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.

Willow (Salix spp.), a major source of biomass and renewable fiber production, is one of the best choices for short-rotation intensive culture (SRIC) in Canada. Since fungal communities play important roles in the plant’s health status, it is vital to understand their interactions with willows and their roles in the sustainability of SRIC. In this study, fungal diversity of the above-ground organs (stem/leaf) of healthy and diseased willow plants in western Canadian Prairies were assessed using cultural and PCR-denaturing gradient gel electrophoresis (DGGE) techniques. Comparison of the mycoprofiles within established plantations vs. newly introduced cuttings revealed differences in the fungal communities. Ascomycota were mainly isolated, followed by Basidiomicota and Zygomycota. Willow genotypes seem have an influence on the abundance of fungal pathogens and disease severity; among them Charlie (Salix alba x gladfelteri) and SV1 (S. eriocephala) cultivars demonstrated superior performances. Photosynthesis measurements and biomass compositions confirmed these findings. Potentially pathogenic fungi (Dothioraceae, Diaporthaceae, Glomeraceae, and Pleosporaceae) dominated in diseased or symptomatic willows, whereas potentially beneficial fungi (Coniochaetaceae, Hypoceraceae, Nectriaceae, Trichocomaceae, and Agaricaceae) prevailed in healthy plants. In-vivo and greenhouse assays showed that inoculation with potentially pathogenic fungi induced leaf necrosis, anthracnose and open cankers. However, suppression of the latter was still possible using fungal antagonists. Hence, assessment of stem/bark and leaf fungal communities with respect to willow genotypes, cuttings origin, and SRIC location, is useful for the design of an effective management strategy to increase the productivity of the SRIC-biomass systems.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references. Also available on the World Wide Web.

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Au cours des vingt dernières années, les études sur les sédiments marins profonds ont révélé la présence et l'activité de communautés microbiennes inattendues. Il est maintenant formellement établi que la biosphère profonde héberge de nombreux représentants des domaines des Archaea et des Bacteria. Cependant,les micro-eucaryotes et plus particulièrement les champignons n’ont été que très peu étudiés dans ces écosystèmes singuliers. Dans ce contexte, des approches moléculaire et culturale ont été utilisées afin de caractériser la diversité des communautés fongiques des sédiments marins profonds en utilisant le bassin de Canterbury comme modèle d’étude. Les résultats principaux obtenus lors de ce travail de thèse sont les suivants : (i) L’approche moléculaire basée sur l’ADN a fourni la preuve directe que les communautés fongiques peuvent persister jusqu’à la profondeur record de 1740 mètres sous la surface du plancher océanique. (ii) Des approches complémentaires ciblant les ARNr et les ARNm ont permis de préciser leur activité métabolique et d’obtenir de premiers indices sur les fonctions de ces champignons à 350m sous la surface du plancher océanique, principalement liées à la croissance, à l’adaptation aux contraintes environnementales in situ et aux interactions entre communautés microbiennes. (iii) L’approche culturale a permis de constituer une collection de culture de 183 isolats fongiques avec des caractéristiques écophysiologiques témoignant leur capacité d’adaptation aux conditions in situ. (iv) Le potentiel biotechnologique des isolats obtenus a été estimé via la recherche de gènes impliqués dans la synthèse de métabolites secondaires et a permis de positionner cette collection d’organismes originaux comme une ressource d’intérêt biotechnologique potentiel. Ce travail qui témoigne de la persistance et de l’activité des communautés fongiques dans les sédiments marins profonds élargit notre vision de la diversité microbienne dans ces milieux et soulève des hypothèses sur le rôle écologique des champignons au sein de la biosphère profonde. / Over the past two decades, investigations on deep marine sediments have revealed the occurrenceand activity of unexpected microbial communities. Many representatives of Archaea and Bacteria were reportedbut micro-eukaryotes and especially fungal communities are still poorly studied in this ecosystem. In this underexplored context, molecular- and culture-based approaches were used to characterize the diversityof fungal communities in deep subsurface sediments using the Canterbury Basin as a model system. The main results of this work are: (i) The molecular DNA-based approach provided direct evidence that the fungal communities persist until the record depth of 1,740 meters below sea floor. (ii) Supplementary approaches targeting rRNA and mRNA revealed their metabolic activity and highlighted first hints into the fungal functions at350 meters below sea floor, mainly related to growth, adaptation to in situ environmental constraints andmicrobial interactions. (iii) The culture based approach allowed establishing a culture collection of 183 fungal isolates with ecophysiological characteristics indicating their ability to adapt to in situ conditions. (iv) This culture collection seems to represent a reservoir of secondary metabolites as many genes involved in secondary metabolites pathways were revealed. The fungal collection established may be considered as an untapped resource to explore for biotechnological applications. This work demonstrating the persistence and activity of fungal communities in deep subsurface sediments (i)broadens our view of microbial diversity in these environments and (ii) raises hypotheses about the ecologicalroles of fungi in the deep biosphere

