Background. Fungal endophytes inhabit symptomless, living tissues of all major plant lineages to form one of earth's most prevalent groups of symbionts. Many reproduce from senesced and/or decomposing leaves and can produce extracellular leaf degrading enzymes, blurring the line between symbiotrophy and saprotrophy. To better understand the endophyte saprotroph continuum we compared fungal communities and functional traits of focal strains isolated from living leaves to those isolated from leaves after senescence and decomposition, with a focus on foliage of woody plants in five biogeographic provinces ranging from tundra to subtropical scrub forest. Methods. We cultured fungi from the interior of surface-sterilized leaves that were living at the time. of sampling (i.e., dophytes), leaves that were dead and were retained in plant canopies (dead leaf fungi,eDn LF), and fallen. leaves (leaf litter.fungi,LLF) from 3-4 species of woody plants in each of five sites in. North America. Our sampling encompassed 18 plant species. representing. two families of Pinophyta.and five families of Angiospermae. Diversity and composition of fungal communities within and among leaf life stages, hosts, and sites were compared using ITS-partial L SU rDNA data. We evaluated substrate use and enzyme activity by a subset of fungi isolated'onlyfrom living tissues vs. fungi isolated only from non-living leaves. Results Across the diverse biomes and plant taxa surveyed here, culturable fungi living leays were isolated less frequently and were less diverse than those isolated from non-living leaves. Fungal communities in living leaves also differed detectably in composition from communities in dead leaves and leaf litter within focal sites and host taxa, regardless of differential weighting of rare and abundant fungi. All focal isolates grew on cellulose, lignin, and pectin as sole carbon sources, but none displayed igninolytic or pectinolytic activity in vitro. Cellulolytic activity differed among fungal classes. Within Dothideomycetes, activity differed significantly between fungi from living vs. non-living leaves, but such differences were not observed in Sordariomycetes. Discussion. Although some fungi with endophytic life stages clearly persist for periods of time in leaves after senescence and incorporation into leaf litter, our sampling across diverse biomes and host lineages detected consistent differences between fungal assemblages in living vs. non-living leaves, reflecting incursion by fungi from the leaf exterior after leaf death and as leaves begin to decompose. However, fungi found only in living leaves do not differ consistently in cellulolytic activity from those fungi detected thus far only in dead leaves. Future analyses should consider Basidiornycota in addition to the Ascomycota fungi evaluated here, and should explore more dimensions of functional traits and persistence to further define the endophytism-to-saprotrophy continuum.

