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 This dissertation includes previously published and unpublished coauthored material.

<p>Endophytes are a unique group of microorganisms that spend at least part of their life cycle within plant tissues. These microbes are increasingly being recognized for their potential to improve the health and productivity of their host plants. Recent studies indicate that endophytes could also influence human health by altering the composition of chemical compounds within plants, thereby affecting their nutritional quality and flavor. In addition, the presence of endophytes in edible plant tissues could directly affect human health by introducing microbes that can stimulate the immune system or act as opportunistic pathogens in people with compromised immune systems. However, despite their potential importance for plant and human health, these plant-associated microbes have been understudied due to a lack of visible symptoms associated with their presence and difficulty in isolating them from plant tissues. In the present studies, we hypothesized that endophytes play an important role in carrot, one of the most important vegetable crops in the world.</p> <p> Carrot is well known as an important source of vitamins, antioxidants and other nutrients in the human diet, and carrot taproots are often consumed raw. Carrot crops are slow to establish and are subject to assault by a wide range of pests that negatively affect the health and productivity of this crop, as well as the storage potential of its taproots. Consequently, the aim of these studies was to examine endophyte dynamics in carrot. Studies were carried out in field, greenhouse and laboratory trials using a diverse set of carrot genotypes with broad genetic backgrounds and physiological characteristics. Endophyte communities were studied using traditional culture-based techniques, along with low and high throughput sequencing technologies.</p> <p>Results of these studies demonstrate that carrot seeds and taproots are colonized by an abundant and diverse set of endophytic microbial taxa. Many of these endophytes could solubilize phosphorous, fix atmospheric nitrogen, produce siderophores and auxin and suppress infection by a key carrot pathogen, <i>Alternaria dauci</i>, demonstrating their potential importance for maintaining carrot health and productivity. Some of the endophytes identified in these trials were vertically transmitted to progeny inside carrot seeds, indicating that they could be part of a core microbiome that evolved alongside carrot plants, and are likely to be critical in early seedling establishment. We also determined that carrot endophytes could be acquired via horizontal transmission from soil with greater soil health in an organic relative to a conventionally managed system resulting in greater populations of endophytes with antagonistic activity against <i>A. dauci</i>. Finally, endophyte communities varied among the genotypes evaluated in this study, with some being more responsive to the presence of greater populations of beneficial endophytes in their environments. This indicates that it could someday be possible to begin selecting for these beneficial plant microbial relationships in breeding programs.</p> <p> Based on the results of these studies, we conclude that endophytes do indeed play an important role in carrot. Additional research aimed at determining how these microbes functionally interact with carrot plants and identifying practical approaches to manipulate these communities to enhance the productivity and quality of carrot taproots, are recommended. A new isolation technique identified in these trials will aid in these efforts.</p>

Fungi continue to serve as valuable sources of a diverse variety of bioactive secondary metabolites, some of which have found applications as important pharmaceuticals and agrochemicals. Our research group employs an ecology-based strategy for the selection of fungi for chemical studies. The research described here involves chemical investigation of fungicolous and endophytic fungi that resulted in the isolation and identification of many new natural products, as well as some previously known metabolites. Mycoparasitic and fungicolous fungi are those that colonize other fungi, and are viewed as potential sources of antifungal agents because of the negative effects exerted on their hosts due to this colonization. Many antifungal compounds have been isolated by members of our research group from such fungi. Fractionation of the ethyl acetate extracts of cultures of thirteen fungal isolates, followed by characterization using various NMR and MS techniques, afforded fifteen new and twenty-six known compounds representing diverse structural classes. Some of these compounds showed antifungal and antiinsectan effects accounting for bioactivities originally observed for the crude extracts. Endophytic fungi are those that inhabit host plants asymptomatically and may or may not cause disease after some time. Studies show that in some cases they may be beneficial to the host plant by providing protection against various pathogens or by improving the resistance of the host in some way. Such advantages may be due, at least in part, to the production of bioactive secondary metabolites by these fungi. Chemical investigations of seven crop endophytes led to the isolation and characterization of eleven new and twenty-eight known compounds. Some of these fungal known fungal metabolites exhibited antifungal, antimicrobial, and/or antiinsectan effects, and may therefore provide insight into the potential protective roles of the corresponding crop endophytes against plant pathogens or insect pests. The fungal secondary metabolites isolated during the course of these studies represent a variety of biosynthetic classes, including peptides, polyketides, terpenoids, and compounds with mixed biogenetic origins. Details of the isolation, structure elucidation, and biological activity of these new compounds are presented in this thesis. Structure elucidation was performed mainly by analysis of various MS and NMR data, although chemical derivatization reactions and/or X-ray diffraction analysis data were employed in some instances. Absolute configuration assignments were made using Mosher's method, X-ray crystallography, and/or by ECD spectral analysis in combination with computational analysis. Details of the isolation, structure elucidation, and biological activity of these new compounds are presented in this thesis.

