Bacterial Endosymbionts of Endophytic Fungi: Diversity, Phylogenetic Structure, and Biotic Interactions Item Type text; Electronic Dissertation Authors Hoffman, Michele Therese Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 23/09/2021 12:40:10 Link to Item http://hdl.handle.net/10150/196079 BACTERIAL ENDOSYMBIONTS OF ENDOPHYTIC FUNGI: DIVERSITY, PHYLOGENETIC STRUCTURE, AND BIOTIC INTERACTIONS by Michele Therese Hoffman _______________________ A Dissertation Submitted to the Faculty of the DEPARTMENT OF PLANT SCIENCES In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY WITH A MAJOR IN PLANT PATHOLOGY In the Graduate College THE UNIVERSITY OF ARIZONA 2010 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Michele T. Hoffman entitled "Bacterial endosymbionts of endophytic fungi: diversity, phylogenetic structure, and biotic interactions.” and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy. A. Elizabeth Arnold _______________________________________________________ Date: 4/21/10 Judith L. Bronstein ________________________________________________________ Date: 4/21/10 Marc J. Orbach ___________________________________________________________ Date: 4/21/10 Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. A. Elizabeth Arnold ____________________________________ Date: 4/21/10 Dissertation Director 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: Michele T. Hoffman 4 ACKNOWLEDGMENTS This research would not have been possible without the assistance of many wonderful individuals. I wish to first thank my advisor A. Elizabeth (Betsy) Arnold for her support and guidance these past years. I am forever grateful to have had this opportunity to participate in this outstanding research program. I appreciated that I was given the freedom to explore my own research questions while being rigorously challenged as a scientist. I am also most grateful to the members of my committee, Judie Bronstein for your thoughtful questions and feedback, Marc Orbach for helping me troubleshoot research problems and Sandy Pierson, for all things prokaryotic. I have appreciated all your time, guidance and patience. I want to thank the School of Plant Sciences and the IGERT program for funding and the staff members in Plant Pathology and Microbiology for your kindness and good will. Numerous friends deserve my thanks and gratitude for their support, cheerful spirit, and camaraderie. I wish to especially thank Cara Gibson and members of her phylogenetics lunch club, Jeff Oliver and Chris Schmidt, along with fellow graduate students and faculty: Fabiola Santos Rodriguez, Anne Estes, David Maddison, Randy Ryan, David Bentley, Periasamy (Ravi) Chitrampalam, Scott Kroken, Mary Jane Epps, Jana U’ren, Rhodesia Celoy, Mariana Del Olmo Ruiz, David Jarvis, Gerard White, Baomin Wang, Mary Shimabukuro, Dylan Grippi, Andrea Woodard, Jacob Russell, Eric McDowell, and Bridget Barker. I could not have accomplished this work without the help and guidance of Mali Gunatilaka. You are a special soul Mali and I owe you one million thanks! Finally, I wish to thank my incredible friends and family for their love and support. 5 DEDICATION This journey represents the culmination of many years of good intentions, perseverance, and the unwavering belief that dreams can and do eventually come true. I dedicate this work to Edie, the person who encouraged me to dream. 6 TABLE OF CONTENTS ABSTRACT.........................................................................................................................7 INTRODUCTION...............................................................................................................9 1.1 Literature review.……………………………….…..................................................9 1.2 Explanation of dissertation format………………………………………………...15 PRESENT STUDY............................................................................................................17 2.1 Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees……………………………………………………………………....17 2.2 Diverse bacterial endosymbionts inhabit living hyphae of phylogenetically diverse fungal endophytes …………………………………………………………...….18 2.3 IAA production by endophytic Pestalotiopsis neglecta is enhanced by an endohyphal bacterium .…………………………………..…………...……………....…18 REFERENCES…………………………………………………………………………..19 APPENDIX A: GEOGRAPHIC LOCALITY AND HOST INDENTITY SHAPE FUNGAL ENDOPHYTE COMMUNITIES IN CUPRESSACEOUS TREES...............24 APPENDIX B: DIVERSE BACTERIAL ENDOSYMBIONTS INHABIT LIVING HYPHAE OF PHYLOGENETICALLY DIVERSE FUNGAL ENDOPHYTES ….….49 APPENDIX C: IAA PRODUCTION BY ENDOPHYTIC PESTALOTIOPSIS NEGLECTA IS ENHANCED BY AN ENDOHYPHAL BACTERIUM..…………….99 7 ABSTRACT This dissertation comprises a series of studies designed to explore the associations between plants and the endophytic fungi they harbor in their above-ground tissues. By viewing endophyte diversity in ecologically and economically important hosts through the lenses of phylogenetic biology, microbiology, and biotechnology, this body of work links plant ecology with newly discovered symbiotic units comprised of endophytic fungi and the bacteria that inhabit them. This work begins with a large-scale survey of endophytic fungi from native and non-native Cupressaceae in Arizona and North Carolina. After isolating over 400 strains of endophytes, I inferred the evolutionary relationships among these fungi using both Bayesian and parsimony analyses. In addition to showing that native and introduced plants contained different endophytes, I found that the endophytes themselves harbor additional microbial symbionts, recovering members of the beta- and gamma- proteobacterial orders Burkholderiales, Xanthomonadales, and Enterobacteriales and numerous novel, previously uncultured bacteria. This work finds that phylogenetically diverse bacterial endosymbionts occur within living hyphae of multiple major lineages of ascomycetous endophytes. A focus on 29 fungal/bacterial associations revealed that bacterial and fungal phylogenies are incongruent with each other and did not reflect the phylogenetic relationships of host plants. Instead, both endophyte and bacterial assemblages were strongly structured by geography, consistent with local horizontal transmission. 8 Endophytes could be cured of their bacterial endosymbionts using antibiotics, providing a tractable experimental system for comparisons of growth and metabolite production under varying conditions. Studies of seven focal fungal/bacterial pairs showed that bacteria could significantly alter growth of fungi at different nutrient and temperature levels in vitro, and that different members of the same bacterial lineages interact with different fungi in different ways. Focusing on one isolate, I then describe for the first time the production of indole- 3-acetic acid (IAA) by a non-pathogenic, foliar endophytic fungus (Pestalotiopsis neglecta), suggesting a potential benefit to the host plant harboring this fungus. I show that this fungus is inhabited by an endohyphal bacterium (Luteibacter sp.) and demonstrate that mycelium containing this bacterium produces significantly more IAA in vitro than the fungus alone. I predict that the general biochemical pathway used by the fungal-endohyphal complex is L-tryptophan-dependent and measure effects of IAA production in vivo, focusing on root and shoot growth in tomato seedlings. 9 INTRODUCTION 1.1 Literature review 1.1.1 Fungal endophytes All lineages of land plants surveyed to date harbor living fungi within their healthy, aboveground tissues (e.g., Carroll, 1988; Petrini, 1996; Davis et al., 2003; Cohen, 2004; Arnold & Lutzoni, 2007; Kauserud et al., 2008; Arnold et al., 2009). These endophytic fungi colonize and persist within living tissues such as leaves and stems without causing overt symptoms of disease (Petrini, 1991; Stone et al., 2000). Fungal endophytes are highly diverse at the species level (Carroll, 1995; Fröhlich & Hyde, 1999; Lu et al., 2004; Rodrigues et al., 2004; Arnold, 2007), represent every major lineage of non-lichenized