Made available in DSpace on 2015-03-26T13:36:32Z (GMT). No. of bitstreams: 1 texto completo.pdf: 623531 bytes, checksum: 3e10a939648d01ae404b0a09438fda4c (MD5) Previous issue date: 2007-08-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The high acidity, the intense intemperism, and high aluminum content are limiting factors to plant development in Brazilian Cerrado Upon abiotic stress conditions the association with mycorrhizal fungi can assist the host plant in absorbing nutrients and water from soil. The high availability of these elements to the host plant improved by arbusculares mycorrhizal fungi (AMF) is determinant in plant survival and structuring of plant ecosystems. The presence of dark septate entophytic fungi in plant roots is another type of symbiotic association related to abiotic conditions such as low humid and day length. The reduced number of publications about these associationsin cerrado species is mainly a function of edaphic conditions and root structural characteristics of native species which difficult the in loco studies of these characters. In this work, it was aimed to study the association between AMF and cerrado native species. It was evaluated and adapted methods for fungal structures observation in symbiosis with Annonaceae, Anacardiaceae, Leguminosae, Melastomataceae, Myrsinaceae, Myrtaceae and Rubiaceae families, which were collected during dry season on senso strictu Cerrado in Floresta Nacional (FLONA) in the municipal district of Paraopeba, Minas Gerais State. Root samples were submitted to three protocols for fungal structures observationg. All evaluated species were colonized by AMF and dark septate fungi, except Xylopia aromatica, which not presented dark septate strutctures. The best diaphanization procedure was obtained when the root samples were autoclaved at 121ºC by 20 minutes in a 2% KOH solution and transferred to a 2% KOH solution by 24 hours at room temperature. This procedure was repeated twice and, after that, the samples were submerged in a 2% KOH solution for 2 hours. The arbuscules were observed with more detail after been included in resin, sectioned and stained with toluidine blue. The non-specific characteristic of AMF observed in sensu stricto Cerrado species was confirmed and suggested for dark septate associations. It indicates that these associations were important as adaptive strategies to cerrado conditions. Roupala montana Aubl. is a species from Proteaceae family which presents a large distribution on different phytophysiognomies of cerrado. Recent studies have been showed that several mycorrhizal fungi structures occurred in these species said to be typically non- mycorrhizal. The low pH and nutrients deficiency in cerrado soils propitiate good conditions for development and establishment of mycorrhizal association in R. montana, as observed in other Proteaceae species. Therefore, in this work it was also aimed to confirm the presence of AMF in R. montana specimens growing at natural cerrado conditions, and correlated the mycorrhizal frequency with nutritional status and edaphic conditions. Three individuals of R. montana were collected in FLONA on cerrado senso strictu, dense cerrado senso strictu, and cerradão during dry and rain seasons. The mycorrhizal occurrence was positively correlated with P, Al, and Mg contents in the soil, and P, K, and Ca in the leaves, and negatively correlated with Ca, Mn, and N contents in the soil. The cerrado soils are acid and have high Al contents which can inhibit the transport of Ca and K to the plants. In this way, the presence of mycorrhizal fungi in R. montana could be a adaptive mechanism for plant surviving under Brazilian cerrado conditions. / A elevada acidez, o intenso intemperismo e os altos teores de alumínio nos solos do Cerrado são fatores limitantes do desenvolvimento vegetal. Em condições de estresses abióticos a associação com fungos micorrízicos auxilia a planta hospedeira na absorção de nutrientes e água do solo. A maior disponibilidades desses elementos para a planta hospedeira pelos fungos micorrízicos arbusculares (FMAs) é determinante na sobrevivência das plantas e na estruturação de formações vegetais. A presença de fungo endofítico do tipo dark septate (DSEF) nas raízes das plantas é outro tipo de associação simbiótica relacionada às condições abióticas como baixa umidade e comprimento do dia. O reduzido número de trabalhos encontrados na literatura dessas associações em espécies de cerrado se deve em parte, pelas condições edáficas e características estruturais radiculares das espécies nativas que dificultam a realização de estudos in loco. Neste trabalho teve-se por objetivo estudar a associação de FMAs em espécies de plantas nativas do cerrado. Para isso, foram testados e adaptados métodos para observação das estruturas fúngicas em simbiose em espécies das famílias Annonaceae, Anacardiaceae, Leguminosae, Melastomataceae, Myrsinaceae, Myrtaceae e Rubiaceae coletadas durante a estação seca em cerrado senso strictu na Floresta Nacional (FLONA) do