Reproduction de : Thèse doctorat : Biologie et physiologie végétales : Reims : 2007. / Titre provenant de l'écran titre. Bibliogr. f. 123-154.

Taxonomic diversity, biology and ecology of leaf endophytes were studied from some evergreen plants including Arbutus unedo, Buxus sempervirens, flex aquifolium, Laurus nobilis, Ligustrum vulgare, Prunus lusitanica, Rhododendron ponticum, Rhododendron sp., and Skimmia sp. from England, and some others from China. It was found: 1. A great number of fungal species, including several new species and new British records, have been isolated. Distribution patterns of endophyte assemblages and their variations between plant species and geographical locations are described. 2. Comparisons of leaf endophytes and saprobes of R. ponlicum at the same locality showed they belonged to two different ecological groups. This was further confirmed by study on endophytes and saprobes from a number of plant species growing in the same locality. 3. Host specificity of endophytic fungi at the species level was rare and this was supported by comparisons of endophyte assemblages from both taxonomically related (same family) and unrelated (different families) plant species. Molecular characterisations of Phyllosticta species confirmed this. 4. Infection and colonisation studies during a two year period showed that leaf endophytes of R. ponlicum were horizontally transmitted. Internal bud material was sterile and became infected by aerial spores. The infection and colonisation level of endophytes were strongly affected by environmental conditions. 5. Phylogenetic studies of Phyllosticta based on ITS 1-5.8s rDNA-ITS2 sequences concluded there was no evidence to show that the evolution of host plants of Phyllosticta species and ITS were related. Most Phyllosticta species from the same locality were found to have a broad host range and occurred on many taxonomically unrelated plants in the same locality. P. concentrica was separated into 4 species including P. concentrica on Hedera, P. arxii on Ilex, P. maxima on Rhododendron and P. taxi on Taxus.

Several fungal endophytes (C. protuberata, F. culmorum, and C. magna) confer stress tolerance to plants in the presence of certain pressures. This relationship is known as habitat-adapted symbiosis; its mechanism is currently unknown. Here, sFTIR, FTIR coupled to an FPA detector, and Raman spectromicroscopy are used to examine whether any biochemical differences exist between different isolates of the same species: one that can confer stress tolerance to plants and the other that cannot. No major differences have been observed in spectra that can differentiate between those endophytes that confer stress tolerance and those that do not. However, some hy-phae from both isolates of C. protuberata have been found to contain mannitol; its pres-ence may be more common in geothermal rather than non-geothermal isolates. Mannitol is a compound involved in providing stress tolerance to fungi. Any role that it may have in the mechanism of habitat-adapted symbiosis will need further investigation.

Several fungal endophytes (C. protuberata, F. culmorum, and C. magna) confer stress tolerance to plants in the presence of certain pressures. This relationship is known as habitat-adapted symbiosis; its mechanism is currently unknown. Here, sFTIR, FTIR coupled to an FPA detector, and Raman spectromicroscopy are used to examine whether any biochemical differences exist between different isolates of the same species: one that can confer stress tolerance to plants and the other that cannot. No major differences have been observed in spectra that can differentiate between those endophytes that confer stress tolerance and those that do not. However, some hy-phae from both isolates of C. protuberata have been found to contain mannitol; its pres-ence may be more common in geothermal rather than non-geothermal isolates. Mannitol is a compound involved in providing stress tolerance to fungi. Any role that it may have in the mechanism of habitat-adapted symbiosis will need further investigation.