Cavity-excavating birds, such as woodpeckers, are ecosystem engineers and are often assumed to rely upon wood decay fungi to assist in softening the wood of potential excavation sites. Endangered red-cockaded woodpeckers (Picoides borealis; RCWs) are the only birds known to solely excavate through the sapwood and into the heartwood of living pine trees and generally take many years to complete their excavations. These birds may have developed a partnership with wood-inhabiting fungi to facilitate the excavation process. Past attempts to understand the complex relationships between cavity excavators and fungi relied on visual surveys of fruiting bodies, or evidence of decay, resulting in a one bird, one fungus paradigm. Using molecular methods, I investigated the relationships between RCWs and fungi, and found that the relationships between cavity-excavators and fungi involve multiple fungal species and are far more complex than previously imagined. Through a field survey, I showed that RCW excavations contain distinct communities of fungi, and propose two hypotheses to explain this result, (1) RCWs select trees with distinct fungal communities (tree selection hypothesis), or (2) RCWs promote distinct fungal communities via their excavations (bird facilitation hypothesis). By swabbing the birds, I found that RCWs carry fungal communities similar to those found in their completed excavations, demonstrating that RCWs may directly facilitate fungal dispersal during the excavation process. Through a test of the bird introduction hypothesis which implemented human-made experimental drilled cavity starts (incomplete excavations), half of which were inaccessible to the birds, I showed that RCW accessibility influences fungal community development in excavations. This experimental evidence demonstrates that the relationship between RCWs and fungal communities is a multipartite symbiosis may be mutualistic. Finally, by tracking fungal community development in experimental cavity starts through time, I also demonstrated that the fungal communities found in RCW excavations undergo succession, and that this process is influenced by the birds. The relationships described in this body of work provide the basis for future studies on cavity excavators and fungi, and also have implications for a diverse community of secondary cavity nesters, wood-inhabiting fungi, forest ecology, and the conservation of biodiversity. / Ph. D.

La dissipation des Hydrocarbures Aromatiques Polycycliques (HAP), polluants persistants majoritaires des sols de friches industrielles, implique l'action de microorganismes bactériens et fongiques. Cependant, leur contribution relative à la dissipation in situ, en fonction des interactions entre ces microorganismes ou avec les plantes et les polluants, est mal connue et les communautés fongiques ont été très peu étudiées dans de tels environnements. L'objectif de cette thèse était de préciser l'écodynamique des HAP dans la rhizosphère sous l'effet des microorganismes et de leurs interactions et d'évaluer la diversité fongique dans des sols contaminés. La dynamique des communautés fongiques a été étudiée in situ pendant 5 années, par PCR en temps réel et Temporal Temperature Gradient Electrophoresis (TTGE), dans un sol historiquement contaminé en présence ou non de plantes et dans le même sol ayant subi un traitement de remédiation par désorption thermique. Les plantes avaient un effet positif sur l'abondance et la diversité des champignons et étaient le facteur déterminant la structure des communautés fongiques, dominées par les Ascomycètes. D'autre part, des souches bactériennes et fongiques ont été isolées de ce sol et testées pour leur capacité à dégrader les HAP in vitro. Parmi celles-ci, la bactérie Arthrobacter oxydans Pyr2MsHM11 et le champignon Fusarium solani MM1 ont été choisies comme souches modèles pour étudier leur action individuelle ou conjointe sur la dissipation de trois HAP, dans différentes conditions, des plus simples (cultures liquides) aux plus complexes (microcosmes de sol planté en présence d'une microflore). Les résultats ont montré l'importance fondamentale des interactions entre microorganismes, notamment des phénomènes de compétition, pour la croissance des souches modèles et pour l'expression de leurs potentiels de dissipation des HAP / The dissipation of Polycyclic Aromatic Hydrocarbons (PAHs), very common and persistant pollutants in soils from industrial wastelands, involve the action of bacterial and fungal microorganisms. However their respective contribution, and the influence of the microbial and plant-microbe interactions on in situ PAH dissipation, are poorly known and fungal communities were scarcely studied in such environments. This work aimed to study the fate of PAHs in rhizosphere under the influence of microorganisms and their interactions and to estimate the fungal diversity in contaminated soils. The dynamic of fungal communities was monitored in situ for 5 years, by real-time PCR and Temporal Temperature Gradient Electrophoresis (TTGE) in an aged PAH-polluted soil and in the same soil treated by thermal desorption. The results showed that plants had a positive effect on fungal abundance and diversity and were the main driver of fungal community structure, dominated by Ascomycetes. Besides, bacterial and fungal strains were isolated from this soil and screened for their ability to dissipate PAHs in vitro. Among them, the bacteria Arthrobacter oxydans Pyr2MsHM11 and the fungus Fusarium solani MM1 were chosen as model strains to study their individual and simultaneous effect on PAH dissipation in different experimental conditions, from liquid cultures to planted soil microcosms with a complex microflora. It was found that interactions between microorganisms, notably competition, had a crucial influence on their growth and on the expression of their PAH dissipation potential

