AN ABSTRACT OF THE THESIS OF V. Eleanor Vandegrift for the degree of Master of Science in Forest Science presented on Date May 1, 2002. Title: Fungal Diversity within Decomposing Woody Conifer Roots in Oregon. -. riçv. Signature redacted fo / V L(.- Abstract approved: Mark E. Harmon ft Signature redacted for privacy. ' V 'I Hua Chen Previous studies have indicated that roots from five tree species (Picea sitchensis, Tsuga heterophylla, Pseudotsuga menziesii, Pinusponderosa, and Pinus contorta) decompose at different rates across an environmentalgradient in Oregon. Measurements of wood chemistry from each tree species as well as moisture and temperature from each location do not explain the differences in decomposition rates. Molecular techniques were employed to generateInternal Transcribed Spacer - Restriction Fragment Length Polymorphism (1TS-RFLP) patterns to examine saprotrophic fungi in roots of these tree species and to see if differences in the fungal communities might explain observed differences in decomposition rates. However, due to a large number of ITS-RFLP patterns recovered and low levels of similarity in ITS-RFLP patterns across samples, we were unable to explain root decomposition based upon the fungal community information. Consequently, the analysis focused on comparing levels of ITS-RFLP similarity at each sampling level, determining the extent to which the sampling methods captured the total fungal biodiversity, and examining samples with microscopy and gene sequencing techniques to identify fungi. Root samples were retrieved from trees cut seven to fifteen years prior to sampling. Two tree species were sampled at each of three locations across Oregon. DNA was extracted from hyphae samples collected directly from roots, and fungi cultured from root chips. DNA was also extracted from fungal herbarium specimens, field collected samples, and cultures of saprotrophic fungi. To analyze genetic diversity of the samples, they were amplified using polymerase chain reaction (PCR) techniques, digested with endonucleases (Hinf I, Dpn II, and Hae III), and ITS-RFLP patterns were evaluated. Nei and Li similarity index analyses were used to compare differences in fungal composition based upon ITS-RFLP patterns between tree species, sites, and harvest stands. Over two hundred distinct ITS-RFLP patterns were recognized from fungal samples. Similarities in ITS-RFLP patterns of hyphal and cultured samples ranged 0 to 1, where 0 indicated no overlap and 1 indicated 100% matching of ITS-RFLPs. When all ITS-RFLPs obtained from each stump were combined, similarities in patterns between sites ranged from 0 to 0.07, from 0 to 0.13 between tree species, from 0 to 0.11 between harvest stands, and from 0 to 0.67 between individual stumps. Linearly increasing JTS-RFLP sampling intensity curves indicate a large diversity of fungi. Using microscopy, cultured samples were examined for hyphae and reproductive structures. In culture, zygomycetous structures were prevalent. DNA gene sequences of the nuclear large and small subunits were used to place unknown ITS-RFLP patterns into family and generic groups. Twenty-three common and five uncommon ITS-RFLP patterns were sequenced; most matched with the Mortierellaceae and Mucoraceae families of the zygomycetes. The large diversity of ITS-RFLP patterns indicates the coarse roots provided habitat to many fungi at the stage of decomposition when samples were collected. © Copyright by V. Eleanor Vandegrift May 1, 2002 All Rights Reserved Fungal Diversity within Decomposing Woody Conifer Roots in Oregon by V. Eleanor Vandegrift A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented May 1, 2002 Commencement June 2002 1 ACKNOWLEDGEMENTS I would like to thank my advisors Mark Harmon and Hua Chen and committee members Tom Horton and Joey Spatafora for all of their help, never- ending ideas, and encouragement during my time at OSU. Thank you to John Bliss for serving as the graduate school representative to my committee. I have been fortunate to have received advice, help with fieldwork, instruction on lab techniques, and lab space to carry out my research from a large number of people. Thanks specifically to Kermit Cromack, Jr., Joyce Eberhardt, Becky Fasth, Camille Frietag, Admir Giachini, Bob Griffiths, Amy Grotta, Jill Hoff, Kathy Jacobs, Michelle Jeffers, Kentaro Hosaka, Dan Luoma, Doni McKay, Jay Sexton, Jane Smith, Pearce Smithwick, Gi-Ho Sung, Wendy Sutton, Kirk Waterstripe, Travis Wooley, and Yuriko Yano for all of your help in steering my project in the right direction. Kerry O'Donnell provided primers for sequencing. Thanks to the Departments of Forest Science and Botany and Plant Pathology at Oregon State University and the Pacific Northwest Research Station of the US Forest Service for use of their facilities. Field research was conducted on the Siuslaw National Forest, Cascade Head Experimental Forest, H.J. Andrews Experimental Forest (Willamette National Forest), and Deschutes National Forest. I am grateful for access to field sites. This research was funded by USDA grant # 99-35107-7783. I wish to thank my extended family and friends for trying to understand the reasons, methods, and results of my research. Thanks to all of my Forest Science cohorts for great discussions in the computer labssometimes related to our theses. Finally, a big thank you to Inky and Deven Holmgren for providing support and lots of study breaks and stress relievers from school and thesis writing. 