Orientador: Dulce Helena Siqueira Silva / Banca: Angela Regina Araujo / Banca: Edson Rodrigues Filho / Resumo: This work dealt with the study of fungal strains Sarocladium strictum (Ps-02) e Coniothyrium sp. (Ps-03) associated to the red macroalga Pyropia spiralis, collected at SP State shore. Their extracts were obtained in different culture media: rice, malt and Czapek, which induced diversified metabolic production, as shown by HPLC-DAD and NMR analyses and evidenced that the metabolic profile depends on culture media for these strains. Extracts and fractions from Sarocladium strictum and Coniothyrium sp. were evaluated in antifungal, anticholinesterasic and cytotoxic assays and presented one or more potential bioactivities. Fraction Ps-03 A-ACN from Coniothyrium sp. showed antifungal and cytotoxic activities, and was selected for reversed phase column chromatography under positive pressure and gradient elution with H2O:MeOH. Fractions purification by HPLC-DAD or normal phase column chromatography led to the isolation of one diketopiperazine, polyketides and steroids, which had their structural elucidation by UV, 1D and 2D NMR, and MS analyses. The polyketides isolated in this study include three aromatic macrolides (1 - 3): (3R,5R)-sonnerlactone and two sonnerlactone analogues: one novel compound and one novel as a natural product; the aromatic butenolide gymnoascolide A (4), and the antiviral tetralone 10-norparvulenone (7). Two steroids: ergosterol-5,8-endoperóxido-∆6,22 (5) and 22E-ergosta-7,22-dieno-3β,5,6-triol (6) were also isolated. They are structurally related, probably ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Este trabalho consistiu no estudo dos micro-organismos Sarocladium strictum (Ps-02) e Coniothyrium sp. (Ps-03) associados à macroalga vermelha Pyropia spiralis, coletada no litoral paulista. Os extratos foram obtidos em diferentes meios de cultivos: arroz, malte e Czapek, e induziram produção metabólica diversificada, conforme apontado pelas análises por CLAE-DAD e RMN de 1H, evidenciando que o perfil metabólico é dependente do meio de cultivo para estas linhagens. Os extratos e frações de Sarocladium strictum e Coniothyrium sp. foram avaliados em ensaios para atividade antifúngica, anticolinesterásica e citotóxica e apresentaram uma ou mais potenciais atividades. A fração Ps-03 A-ACN de Coniothyrium sp. apresentou atividade antifúngica e citotóxica, e foi selecionada para fracionamento por cromatografia em coluna sob pressão em modo reverso (C18) com eluição em gradiente H2O:MeOH. A purificação das frações por CLAE-DAD ou cromatografia flash em modo normal levou ao isolamento de uma dicetopiperazina, policetídeos e esteroides, que tiveram suas estruturas determinadas por análises na região do UV, RMN 1D e 2D, e por espectrometria de massas. Os policetídeos isolados neste estudo incluem três macrolídeos aromáticos (1 - 3): a (3R,5R)-sonnerlactona e dois análogos à sonnerlactona, sendo um inédito na literatura e outro, inédito como produto natural; o butenolídeo aromático gymnoascolídeo A (4), e a tetralona antiviral10-norparvulenona (7). Foram também obtidos os esteroides erg... (Complete abstract click electronic access below) / Mestre

Plants can be colonized by complex communities of endophytic fungi. This thesis presents two studies, both of which investigate biotic filtering in endophytic fungal communities. Chapter 1. Endophytic fungi can be acquired horizontally via propagules produced in the environment such as in plant litters of various species. Given that litters from different plant species harbor distinct endophytic fungal communities and that endophytic fungi may be dispersal-limited, the structure of the endophytic fungal community of a given plant may be determined by proximity to particular inoculum sources. Community assembly may also be affected by biotic filtering by the plant. Therefore, a plant may be able to select particular fungal taxa from among the available pool. In that case, the structure of the endophytic fungal community in the plant could be somewhat independent of the structure of the inoculum community. We tested the hypothesis that biotic filtering of endophytic fungal communities occurs in Bromus tectorum by exposing it to a variety of inoculum sources including litters from several co-occurring plant species. The inoculum sources differed significantly from each other in the structures of the communities of endophytic fungi they harbored. We characterized the structures of the resulting leaf and root endophytic fungal communities in Bromus tectorum using high-throughput sequencing. All tested inoculum sources successfully produced complex communities of endophytic fungi in Bromus tectorum. There was significantly more variation in the structures of the communities of endophytic fungi among the inoculum sources than in the resultant endophytic fungal communities in the leaves and roots of Bromus tectorum. These results suggest that biotic filtering by Bromus tectorum played a significant role in the assembly of the endophytic fungal communities in tissues of Bromus tectorum. Because endophytic fungi influence plant fitness, it is reasonable to expect there to be selective pressure to develop a uniform, desirable endophytic fungal community even from disparate inoculum sources via a process known as biotic filtering. Chapter 2. Frequently one finds that different plant species harbor communities that are distinct. However, the nature of this interspecific variation is not clear. We characterized the endophytic fungal communities in six plant species from the eastern Great Basin in central Utah. Four of the species are arbuscular mycorrhizal (two in the Poaceae and two in the Asteraceae), while the other two species are nonmycorrhizal (one in the Brassicaceae and one in the Amaranthaceae). Our evidence suggests that both host mycorrhizal status and phylogenic relatedness independently influence endophytic fungal community structure.