município de Paraopeba, Minas Gerais. Amostras radiculares foram submetidas a três tipos de protocolos para observação de estruturas fúngicas. Todas as espécies investigadas encontravam-se colonizadas por FMAs e DSEF, exceto DSEF em Xylopia aromática. O melhor método de diafanização foi observado quando as raízes foram autoclavadas a 121 °C em KOH 2 %, por 20 min e, subseqüentemente transferidas para solução nova de KOH 2 % por 24 horas à temperatura ambiente. Este procedimento foi repetido por duas vezes e, em seguida, essas amostras foram imersas em H2O2 2 % por 2 horas. Os arbúsculos foram observados com maiores detalhes após as raízes serem incluídas em resina, seccionadas e coradas com azul de toluidina. O caráter generalista dos FMAs observado nas espécies vegetais do cerrado sensu stricto foi confirmado e sugerido para DSEFs, indicando a importância destas simbioses como estratégia adaptativa às condições de cerrado. Roupala montana Aubl. é uma espécie da família Proteaceae com ampla distribuição nas diferentes fitofisionomias do cerrado. Estudos recentes têm relatado estruturas típicas de fungos micorrízicos em condições de estresses abióticos em espécies dessa família tipicamente não micorrizável. A acidez e deficiência dos solos de cerrado provem apropriadas condições para o desenvolvimento e formação de associações micorrízicas em R. montana, como observado em outros espécies da família Proteaceae. Assim, este trabalho também teve por objetivo confirmar a presença de fungos micorrízicos arbusculares em R. montana crescidas naturalmente em cerrado, correlacionando a freqüência de micorrização com o estado nutricional e condições edáficas. Em três repetições, indivíduos de R. montana foram coletados na FLONA em cerrado senso strictu, cerrado senso strictu denso e cerradão nas estações seca e chuvosa. A micorrização em R. montana foi correlacionada positivamente com o teor de P, Al, e Mg, no solo e de P, K e Ca nas folhas, e negativamente com Ca, Mn e N no solo. Os solos de Cerrado são ácidos com altas concentrações de Al, os quais podem inibir o transporte de Ca e K para as plantas, sugerindo que a presença de fungos micorrízicos em R. montana é um mecanismo adaptativo de sobrevivência as condições do cerrado brasileiro.

A busca por medidas sustentáveis para melhorar e suprir a demanda por alimentos que o crescimento populacional exige, aliado a escassez de terras produtivas, além do acúmulo excessivo de metais no ambiente devido a atividades como mineração e aplicação de fertilizantes no solo, foram as duas problemáticas que motivaram o presente estudo. O uso de fungos promotores do crescimento vegetal: Beauveria bassiana, Metarhizium anisopliae, Pochonia chlamydosporia, Purpureocillium lilacinum e Trichoderma asperella, pode tanto vir a influenciar o aumento da produtividade como potencializar a fitorremediação de metais pesados no solo. Esses fatos são explicados pela capacidade do consórcio fúngico de sintetizar e induzir as plantas a produzir fitohormônios direcionados para o melhor desenvolvimento das raízes do hospedeiro. O maior desenvolvimento de raízes pode estimular a fitoextração de metais do solo e conferir tolerância das plantas à contaminação. Na intenção de avaliar a influência dos fungos no desenvolvimento das plantas, foram realizados tratamentos das sementes de soja e milho e aplicação na parte aérea da cultura da cana-de-açúcar. Já para analisar a influência desses organismos no processo de fitorremediação de Cu, Zn e Mn em colunas de solos cultivados com Jacaranda mimosifolia D. Don, os fungos foram inoculados no solo e na parte aérea das plantas. Todos os tratamentos tiveram 6 repetições e suas médias foram submetidas à análise de variância e teste de média ao nível de 5% de probabilidade. O consórcio fúngico condicionou o crescimento de raízes de soja e milho, aumentou a colonização micorrízica arbuscular em soja e cana-de-açúcar. No jacarandá, os fungos melhoraram o desenvolvimento de raízes e parte aérea, maior acúmulo de Mn e Zn na planta, melhor translocação de Mn e Zn das raízes para a parte aérea, além de diminuir o estresse fisiológico. Além disso, a aplicação conjunta de fungos e biochar nas mudas proporciona proteção à sua atividade fotossintética e reduz o risco ambiental de contaminação por metais por diminuir o total de Cu, Mn e Zn lixiviado. O biochar foi adquirido por meio da pirólise lenta do resíduo do beneficiamento da madeira de eucalipto e aplicado superficialmente no solo. Com isso, esse estudo mostrou que os fungos utilizados apresentam potencial para colaborar com o aumento da produtividade de culturas comerciais e colaborar com a melhoria na remediação de solos contaminados com íons metálicos. / The search for sustainable measures to improve and meet the demand for food that population growth requires, combined with the scarcity of productive land, in addition to the excessive accumulation of metals in the environment due to activities such as mining and application of soil fertilizers, were the two problems that motivated