Fungal endophytes are highly diverse, cryptic plant endosymbionts that form asymptomatic infections within host tissue. They represent a large fraction of the millions of undescribed fungal taxa on our planet with some demonstrating mutualistic benefits to their hosts including herbivore and pathogen defense and abiotic stress tolerance. Other endophytes are latent saprotrophs or pathogens, awaiting host plant senescence to begin alternative stages of their life cycles. Most, however, are likely plant commensals with no observable benefits to their hosts while under study. Yet, when considering the context-dependence that may determine plant resistance to pathogen attack, the consortium of endophytes present in the host could alter these outcomes, thereby affecting plant evolution. Despite the excitement of exploiting endophytes for their potential to produce bioactive compounds that are useful to medicine and agriculture, fungal endophyte community ecology is a lagging field. Much remains unknown regarding their colonization patterns within hosts, their spatial and temporal turnover and their diversity. Further, a severe deficiency exists in work on endophytes in seed-free plants, particularly ferns. Since ferns exist in free-living forms in both the haploid and diploid stages, are the second largest group of vascular plants, occupy important ecological niches and represent an older lineage of land plants, this is a major shortcoming in our understanding of plant-fungal ecology and evolution. For these reasons, I have examined endophyte community ecology in a widespread fern host in the Pacific Northwest, Polystichum munitum, addressing several gaps in the endophyte literature. Since an understanding of the degree of early endophyte colonization, temporal turnover and the relative contribution of priority effects to community composition are unknown, I conducted a temporal survey of fern endophyte communities utilizing culture-independent, next-generation sequencing on a monthly basis for an entire growing season. A high degree of temporal turnover was observed early in the growing season, where a late colonist rapidly took over the fern population and persisted throughout the year. This colonist, which was isolated from several of the same plants, appears to support growth rates of the host plant when in the gametophytic stage, but is not highly competitive against other endophytes in vitro. Additionally, in an effort to examine host and habitat specificity I examined the spatial turnover of endophytes across four distinct habitat types: a coastal site, a coniferous understory site, a montane site near Mount Saint Helens but not impacted by the 1980 eruption, and a secondary succession site at Mount Saint Helens, spanning 150-km at a single point in time. A high degree of host specificity was found when compared to endophyte communities in neighboring plant taxa and a lack of distance decay was also observed contrasting with other work examining endophyte biogeographic patterns. We conclude that a high degree of host filtering, combined with an abundance of senescent fern material around the base of the plant--which potentially serves as a reservoir of endophyte inoculum--is likely responsible for the observed results. In the process of the ecological studies described above, I isolated over 500 strains of endophytes that corresponded to ca. 100 operational taxonomic units (OTUs). Four of these OTUs are previously undescribed and form a new family and genus, Catenosporaceae and Catenospora, respectively. One of these taxa is responsible for the strong spatial and temporal signals found in the ecological studies. We emphasize that future work should examine if the same phenomena are observed in other fern systems and further encourage endophyte researchers to expand the scope of their investigations into non-traditional plant lineages, as exciting ecological interactions that contribute to our understanding endophyte ecology--and community ecology as a whole--are waiting to be discovered.

Many years of study have revealed that fungi are excellent sources of novel bioactive secondary metabolites. Some of these secondary metabolites possess therapeutic qualities that improve the quality of life for millions of people. Such metabolites include well known classes such as the penicillins, cephalosporins, and statins, yet many fungi remain underexplored as sources of biologically active metabolites. The research described in this thesis employs an ecology-based approach to targeting fungi for chemical investigation, and describes studies of fungi from two niche groups, fungicolous/mycoparasitic and endophytic fungi, as possible sources of new secondary metabolites with biological activities. In a parallel project, the structures of bioactive compounds isolated from peanut seeds that had been subjected to fungal attack were elucidated in the pursuit of compounds with beneficial bioactivities. Mycoparasitic fungi are those that colonize other fungi by parasitizing the host, often leading to damage to the host fungus. Fungicolous fungi are those that colonize other fungi, but have not been proven to be true mycoparasites. The damage often caused by colonization of host fungi indicates that mycoparasitic and fungicolous fungi can produce antifungal compounds. Chemical investigations of such fungi described in this thesis afforded 37 compounds of various biosynthetic types, seven of which were new. Many of these compounds show antifungal, antimicrobial, and/or cytotoxic effects. Endophytic fungi live asymptomatically within plant tissues and in some cases may provide benefits to the host plant through the production of secondary metabolites. Chemical investigations of corn, wheat, and sorghum endophytes led to the isolation and characterization of 21 compounds, seven of which were new. Many of the endophyte metabolites encountered in this work showed antifungal, antimicrobial, and/or cytotoxic effects. The compounds isolated from peanut seeds were produced in response to fungal attack by an Aspergillus caelatus strain. All of these compounds were stilbene-derived phytoalexins, which are considered to be inducible chemical defenses whose production is elicited or enhanced upon microbial attack. Further studies of these newly identified compounds and their production could lead a a better understanding of how the plant defends itself. Such knowledge could enable researchers to manipulate this mechanism to obtain greater peanut resistance to invasion by pests. Additionally, the health benefits from related stilbene-derived compounds (e.g. resveratrol) from peanuts and other plants have been widely established. Knowledge about the presence of compounds of this type could add to the importance of peanut crop production. The compounds identified in this work were isolated using multiple chromatographic techniques, and the structures were established based on analysis of 1D and 2D NMR data combined with MS, chemical derivatizations, and/or optical measurement data. Absolute configuration assignments were achieved by application of Mosher's Method, CD spectral analysis, and/or chemical derivatizations. Details of the isolation, structure elucidation, and biological activity of these compounds are presented in this thesis.

Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.

Western Oregon is home to native Rhododendron species and is the center for cultivated Rhododendron production in the United States. These Rhododendron spp. are known to be infected with fungal endophytes. However, the community structure of these endophytes in native and cultivated Rhododendron is poorly understood. Our study targeted the foliar fungal endophyte communities of two native Rhododendron spp. and two non-native commercially cultivated Rhododendron varieties. Parallel culturedependent (fungal isolation and identification based on ITS sequencing) and cultureindependent sequencing approaches (metagenomic sequencing of the ITS region using 454 pyrosequencing) were employed, and results provided evidence of distinctly different community structure in each host species. Additionally, results indicated higher diversity among cultivated and nursery grown Rhododendron. This suggests that the close proximity of the nurseries sampled to the forest environment allows exposure to two distinct sources of endophyte infection. Together, our results show the importance of host identity and environment in structuring the associated endophyte communities. / Graduation date: 2013

Fungal communities of aquatic ecosystems are understudied, leaving major gaps in estimating global fungal biodiversity. In particular, little is known regarding the distinctiveness of (1) aquatic endophytes vs. those in terrestrial plants, and (2) waterborne fungi vs. those inhabiting aquatic plants or sediments. I assessed the diversity, composition, host affiliations, and geographic structure of culturable endophytes associated with aquatic angiosperms in reservoirs in northern Arizona, and their distinctiveness relative to waterborne- and sediment fungi. Endophytes were low in abundance yet extremely diverse. Communities differed significantly in composition among reservoirs and tissues. Aquatic endophytes were largely distinct from those in terrestrial plants, and from those in water and sediment, but did not demonstrate host specificity. This work reveals the uncommon diversity and distinctiveness of endophytes in aquatic plants, and provides a first quantitative estimation of endophytic associations in diverse, ecologically important, and economically relevant aquatic plants.

Bacterial endophytes, bacteria residing within plants, play an important role in the growth and development of plants and their ability to thrive under adverse conditions. The endophytes of Acer negundo, Ulmus pumila and Ulmus parvifolia trees sampled from a hydrocarbon-contaminated site were analyzed for variation between seasons and plant species. Branches from the same trees over a span of 3 seasons were collected and analyzed via culture dependent and culture independent methods. Numerous culture independent approaches were tested, culminating in the development of a new method for the amplification of endophytic bacterial ribosomal DNA that excludes plastid DNA. Community analyses using this new method in combination with T-RFLP showed significant differences between the endophytic communities of different plants species and of the same species growing in different seasons. The proposed technique can be used for the future study of endophytic communities of plants.

Bacterial endophytes, bacteria residing within plants, play an important role in the growth and development of plants and their ability to thrive under adverse conditions. The endophytes of Acer negundo, Ulmus pumila and Ulmus parvifolia trees sampled from a hydrocarbon-contaminated site were analyzed for variation between seasons and plant species. Branches from the same trees over a span of 3 seasons were collected and analyzed via culture dependent and culture independent methods. Numerous culture independent approaches were tested, culminating in the development of a new method for the amplification of endophytic bacterial ribosomal DNA that excludes plastid DNA. Community analyses using this new method in combination with T-RFLP showed significant differences between the endophytic communities of different plants species and of the same species growing in different seasons. The proposed technique can be used for the future study of endophytic communities of plants.