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.

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.

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.

O presente trabalho teve por objetivo avaliar o efeito de três ecossistemas sobre, a ocorrência de fungos rizosféricos, endofíticos e epifíticos. As áreas de estudo estão localizadas em uma propriedade rural, situada na cidade de Venâncio Aires, RS. Os fungos endofíticos e epifíticos foram isolados de amostras de raízes de guajuvira, (Patagonula americana L.), coletadas na mata (área 1), de uva-do-japão (Hovenia dulcis Thunb.), na área intermediária (área 2) e de fumo ou de milho, na lavoura (área 3) e, os fungos rizosféricos isolados de solo coletado junto as raízes dos vegetais citados. As amostras foram coletadas nos meses de janeiro, maio, setembro e novembro de 2004 e 2005. Os fungos foram identificados segundo características morfológicas com auxílio de chaves de identificação. As áreas 2 e 3 foram mais similares com relação aos fungos rizosféricos e epifíticos, enquanto, que, as áreas 1 e 2 foram mais similares com relação aos fungos endofíticos. A diversidade dos fungos isolados das três áreas não diferiu ao longo dos dois anos de coleta. A presença de Fusarium spp., em vegetais sem sintomas de doença indica que, as mesmas podem ser raças avirulentas ou patógenos latentes em equilíbrio com o hospedeiro e o ambiente. A presença de fungos antagonistas, principalmente Trichoderma spp. também, são responsáveis por esse equilíbrio, o qual ocorre nas três áreas. / The present paper aimed at assessing the effect of three ecosystems, on the occurrence of rhizospheric, endophytic and epiphytic fungi. The study areas are located in a farm in Venâncio Aires, Rio Grande do Sul. Samples were collected in the months of January, May, September and November 2004 and 2005. The endophytic and epiphytic fungi were isolated from samples of guajayvi roots (Patagonula americana L.), picked in the forest (area 1), of Japanese raisin tree (Hovenia dulcis Thunb.), in the intermediate area (area 2), and tobacco or corn, in the cultivated area (area 3) and rhizospheric fungi isolated from soil. The fungi were identified according to morphological characteristics, with the help of identification keys. Areas 2 and 3 were more alike concerning rhizospheric and epiphytic fungi; Areas 1 and 2, in turn, were more alike concerning endophytic fungi. The diversity of the isolated fungi from these three areas did not change during the two years of collection. The abundance of fungi with phytopathogenic potential such as the Fusarium spp. and Macrophomina phaseolina, mostly as endophytic in area 3 shows the influence of the host on them. The presence of antagonistic fungi, principally Trichoderma spp. also, are responsable for this equilibrium, which occured in the three areas.

