Genetics of Sexuality and Population Genetics of Phellinus Weirii
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AN ABSTRACT OF THE THESIS OF Peter Andrew Angwin for the degree of Doctor of Philosophy in Botany and Plant Pathology presented onJune 2, 1989 Title: Genetics of Sexuality and Population Genetics of Phellinus weirii Abstract approve dRedacted for Privacy Phellinus weirii (Murr.) Gilbertson, cause of laminated root and butt rot, is a heterothallic basidiomycete lacking clamp connections. Two biological species groups of the fungus have been hypothesized, the Douglas-fir and cedar-types, on the basis of physiological, morphological and epidemiological differences. Single-spore and vegetative isolates differ in cultural morphology, nuclear composition and growth rates, but variation among isolates makes it impossible to accurately identify the ploidy of individuals. Pairings of single-spore isolates in culture produce a wide range of reactions, from formation of darkly pigmented barrage lines to no visible change in morphology. Back-pairings of interacted mycelium from single-spore pairings with completely compatible homokaryons reveal that P. weirii follows a bipolar, multiallelic system of mating compatibility. Single-spore and homokaryon-heterokaryon pairings followed by back-pairings demonstrate the genetic isolation between Douglas-fir and cedar-type isolates, confirming the biological species hypothesis. Existence of a third Asian species is suggested by the lack of interaction of two Japanese isolates with both Douglas-fir and cedar-type tester homokaryons. Protein banding patterns obtained by SDS polyacrylimide gel electrophoresis show overall similarity between the three types but differ at several band locations. The Douglas-fir-type isolates were found on Douglas-fir and six other host species, including western redcedar. The cedar-type isolates were mostly confined to western redcedar, but were also identified on western hemlock and grand fir. The geographic ranges of the Douglas-fir and cedar-type Phellinus weirii overlap in much of the Douglas-fir region, indicating that the emerging biological species groups are likely the result of host specialization rather than geographic separation. Genetics of Sexuality and Population Genetics of Phellinus weirii by Peter Andrew Angwin A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed June 2, 1989 Commencement June, 1990 APPROVED: Redacted for Privacy Professor of Botany and Plant Pathology in Charge of Major Redacted for Privacy Head of the Department of Botany nand Plant Pathology Redacted for Privacy Dean of Graduate.. hool Date thesis is presented June 2, 1989 Typed by Maria M.A. Angwin for Peter Andrew Angwin ACKNOWLEDGEMENTS I would like to thank the following people for their help and support during my years of work and study at OregonState University: Dr. Everett Hansen, for his help, patience and guidance through good times and bad. It has been a pleasure to work in his lab! Dr. Lewis Roth, Dr. Earl Nelson and Dr. Walt Thies, for their advice and help through the years. Dr. Michael Larsen (USDA Forest Service, Center for Forest Mycology Research, Madison, Wi.) and Dr. Alan Dickman (Dept. of Biology, University of Oregon), for contributingmany of the Phellinus weirii isolates that helped make these studies possible. Dr. Neil Martin (USDA Forest Service, Intermountain Research Station, Moscow, Id.), for his help during the collecting trips to Idaho. Thanks and grudging respect to Phil Hamm, for keeping the lab running smoothly and for showing me the ins and outs of electrophoresis. My fellow graduate students in forest pathology: Paul Hennon, Paul Hessburg, John Kalafarski, Jeff Witcosky, Barry Goldfarb, Kathy Lewis, Ellen Michaels Goheen and Jim Entry a truly unique group of individuals! All the friends and aquaintances in Corvallis, who have helped make Oregon such a wonderful place to live! My wife Maria, a constant source of love and support, who has had to put up with a lot as I've made my way through graduate school. I don't know if I could have made it without her! My parents, Robert and Pam Angwin, and my "other" parents, David and Eunice Aro, for helping to show Maria and me the way... And finally, to my brother Tim ("I understand most of this!"), to whom this work is dedicated - We Miss You Bro! Table Of Contents Page I. Introduction 1 II. Literature Review 4 Taxonomy 4 Geographic and Host Distribution 6 Epidemiology and Pathology 9 Basidiomycete Life Cycles and Mating Systems 12 Genetics of Sexuality and Population Genetics 14 Biological Species Concept 18 Species Relationships in Phellinus weirii 21 III. Geneticsof Sexuality of Phellinus weirii 26 A. Materials and Methods 26 1. Sources and isolation of vegetative and single-spore isolates 26 2. Distinguishing homokaryons from heterokaryons 27 3. Pairing studies 32 B. Results 35 1. Distinguishing homokaryons and heterokaryons 35 2. Pairing studies 59 a. Intra-basidiocarp pairings 59 b. Inter-basidiocarp, intra-species pairings 68 c. Di-mon pairings 71 d. Back-pairings 74 C. Discussion 82 IV. Biological Species Concept 99 A. Materials