the present study. The use of fungi plant growth promoters: Beauveria bassiana, Metarhizium anisopliae, Pochonia chlamydosporia, Purpureocillium lilacinum and Trichoderma asperella can both influence on increasing productivity as the phytoremediation of heavy metals in the soil. These facts are explained by the capacity of the fungal Consortium to synthesize and induce plants to produce phytohormone targeted for the better development of the roots of the host. The further development of roots can stimulate phytoextraction of metals from the soil and give plants tolerance to contamination. In order to evaluate the influence of fungi on the development of plants, were done seed treatments of soybeans and corn and application in aerial part of the culture of sugar cane. To analyze the influence of these organisms in the process of phytoremediation of Cu, Zn and Mn in soil columns cultivated with Jacaranda mimosifolia D. Don, fungi were inoculated in soil and aerial part of plants. All treatments had 6 reptitions and their averages were subjected to analysis of variance and average test at 5% probability. The fungal consortium has conditioned the growth of roots of soybeans and corn, mycorrhizal colonization increased in soybeans and sugar cane. In jacarandá, fungi have improved the development of roots and shoots, greater accumulation of Mn and Zn in plant, best of Mn and Zn translocation from roots to shoots, in addition to decrease physiological stress. In addition, the joint application of fungus and biochar in seedlings provides protection to photosynthetic activity and reduces the risk of environmental contamination by metals by decreasing the amount of Cu, Mn and Zn leached. The biochar was acquired through the slow pyrolysis of the eucalyptus wood processing residue and applied superficially to the soil. With that, this study showed that fungi used present potential to contribute to the increased productivity of commercial crops and contribute to the improvement in the remediation of soils contaminated with metal ions. / Dissertação (Mestrado)

O estudo de fontes naturais pouco exploradas e que exibem interações ecológicas específicas no seu habitat tem sido enfatizado como estratégico para a descoberta de novas substâncias bioativas. Microrganismos endofíticos são fontes de produtos naturais relativamente pouco estudadas. Estes microrganismos vivem no interior de um vegetal sem causar danos aparentes ao hospedeiro. A interação ecológica específica entre os microrganismos endofíticos e sua planta hospedeira pode estimular a biossíntese de novos produtos naturais bioativos. Os fungos endofíticos \"Glomerella cingulata\" e \"Guignardia mangiferae\" foram isolados da folhas de \"Viguiera arenaria\". O fungo \"G. mangiferae\" foi cultivado em diferentes condições: meios líquidos Czapek, extrato de malte e caldo batata-dextrose e meio sólido de arroz. Extratos e frações obtidas com o cultivo de \"G. mangiferae\" foram avaliados em ensaios antimicrobianos, citotóxicos e enzimáticos. Foram realizados procedimentos cromatográficos com os extratos brutos, obtendo-se 13 substâncias puras, sendo cinco delas inéditas na literatura. Entre as novas substâncias, quatro são derivados tricicloalternarenos. O extrato bruto etanólico oriundo do cultivo em extrato de malte foi o mais promissor nos ensaios biológicos realizados. A partir desse extrato foi isolado a chaetoglobosina D, um composto já descrito na literatura como citotóxico em células cancerígenas. Os demais extratos, frações e substâncias ensaiadas apresentaram apenas atividade fraca ou moderada. O fungo \"G. cingulata\" foi investigado sobre a produção do metabólito secundário tirosol, previamente isolado de várias linhagens de fungos endofíticos. Devido à ampla ocorrência em fungos endofíticos, o tirosol pode estar correlacionado com uma função sinalizadora para a produção de outros metabólitos secundários. Para a determinação da quantidade de tirosol produzido por \"G. cingulata\", um método analítico quantitativo foi desenvolvido obedecendo-se padrões descritos pelo Guidance of Industry. Foi realizada a análise dos fluídos das culturas de \"G. cingulata\" em diferentes condições de cultivo em meio líquido Czapek. Observou-se que a melhor condição para produção do tirosol ocorre em pH 5,0, 120 rpm, 30 ºC, seis dias de incubação. O cultivo de \"G. cingulata\" com e sem adição de tirosol no meio líquido Czapek não permitiu resultados conclusivos sobre a possível função sinalizadora do tirosol na produção de metabólitos secundários em fungos endofíticos. Durante o período de estágio sanduíche no Departament of Molecular Biology do Massachusetts General Hospital (Harvard Medical School), sob a supervisão do Prof. Dr. Frederick M. Ausubel, foram avaliadas cerca de 170 amostras de origem natural do Brasil, sendo a maioria delas oriundas de fungos endofíticos, frente ao nematóide \"Caenorhabditis elegans\" infectado com linhagem patogênica de \"Enterococcus faecalis\". Cerca de 29% das substâncias puras e 2% dos extratos ensaiados