11 TABLE OF CONTENTS Page CHAPTER 1 INTRODUCTION 1 CHAPTER 2 SAPROTROPHIC FUNGAL DIVERSITY 5 DECOMPOSITION OF COARSE ROOTS, LOGS, AND STUMPS 6 SAPTROTROPHIC FUNGI 7 White-rot basidiomycetes 9 Brown-rot basidiomycetes 10 Zygomycota 12 MYCODIVERSITY 13 MOLECULAR TECHNIQUES 16 FUNGAL COMMUNITIES AND SUCCESSION 19 LITERATURE CITED 23 CHAPTER 3 FUNGAL DIVERSITY WITHIN DECOMPOSING WOODY CONIFER ROOTS IN OREGON 29 ABSTRACT 30 INTRODUCTION 31 METHODS 33 Study sites and trees 33 Year 2000 root selection and collection 35 Culturing 37 Reciprocal transplant roots (5-year) 38 Known fungi 39 Year 2001 root resampling and culture 39 DNA extraction, PCR, and RFLPs 40 Sequencing 41 Analysis 42 111 TABLE OF CONTENTS (Continued) Page RESULTS 43 Brown-and white-rot on roots 43 Decay classes of stump 44 Microscopic evidence of zygomycetes in culture 44 RFLPs 46 Biodiversity results for year 2000 culture, room temperature, and reciprocal transplant roots 49 RFLP similarity from year 2000 cultured and room temperature samples combined 50 RFLP similarities for year 2000 culture samples 54 RFLP similarity of year 2000 room temperature samples 57 RFLP similarity of year 2000 room temperature and culture samples compared separately 57 RFLP similarity of reciprocal transplant roots (5-year) 62 RFLP similarity of common patterns from year 2000 culture, room temperature, and reciprocal transplant roots 62 Biodiversity results for year 2001 resampled roots 64 RFLP similarity from year 2001 resampled roots 65 Sequences 66 DISCUSSION 69 LITERATURE CITED 74 CHAPTER 4 CONCLUSIONS 78 BIBLIOGRAPHY 81 APPENDICES 88 iv LIST OF FIGURES Figure Page Flow chart of methods for root collection, culturing, hyphae collection, and molecular techniques. 36 ITS-RFLP sampling intensity curve. Represents the number of new RFLP patterns gained for each new stump sampled. Linear curve indicates an underestimation of biodiversity. 49 ITS-RFLP sampling intensity curve for reciprocal transplant roots (5-year). 50 Unrooted neighbor joining tree for similarity for stumps from combined room temperature and culture results. Branches on the tree that are closer together represent stumps with greater similarity in RFLP patterns than stumps on branches that are further apart. The first letter in the code represents the site, the second letter indicates the species, the third number is the year the stand was harvested, and the last digit in code refers to stump 1, 2, or 3 from each species at each stand. 53 Unrooted neighbor joining tree for similarity in RFLP groups for cultured samples by stump. See Figure 4 for explanation of codes 56 Unrooted neighbor joining tree for similarity in RFLP groups for room temperature and cultured samples separately by stump. See Figure 4 for explanation of codes. 61 ITS-RFLP sampling intensity curve for year 2001 resampled roots 64 ITS-RFLP sampling intensity curve for resampled roots from each root and media type 65 V LIST OF TABLES Table Page Reciprocal transplant root samples from each species and location. 38 Number samples with evidence of white- and brown-rot in roots sampled from each stump. Numbers in parenthesis are percentages of the total fifty-four samples. 44 Number of stumps out of a possible total of three per stand of each decay class for each species and stand age 45 Occurrence of fungal hyphal structures in year 2000 cultures, common RFLP groups from year 2000 cultures, reciprocal 5-year samples, and year 2001 resampled cultures. Numbers in parenthesis are the percentage occurrence of each structure for each type of sample. 46 Number of samples collected and examined by RFLP analysis. Percentage success is the number of samples that could be sampled for RFLP patterns divided by the number of samples collected for each category. Totals for each category are in bold. 2000 cultures, 2000 room temperature, and reciprocal 5-year samples are split into subcategories for site and collection methods shown above each total in regular type. Unknown totals are based on 2000 cultures, 2000 room temperature, reciprocal 5-year roots, and 2001 resampled roots. Known totals are based on field collected, herbarium, and WSE cultures 48 Similarity matrix of room temperature and culture samples combined for each species combination 51 Similarity matrix of room temperature and culture samples combined for each stand combination. 52 Similarity matrix of culture samples for each species combination 55 Similarity matrix for culture samples for each stand combination 55 vi LIST OF TABLES (Continued) Table Page Similarity of room temperature and culture samples separately for each site combination 58 Similarity matrix of room temperature and culture samples separately for each species combination 59 Similarity matrix of room temperature and culture samples separately for each site combination 60 Similarity matrix of common RFLP patterns for each species combination 63 Similarity matrix of common RFLP patterns for each stand combination. 63 Similarity matrix for resampled roots (1, 2, 3) with Goldfarb's (GF) and Malt-agar (MA) media 66 Sequence matches for RFLP groups in the large subunit and small subunit based upon sequence blasts with GenBank. Information includes GenBank accession number of potential matches, number of base pairs compared, percent matched basepairs (higher number indicates better match), error rating (lower number indicates better match), and score in bits (higher number indicates better match) 67 RFLP groups where one sample was sequenced and others within the group were found on additional tree species 68