Hydrolytic enzymes degrade macromolecules into smaller components. These enzymes are important in fungal nutrition and have been implicated in the pathogenicity and virulence of pathogenic fungi towards their hosts. However, it is unknown if hydrolytic enzymes play important roles in mutualistic symbioses. In this study, the function of two different classes of hydrolytic enzymes was examined in the mutualistic symbiosis between the fungal endophyte Epichloë festucae and perennial ryegrass (Lolium perenne cv. Nui). Nine members of a gene family encoding subtilisin-like proteases were identified in E. festucae. The prt2, prt3 and prt5 genes encode putative extracellular proteins belonging to the proteinase K subfamily 1, and prt1 and prt6 encode putative extracellular proteins belonging proteinase K subfamily 2. The prt7 and prt8 genes encoded pyrolysin-like enzymes from subfamilies 1 and 2. The prt4 gene encodes a putative vacuolar protease, while the kex2 gene encodes a putative proprotein convertase. Expression analysis showed that the prt1, prt3, prt5, prt4 and kex2 genes, but not the prt2 gene, were expressed in culture. The prt1 and prt3 genes appeared to be up-regulated in planta compared to culture. The function of prt1 and prt2 in the symbiotum between E. festucae and perennial ryegrass was characterised by expressing these genes under the control of the Aspergillus nidulans gpdA or the E. festucae F11 ltmM promoters. No major differences in hyphal or plant morphology were observed between symbioses containing wild type E. festucae or endophyte strains containing the prt1 or prt2 transgenes. The gcnl gene, which encodes a β-1,6-glucanase, was identified immediately downstream of the prt2 gene. The function of the gcnl gene was characterised by gene replacement and testing the phenotype during growth in culture and in planta. E. festucae ∆gcnl strains grew normally on glucose-containing media. On media containing the β-1,6-glucan pustulan, ∆gcnl strains did not form aerial hyphae or hydrolyse pustulan, which the wild type strain did. This phenotype was partially complemented by growth of the ∆gcnl mutant in close proximity to wild type strains, and fully complemented by insertion of the gcnl gene. This suggests that the gcnl gene encodes the major β-1,6-glucanase activity of E. festucae.

Appendix content (raw data and statistics; sequences) unavailable online, but available with print copy / Epichloë/Neotyphodium endophytes are a group of clavicipitaceous fungi that form symbiotic associations with temperate grasses. The asexual N. lolii form asymptomatic mutualistic associations with ryegrass whereas the sexual E. typhina behaves similar to a mutualist during the vegetative phase of plant growth but switches to epiphytic growth and formation of an external stroma upon development of the floral inflorescence. The aim of this project was to study the metabolic interaction between these endophytes and their perennial ryegrass host. The role of endophyte thiamine biosynthesis in host colonisation and stroma development was chosen, because of the key role this coenzyme plays in primary cellular metabolism and because thiamine biosynthetic genes are induced in several fungal-plant interactions. The orthologue (thil) of Saccharomyces cerevisiae THI4 was isolated from N. lolii and E. typhina by PCR using degenerate primers designed to conserved regions of known thiazole biosynthetic genes. This gene is expressed in planta and in culture, and is alternatively spliced, with distinct patterns of the isoforms expressed under different nutritional conditions. Mutant with a deletion in the E. typhina thil gene was constructed and shown to have reduced hyphal density and branching compared to the wild-type on defined media lacking thiamine. Both thiamine and thiazole complemented this defect. Artificial inoculation of the mutants into plants showed that the thil mutant retained the ability to colonise the perennial ryegrass host and form stromata. However, the mutant had some differences in host colonisation and growth, including reduced hyphal branching and reduced detrimental effects on the host. In addition, glycogen-like deposits, which were abundant in the wild-type hyphae, were not evident in the mutants. Unexpectedly, both the thil mutant and wild-type strains formed some stromata on vegetative tissue. Electron microscopic examination revealed that the cells of epiphytic hyphae found on the vegetative tillers typically were enlarged, lacking in cytoplasm and highly vacuolated, an ultrastructure similar to that found for hyphae growing in reproductive tillers. The mutants retained the ability to form conidia on the outer layer of the stromata. Extensive vascular colonisation and hyphal ramification in the mesophyll were common characteristics of stromata bearing regions. Although the morphology and ultrastructure of stromata formed on vegetative tillers is very similar to those on reproductive tillers, one significant difference was the presence of abundant glycogen-like deposits in hyphae of vegetative tillers. Furthermore, there were dramatic differences in the levels of glycogen-like deposits in hyphae in different regions of the vegetative tillers, indicating that the energy demand changes during stroma development. This is the first report of E. typhina forming stromata on non-inflorescence tillers.

Thesis (M.S.)--Brigham Young University. Dept. of Chemistry and Biochemistry, 2005. / Includes bibliographical references.