apresentaram boa capacidade de recuperação do nematóide infectado. Os policetídeos chaetoviridinas foram os compostos mais ativos neste modelo de ensaio antibacteriano \"in vivo\". / The search for small bioactive molecules from under-explored natural sources that possess specific ecological interaction in their respective habitat has been emphasized as strategic for the discovery of bioactive compounds. Endophytic microorganisms are still relatively under-explored sources of natural products. These microorganisms live inside the host plant tissues without causing symptoms of disease to the host. The specific ecological interaction between the endophytes and their host plants may trigger the biosynthesis of new small bioactive molecules. The endophytic fungi \"Glomerella cingulata\" and \"Guignardia mangiferae\" were isolated from \"Viguiera arenaria\" leaves. G. mangiferae was cultured using different conditions: Czapek, malt extract and potato-dextrose broth liquid media and rice solid medium. Crude extracts and fractions obtained after \"G. mangiferae\" cultivation were evaluated in antimicrobial, cytotoxic and enzymatic assays. Chromatographic procedures were carried out with crude extracts and led to the isolation of 13 compounds, five of them new in the literature. From the novel compounds four are tricycloalternarene derivatives. The crude ethanolic extract obtained from malt extract cultivation was the most promising extract in the biological assays. Chaetoglobosin D, a cytotoxic compound against cancer cell lines previously described in the literature, was isolated from this bioactive extract. The other extracts, fractions and compounds assayed showed weak or moderate activity in the performed assays. The production of tyrosol by \"G. cingulata\" was investigated, since several endophytic fungal strains from Asteraceae species had already produced this compound. Tyrosol may act as a signaling molecule for the production of other metabolites in endophytic fungi. In order to quantify the production of tyrosol by \"G. cingulata\", an analytical method was validated under Guidance of Industry recommendations. Fluid culture analyses were performed after the cultivation of \"G. cingulata\" under different conditions using Czapek liquid medium. The best condition for tyrosol production occurred at pH 5.0, 120 rpm, 30 ºC, six days of incubation. Cultivation of \"G. cingulata\" with and without tyrosol addition in the Czapek liquid medium did not allow the conclusion that tyrosol act as a signaling molecule for metabolite production by endophytic fungi. During sandwich training at Department of Molecular Biology of Massachusetts General Hospital (Harvard Medical School), under Dr. Frederick M. Ausubel supervision, 170 samples from Brazil, the most part obtained from endophytic fungi, were assayed using the nematode \"Caenorhabditis elegans\" infected with the pathogen \"Enterococcus faecalis\". About 29% of pure compounds and 2% of extracts assayed were able to promote the survival of the infected nematode. The polyketides chaetoviridins were found to be the most active compounds in this \"in vivo\" antibacterial assay.

The biological control of plant pests with beneficial microbes has become increasingly important over the last decades. Soil microbes such as fungi and bacteria colonise the roots of plants and promote their growth. Some beneficial microbes can trigger a weak plant defence response that enhances the immune response of the plant at subsequent pathogen attacks and therefore increase the resistance of the plant to other invaders. This mechanism is called “priming”. While biocontrol agents are applied against a variety of plant pests fundamental knowledge of the molecular mechanisms of plant-microbe interactions is still lacking. Especially molecular studies on the role of resistance genes in the interaction of plants with beneficial endophytic fungi are rare. In this study it was investigated how the fungal biocontrol agent Acremonium alternatum affects the development of the clubroot pathogen Plasmodiophora brassicae within the plant host Arabidopsis thaliana. Clubroot is a devastating disease in crop plants such as cabbage and rapeseed and causes abnormal root growth that leads to so called “club roots”. P. brassicae develops within the plant roots and forms resting spores that are very durable and stay infective in soils for up to 2 decades. The control of clubroot by chemical means is difficult and the disease continues to spread on all continents and was also found in Saxony, Germany in recent years. In 2 preliminary studies the co-inoculation of clubroot plants with the fungus A. alternatum resulted in reduced clubroot symptoms in Chinese cabbage and Arabidopsis. It was therefore hypothesised that A. alternatum induces resistance mechanisms in the plant and thus enhances immunity. The focus of this study was to test this hypothesis by carrying out expression analyses on root tissue of infected Arabidopsis plants. For this the plants were inoculated with spores of