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Endophytic fungi live within tissues of plant hosts without causing symptoms of disease. These fungi are broadly split into the taxonomically and ecologically cohesive Clavicipitaceous endophytes, which infect grasses, and the taxonomically diverse non-Clavicipitaceous endophytes, which are found in nearly all plants and have diverse ecological strategies. My dissertation has two sections: Section A investigates the intersection of Clavicipitaceous endophyte ecology with other ecological theory, including invasion ecology (Chapter II) and community ecology and climate change (Chapter III); Section B investigates the ecology of one group of non-Clavicipitaceous endophytes, the Xylariaceae, using a culture-based study in Ecuador (Chapter IV) and a next-generation sequencing based endophyte survey in Taiwan (Chapter V). Section B is centered on testing the Foraging Ascomycete (FA) hypothesis—the idea that some decomposer fungi may adapt an endophytic lifestyle to escape limitations in primary substrate in both time and space. In Chapter II, I utilized a host-specific Epichloë endophyte present ubiquitously in the European native range of the Pacific Northwest (PNW) invasive grass Brachypodium sylvaticum to test theories of invasion. In Chapter III, I examined the grass Agrostis capillaris in the context of a climate manipulation experiment in prairies in the PNW to elucidate patterns of interaction between multiple symbionts (Epichloë endophytes, dark septate root endophytes, and arbuscular mycorrhizal fungi) within single hosts across climatic variation. In Chapter IV, I began to test the FA hypothesis by examining spatial relationships of Xylaria endophytic fungi in the forest canopy with Xylaria decomposer fungi on the forest floor in a remote Ecuadorian cloud forest. In Chapter V, I build on the results from the previous study, using a novel technique to examine spatial ecology of the Xylariaceae, pairing traditional mycological collection with the preparation of a next-generation sequencing metabarcode library of endophytes over a much greater area. This dissertation includes previously published and unpublished coauthored material.

Sixty-two natural cankers on branches and main stems of three 16-year-old grafted American chestnut trees at the Lesesne State Forest were sampled for Cryphonectria parasitica. Cankers were sampled in 1996 and 1997 at various distances from the main stem zone on the grafts (ground to 183 cm) that was inoculated in 1982 and 1983 with a mixture of dsRNA-containing white and pigmented hypovirulent strains. Grafted trees exhibited a high level of blight control, and all bark cores extracted from cankers on the grafted trees showed superficial necrosis. Bark cores extracted from these cankers yielded 156 isolates of C. parasitica. Fifty-three of these isolates were white, and 103 were pigmented. The farthest canker containing a white isolate was located 564 cm from the zone inoculated with hypovirulent strains (H-inoculated zone). The number of white isolates recovered per canker on the grafted trees near the H-inoculated zone (< 0.5 maximum sampling distance) was significantly greater (P=0.0039) than the number of white isolates recovered per canker on the grafted trees far from the H-inoculated zone (>0.5 maximum sampling distance). Lloyd's index of patchiness value for the frequency of white isolates in cankers was 1.36, indicating that white isolates were slightly aggregated in cankers. White isolates of C. parasitica were found in two of seven artificially established cankers 5 months after inoculation with a pigmented virulent strain (WK). Thirteen of 14 pigmented isolates collected from these cankers after 5 months were compatible with WK in vegetative compatibility (VC) tests. Eight of 25 white isolates recovered 5, 11, and 50 months after WK inoculation converted the pigmented WK strain to the white hypovirulent phenotype in vitro. Sixty-five pigmented isolates collected from natural cankers were paired in VC assays, revealing 28 VC groups. All 11 white isolates of C. parasitica assayed contained a 12.7 kb dsRNA in high concentrations. None of 48 pigmented isolates assayed contained dsRNA. All white isolates tested in virulence trials on American chestnut stems in a forest clearcut were hypovirulent, based on low canker severity indices. Little or no dissemination of white strains to cankers on the American chestnut stump sprout clusters, which surround the grafted trees, was found. In the future, to maximize spread of white hypovirulent strains on American chestnut trees, it may be beneficial to re-inoculate trees with hypovirulent strains farther up the main stem after substantial tree growth has occurred. / Master of Science