No description available.

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-09-08T19:29:03Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese definita com folha de aprovação CORREÇÃO BIBLIOTECA data imprimir gravar.pdf: 3647168 bytes, checksum: 1df93fa61cfc9c0e2d172eb9eb4b645e (MD5) / Made available in DSpace on 2016-09-08T19:29:03Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) tese definita com folha de aprovação CORREÇÃO BIBLIOTECA data imprimir gravar.pdf: 3647168 bytes, checksum: 1df93fa61cfc9c0e2d172eb9eb4b645e (MD5) Previous issue date: 2015-11-15 / A busca por novos metabólitos secundários de interesse biotecnológicos tem aumentoado nos últimos anos. O objetivo foi isolar micro-organismos endofíticos e da rizosfera, avaliar o potencial biotecnológico dos micro-organismos endofíticos e da rizosfera de Ipomoea pes-caprae. Os endófitos isolados foram inoculados em meio específico para bactérias, actinobactérias (ALA, Meio Completo, ISP2) e fungos (BDA e Agar Sabouraud) à temperatura de 30°C. Para o isolamento das actinobactérias da rizosfera foi utilizado 10 g da amostra do solo rizosférico e este foi misturado a 90 mL de NaCl a 0,9%(m/v) a pH (5,6). Estes micro-organismos foram cultivados em meios de cultura ISP2 e Czapeck Dox a 30 ºC por 7 dias. Foi realizada atividade antimicrobiana de todos os micro-oranismos isolados tanto endofítico como rizosféricos. Para a enzima L- asparaginase foi realizada análise qualitativa e cinética através de Fermentação Semi-Sólida com farinha de soja como substrato. Além da análise qualitativa da L-asparaginase foi verificada a presença de hidrolase (amilase, protease, celulase e lipase. A análise química do extrato metanólico do endófito Streptomyces sp, fungos e actinobactéria rizosférica Nocardia sp foi realizada utilizando a cromatografia de camada delgada (CCD). Das bactérias endofíticas foi avaliada sua capacidade de produção de Ácido Indol Acético. As frequências de endófitos isolados foram: fungos-folha 44.71% (55/123), fungos raízes 13% (16/123), bactérias-folhas 41.46% (51/123), bactérias raízes 1.62% (2/123), actinobactéria 0,81% (1/123). Da rizosfera foram isolados 81 actinobactérias. Na atividade antimicrobiana a maioria dos micro-organismos da rizosfera e endofíticos apresentou atividade antimicrobiana para bactérias Gram-positivas. No extrato dos micro-organismos endofíticos e rizosférico como no extrato da planta foram verificadas a presença de composto mono-sesquiterpenos. A produção de L-aspraginase foi verificada nos micro-organismos endofíticos e nas actinobactérias rizosférica. Na cinética de produção de L-asparaginase pelo endófito Streptomyces sp, verificou-se que o melhor dia de produção foi de 10° dia (0,0702 U/mL de L-asparaginase). Com relação as hidrolase foi verifacdo a presença de amilase e protease na maioria dos endofíticos. Das 53 bactérias endofíticas isoladas, 8 linhagens são produtoras de ácido indol acético. Por tanto, a prospecção dos micro-organismos rizosféricos e endofíticos isolados da planta I. pes-caprae (L.) R. Br. é uma promissora fonte de substâncias de interesse biotecnológico. / The search for new secondary metabolites of biotechnological interest has stepped up in recent years. The aim of present study was to isolate endophytic and rhizosphere micro-organisms to evaluate the secondary metabolites produced by micro-organisms associated with the leaves, roots and rhizosphere of Ipomoea pes-caprae. The endophytes isolated were inoculated in media specific to actinobacteria, bacteria (ALA, full medium, ISP2) and fungi (BDA and Agar Sabouraud) at a temperature of 30°C. The isolation of actinobacteria from the rhizosphere used 10 g of the rhizosphere soil sample mixed with 90 mL of NaCl at 0.9% (m/v) and pH 5.6. These micro-organisms were cultivated in ISP2 and Czapeck Dox culture media at 30 ºC for 7 days. Antimicrobial activity was analyzed. Qualitative analysis, 23 experimental planning and kinetic analysis of the enzymatic activity of L-asparaginase was carried out using semi-solid fermentation with soya flour as a substrate. Chemical analysis of the methanolic extract of the endophyte Streptomyces sp, fungi and the rhizosphere actinobacteria Nocardia sp was conducted using thin layer chromatography (TLC). The capacity of the endophytic bacteria to produce indole acetic acid was measured. The frequencies for isolated endophytes were: fungi-leaves 44.71% (55/123), fungi-roots 13% (16/123), bacteria-leaves 41.46% (51/123), bacteria-roots 1.62% (2/123), and actinobacteria 0.81% (1/123). Eighty-one actinobacteria were isolated from the rhizosphere. Most of the rhizosphere and endophytic micro-organisms exhibited antimicrobial properties against Gram-positive bacteria. Mono-sesquiterpene compounds were found in the endophytic and rhizospheric micro-organisms and in the plant extract. Production of L-asparaginase was found in endophytic micro-organisms and in actinobacteria from the rhizosphere. 23 experimental planning of the production of the L-asparaginase enzyme by the Streptomyces sp endophyte suggests that the lower the quantity of micro-organism inoculate, the concentration of L- asparagine and temperature, the better the production of the L-asparaginase enzyme. The kinetics of L-asparaginase production by the Streptomyces sp endophyte showed that the best day for production was the 10th (0.0702 U/mL of L-asparaginase). Of the 53 endophytic bacteria isolated, eight strains produced indole acetic acid. The prospection of rhizosphere and endophytic micro-organisms isolated from I. pes-caprae (L.) R. Br. is thus a promising source of substances of biotechnological interest.