Submitted by MAIARA STEFANINI BORGES null (borges.maiara@iq.unesp.br) on 2016-03-22T21:06:41Z No. of bitstreams: 1 MAIARA STEFANINI BORGES.pdf: 2921643 bytes, checksum: 71bed0595a70fef8c32665cc82adc905 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-23T13:30:07Z (GMT) No. of bitstreams: 1 borges_ms_me_araiq.pdf: 2921643 bytes, checksum: 71bed0595a70fef8c32665cc82adc905 (MD5) / Made available in DSpace on 2016-03-23T13:30:07Z (GMT). No. of bitstreams: 1 borges_ms_me_araiq.pdf: 2921643 bytes, checksum: 71bed0595a70fef8c32665cc82adc905 (MD5) Previous issue date: 2016-03-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Micro-organismos endofíticos, geralmente fungos e bactérias, vivem sistematicamente no interior das plantas sem causar, aparentemente, danos a seus hospedeiros. Fungos endofíticos representam um importante grupo de fungos com potencial fármaco e biotecnológico e podem ser isolados de diversas plantas medicinais. A espécie vegetal Eugenia jambolana Lam. tem sido intensamente explorada química e biologicamente devido a seus usos populares e por apresentar diversas atividades biológicas. Logo, o estudo de fungos endofíticos, associados a esse espécie vegetal, apresenta-se como uma estratégia promissora na obtenção e identificação de substâncias potencialmente bioativas. Dentro deste contexto, este trabalho teve como objetivo principal a avaliação química do fungo endofítico Saccharicola sp. isolado de Eugenia jambolana Lam. Este endófito foi cultivado em pequena escala nos meio de cultura liquido (MDB) e sólido (Milho) e em escala ampliada no meio Czapek. O caldo foi separado dos micélios após 28 dias (meios líquidos) e 21 dias (meio sólido) por filtração e o filtrado foi submetido à partição líquido/líquido com AcOEt fornecendo, após a evaporação do solvente, os respectivos extratos brutos. Os extratos foram avaliados frente a produção metabólica por CCDC, CLAE-DAD e RMN de 1H e também avaliados frente aos ensaios biológicos. Portanto para os extratos MDB, Milho Fr.CH3CN, Milho Fr. aquosa e Milho Fr.Hexânica, foram realizados ensaios antifúngico, antioxidante e anticolinesterásico, onde estes extratos demonstraram ser promissores. O extrato obtido em escala ampliada (Czapek) foi fracionado em cartucho SPE (extração em fase sólida), resultando em seis frações (Ss.C.Fr1- Ss.C.Fr6). Do estudo da fração Ss.C.Fr1 foi possível o isolamento de uma substância o Ácido fusárico (1) e a fração Ss.C.Fr3 resultou no isolamento de 6,7 dihidroxi- 4,6 dimetil octanoato de metila (2), substância inédita. O extrato obtido em escala reduzida (Milho Fr.CH3CN) foi submetido a fracionamento em coluna de sílica gel de fase reversa (C18), resultando em oito frações (Ss.M. CH3CN Fr1 - Ss.M. CH3CN Fr8). O estudo da fração Ss.M. CH3CN Fr5 resultou no isolamento da substância 4-hidroximeleina (3). O estudo da fração Milho Fr.Aquosa-XAD resultou no isolamento de duas substâncias Timidina (4) e Adenosina (5). Portanto, o estudo químico com o fungo endofítico Saccharicola sp. resultou no isolamento de cinco substâncias puras. Sendo assim, este trabalho visou isolar substâncias potencialmente bioativas através da investigação dos extratos e frações de Saccharicola sp.. / Endophytic microorganisms, often bacteria and fungi systematically live within the plant without apparent harm their hosts. Endophytic fungi represent an important group of fungi with biotechnological potential and may be isolated from various medicinal plants. The plant species Eugenia jambolana Lam. has been intensively explored chemically and biologically due to its popular uses and it has several biological activities. Thus, the study of endophytic fungi associated with this plant species, presents itself as a promising strategy in obtaining or identifying potentially bioactive substances. Within this context, this study aimed to evaluate the chemical endophytic fungus Saccharicola sp. isolated from Eugenia jambolana Lam., encoded as Ej-c04. This endophyte was cultivated on a small scale to a liquid culture medium PDB solid Corn and enlarged scale for the Czapek medium. The broth was separated from the mycelium after 28 days (liquid media) and 21 days (solid medium) by filtration and the filtrate was subjected to liquid / liquid partition with ethyl acetate providing after evaporation of the solvent, their crude extracts. The extracts were evaluated against metabolic production by TLC, CLAE-DAD and 1H NMR also evaluated and compared to the bioassay. So for MDB extracts, Fr.CH3CN Corn, aqueous Fr. Corn and Corn Fr.Hexânica, tests were performed antifungal, antioxidant and anticholinesterase where these extracts have shown promise.The extract obtained in enlarged scale (Czapek) was fractionated on SPE cartridge (solid phase extraction), resulting in six fractions (Ss.C.Fr1- Ss.C.Fr6). From Ss.C.Fr1 fraction was possible to isolate a substance fusaric acid (1) and Ss.C.Fr3 fraction resulted in the isolation of (2E, 4E) - 6,7 dihydroxy-4,6-dimethyl octanoate (2) novel substance. The extract obtained in small scale (Corn Fr.CH3CN) was subjected to fractionation column of silica gel reversed phase (C18) resulting in eight fractions (Ss.M. CH3CN- Fr1 - CH3CN Ss.M. Fr8). The study fraction Ss.M. CH3CN Fr5 resulted in the isolation of 4-hidroximeleina substance (3). The study of fraction Corn Fr. Aqueous -XAD resulted in the isolation of two substances Thymidine (4), adenosine (5). Therefore the chemical study of the endophytic fungus Saccharicola sp. resulted in the isolation of five pure substances Thus, this study aimed to isolate potentially bioactive substances through the investigation of extracts and fractions of Saccharicola sp..