Molecular genetic analysis of fescue-derived fungal endophytes

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Life Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The research described in this thesis was focused on assessment of genetic diversity, metabolic profile analysis and reconstruction of phylogenetic relationships between fungal endophytes of fescue pasture grasses. Incidence and genetic diversity were evaluated for tall fescue- and meadow fescue-derived endophytes, that were collected from diverse geographical origins throughout the range of natural growth and domesticated cultivation, using a set of endophyte-specific simple sequence repeat (SSR) genetic markers. Moderate (37 percent) endophyte incidence was demonstrated in 760 fescue tested accessions. Subsequent analysis of endophyte diversity identified a high proportion of novel endophyte genotypes that were attributed to six distinct taxa. The majority of the identified endophytes belonged to the taxon Neotyphodium coenophialum, while representatives of other known taxa, such as Neotyphodium uncinatum, Festuca arundinacea taxonomic group 2 and 3 (FaTG-2 and FaTG-3) were also observed. Furthermore, diversity analysis revealed the occurrence of two putative novel endophyte taxa which were designated as ‘FaTG-3-like’ and ‘uncharacterised Neotyphodium species’ (UNS) in this study. Following diversity analysis, novel endophytes suitable for metabolic profile analysis were selected, and alkaloid profiles were determined in the endogenous genetic background. Based on identified alkaloid profiles and genetic diversity, a set of 20 fescue endophytes were subjected to genome survey sequencing. Comparison with genomic regions containing gene clusters involved in alkaloid biosynthesis demonstrated that gene loss could account for some of the differences observed between toxin profiles among the different endophyte strains. Furthermore, phylogenetic analysis based on nuclear genes (tub2, tefA and perA) revealed putative ancestral sub-genome contributions from known Epichloë species to the putative novel endophyte taxa identified from this study, while also confirming the proposed progenitors of known endophyte taxa. In addition to the nuclear genome, the complete mitochondrial genome (mt genome) sequence was determined for each of the sequenced endophyte, and phylogenetic relationships among mt genome protein coding gene complement were reconstructed. Novelty of the FaTG-3-like and UNS taxa was confirmed by the results of the phylogenetic analysis. The results obtained in this study will provide the basis for longer-term manipulation of the beneficial associations between host grasses and endophytes.