Arbuscular mycorrhizal fungi, colonizing over 80% of all plants, were long thought absent in wetlands; however, recent studies have shown many wetland plants harbor arbuscular mycorrhizae (AM) and dark septate endophytes (DSE). Wetland services such as biodiversity, shoreline stabilization, water purification, flood control, etc. have been estimated to have a global value of $14.9 trillion. Recognition of these vital services is accompanied by growing concern for their vulnerability and continued loss, which has resulted in an increased need to understand wetland plant communities and mycorrhizal symbiosis. Factors regulating AM and DSE colonization need to be better understood to predict plant community response and ultimately wetland functioning when confronting natural and human induced stressors. This study focused on the effects of water quality, hydrology, sedimentation, and hurricanes on AM and DSE colonization in three wetland species (Taxodium distichum, Panicum hemitomon, and Typhal domingensis) and plant communities of coastal wetlands in Southeast Louisiana and effects of an antimicrobial biocide, triclosan (TCS), on AM (Glomus intraradices) spore germination, hyphal growth, hyphal branching, and colonization in fresh water wetland plants (Eclipta prostrata, Hibiscus laevis, and Sesbania herbacea) from bottom land hardwood forest in north central Texas. The former, mesocosm studies simulating coastal marsh vegetation ran for five years. In the latter studies, AM spores and wetland plants were exposed to 0 g/L, 0.4 g/L, and 4.0 g/L TCS concentrations in static renewal and flow through exposures for 21 and 30 days, respectively. AM and DSE colonization was significantly affected by individual and interactions of four independent variables in mesocosm experiments. Similarly, spore germination, hyphal growth, hyphal branching, and AM colonization in selected wetland plants were significantly lowered by exposure to the TCS at environmentally relevant concentrations. However, levels of effects were plant species and fungal propagules specific. My results showed that natural and human induced alterations in environmental factors and chemical contaminants can significantly impact levels of mycorrhizal spore germination, colonization, and spore density in coastal and freshwater wetland plants. The resulting impacts on plant community structure and ecosystem function require further study.