ABSTRACT Title of Document: EVALUATING THE ENDOPHYTIC FUNGAL COMMUNITY IN PLANTED AND WILD RUBBER TREES (Hevea brasiliensis) Romina O. Gazis, Ph.D., 2012 Directed By: Assistant Professor, Priscila Chaverri, Plant Science and Landscape Architecture The main objectives of this dissertation project were to characterize and compare the fungal endophytic communities associated with rubber trees (Hevea brasiliensis) distributed in wild habitats and under plantations. This study recovered an extensive number of isolates (more than 2,500) from a large sample size (190 individual trees) distributed in diverse regions (various locations in Peru, Cameroon, and Mexico). Molecular and classic taxonomic tools were used to identify, quantify, describe, and compare the diversity of the different assemblages. Innovative phylogenetic analyses for species delimitation were superimposed with ecological data to recognize operational taxonomic units (OTUs) or ―putative species‖ within commonly found species complexes, helping in the detection of meaningful differences between tree populations. Sapwood and leaf fragments showed high infection frequency, but sapwood was inhabited by a significantly higher number of species. More than 700 OTUs were recovered, supporting the hypothesis that tropical fungal endophytes are highly diverse. Furthermore, this study shows that not only leaf tissue can harbor a high diversity of endophytes, but also that sapwood can contain an even more diverse assemblage. Wild and managed habitats presented high species richness of comparable complexity (phylogenetic diversity). Nevertheless, main differences were found in the assemblage‘s taxonomic composition and frequency of specific strains. Trees growing within their native range were dominated by strains belonging to Trichoderma and even though they were also present in managed trees, plantations trees were dominated by strains of Colletotrichum. Species of Trichoderma are known for their biocontrol properties, whereas species of Colletotrichum have been always associated with plant disease. EVALUATING THE ENDOPHYTIC FUNGAL COMMUNITY IN PLANTED AND WILD RUBBER TREES (Hevea brasiliensis) By Romina O. Gazis Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2012 Advisory Committee: Dr. Priscila Chaverri, Chair Dr. Stephen Rehner Dr. Daniel Gruner Dr. Yilmaz Balci Dr. Maile Neel Dr. Charles Mitter Foreword This thesis is based on the following articles, which are referred in the text as Chapters: 1. Gazis R., Chaverri, P. 2010. Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis) in Peru. Fungal Ecology 3: 240-254 2. Gazis R., Rehner S., Chaverri P. 2011. Species delimitation in fungal endophyte diversity studies and its implications in ecological and biogeographic inferences. Molecular Ecology 20: 3001-3013. 3. Gazis R., Gruner D.S., Chaverri P. 2012. Sampling effect in estimating tropical fungal endophyte diversity: Are we undersampling? 4. Gazis R., Miadlikowska J., Arnold B., Lutzoni F., Chaverri P. 2012. Culture- based study of endophytes associated with rubber trees in Peru reveals a new class of Pezizomycotina (Xylonomycetes). 5. Gazis R., Chaverri P. 2012. Amazonian forests are a reservoir of natural enemies against plant diseases: A comparison of fungi associated with wild and planted rubber trees (Hevea spp.). ii Other publications deliverable from this study: • Chaverri P., Gazis R. 2010. Perisporiopsis lateritia, a new species on decaying leaves of Hevea spp. from the Amazon basin in Peru. Mycotaxon 113: 163- 169. • Chaverri P., Gazis R., Samuels, G.J. 2010. Trichoderma amazonicum, a new endophytic species on Hevea brasiliensis and H. guianensis from the Amazon basin. Mycologia 103: 139-151. • Chaverri P., Gazis R. 2010. Linking ex planta fungi with their endophytic stages: Perisporiopsis, a common leaf litter and soil fungus, is a frequent endophyte of Hevea spp. and other plants. Fungal Ecology 4: 94-102. iii Dedication To my grandparents Julia and Jose Olivas, whom I miss every day. iv Table of Contents Foreword ....................................................................................................................... ii Dedication .................................................................................................................... iv Table of Contents .......................................................................................................... v List of Tables ............................................................................................................. viii List of Figures .............................................................................................................. ix Introduction ................................................................................................................... 1 Endophytes as mutualistic facilitators ...................................................................... 7 Endophytes as potential biological control agents .................................................... 7 Hevea brasiliensis: the study system....................................................................... 10 Objectives ............................................................................................................... 13 References ............................................................................................................... 14 Summary of chapters .................................................................................................. 31 Importance of the study .............................................................................................. 33 Chapter 1: Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis) in Peru ........................................................................................ 36 Introduction ............................................................................................................. 37 Materials and Methods ............................................................................................ 40 Study Site and Sampling Strategy............................................................................... 40 Isolation of Endophytic Fungi .................................................................................... 41 Morphological Characterization of Endophytes ......................................................... 42 Phylogenetic Analyses ................................................................................................ 43 Endophyte Diversity Analyses .................................................................................... 46 Results ..................................................................................................................... 47 Diversity of Endophytic Fungi.................................................................................... 47 Characterization and Comparison of Endophytic Fungi from H. brasiliensis............ 48 Discussion ............................................................................................................... 50 Diversity of Fungal Endophytes in Hevea brasiliensis............................................... 50 Community Composition and Assemblage ................................................................ 52 Endophytic Fungi as Potential Biocontrol Agents against H. brasiliensis diseases ... 58 Acknowledgments................................................................................................... 59 References ............................................................................................................... 60 Chapter 2: Species delimitation in fungal endophyte diversity studies and its implications in ecological and biogeographic inferences ........................................... 77 Abstract ................................................................................................................... 78 Introduction ............................................................................................................. 79 Materials and Methods ............................................................................................ 83 Study Taxa .................................................................................................................. 83 Source of Endophytic Isolates .................................................................................... 83 DNA Extraction, Amplification, and Sequencing ...................................................... 84 Diversity and Phylogenetic Analyses ......................................................................... 84 Species Recognition/Delimitation .............................................................................. 86 v Effects of Species Delimitation on Endophyte Diversity Measures: A Case Study ... 88 Results ..................................................................................................................... 88 Phylogenetic Analyses: Single Locus ......................................................................... 88 Phylogenetic Analysis: Multi-Locus Approach and Species Delimitation................. 89 Estimation of Intra- and Interspecific Divergence ...................................................... 90 Effects of Species Delimitation on Ecological and Biogeographic Inferences .........