Microrganismos endofíticos apresentam grande diversidade genética e metabólica, com enorme potencial biotecnológico para aplicações em diferentes áreas. A bioprospecção desses microrganismos possibilita a obtenção de novos bioprodutos agrícolas. Endofíticos podem ser aplicados, por exemplo, para a biorremediação de áreas contaminadas ou para o controle de insetos pragas pela produção de compostos entomotóxicos. Apesar de sua importância, ainda existem poucos estudos voltados à bioprospecção da microbiota endofítica em regiões tropicais. Assim, o presente trabalho pretendeu verificar a diversidade de microrganismos endofíticos cultiváveis em milho e a comparar a diversidade de metabólitos desses endofíticos ao de isolados de Citrus. Também foi explorado o potencial dos diferentes endófitos em degradar os principais inseticidas (lufenuron, spinosad, chlorpyrifos ethyl, lambda-cyhalothrin e deltamethrin) utilizados em lavouras de milho para o controle de lagartas de Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae), para exploração dos mesmos em programas de biorremediação. Finalmente, foi avaliado o potencial entomotoxida dos extratos orgânicos dos endofíticos estudados até a identificação das moléculas ativas. Foram isolados 30 microrganismos endofíticos de milho (27 bactérias e 3 fungos), os quais foram comparados aos endófitos de citros do acervo do Laboratório de Genética de Microrganismos \"Prof. João Lúcio de Azevedo\". Dos isolados obtidos, sete apresentaram capacidade de metabolização dos cinco inseticidas testados, sendo a maioria deles capaz de metabolizar pelo menos um dos inseticidas testados. Ensaios de bioatividade dos extratos orgânicos do cultivo dos endofíticos contra lagartas de S. frugiperda, levaram à identificação de extratos que induziram 100% de mortalidade de lagartas do terceiro ínstar. O isolamento e a caracterização da molécula ativa do extrato produzido pelo isolado LGM-CR-JM-02 - Streptomyces badius, permitiu a identificação da valinomicina (CL50 = 4,38 Mi g/mL) como molécula ativa. Assim, demonstramos que a comunidade de endofíticos pode reunir grande diversidade biológica e, principalmente, metabólica, sendo uma excelente oportunidade para exploração de microrganismos com potencial para biorremediação e produção de compostos com atividade inseticida / Endophytes are microorganisms with great genetic and metabolic diversity, and with a huge biotechnological potential for applications in different areas. The bioprospection of these microorganisms makes possible to obtain new agricultural bioproducts. Endophytes may be applied, for example, to bioremediation of contaminated areas or to the control of pest insects with entomotoxic compounds. Despite its importance, there are few studies focused on the bioprospection of endophytic microbiota in tropical regions. Thus, we investigated the biological diversity of culturable endophytes from maize and compared the diversity of metabolites endophytes from maize and Citrus produced. We also explored the potential of different endophytes to grow in minumum medium containing one of the main insecticides (lufenuron, spinosad, chlorpyrifos ethyl, lambda-cyhalothrin and deltamethrin) applied in maize crops to control the caterpillars Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae) as the only source of carbon in order to evaluate their potential use in bioremediation programs. Finally, we evaluated the entomotoxic potential of the organic extracts of the endophytes and the fractions up to the identification of the active molecules. In total, thirty maize endophytes (27 bacteria and 3 fungi) and five endophytes isolated from citrus that were deposited at the collection of the Laboratory of Genetics of Microorganisms \"Prof. João Lúcio de Azevedo\" were investigated. Seven out of 35 endophytes tested metabolized the five insecticides used, and most of them were able to metabolize at least one of the tested insecticides. Bioactivity assays of the organic extracts produced from cultured endophytes were fed to third instars of S. frugiperda causing 100% mortality. The isolation and characterization of the active molecule produced by one of these isolates (LGM-CR-JM-02 - Streptomyces badius) led to the identification of valinomycin (LC50 = 4.38 Mi g / mL) as the active molecule. Thus, we demonstrated that endophytes can gather great biological and metabolic diversities, demonstrating endophytes are an excellent opportunity for the identification of microorganisms with potential for bioremediation and production of compounds with insecticidal activity