Les champignons de la rhizosphère ont une grande influence sur le développement et la croissance des plantes. Certains de ces micro-organismes protègent les plantes contre les pathogènes, atténuent l'impact des stress abiotiques ou facilitent la nutrition des plantes. Ces organismes s'influencent mutuellement et forment des réseaux complexes d'interactions. Déterminer le fonctionnement du microbiome fongique de la rhizosphère des plantes cultivées est une étape nécessaire pour optimiser l'efficacité de la production végétale. Nous avons testé les hypothèses suivantes : (1) la diversification des systèmes de culture influe sur le microbiome fongique de la rhizosphère du canola; (2) le canola a un core microbiome, soit un ensemble de champignons toujours associés au canola quelles que soit les conditions du milieu; et (3) que certains de ces taxons ont une influence déterminante sur la structure des communautés (taxons nodaux) dans le core microbiome. Pour ce faire, en 2013 et 2016, nous avons échantillonné à la floraison, la phase de canola (Brassica napus) du système de culture, qui est l'un des deux types de canola (Roundup Ready® et Liberty Link®), utilisés dans le cadre d'une expérience de terrain à long terme (6 ans). Lacombe (Alberta), Lethbridge (Alberta) et Scott (Saskatchewan). En utilisant le séquençage d’amplicon par illumina, nous avons obtenus des résultats qui montrent que la diversification des cultures a un impact significatif sur la structure des communautés fongiques de la rhizosphère. Nous avons également découvert et décrit un core microbiome constitué de 47 OTU (Unité Taxonomique Opérationnelle) en 2013 et identifié Preussia funiculata, Schizothecium sp., Mortierella sp., Nectria sp. ainsi que deux taxons inconnus (OTU12 et OTU298) comme taxons nodaux parmi ce core microbiome. Cependant ce core microbiome s’est montré variable, et nous n’avons pu identifier qu’un OTU y appartenant en 2016 : Olpidium Brassicae. Nos résultats permettent de confirmer l’impact de la diversité culturale sur le microbiome fongique du canola et sont présentés comme une base pour le développement de stratégies d'ingénierie écologique pour l'amélioration de la production de canola. / The fungi in the rhizosphere have a large influence on plant development and growth. Some of these micro-organisms protect plants against pathogens, mitigate the impact of abiotic stress, or facilitate plant nutrition. These organisms influence each other and form complex webs of interactions. Deciphering the structure and function of the fungal microbiome of crop plant rhizosphere is a necessary step toward optimizing the efficiency of plant production. We tested the hypotheses that (1) the diversification of cropping systems influences the fungal microbiome of canola rhizosphere, (2) canola has a fungal core microbiome, i.e. a set of fungi that are always associated with canola, and (3) that some taxa have a determining influence on the structure of the communities (hub-taxa) within the core microbiome. In 2013 and 2016 we used the canola (Brassica napus) phase of five cropping system at blooming stage, from the less to the most diversified, that included one of two types of canola (Roundup Ready® and Liberty Link®), in an existing long-term (6 years) field experiment. The experiment has a randomized complete block design with four blocks, and is replicated at three locations: Lacombe (Alberta), Lethbridge (Alberta) and Scott (Saskatchewan). Our results show that crop diversification has significant impact on the structure of rhizosphere fungal communities. We also discover and described a canola core microbiome made of 47 OTUs in 2013 and identified Preussia funiculata, Schizothecium sp., Mortierella sp., Nectria sp. and two other unidentified taxa (OTU12 and OTU298) as the hub-taxa among this core. However this core microbiome was variable and could identify only one member in 2016 : Olpidium brassicae. Our results confirmed the effect of crop diversification upon the fungal microbiome of canola and are presented as a basis for the development of ecological engineering strategies for the improvement of canola production.