P. brassicae and A. alternatum before RNA was extracted from the roots, followed by cDNA synthesis and quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). A microarray of root tissue of infected Arabidopsis plants was carried out to depict the events at the stage of initial root hair infection with the clubroot pathogen. The findings from the gene expression analyses were verified for 2 genes with Arabidopsis mutants that are defective in the respective gene and with 2 overexpressor lines. Clubroot symptoms were assessed by rating the root galls according to their stage of development. The overall plant health was further evaluated by recording the developmental stage of the plants (generative vs. vegetative), stem lengths and plant biomass. In addition, 2 local varieties of the economically important crop plant rapeseed (Brassica napus var. Ability and var. Visby) were investigated with qRT-PCR and by recording the disease parameters just described. A second goal of this study was to assess the general biocontrol potential of the yet relatively unknown endophyte A. alternatum in terms of enzymatic activity and competitive behaviour against other phytopathogenic fungi. The potential of this fungus for the use in integrative pest management was investigated. The results presented here are novel findings for this fungus and have not been studied before. The microarray from Arabidopsis roots revealed that the clubroot pathogen P. brassicae suppresses its recognition by pathogen receptors of the plant and thus prevents the host to induce resistance mechanisms. The fungus A. alternatum boosted the level of the pathogen recognition-related genes BAK1 and FLS2 and thus helped to establish early plant defence responses. PCR analyses confirmed that these early responses led to salicylic acid-dependent resistance in the plants which was maintained for several days as shown by elevated levels of the PATHOGENESIS-RELATED gene PR1. Marker genes for an alternative resistance pathway that is mediated over the plant signals jasmonate and ethylene were not activated in Arabidopsis. The co-inoculation of Arabidopsis plants with the endophyte A. alternatum resulted in a significant reduction of clubroot symptoms by up to 24%. In rapeseed the reduction of disease symptoms was 19% and 28% when the plants were treated with a crude cell wall extract of A. alternatum before inoculation with the clubroot pathogen. PCR analyses from Arabidopsis showed a strong response of pathogen recognition genes to the cell wall extract and spores of the endophytic fungus. In rapeseed all of the investigated pathogen recognition genes were upregulated after the endophyte treatment but not with the clubroot pathogen. Together with the PCR results from the microarray these findings suggest that A. alternatum primes its host plant and enhances the resistance of the plant towards P. brassicae. In addition, the fungus increased biomass, stem lengths and survival rates of clubroot-infected plants. In vitro tests revealed that the endophyte can solubilise phosphate and is not very competitive against other phytopathogenic fungi such as Aspergillus or Fusarium which is likely an effect of the relatively slow growth of the endophyte on agar plates. From this study it can be concluded that i) the fungus Acremonium alternatum induces resistance mechanisms in Arabidopsis and 2 Brassica napus cultivars and facilitates the recognition of the clubroot pathogen Plasmodiophora brassicae; ii) that Arabidopsis and Brassica react differently to this beneficial microbe, a fact that has been observed for Plasmodiophora and other microorganisms as well; iii) living spores are not necessary for clubroot biocontrol in rapeseed as a crude cell wall extract reduces symptoms more efficiently. Overall the endophyte A. alternatum is a very promising candidate for the use in integrative pest management in plant strengtheners or as biocontrol agent. / Die biologische Kontrolle von Pflanzenkrankheiten gewinnt zunehmend an Bedeutung. Bodenbewohnende Mikroben wie Pilze oder Bakterien kolonisieren die Wurzeln von Pflanzen und fördern deren Wachstum. Einige dieser förderlichen Mikroben aktivieren eine schwache Abwehrreaktion in der Pflanze die sich verstärkt bei einer weiteren Infektion mit einem Krankheitserreger. Dieser Mechanismus, den man “Priming” nennt, führt zu einer verbesserten Resistenz der Pflanze gegenüber Pflanzenpathogenen. Obwohl natürliche Schädlingsbekämpfer bereits gegen eine Vielzahl an Krankheiten eingesetzt werden, weiss man über grundsätzliche molekulare Mechanismen dieser Pflanzen-Mikroben-Interaktionen nur wenig. Besonders die Rolle von Resistenzgenen ist bisher wenig erforscht, welche bei der Beziehung zwischen Pilzen und Pflanzen eine Rolle spielen. In der hier vorliegenden Arbeit wurde untersucht, wie der endophytische Pilz Acremonium alternatum die Entwicklung des Krankheitserregers Plasmodiophora brassicae in der Pflanze Arabidopsis thaliana beeinflusst. Die Kohlhernie, ausgelöst von P. brassicae, ist eine verheerende