FUNGAL ENDOPHYTES ISOLATED FROM LARGE ROOTS OF DOUGLAS-FIR (PSEUDOTSUGA MENZIESII) AND PONDEROSA PINE (PINUS PONDEROSA) By John Richard Goetz III A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN PLANT PATHOLOGY WASHINGTON STATE UNIVERSITY Department of Plant Pathology December 2006 To the Faculty of Washington State University: The members of the Committee appointed to examine the thesis of John Richard Goetz III find it satisfactory and recommend that it be accepted. _______________________ Chair _______________________ _______________________ _______________________ ii ACKNOWLEDGEMENT I would first like to thank Dr. Jack Rogers for taking a chance and supplying me with this amazing opportunity, for his priceless experience, expert training, understanding and all the stories and jokes. I thank Dr. Paul Hessburg for funding and coordination of sample collection. A special thanks to Dr. Mee- Sook Kim for her experience, patience and diligence in teaching molecular techniques to a guy like me. Dr. Ned Klopfenstein for constant encouragement, guidance, conversation, friendship, for believing in me the first time we met and for every time he bought me lunch. Alison Sigler, for being my best friend and agreeing to live and work in Pullman in support of my goals, her love, laughter and ability to keep me focused. I have to give an extra special thanks to Dr. Lori Carris for training in fungal identification, the encouraging conversations, the mushroom hunts and, most importantly, being my friend. Momentous thanks goes to Marion Andrew and Jane Stewart for the daunting task of working with me, for taking time to listen, all the support and being two of my greatest friends. It is beyond my capabilities to string together the words that can adequately express how each of you have impacted my life aside from saying that I love each of you and that I miss you all already. This project was funded by the USDA Forest Service, Pacific Northwest Research Station-Managing Natural Disturbance Regimes (PNW-4577), Wenatchee, WA, USA, the USDA Forest Service, Rocky Mountain Research Station-Microbial Processes as Ecosystem Regulators in Western Forests (RM- iii 4552) Moscow, ID, USA and Washington State University Department of Plant Pathology. All field work and root core samples were provided by the PNW Research Station, Wenatchee Forestry Sciences Laboratory. This is Contribution Number 86 of the National Fire and Fire Surrogate Project (FFS), funded by the U.S. Joint Fire Science Program. Last but not least, thanks to Ann Abbott for statistical support, Anne Conway, Jordana Cassel, Rebecca Guenther and Andy “Woody” Wood for their efforts in sample collection, without which, none of this would’ve come to fruition. iv FUNGAL ENDOPHYTES ISOLATED FROM LARGE ROOTS OF DOUGLAS-FIR (PSEUDOTSUGA MENZIESII) AND PONDEROSA PINE (PINUS PONDEROSA) Abstract by John Richard Goetz III, M.S. Washington State University December 2006 Chair: Jack D. Rogers Little is known about the diversity and ecological roles of endophytes in woody roots of forest trees in the inland northwestern USA, especially as related to fire ecology. To assess fungal endophytes in large woody roots of Douglas-fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa), we conducted a mycobiotic survey of root-core samples from 12 sites in north-central Washington State. Each site was subjected to one of the following fuel treatments: 1) untreated control; 2) burn only 3); thin only; and 4) thin and burn. Fungal endophytes were cultured and identified by morphology and sequencing of ITS1, 5.8S, ITS2 rDNA. In total, 1130 surface-disinfected, root-cores pieces were processed and 43% yielded one or more fungal isolates. From the 1130 surface-disinfected, root-cores pieces, 365 (72%) of 509 fungal isolates were identified comprising 20 genera (224 isolates) of hyphomycetes, 5 genera (79 isolates) of Ascomycota, 3 genera (48 isolates) of Zygomycota, and 14 isolates of v Basidiomycota. Among the more interesting findings were 1) yeast taxa usually associated with above-ground bark beetles including two undescribed species of Candida; 2) a putatively undescribed eurotiaceous fungus that produces ascomata in culture; and 3) multiple genera of dematiaceous hyphomycetes that have been reported from above-ground plant tissues and grass roots. Fuel treatment, host species and culture media all significantly influenced fungal endophytes recovered. Compared to controls, units subjected to burn-only showed reduced numbers of fungal endophytes; whereas recovery of endophytic fungi increased in the thin and burn units and thin-only units exhibited no change. Some associations are noted among tree root endophytes, grass root endophytes, and above-ground saprobes and facultative parasites, because many of the same species are found across all of these habitats and environments. Possible roles and functions of endophytic fungi of woody coniferous roots are discussed. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENT...............................................................................................iii ABSTRACT........................................................................................................................v LIST OF TABLES...............................................................................................................x LIST OF FIGURES............................................................................................................xi CHAPTER ONE 1. INTRODUCTION AND REVIEW OF LITERATURE..................................1 Definition of Fungal Endophyte..................................................................1 Ecological Roles of Fungal Endophytes.....................................................3 Protection of Host Plants..............................................................................5 Altering Host Tolerance to the Environment.............................................7 Heat-Tolerant Fungi as Endophytes...........................................................7 Previous investigation..................................................................................9 Present Investigation and Objectives.......................................................10 2. MATERIALS AND METHODS.....................................................................11 Sampling sites..............................................................................................10 Fuel Treatments...........................................................................................12 Sample Collection........................................................................................12 Fungal Isolation and Cultures...................................................................13 vii Characterization of Fungal Morphology.................................................14 DNA Sequencing.........................................................................................18 Statistical Analysis......................................................................................19 3. RESULTS...........................................................................................................21 Overall Results.............................................................................................21 Identified Fungal Taxa by Treatment.......................................................21 Overall Effect of Fuel Treatments on Fungal Endophytes....................25 Identified Fungal Taxa by Host.................................................................25 Identified Fungal Taxa by Culture Media...............................................26 4. DISCUSSION....................................................................................................59 Dematiaceous Hyphomycetes or Dark Septate Endophytes (DSE).....59 Ascomycetous and Basidiomycetous Yeasts...........................................64 Penicillia as Potential Endophytes............................................................65 Novel Eurotiaceous Taxon and Associated Questions..........................66 Vertical or Horizontal Transmission of Fungal Endophytes.................66 Fungi Isolated by Overall Treatment........................................................68 Fungi Isolated by Host................................................................................68 Fungi Isolated by Culture Medium...........................................................70 Endophytic Fungi Isolated from Thin-Only Units..................................71 Endophytic Fungi Isolated from Burn-Only Units.................................73 Endophytic Fungi from the Thin and Burn Units...................................77 viii Present and Previous Investigation Comparison...................................78 BIBLIOGRAPHY.............................................................................................................79 APPENDIX.......................................................................................................................86 A. Alternaria malorum: A Mini-Review with New Records for....................86 Hosts and Pathogenicity B. Microwave Processing for Scanning Electron Microscopy (SEM).........97 C. Inventory of Root-Core Samples with Host Status, Collection............101 Data and Treatment. D. Summary of Treatment Units and Plant Associations..........................116 ix LIST OF TABLES 1. Percent of cores by treatment that yielded one or more fungal isolates.....28