Endófitos são todos os microrganismos, cultiváveis ou não, que habitam o interior dos tecidos vegetais, não causando danos ao hospedeiro e nem o desenvolvimento de estruturas externas, excluindo, dessa forma, bactérias noduladoras e micorrizas. Theobroma cacao L. é importante para as indústrias alimentícia e cosmética, por constituir a matéria prima para a produção de manteiga de cacau. Considerando a importância do estudo do cacaueiro e sua comunidade fúngica endofítica associada, os objetivos desse trabalho foram: i) estudar a diversidade da comunidade endofítica de cacau; ii) avaliar o potencial dessa comunidade para o controle biológico de Crinipellis perniciosa, o agente causal da vassoura de bruxa e outros fitopatógenos e iii) estudar a interação endófito-patógeno e endófito-hospedeiro. O trabalho foi iniciado com o isolamento de fungos endofíticos de cacau, que foram caracterizados com base em características morfológicas e moleculares. A diversidade foi avaliada por análises de rDNA (regiões ITS1, ITS2 e 5.8) (ARDRA e sequenciamento). A análise de ARDRA gerou 64 haplótipos, os quais incluíram no mínimo, 16 espécies. A seguir, fungos endofíticos antagônicos de cacau foram selecionados in vitro contra C. perniciosa. Foi observado que, dentre os 145 isolados avaliados, 38.6% foram classificados como antagônicos, enquanto 13.1% foram classificados como parasitas. Após a seleção, os isolados foram usados para extração de metabólitos secundários e outras caracterizações. Também foi feita a detecção do gene da δ-(L-α- aminoadipil)-L-cisteinil-D-valin (ACV), que é a precursora das β-lactanas, antibióticos como as penicilinas e cefalosporinas. A amplificação desse gene gerou um fragmento de 600 pb em 9.1% dos isolados avaliados. Alguns isolados foram testados contra fungos fitopatogênicos (Colletotrichum falcatum, C. sublineorum, Erythricium salmonicolor, Rhizoctonia solani, Ceratocystis paradoxa, Phytophthora sp., P. parasitica, P. palmivora e Fusarium moniliforme). Apenas C. sublineorum de sorgo e Ceratocystis paradoxa de cana-de-açúcar foram inibidos in vitro pelos endófitos 42.3 e 2, respectivamente. Foi feita a investigação da habilidade de penetração e colonização do fungo endofítico 42.3 em cacau comum suscetível, usando as técnicas de RAPD e SEM. A penetração do fungo foi iniciada entre 3 e 6 horas após a inoculação. A interação entre o endófito de cacau 42.3 com C. perniciosa 281 (patogênico), foi avaliada por SDS-PAGE, visando a detecção de proteínas expressas pelo patógeno e pelo endófito. A SDSPAGE permitiu a visualização de bandas expressas na interação, quando os microrganismos foram crescidos juntos. Na presença do substrato de crescimento do endófito, C. perniciosa não expressou proteínas visíveis, ou distinguíveis em SDS-PAGE, mas o co-cultivo desses fungos, geraram bandas não observadas em análises prévias. / Endophytes are all microorganisms, culturable or not, that inhabit the interior of plant tissues, causing no harm to the host, and that do not develop external structures, excluding in this way, nodulating bacteria and mycorrhizal fungi. Theobroma cacao L. is important to food and cosmetic industry due the fact that it is used for production of cacao butter. Considering the importance of cacao and associated endophytic fungal community studies, the objectives of this work were to: i) study the diversity on endophytic community of cacao; ii) evaluate the potential of this community to biological control of Crinipellis perniciosa, causal agent of witches’ broom disease and other phytopathogens and iii) study the interaction endophyte-pathogen and endophyte-host. The work began with isolation of the cacao endophytic fungi that was characterizated based on morphological and molecelar characters. The diversity was evaluated by rDNA (ITS1, ITS2 e 5.8 subunit regions) analyses (ARDRA and sequencing). The ARDRA analysis generated 64 haplotypes, which included at least 16 species. Following, antagonistic endophytic fungi from cacao were selected in vitro against C. perniciosa. It was observed that in 145 evaluated isolates, 38.6% was classified as antagonic, while 13.1% were classified as parasitic. After the selection, isolates were used for secondary metabolites extraction and further characterization. Also, detection of δ-(L-α-aminoadipil)-L-cisteinil-D-valin (ACV) gene, which is the precursor of β-lactans, antibiotics like penicilins and cephalosporins, was carried out. The amplification of this gene generated a fragment of 600pb in 9.1% of the isolates evaluated. Some isolates were further evaluated against phytophathogenic fungi (Colletotrichum falcatum, C. sublineorum, Erythricium salmonicolor, Rhizoctonia solani, Ceratocystis paradoxa, Phytophthora sp., P. parasitica, P. palmivora and Fusarium moniliforme). Only C. sublineorum of sorghum and Ceratocystis paradoxa of sugarcane were in vitro inhibited by the endophytes 42.3 and 2, respectively. The investigation of the penetration and colonization ability of endophytic fungus 42.3 on susceptible cacao comum was carried out, using RAPD and SEM techniques. The penetration of the fungus was initiated between 3 and 6 hours after inoculation. The interaction between cacao endophyte 42.3 with C. perniciosa 281 (pathogenic), was evaluated by SDSPAGE, aiming the detection of expressed proteins by pathogen and endophyte. The SDS-PAGE allowed the visualization of bands expressed in the interaction, when the microorganisms were grown together. In the presence of the endophyte growth substrate, C. perniciosa do not express visible proteins or, at least, distinguishable in SDS-PAGE, but the co-cultive of these fungi generated non observed bands in previously analysis.