and Methods 99 1. Homokaryon-homokaryon pairings 99 2. Homokaryon-heterokaryon pairings 101 3. Electrophoresis 102 4. Cultural morphology and growth rates 104 B. Results 104 1. Homokaryon-homokaryon pairings 104 2. Homokaryon-heterokaryon pairings 115 3. Electrophoresis 125 4. Cultural morphology and growth rates 130 C. Discussion 135 V. Summary and Conclusions 148 VI. Literature Cited 155 Table Of Contents (Cont.) Page VII. Appendices A. Identification name, host species, source location, name of collector and date collected of the Phellinus weirii isolates assembled as stock cultures 164 B. List of isolates used in experiments to examine the genetics of sexuality and population genetics of Phellinus weirii 169 List of Figures Figure Page 1 ISCC-NBS method of determining pigmentation colors of 29 Phellinus weirii isolates. 2 Rating scheme to assess the strength of interaction 34 lines in pairings of Phellinus weirii isolates. 3 Color analysis at 13 and 42 days of 47 homokaryotic 36 and 29 heterokaryotic isolates of Phellinus weirii grown on 3% malt agar at 22 C. 4 Electrophoresis Gel #1 Electrophoretic banding 50 patterns of total protein extracts of single-spore and vegetative isolates of Phellinus weirii. 5 Electrophoresis Gel #2 - Electrophoretic banding 51 patterns of total protein extracts of single-spore and vegetative isolates of Phellinus weirii. 6 Electrophoresis Gel #3 - Electrophoretic banding 52 patterns of total protein extracts of single-spore and vegetative isolates of Phellinus weirii. 7 Electrophoresis Gel #4 - Electrophoretic banding 53 patterns of total protein extracts of single-spore and vegetative isolates of Phellinus weirii. 8 Variety of line and pigmentation reactions observed 67 in single-spore isolate pairings of Phellinus weirii. 9 Interaction lines obtained in di-mon pairings of 73 Phellinus weirii. 10 Heterokaryon formation in selected S8 x 134696 83 pairings of Phellinus weirii as revealed by back-pairing with the same homokaryons used in the initial pairings. 11 Drawing of DAPI-stained nucleus of Phellinus weirii 86 homokaryon MP2-2 passing through a cytoplasmic bridge between adjacent cells. 12 Homokaryon-homokaryon pairing combinations performed 100 among and between host/geographic groups of Phellinus weirii. List of Figures (Cont.) Figure Pa e 13 Electrophoresis Gel #5 - Electrophoretic banding 126 patterns of total protein extracts of Douglas-fir and cedar-type vegetative isolates of Phellinus weirii. 14 Electrophoresis Gel #6 - Electrophoretic banding 127 patterns of total protein extracts of Douglas-fir, cedar and Japanese-type vegetative isolates of Phellinus weirii. 15 Electrophoresis Gel #7 - Electrophoretic banding 128 patterns of total protein extracts of Douglas-fir, cedar and Japanese-type vegetative isolates of Phellinus weirii. 16 Electrophoresis Gel #8 - Electrophoretic banding 129 patterns of total protein extracts of Douglas-fir, cedar and Japanese-type vegetative isolates of Phellinus weirii. 17 Color analysis at 13 days of 26 Douglas-fir, 13 132 cedar, and 3 Japanese-type isolates of Phellinus weirii grown on 3% malt agar at 22 C. 18 Color analysis at 42 days of 26 Douglas-fir, 13 133 cedar, and 3 Japanese-type isolates of Phellinus weirii grown on 3% malt agar at 22 C. 19 Results of homokaryon-homokaryon pairings among 137 and between host/geographic groups of Phellinus weirii. 20 Geographic distribution of Douglas-fir and cedar- 143 type vegetative isolates of Phellinus weirii. 21 Proposed phylogenetic scheme for the evolution of 147 the biological species groups of Phellinus weirii. List of Tables Table Page 1 Colony morphology of homokaryons and heterokaryons 38 of Phellinus weirii grown on 3% agar at 22 C. 2 Growth rates of homokaryons and heterokaryons of 39 Phellinus weirii grown on 3% agar at 22 C. 3 Nuclear condition of vegetative cells of homokaryons 41 and heterokaryons of Phellinus weirii treated with the fluorescent stains DAPI and calcifluor. 4 Distribution of nuclei in vegetative cells of 42 homokaryons and heterokaryons of Phellinus weirii treated with the fluorescent stains DAPI and calcifluor. 5 Nuclear condition of tip cells of homokaryons and 43 heterokaryons of Phellinus weirii treated with the fluorescent stains DAPI and calcifluor. 6 Distribution of nuclei in tip cells of homokarycns 44 and heterokaryons of Phellinus weirii treated with the fluorescent stains DAPI and calcifluor. 7 Distribution of nuclei tip cells of homokaryons and 45 heterokaryons of Phellinus weirii from various hosts as revealed by treatment with the fluorescent stains DAPI and calcifluor. 8 Numbers of nuclei in DAPI-stained basidiospores from 46 basidiocarps on western redcedar collected at Solo Creek and Priest Lake, Idaho. 9 Identification of the karyology of 28 Phellinus weirii 49 isolates by counting nuclei in 10 tip cells treated with DAPI and calcifluor. 10 Similarity of protein banding patterns of total 55 protein extracts of Phellinus weirii homokaryons and heterokaryons as revealed by SDS-polyacrylimide gel electrophoresis. 11 Single-spore and di-mon pairings to determine the 60 genetics of sexuality of Phellinus weirii.