This thesis investigates whether there are alternative mechanisms of tip growth in invasive and non-invasive hyphae of the fungus Neurospora crassa. The cytoskeleton protein actin is thought to play a pivotal role in hyphal tip growth, performing a multitude of tasks, one of which may be the provision of a resistive force to counter turgor pressure. An Actin depleted zone (ADZ) was the dominant feature of invasive hyphal tips, which was largely absent from non-invasive hyphae. The Spitzenkörper was slightly larger in invasive hyphae but this size difference alone was thought insufficient to account for the exclusion of filamentous actin (F-actin) from the tip. The actin nucleating protein formin was found at sites where actin nucleation is occurring, while cofilin, a protein that severs F-actin, was found to localise where F-actin disassembly was likely to be occurring. It is suggested that these proteins are likely to play a role in controlling a dynamic cytoskeleton, rearrangements of which are required for the two modes of growth. Invasive hyphae were found to generate a higher turgor than non-invasive hyphae. These results suggest that the F-actin rearrangements facilitated by cofilin give an ADZ that may play a role in invasive hyphal tip growth; possibly through a reduction of tip resistance; thus enabling the provision of a greater protrusive force by turgor.

Autophagie ist ein Degradationsprozess der streng reguliert ist und in welchem eine eukaryotische Zelle zelleigene Organellen und Proteine bei Nährstoffmangel abbaut. Außerdem konnte gezeigt werden, dass dieser Prozess auch in verschiedene Entwicklungsprozesse involviert ist. Die molekulare Entschlüsselung der Autophagie wurde hauptsächlich in der Bäckerhefe S. cerevisiae vorgenommen. Allerdings ist Beteiligung der Autophagie an Entwicklungsprozessen in multizellulären filamentösen Ascomyceten weitestgehend unbekannt. Die Fruchtkörperentwicklung von Pilzen ist ein komplex gestalteter Differenzierungsprozess der von einem zwei-dimensionalem Pilzgeflecht ausgeht das sich zu einem dreidimensionalem Perithezium entwickelt. Die Fruchtkörperentwicklung erfordert spezifische Umgebungsbedingungen und wird durch viele entwicklungsassoziierten Genen reguliert. In dieser Studie diente der Modellorganismus Sordaria macrospora zur Untersuchung des Einflusses der Autophagie auf die Fruchtkörperentwicklung. Der coprophytische filamentöse Ascomycet S. macrospora pflanzt sich lediglich sexuell fort, was ihn ideal für die Fragestellung dieser Arbeit macht. Für diese Arbeit wurden eine Reihe konservierter Autophagie bezogener Gene auserwählt. Folgende Gene die homolog zu denen anderer Ascomyceten sind wurden isoliert: Smvps34, Smvps15, Smatg8, Smatg4, und Smjlb1. Durch die Deletion dieser Gene sollte geklärt werden wie Autophagie in die Fruchtkörperentwicklung involviert ist. Die Deletion des Phospolipidkinase Gens Smvps34 und des Proteinkinase Gens Smvps15 führte zur Lethalität von S. macrospora was durch eine Auskeimungsuntersuchung belegt wurde. Die Deletion des Gens Smatg8, welches eine autophagosomale Strukturkomponente kodiert und des Gens Smatg4, das eine Cystein-Protease kodiert, die SmATG8 prozessiert, beeinträchtigte ebenfalls die Fruchtkörperentwicklung und das vegetative Wachstum. Durch Fluoreszenzmikroskopie konnte gezeigt werden, daß SmATG8 in Autophagosomen lokalisiert und SmATG4 vorwiegend im Zytoplasma lokalisiert ist. Die Prozessierung von SmATG8 durch SmATG4 wurde ebenfalls durch Fluoreszenzmikroskopie und Western-blot Analyse bestätigt. Die heterologe Expression von Smatg8 und Smatg4 in S. cerevisiae und der Ape1 Reifungsuntersuchung zeigte, das die cDNA von Smatg8 und Smatg4 den Deletionsphenotyp der jeweiligen Hefedeletionsmutanten aufheben konnte. Somit konnte die Konservierung dieser beiden Gene innerhalb der Ascomyceten gezeigt werden. Die Blockade der Fruchtköperentwicklung wurde durch die Deletion des bZIP Transkriptionsfaktor Gens Smjlb1 verursacht genauso wie die Beeinträchtigung des vegetativen Wachstums. SmJLB1 ist im Kern lokalisiert und durch qRT-PCR Experimente wurde gezeigt, dass die Autophagiegene Smatg8 und Smatg4 durch Smjlb1 reguliert werden. Dies läßt vermuten, dass Smjlb1 in den Prozess der Autophagie involviert ist. Die Ergebnisse dieser Arbeit weisen darauf hin, dass Autophagie und Fruchtkörperentwicklung des filamentösen Pilzes S. macrospora streng miteinander verknüpft sind.