Remediation of trichloroethylene (TCE) is a major priority for many contaminated sites all over the industrialized world including Hill Air Force Base, UT (HAFB). Phytoremediation as part of a cleanup strategy is an appealing option, and trees at HAFB have been studied to this end. Trees have also been used to delineate groundwater plumes because the passive uptake of organic contaminants by trees generally results in a direct relationship between tree and groundwater TCE concentrations if the trees are using the contaminated groundwater. However, the concentrations of plant-produced TCE metabolites can vary greatly. It was hypothesized that the endophytic microbial community present may be affecting the fate of TCE within these trees. This study was designed to determine if the microbial community present within hybrid poplar trees would change in response to TCE exposure. Trees were grown in a greenhouse to reduce environmental variability. Concentrations of TCE, its degradation products, and its metabolites were then measured in these trees. DNA was extracted from the tree tissues and amplified to determine the quantity of microbial DNA. Diversity of this DNA was determined by fragment analysis. Data were analyzed to determine if there was an effect of TCE treatment on the microbial community composition in the trees. Results showed that all tissues of dosed trees contained TCE. Metabolism of TCE to trichloroacetic acid (TCAA) and trichloroethanol (TCEtOH) in tree tissues was observed by the accumulation of these metabolites. Microbial DNA results revealed that TCE treatment reduced both the quantity and diversity of endophytic bacteria and fungi in several cases. Multivariate statistical analyses also showed that the endophytic microbial community shifted in response to TCE treatment. The endophytic microbial communities present in the hybrid poplar trees of this study were high in concentration and diversity, both of which were affected by TCE treatment. Endophytic bacterial concentrations were observed at over 109 copies/g with diversities of 70+ genetically distinct organisms. Decreases in these values with the addition of TCE showed that the community dramatically changed in some cases, but was able to more quickly adapt to TCE addition in other cases. The effects of these endophytic microorganisms associated with plants should therefore be included when phytoremediation is considered.

Orientadora: Dulce Helena Siqueira Silva / Banca: Cintia Duarte de Freitas Milagre / Banca: Mariana Helena Chaves / Resumo: Sabe-se que os fungos exercem importantes papéis no cotidiano, sendo utilizados na produção de alimentos fermentados e de enzimas de interesse industrial, em processos de biodegradação e tratamento biológico de efluentes, além de auxiliar na agricultura como parte do corpo de decompositores, e ainda na defesa e crescimento de plantas em que vivem em simbiose. Adicionalmente, nos últimos anos, as pesquisas com fungos e outros micro-organismos têm-se intensificado pelo surgimento de evidências crescentes de seu potencial como fonte de substâncias bioativas para o desenvolvimento de agentes terapêuticos, como as penicilinas e cefalosporinas - antibióticos, a mevastatina e lovastatina - redutores de colesterol, ciclosporina e rapamicina-imunossupressores, entre outros. Muitos fungos endofíticos possuem potencial farmacológico, demonstrando atividades como citotóxica, antibacteriana, anticolinesterásica, antifúngica, antiplasmódica, entre outras. Além de indicarem ser fonte de uma grande variedade de novos metabólitos secundários, decorrente de sua quimiodiversidade marcante. Este contexto levou à investigação de uma linhagem fúngica isolada da alga marinha Asparagopsis taxiformis (Delile) Trevisan, identificada como Microascus intricatus. O cultivo desta linhagem em meio PDB por 28 dias, 65 frascos de Erlenmeyer forneceu o extrato AT-07, que foi submetido a partição com acetato de etila. As frações AT-07 F3, AT-07 F4 e AT-07 F6 foram submetidas a procedimentos cromatográficos e l... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fungi play important roles in our daily life as in the production of fermented food and enzymes for industrial applications, biodegradation processes and biological treatment of effluents, in addition to its roles in agriculture, enhancing decomposition, and defense and growth of plants living in symbiosis.Research involving fungi and other microorganisms has been intensified in the last decades owing to growing evidence of their potential as important sources of novel bioactive compounds for the development of therapeutic agents as penicillins, cephalosporins (antibiotics), mevastatin and lovastatin (cholesterol lowering agents), cyclosporine and rapamycin (immunosuppressors), among others.Several endophytic fungal strains exhibit pharmacological potential as cytotoxic, antibacterial, anticholinesterase antifungal, antiplasmodial activities, which has been markedly associated to the huge chemodiversity of their secondary metabolites.Such context prompted us to investigate one fungal strain isolated from marine red alga Asparagopsis taxiformis (Delile) Trevisan, which has been identified as Microascus intricatus.Cultivation of the fungal strain in PDB media for 28 days, Erlenmeyr flasks yielded extract AT-07 partition with etoac extract which was submitted to chromatographic procedures and led to the isolation of natural products belonging to diketopiperazines classes and one isoflavone.The isolated compounds were identified as cyclo-(Pro-Tir), cyclo-(ProPhe), cyclo-(Phe-Phe)... (Complete abstract click electronic access below) / Mestre