Krankheit die u. a. bei Kohl und Raps auftritt und Wurzelgallen, so genannte “Hernien”, hervorruft. Der Krankheitserreger entwickelt sich im Wurzelsystem der Pflanze und bildet Dauersporen, die bis zu 20 Jahre lang im Boden infektiös überdauern können. Ein Eindämmen der Krankheit mit Pflanzenschutzmitteln ist durch den komplexen Lebenslauf des Erregers sehr schwierig, das führte zu einer weltweiten Verbreitung der Kohlhernie. Auch in Sachsen wurden in den letzten Jahren Fälle von Kohlhernie gemeldet. Wie 2 Studien zeigen, führt die Ko-Inokulation von Kohlhernie-erkrankten Pflanzen mit A. alternatum zu einer Verringerung der Symptome in Chinakohl und Arabidopsis. Es wurde daher die Hypothese aufgestellt, dass der Pilz Resistenzmechanismen in der Pflanze anschaltet und damit ihre Immunität erhöht. Um diese Hypothese zu testen, wurden in der hier vorliegenden Studie Genexpressionsanalysen an infizierten Arabidopsiswurzeln durchgeführt. Dafür wurden die Pflanzen zunächst mit Sporen des Kohlhernieerregers und des Pilzes inokuliert, es wurde RNA aus den Wurzeln extrahiert, in cDNA umgeschrieben und diese mittels quantitativer Reverse-Transkriptase-Polymerasenkettenreaktion (RT-qPCR) untersucht. Ein Microarray von Wurzeln infizierter Pflanzen wurde durchgeführt um die Ereignisse abzubilden, die sich zeitnah nach der Infektion in den Wurzeln abspielen. Die Ergebnisse der Genexpressionsanalysen wurden dann an Arabidopsismutanten, die einen Gendefekt im jeweiligen Gen haben, und an Überexprimierer-Pflanzen verifiziert. Kohlherniesymptome an Pflanzen wurden durch eine Kategorisierung der Schadsymptome erfasst. Die allgemeine Pflanzengesundheit sowie der Entwicklungsstand der Pflanze, Stengellängen und das Frischgewicht wurden bestimmt. Zusätzlich wurden 2 Rapssorten, die in Sachsen angebaut werden, untersucht im Hinblick auf die Krankheitsenwicklung und die Reguation von Abwehrgenen. Ein weiteres Ziel dieser Arbeit war es das Biokontrollpotential des bisher schlecht untersuchten Pilzes A. alternatum zu bestimmen. Dazu wurde in vitro die Enzymaktivität des Pilzes getestet sowie seine Konkurrenzfähigkeit gegenüber anderen pflanzenpathogenen Pilzen. Das Potential des Pilzes für die Anwendung im integrierten Pflanzenschutz wurde getestet. Die hier präsentieren Ergebnisse stellen neue Erkenntnisse dar, die für diesen Pilz noch nie untersucht wurden. Der Microarray von Arabidopsiswurzeln zeigte, dass der Kohlhernieerregers die Erkennung durch die Pflanze verhindert und damit Abwehrmechanismen verhindert. Der Pilz A. alternatum förderte die Aktivität der pflanzlichen Erkennungsrezeptoren FLS2 und BAK1 und setzte damit die Erkennung von P. brassicae in Gang. PCR-Analysen ergaben, dass diese früh induzierten Abwehrmechanismen zu einer systemischen Resistenz in der Pflanze führte durch die Aktivierung des Pathogenese-relevanten Gens PR1. Genmarker, die die Aktivität eines alternativen, von Jasmonat und Ethylen vermittelten Abwehrweges anzeigen, waren nicht ativiert. Die Ko-Inokulation von Arabidopsis mit dem Endophyten führte zu einer signifikanten Reduktion der Krankheitssymptome um 24%. In Raps betrug die Reduktion 19% und 24% wenn die Pflanzen vor der Kohlhernie-Infektion mit einem Zellwandextrakt des Pilzes behandelt wurden. Mittels PCR konnte gezeigt werden, dass Gene für das Erkennen von Pathogenen in der Wurzel von Arabidopsis auf den Zellwandextrakt und Sporen des Pilzes reagieren. In Raps wurden alle der untersuchten Erkennungsgene aufreguliert nach der Infektion mit A. alternatum, nicht jedoch bei der Infektion mit P. brassicae. Zusammenfassend lässt sich sagen, dass der endophytische Pilz A. alternatum die Wirtspflanze auf eine folgende Infektion vorbereitet (Priming) und systemische Abwehr-mechanismen in der Pflanze induziert, wenn diese mit Kohlhernie infiziert ist. Außerdem treibt der Pilz das Sprosswachstum voran, erhöht die Biomasse und fördert das Überleben von Kohlhernie-infizierten Pflanzen. In vitro-Tests ergaben, dass der Endophyt Kalziumphosphat löslich machen kann und wenig kompetitiv gegenüber Pflanzenpathogenen wie Aspergillus oder Fusarium ist. Dies ist vermutlich mit dem langsameren Wachstum des Endophyten im Gegensatz zu den anderen Pilzen zu erklären. Aus den Ergebnissen dieser Arbeit lassen sich folgende Schlüsse ziehen: i) der endophytische Pilz Acremonium alternatum induziert Resistenzmechanismen in Arabidopsis und Raps und und fördert die Erkennung des Kohlhernieerregers Plasmodiophora brassicae; ii) Arabidopsis und Raps reagieren unterschiedlich auf diesen förderlichen Pilz, ein solcher Unterschied wurde bereits für Plasmodiophora und andere Mikroben beschrieben; iii) lebende Sporen des Pilzes sind nicht notwendig um Krankheitssymptome der Kohlhernie in Raps zu verringern, ein Zellwandextrakt von A. alternatum ist dafür besser geeignet. Ganz allgemein lässt sich sagen, dass der endophytische Pilz Acremonium alternatum ein sehr vielversprechender Kandidat ist für den Einsatz im integrierten Pflanzenschutz in Pflanzenstärkungsmitteln oder als Biokontrollorganismus.