Mat-forming ectomycorrhizal (EcM) fungi represent a prevalent constituent of many temperate forest ecosystems and create dramatic changes in soil structure and chemistry. EcM mat soil have been shown to have increased microbial respiration rates and have been hypothesized to harbor unique assemblages of fungi and bacteria. The objectives of this dissertation were to characterize and examine the fungal and bacterial communities associated with EcM mats in old-growth forests of the H.J. Andrews Experimental Forest located in the Oregon Cascades. Additionally, this work assessed the application of traditional, emerging, and novel molecular sampling techniques for determining microbial communities of environmental samples. This research investigated the microbial communities associated with two common EcM mat genera found in old-growth Douglas fir stands in the Pacific Northwest; Piloderma (Atheliales, Basidiomycota) and Ramaria (Gomphales, Basidiomycota). Soil samples were collected from Piloderma and Ramaria mats and surrounding non- mat soil for molecular analysis of nucleic acids. First, a comparative study was conducted to determine the most appropriate rDNA molecular sampling technique for microbial community characterization. Two next-generation sequencing methods, Roche 454 pyrosequencing and Illumina-based environmental sequencing, the latter developed by the author, were compared to a more traditional sequencing approach, i.e., Sanger sequencing of clone libraries. These findings informed the subsequent sampling of the fungal ITS and bacterial 16S rDNA fragment with 454 pyrosequencing to determine the microbial communities within mat and non-mat soils. Second, this work utilized a pyrosequencing approach to explore fungal community structure in EcM mat and non-mat soils. This work concluded that differences in microbial communities do exist between Piloderma mat, Ramaria mat, and non-mat soils, but the differences are largely quantitative with relatively few distinct taxonomic shifts in microbial constituents. Piloderma, Ramaria and Russula, in addition to being the dominant taxa found on mycorrhizal root tips, were found to be the most abundant taxa in bulk soils within their respective mat types or non-mat sample. The background fungal communities within the EcM mats in this study exhibited considerable taxonomic overlap with the exception of Piloderma vs. non-mat comparisons; Russula species dominated nonmat soils but tended to be excluded or significantly underrepresented in Piloderma mats. Lastly, this study explored the bacterial communities associated with Piloderma and Ramaria mats using lower- coverage 454-Jr pyrosequencing. Bacterial communities exhibited significant structure as a function of mat-type, soil horizon and pH, but this finding should be interpreted with respect to the nonrandom distribution of Piloderma-mats in the O- horizon and the Ramaria-mats in the A-horizon, and the tendancy for EcM mats to be more acidic than surrounding soils. Nonetheless, the total microbial (bacterial and fungal) community was typically dominated by the mat-forming taxa, or Russula, in the case of non-mat soils. While the presence of Piloderma mats did enrich or restrict some bacterial groups, soil pH was also found to be a significant driver of bacterial richness and taxonomic diversity. Fungal and bacterial richness were also found to be positively related to one another, regardless of soil horizon or EcM mat type. This work, taken together, contributes to the understanding of hyperdiversity and heterogeneity of microbial communities of temperate forest soils and highlights the potential for fungal and bacterial communities to be influenced by the presence of EcM mats. / Graduation date: 2012

The vast majority of terrestrial plant species live in symbiosis with arbuscular mycorrhizal fungi (AMF). AMF and plants live in complex networks, with roots of individual plants hosting multiple AMF, and single AMF colonizing multiple plants concurrently. Through the exchange of resources, the two partners of this symbiosis can have great effects on each other, effects which can ripple through both communities. What determines the patterns of associations between the partners is still largely unknown. In this dissertation, I examine a variety of factors, and in particular host identity, that could drive the community composition of AMF in roots. I began by surveying the diversity of AMF in roots of 12 plant species at a remnant bunchgrass prairie in Oregon, U.S.A. (Chapter 2). To do that, I first designed new primers for use in polymerase chain reaction (PCR) to specifically amplify DNA from all Glomeromycota species. Using those primers, I found 36 distinct AMF phylogenetic groups, or operational taxonomic units (OTUs) in the roots from the prairie. The proportion of OTUs in the basal order Archaeosporales was greater than in many other environmental surveys. I also conducted an in silico analysis to predict how effectively previously published primers would detect the whole diversity of OTUs I detected. I then assayed AMF community composition in the roots of 50 plants from nine plant species (Chapter 3). To do that, I designed primers specific to 18 of the OTUs detected in the initial field survey and used them to test for the presence of each OTU in the roots individual plants. I used that data to test if AMF community composition in individual roots correlated with host identity, spatial distribution, or soil characteristics. I found host identity was associated with both the richness and the structure of root AMF communities, while spatial distribution and soil characteristics were not. Finally, I performed an experimental test of the effect of host identity and community context on AMF community assembly (Chapter 4). I grew plants from four native perennial plant species, including two common and two federally endangered plants, either individually or in a community of four plants (with one plant of each species). I analyzed the AMF community composition in the roots of all plants after 12 weeks of growth with exposure to a uniform mix of field soil as inoculum. I found that host species identity affected root AMF richness and community composition, and community context affected AMF richness. Only one of the endangered species was highly colonized by AMF, and I did not detect unique AMF communities associated with it. This dissertation provides information on the diversity of AMF at a remnant bunchgrass prairie, an ecosystem which has been the subject of very few studies of AMF. Although a complex mix of factors interact to determine AMF community composition in roots, this work provides strong evidence that host identity plays a major role in that process. / Graduation date: 2013