughing when times were tough. My friends in Prince George never did understand what I was doing, but they bought the groceries on occasion, helped me with "the books", and even worked on stumps. Special thanks to Kathi Hughes, Lynne and Chris Peppler, Cathleen Boyle, and Jay Dahlgren. During the development of my research and thesis, I also developed some friendships in Corvallis which I hope will be long- lasting despite the miles separating us. I am indebted to many people, too many to list, but would especially like to thank Phil Hamm and the rest of "the lab", Kevin McCluskey, Ellen and Don Goheen, Allan Kanaskie, Cheryl Ingersoll, Dean DeNicola and Maria Finckh. Finally to John, without whom I certainly would have completed my doctorate, and probably would have doneit sooner, but it wouldn't have been so much fun! TABLE OF CONTENTS INTRODUCTION 1 I. LITERATURE REVIEW 4 II. SURVIVAL OF INONOTUS TOMENTOSUS IN STUMPS AND SUBSEQUENT INFECTION OF YOUNG STANDS 29 Introduction 29 Materials and Methods -Stand selection and survey of incidence of disease in clearcuts 32 -Distribution of I. tomentosus in stands selected for stump excavation 32 Longevity and location of mycelium in stumps and frequency of fungus transfer to surrounding regeneration 34 Results -Stand selection and survey of incidence of disease in clearcuts 36 -Distribution of I. tomentosus in stands selected for stump excavation 36 -Longevity and location of mycelium in stumps and frequency of fungus transfer to surrounding regeneration 39 Discussion - Stand selection and survey of incidence of disease in clearcuts 58 -Distribution of I. tomentosus in stands selected for stump excavation 59 Longevity and location of mycelium in stumps and frequency of fungus transfer to surrounding regeneration 62 III. VEGETATIVE COMPATIBILITY AND ELECTROPHORETIC GROUPS OF INONOTUS TOMENTOSUS AND THE ROLE OF SPORES IN DISEASE SPREAD 72 Introduction 72 Materials and Methods -Collection of isolates 76 -Vegetative compatibility analysis 79 -Protein electrophoresis 80 -Analysis 81 Results -Vegetative compatibility 82 -Protein Electrophoresis 87 Discussion 98 IV. VEGETATIVE SPREAD OF INONOTUS TOMENTOSUS (Fr.)Teng AND DISEASE DEVELOPMENT IN SPRUCE (PICEA GLAUCA X ENGELMANNII ENGELM 105 Introduction 105 Materials and Methods 108 Results 111 Discussion 124 V. INOCULATION OF PICEA GLAUCA X ENGELMANNII ENGELM. AND PINUS CONTORTA DOUGL. WITH INONOTUS TOMENTOSUS (Fr.)Teng. AND INONOTUS CIRCINATUS (Fr.)Gilbn.: INFECTION AND HOST RESPONSE 128 Introduction 128 Materials and Methods Natural inoculation test 132 -Artificial inoculation test 132 Results 140 Discussion 154 VI. COMPARISON OF INONOTUS TOMENTOSUS (Fr.)Teng and INONOTUS CIRCINATUS (Fr.)Gilbn. BY TOTAL PROTEIN ELECTROPHORESIS AND RESTRICTION FRAGMENT LENGTH POLYMORPHISMS 162 Introduction 162 Materials and Methods Isolates 165 -Protein electrophoresis 165 DNA extraction, electrophoresis and southern hybridizations 167 Results -Protein electrophoresis 169 -DNA restriction fragments 169 Discussion 172 SUMMARY 175 LITERATURE CITED 184 APPENDICES 194 A. 1. Frequency distribution of the number of sampling units with 1,2, 3, 4,5, and 6+ diseased stumps 194 2. The number of run lengths of 1,2, 3, 4, 5, and 6+ diseased stumps by site 194 B. Example of stump excavation maps 195 C. Isolate collection maps for the Jerry Ck, Bobtail and Pelican Rd locations 197 D. Similarity coefficients of protein profiles between isolates from Jerry Ck., Bobtail, and Pelican Rd locations 199 LIST OF FIGURES Figure Page Chapter II II.1 Location in British Columbia of stump excavation sites 38 11.2 Percentage of spruce and pine stumps with butt rot 41 11.3 Mean root length and colonized root length in spruce stumps 45 11.4 Percentage of spruce stump root length colonized by decay category 47 II.5a Spruce stump root with advanced decay 48 II.5b Pine stump root with advanced decay 48 11.6 Mean root length and colonized root length in pine stumps 51 11.7 Percentage of spruce stump root length colonized by decay category 53 Chapter III III.1 Location of collection sites in British Columbia 77 III.2a Pairing reactions between vegetative I. tomentosus isolates 83 III.2b Pairing reactions between vegetative I. tomentosus isolates 83 111.3 Map of the Smithers isolate collection site 85 111.4 Map of the Averil isolate collection site 86 111.5 BV2-4 and BV2-8 protein polymorphisms 89 111.6 Smithers isolates 4, 5,6, 7 and 2, 11, 12 protein profiles 93 111.7 Protein profiles and Vc reactions of Averil isolates 12, 13, and 29 94 111.8 Protein profiles and Vc Reactions of Averil isolates 8 and 30, 10 and 11 95 Chapter IV IV.1 Excavation, grid layout, and labelling of excavated plots 109 IV.2 Lesion and light brown stain caused by I. tomentosus infection 110 IV.3 Intrabark mycelium and stain in root wood caused by I. tomentosus 113 IV.4 Plot 1 root map 115 IV.5 Plot 2 root map 117 IV.6 Plot 3 root map 119 IV.7 Plot 4 root map 120 IV.8 Cambial lesion from penetration by I. tomentosus at root junction 122 IV.9 Plot 5 root map 123 Chapter V V.1 Inoculation sites in British Columbia 133 V.2 Inoculation procedure: root excavation, wounding and inoculum attachment 136 V.3 Location of samples from inoculated roots for isolation and sectioning 138 V.4 Inoculum blocks one year after harvest 141 V.5 Phenol accumulation at a wound 147 V.6 Dehydrogenase extruded from hyphae 148 V.7 Glucosidase in hyphae 150 V.8 Accumulation of starch 151 V.9 Phenolic deposition caused by fungal laccase activity 153 Chapter VI VI.1 Protein electrophoresis band patterns of I.

Load more