This thesis investigates whether there are alternative mechanisms of tip growth in invasive and non-invasive hyphae of the fungus Neurospora crassa. The cytoskeleton protein actin is thought to play a pivotal role in hyphal tip growth, performing a multitude of tasks, one of which may be the provision of a resistive force to counter turgor pressure. An Actin depleted zone (ADZ) was the dominant feature of invasive hyphal tips, which was largely absent from non-invasive hyphae. The Spitzenkörper was slightly larger in invasive hyphae but this size difference alone was thought insufficient to account for the exclusion of filamentous actin (F-actin) from the tip. The actin nucleating protein formin was found at sites where actin nucleation is occurring, while cofilin, a protein that severs F-actin, was found to localise where F-actin disassembly was likely to be occurring. It is suggested that these proteins are likely to play a role in controlling a dynamic cytoskeleton, rearrangements of which are required for the two modes of growth. Invasive hyphae were found to generate a higher turgor than non-invasive hyphae. These results suggest that the F-actin rearrangements facilitated by cofilin give an ADZ that may play a role in invasive hyphal tip growth; possibly through a reduction of tip resistance; thus enabling the provision of a greater protrusive force by turgor.

The fungal microbiome, or “mycobiome” of plants is diverse and important to host health, but the fluxes of fungi among plant hosts and with the surrounding environment are poorly understood. In chapter two, we employed sterile culture techniques and spatial sampling to examine leaves as possible vectors for transfer of their endophytic fungi from the canopy to substrate on the forest floor, as predicted by the Foraging Ascomycete hypothesis. Some foliar endophytic fungal species are also present as wood-decomposing fungi on the forest floor, that transfer of mycelium across these two life history stages can occur, that endophytic life history stages are buffered from environmental conditions in comparison to wood-decomposing fungi, and that spatial linkages between the two life history stages can be observed. In another study, described in chapter 3, wood and leaf wood endophytes were sampled across a 25 ha plot, to explore landscape patterns of mycobiomes, and to explore the concept of a core microbiome in aerial plant tissues. We found that core microbiomes may be observed in a real ecological setting, but that the concept of core must be carefully defined and that some level of buffering from disturbance may be necessary to allow core microbiomes to assemble. In chapter four, we return to examine some of the assumptions and implications of the Foraging Ascomycete hypothesis, with an agent-based model. We model the conditions under which dispersal through falling leaves may represent a fitness-enhancing dispersal strategy for fungi, and that deforestation as is currently underway throughout the world may have impacts on fungi that rely upon a canopy- inhabiting life stage for dispersal. In chapter five, some challenges associated with environmental sampling of microbes using illumina© MiSeq sequences are critically examined. We find that biases introduced by random sampling at various stages of IVenvironmental DNA extraction and illumina© MiSeq sequencing are not well corrected by currently accepted bioinformatic algorithms. In addition, information loss from differential extraction, PCR amplification, and sequencing success, requires that users of MiSeq read libraries to interpret read abundances carefully. This dissertation includes previously published, co-authored material.

Lecythophora (Coniochaeta) hoffmannii, a soil- and lignocellulose-inhabiting sordariomycete (Ascomycota) that can also live as a facultative tree pathogen causing soft rot, belongs to the family Coniochaetaceae. The strain CBS 245.38 sequenced here was assembled into 869 contigs, has a size of 30.8 Mb, and comprises 10,596 predicted protein-coding genes.

We report here the draft genome of Kretzschmaria (Ustulina) deusta, an ascomycetous fungus that colonizes and substantially degrades hardwood and can infest living broad-leaved trees. The genome was assembled into 858 contigs, with a total size of 46.5 Mb, and 11,074 protein-coding genes were predicted.

We report here the draft genome of Kretzschmaria (Ustulina) deusta, an ascomycetous fungus that colonizes and substantially degrades hardwood and can infest living broad-leaved trees. The genome was assembled into 858 contigs, with a total size of 46.5 Mb, and 11,074 protein-coding genes were predicted.