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A Molecular Characterization of the Fungal Endophytes Within the Needles of Ponderosa Pine (Pinus Ponderosa) Amy E

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A Molecular Characterization of the Fungal Endophytes Within the Needles of Ponderosa Pine (Pinus Ponderosa) Amy E Eastern Washington University EWU Digital Commons EWU Masters Thesis Collection Student Research and Creative Works 2016 A molecular characterization of the fungal endophytes within the needles of ponderosa pine (Pinus ponderosa) Amy E. Gray Eastern Washington University Follow this and additional works at: http://dc.ewu.edu/theses Recommended Citation Gray, Amy E., "A molecular characterization of the fungal endophytes within the needles of ponderosa pine (Pinus ponderosa)" (2016). EWU Masters Thesis Collection. 397. http://dc.ewu.edu/theses/397 This Thesis is brought to you for free and open access by the Student Research and Creative Works at EWU Digital Commons. It has been accepted for inclusion in EWU Masters Thesis Collection by an authorized administrator of EWU Digital Commons. For more information, please contact [email protected]. A MOLECULAR CHARACTERIZATION OF THE FUNGAL ENDOPHYTES WITHIN THE NEEDLES OF PONDEROSA PINE (PINUS PONDEROSA) A Thesis Presented to Eastern Washington University Cheney, Washington ______________________________________________________________________________ A Partial Fulfillment of the Requirements of the Master of Science in Biology ______________________________________________________________________________ By Amy E. Gray Winter 2016 ii THESIS OF AMY E. GRAY APPROVED BY: ____________________________________________________________ DATE____________ Suzanne M. Schwab, Ph.D., Professor, Graduate Study Committee Chair ____________________________________________________________ DATE____________ Luis M. Matos, Ph.D., Assistant Professor, Graduate Study Committee ____________________________________________________________ DATE____________ Michael Folsom, Ph.D., Professor, Graduate Study Committee iii MASTER’S THESIS In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Eastern Washington University, I agree that the JFK Library shall make copies freely available for inspection. I further agree that copying of this project in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this thesis for commercial purposes, or for financial gain, shall not be allowed without my written permission. Signature _______________________________________ Date __________________________________________ iv ABSTRACT Fungal endophytes (microbial fungi that live in plant tissues without causing disease) are ubiquitous in plants studied to date. Many conifers host endophytes that produce important bioactive secondary metabolites. While the endophytes in Pinus ponderosa roots have been studied, the endophyte communities within the needles are not well characterized by molecular means: thus, the goal of this study was to do so. Needles were sampled at two sites located five kilometers apart along a slight precipitation gradient in Eastern Washington State. Fungal DNA (along with pine DNA) was isolated directly from needles of two ages. Internal transcribed spacer regions of the ribosomal RNA gene form fungi were amplified by PCR, cloned, and sequenced. Many of the sequences described have high homology matches in GenBank with known fungi, though most have not previously been directly associated with ponderosa pine. Some sequences were highly homologous with previously published sequences of fungi that have yet to be assigned a taxonomic designation , which suggests some previously undescribed fungi occupy ponderosa pine needles. Despite the proximity of sites, only 18% of the endophytes sequenced were common to both sites. Forty one percent of endophytes were unique to needles from 2012, while 45% of endophytes were unique to needles that emerged in 2014. Although more data from a deep sequencing project will be required to confirm this, the results presented here suggests that fungal endophyte communities in ponderosa pine are fairly diverse geographically, and that communities in the same tree differ depending on the age of the needles. v ACKNOWLEDGEMENTS Suzanne Schwab provided guidance throughout this project- from assistance with experimental design, generosity with consumables and lab space, and her aid in editing. Luis Matos opened up his lab to me, and (in addition to a long-time mentorship), provided guidance through the molecular work upon which this project hinged, access to all of his equipment, and considerable time and patience throughout the writing process. Mike Folsom lent me a new set of eyes through which to gauge a landscape. Andrea Castillo was very kind in her assistance with cloning media and plasmid extractions. Most of the EWU Biology Faculty offered insight into the project, and all offered support. Melissa Rook assisted with needle plating, PCR, and electrophoresis troubleshooting. Sarah Chapman kept an eye on culture media to prevent free- range fungi. My husband Caleb provided support, late-night meals, and humor. I thank you all very much! A Swartz Graduate in Biotechnology grant and a Swartz-Narrance Graduate in Biotechnology fellowship were greatly appreciated, and vital to the completion of this work. vi TABLE OF CONTENTS Abstract ------------------------------------------------------------------------------------------------------- iv Acknowledgements ------------------------------------------------------------------------------------------ v Table of contents -------------------------------------------------------------------------------------------- vi List of figures and tables ---------------------------------------------------------------------------------- vii Introduction --------------------------------------------------------------------------------------------------- 1 Endophyte-produced compounds ----------------------------------------------------------------- 4 Describing endophyte communities -------------------------------------------------------------- 5 Materials and Methods -------------------------------------------------------------------------------------- 7 Sampling site description -------------------------------------------------------------------------- 7 Endophyte culturing -------------------------------------------------------------------------------- 7 DNA extraction and PCR--------------------------------------------------------------------------- 8 Cloning of PCR products ------------------------------------------------------------------------- 11 Results --------------------------------------------------------------------------------------------------------11 Needle plating --------------------------------------------------------------------------------------11 Plasmid sequencing ------------------------------------------------------------------------------- 12 Discussion --------------------------------------------------------------------------------------------------- 13 References Cited --------------------------------------------------------------------------------------------18 Appendices -------------------------------------------------------------------------------------------------- 30 Appendix 1. -----------------------------------------------------------------------------------------30 Appendix 2. -----------------------------------------------------------------------------------------34 Appendix 3. -----------------------------------------------------------------------------------------35 Appendix 4. -----------------------------------------------------------------------------------------38 Vita------------------------------------------------------------------------------------------------------------ 40 vii FIGURES AND TABLES Figure 1: Spokane County Map----------------------------------------------------------------------------25 Figure 2: Endophyte community chromatogram ------------------------------------------------------- 26 Figure 3: Sequenced plasmid chromatogram ------------------------------------------------------------27 Table 1: BLAST results from sequenced clones --------------------------------------------------------28 Table 2: Host information for sequenced clones --------------------------------------------------------29 INTRODUCTION The kingdom Fungi is remarkably rich, with more than 100,000 described species, and as many as five million projected undescribed species (BLACKWELL 2011; GAMBOA et al. 2002; TOJU et al. 2012). Fungi range from the largest known organism on Earth, to microbial elements of soil, terrestrial and aquatic ecosystems (CLAY 1988). They exist as saprophytes and colonize both plans and animals. Fungi produce a wealth of enzymes that break down cellulose, proteins, lignin, and chitin into absorbable components (BOYLE et al. 1992; MARTINEZ et al. 2009). Fungal interactions with plants span the spectrum from mutualistic associations that are obligate for plant survival through apparently commensal relations to lethal pathogenic infections. Plant/fungi interactions Most vascular plants have mutualistic mycorrhizal relationships with fungi (WANG and QIU 2006), and arbuscular mycorrhizae can be clearly observed in fossil samples of the earliest known vascular plants from the Rhynie chert bed in Scotland (REMY et al. 1994). Pathogenic interactions between plants and fungi are also well documented (GRZYWACZ and WAZNY 1973; KNOGGE 1996; VON SCHÜTT 1971). While some calculations suggest that fewer than 10% of known fungi are capable of inhabiting living plant tissue, all plant species are attacked by pathogenic fungi (KNOGGE 1996). Fungal pathogenicity in plants is so widespread that it is nearly impossible
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