Bacterial endophytes are the organisms that live inside the plant for a full or a part of their life cycle. Endophytic bacteria have captured the interest of agriculture industry due to their plant beneficial properties, such as synthesis of phytohormones, solubilization of soil nutrients, and alleviation of biotic and abiotic stresses. Several studies have reported that stress tolerant endophytic bacteria can work with a similar performance as non-stressed conditions when inoculated to the plants under stressed conditions. Combination of abiotic stresses such as salinity, drought and low nitrogen stress can have additive or agonistic effects on bacterial and plant growth, and their interactions. However, very few studies have reported the impact of combined stress on endophytic bacterial assisted plant growth promotion. Therefore, understanding the underlying mechanisms of endophytic bacterial assisted plant’s tolerance abiotic stresses may provide the means of better exploiting the beneficial abilities of endophytic bacteria in agricultural production. Thus, the aim of this thesis was to study the stress tolerance mechanisms, beneficial characteristics, and plant growth promotion characteristics of endophytic bacteria under individual and combined abiotic stresses. Transcriptome analysis of endophytic bacteria revealed that tolerance mechanisms to deal with one kind of stress is different than concurrent stresses. Salinity and drought stress largely modulated the genes involved in flagellar assembly and membrane transport, showing reduced motility under stress conditions to preserve the energy. Additionally, bacterial endophyte that can fix nitrogen was studied with maize plant growth promotion under drought and low nitrogen stress conditions. The results suggested that diazotrophic bacterial endophyte can promote plant growth under moderate individual and combined stress conditions. Plant growth promoting endophytic bacteria can be utilized as an efficient tool to increase crop production under individual and concurrent abiotic stresses.

A partir de 280 isolados fúngicos de cana-de-açúcar, 18 cepas foram avaliadas quanto á presença de genes da policetídeo sintase por meio da técnica do PCR. Estes fungos foram identificados taxonomicamente por uma abordagem polifásica, classificando-os dentro de quatro ordens e nove gêneros. A avaliação da atividade biológica demonstrou a presença de metabolitos com propriedades antibióticas quando enfrentados a micro-organismos patogênicos. Segundo a análise de correspondência múltipla, esta atividade poderia estar associada com a local de isolamento dos fungos. Foram detectadas 36 seqüências similares a genes PKS a partir de 17 destes fungos. A análise filogenética do domínio KS, conduzida pelo método de neighbor-joining, indicou que 16 seqüências se acomodaram dentro do grupo monofilético dos PKS envolvidos na produção de policetídeos não reduzidos e as outras 10 seqüências se acomodaram dentro do grupo monofilético dos PKS envolvidos na produção de policetídeos reduzidos. A análise do domínio CMT também apontou que as seqüências podiam se acomodaram em grupos de PKS dependendo do grau de redução do policetídeo, todas as seqüências CMT se relacionaram com PKS envolvidos na produção de policetídeos reduzidos. As análises dos modelos estruturais também demonstraram que as seqüências estavam altamente relacionadas com estruturas protéicas da família das enzimas de condensação, destacando a presença de uma hélice característica que carrega o resíduo de cisteína, responsável pela atividade de condensação. Extratos orgânicos obtidos de cultivos dos fungos foram avaliados parar detectar a presença de compostos tipo lovastatina. Por meio de cromatografia CCDS, detectaram-se bandas de 10 extratos com o mesmo deslocamento que a lovastatina padrão, mas apenas 6 destas foram confirmados por CLAE. O isolado A. flavus CBMAI 1023, foi selecionado para a realização de experimentos de produção a maior escala onde foi possível isolar e caracterizar um novo policetídeo. / From a group of 280 sugarcane-derived fungi 18 strains were assessed for the presence of polyketide synthase genes by PCR approaches. These fungi were identified taxonomically by a polyphasic approach classifying into four orders and nine genres. Biological activity tests showed the presence of antibiotic metabolites against pathogenic microorganisms and the relationship of this activity might be linked with the fungal isolate location by multiple correspondence analyses. 36 sequences similar to PKS genes fragments were detected from 17 of these fungi. A neighbor-joining phylogenetic analysis of the KS domain showed that 16 sequences fit on the monophyletic group of PKS evolved with production of non reduced polyketides, and the other 10 sequences fit on the monophyletic group of PKS evolved with the production of reduced polyketides. CMT domain analysis also pointed that the sequences fit with groups of PKS depending on polyketide reduction grade, all ten related to PKS evolved with the synthesis of reduced polyketides. Protein structural analysis also pointed out that these sequences are closely related with proteins from condensing enzyme family, highlighting the presence of a characteristic helix elbow that bears the cysteine residue responsible for the condensation activity. The fungi were also tested for their capacity of producing lovastatin compounds where chromatographic TLC detected bands from 10 extracts with the same dislocation compared to a lovastatin, but only 6 were confirmed by HPLC. The A. flavus CBMAI (1023) were selected for upscale production experiments, from where it was possible isolate and characterize a new polyketide compound.