Although plant growth-promoting rhizobacteria (PGPR) have shown tremendous potential to be used as inoculants for many agricultural crops, they may not survive severe environmental conditions in the field which could limit their large scale applications. Endophytic bacteria, which can be recovered from inside plant tissues such as roots, stems and leaves, might overcome this limitation due to their unique ecological niche inside plant roots where they are sheltered from external environmental disturbances. Some of these bacterial endophytes have beneficial effects on their host plants and stimulate plant growth or reduce disease symptoms, apparently through mechanisms that are similar to those proposed for PGPR. The objective of this study was to assess a collection of endophytic bacteria for PGPR traits and potential use to enhance the rhizobial-legume symbiosis. Forty isolates obtained from the roots of various plants were identified by fatty acid methyl ester (FAME) analysis, and 16S RNA gene sequencing analysis. The majority (i.e., 75%) were identified as Pseudomonas species. Many of these isolates were able to solubilize phosphate, produce indole-3-acetic acid (IAA), produce aminocyclopropane-1-carboxylic acid (ACC) deaminase, synthesize siderophores and show antagonistic activities against several soil-borne plant pathogenic fungi under in vitro conditions. Selected isolates were further evaluated for the ability to enhance plant growth and nodulation of alfalfa when co-inoculated with Sinorhizobium meliloti under growth chamber conditions using growth pouch and potted soil assays. Results revealed that P. putida strain EB EE 4-25, P. syringae strain EB XDE 4-48, and P. fluorescens strain EB EE 2-23 significantly increased shoot length, root length, enhanced nodulation and increased lateral root formation of alfalfa plants in growth pouch and potted soil assays when co-inoculated with S. meliloti strain P102 compared to plants inoculated with S. meliloti strain P102 alone. Results also suggested that expression of one or more of the mechanisms, such as solubilization of phosphate, production of IAA, production of siderophores, and ACC deaminase production might have played a role in the enhancement of the alfalfa- Sinorhizobium symbiosis. These results suggest that some endophytic bacterial strains may be useful as biofertilizers and/or biocontrol agents in sustainable agricultural practices.

Although plant growth-promoting rhizobacteria (PGPR) have shown tremendous potential to be used as inoculants for many agricultural crops, they may not survive severe environmental conditions in the field which could limit their large scale applications. Endophytic bacteria, which can be recovered from inside plant tissues such as roots, stems and leaves, might overcome this limitation due to their unique ecological niche inside plant roots where they are sheltered from external environmental disturbances. Some of these bacterial endophytes have beneficial effects on their host plants and stimulate plant growth or reduce disease symptoms, apparently through mechanisms that are similar to those proposed for PGPR. The objective of this study was to assess a collection of endophytic bacteria for PGPR traits and potential use to enhance the rhizobial-legume symbiosis. Forty isolates obtained from the roots of various plants were identified by fatty acid methyl ester (FAME) analysis, and 16S RNA gene sequencing analysis. The majority (i.e., 75%) were identified as Pseudomonas species. Many of these isolates were able to solubilize phosphate, produce indole-3-acetic acid (IAA), produce aminocyclopropane-1-carboxylic acid (ACC) deaminase, synthesize siderophores and show antagonistic activities against several soil-borne plant pathogenic fungi under in vitro conditions. Selected isolates were further evaluated for the ability to enhance plant growth and nodulation of alfalfa when co-inoculated with Sinorhizobium meliloti under growth chamber conditions using growth pouch and potted soil assays. Results revealed that P. putida strain EB EE 4-25, P. syringae strain EB XDE 4-48, and P. fluorescens strain EB EE 2-23 significantly increased shoot length, root length, enhanced nodulation and increased lateral root formation of alfalfa plants in growth pouch and potted soil assays when co-inoculated with S. meliloti strain P102 compared to plants inoculated with S. meliloti strain P102 alone. Results also suggested that expression of one or more of the mechanisms, such as solubilization of phosphate, production of IAA, production of siderophores, and ACC deaminase production might have played a role in the enhancement of the alfalfa- Sinorhizobium symbiosis. These results suggest that some endophytic bacterial strains may be useful as biofertilizers and/or biocontrol agents in sustainable agricultural practices.

Thesis (Ph.D.) -- Flinders University, Dept. of Medical Biotechnology. / Typescript (bound). Includes bibliographical references (leaves 256 - 283). Also available online.

Thesis (M.Sc.)--York University, 2006. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 76-81). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR19648

Thesis (M.S.)--University of Tennessee, Knoxville, 2004. / Title from title page screen (viewed Oct. 04, 2004). Thesis advisor: F. Neal Schrick. Document formatted into pages (xiii,105 p. : ill.). Vita. Includes bibliographical references (p. 86-104).