No description available.

No description available.

No description available.

A partir de 280 isolados fúngicos de cana-de-açúcar, 18 cepas foram avaliadas quanto á presença de genes da policetídeo sintase por meio da técnica do PCR. Estes fungos foram identificados taxonomicamente por uma abordagem polifásica, classificando-os dentro de quatro ordens e nove gêneros. A avaliação da atividade biológica demonstrou a presença de metabolitos com propriedades antibióticas quando enfrentados a micro-organismos patogênicos. Segundo a análise de correspondência múltipla, esta atividade poderia estar associada com a local de isolamento dos fungos. Foram detectadas 36 seqüências similares a genes PKS a partir de 17 destes fungos. A análise filogenética do domínio KS, conduzida pelo método de neighbor-joining, indicou que 16 seqüências se acomodaram dentro do grupo monofilético dos PKS envolvidos na produção de policetídeos não reduzidos e as outras 10 seqüências se acomodaram dentro do grupo monofilético dos PKS envolvidos na produção de policetídeos reduzidos. A análise do domínio CMT também apontou que as seqüências podiam se acomodaram em grupos de PKS dependendo do grau de redução do policetídeo, todas as seqüências CMT se relacionaram com PKS envolvidos na produção de policetídeos reduzidos. As análises dos modelos estruturais também demonstraram que as seqüências estavam altamente relacionadas com estruturas protéicas da família das enzimas de condensação, destacando a presença de uma hélice característica que carrega o resíduo de cisteína, responsável pela atividade de condensação. Extratos orgânicos obtidos de cultivos dos fungos foram avaliados parar detectar a presença de compostos tipo lovastatina. Por meio de cromatografia CCDS, detectaram-se bandas de 10 extratos com o mesmo deslocamento que a lovastatina padrão, mas apenas 6 destas foram confirmados por CLAE. O isolado A. flavus CBMAI 1023, foi selecionado para a realização de experimentos de produção a maior escala onde foi possível isolar e caracterizar um novo policetídeo. / From a group of 280 sugarcane-derived fungi 18 strains were assessed for the presence of polyketide synthase genes by PCR approaches. These fungi were identified taxonomically by a polyphasic approach classifying into four orders and nine genres. Biological activity tests showed the presence of antibiotic metabolites against pathogenic microorganisms and the relationship of this activity might be linked with the fungal isolate location by multiple correspondence analyses. 36 sequences similar to PKS genes fragments were detected from 17 of these fungi. A neighbor-joining phylogenetic analysis of the KS domain showed that 16 sequences fit on the monophyletic group of PKS evolved with production of non reduced polyketides, and the other 10 sequences fit on the monophyletic group of PKS evolved with the production of reduced polyketides. CMT domain analysis also pointed that the sequences fit with groups of PKS depending on polyketide reduction grade, all ten related to PKS evolved with the synthesis of reduced polyketides. Protein structural analysis also pointed out that these sequences are closely related with proteins from condensing enzyme family, highlighting the presence of a characteristic helix elbow that bears the cysteine residue responsible for the condensation activity. The fungi were also tested for their capacity of producing lovastatin compounds where chromatographic TLC detected bands from 10 extracts with the same dislocation compared to a lovastatin, but only 6 were confirmed by HPLC. The A. flavus CBMAI (1023) were selected for upscale production experiments, from where it was possible isolate and characterize a new polyketide compound.