Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-19T10:15:55Z No. of bitstreams: 2 Dissertação - Amanda Rafaela Rodrigues - 2014.pdf: 1648839 bytes, checksum: 9a00f301e577d05e94214afe813688c5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-19T10:17:31Z (GMT) No. of bitstreams: 2 Dissertação - Amanda Rafaela Rodrigues - 2014.pdf: 1648839 bytes, checksum: 9a00f301e577d05e94214afe813688c5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-19T10:17:31Z (GMT). No. of bitstreams: 2 Dissertação - Amanda Rafaela Rodrigues - 2014.pdf: 1648839 bytes, checksum: 9a00f301e577d05e94214afe813688c5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-10-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The genus Trichoderma is represented by non-pathogenic soil fungi that have been studied by act as biological control agents against fungal pathogens, such as Sclerotinia sclerotiorum, the fungus that causes white mold, besides that, reduce environment and human health risks by being able to replace agrochemicals. Proteomic strategies with MS techniques have been important tools in studies of patterns identification for protein expression in different growth conditions. This work aims to development new strategies that enable the detection and identification of proteins secreted by Trichoderma asperellum (TR356) and Sclerotinia sclerotiorum when inoculated together and separated. The results obtained by MALDI / TOF analysis allowed the identification of a β-1.3--glucanosiltransferase and α-1.2-Dmannosidase, demonstrating the possibility of proteins identification for better comprehension about interaction between these organisms. It was therefore possible to identify the proteins through strategies used, but further analysis are required in order to elucidate the function and interaction of proteins secreted by the fungus Trichoderma asperellum TR356 against S. sclerotiorum. / OgêneroTrichodermaérepresentadoporfungosdesolonãopatogênicosquesãoestudad osporsuaaçãocomoagentesdecontrolebiológicocontrafungosfitopatógenos,comoScler otiniasclerotiorum,ofungocausadordomofobranco.Osfungos Trichoderma spp.atuamcomoimportantesagentesdecontrolebiológico,diminuindoosriscosàsaúdehu manaeaomeioambienteporseremcapazesdesubstituirosagroquímicos.Estratégiasprot eômicas juntamente com técnicas de MSsãoimportantesferramentasemestudosdepadrão, identificação e expressãodeproteínasemdiferentescondiçõesdecrescimento.Levando em consideração a importância de tais estudos, o presente trabalho tem por objetivo o desenvolvimento de novas estratégias que possibilitem a detecção e identificação de proteínas secretadas por Trichoderma asperellum (TR356) e Sclerotinia sclerotiorum quando inoculados juntos e/ou separados os dois organismos vivos. Os resultados obtidos através das análises por MALDI/TOF permitiram a identificação dasproteínas, β-1,3-glucanosiltransferase e α-1,2-D-mannosidase,demonstrando a possibilidade de identificaçãopara o entendimento futuro acerca da interação destes organismos. Foi possível, portanto, a identificação das proteínas através das estratégias utilizadas, porém serão necessárias futuras análises afim de elucidar a interação e função das proteínas secretadas através do fungo T. asperellum TR356sobre S. sclerotiorum.

<p>  </p> <p><em>Sclerotinia sclerotiorum </em>is a soilborne pathogen of soybean that causes Sclerotinia stem rot, alternatively called white mold. Sclerotinia stem rot can cause significant yield losses under cool and wet environmental conditions. Two biofungicides, <em>Coniothyrium minitans </em>and <em>Bacillus amyloliquefaciens, </em>are currently available and labeled to limit or suppress <em>S. sclerotiorum</em> in soybean. These biofungicides can be applied in place of synthetic foliar fungicides to provide an alternative mode of action for the control of Sclerotinia stem rot. However, limited information is available regarding the efficacy of <em>C. minitans </em>and <em>B. amyloliquefaciens </em>as biocontrol agents of <em>S. sclerotiorum </em>in soybean and the sensitivity of the biofungicides biological activity on <em>S. sclerotiorum </em>to pesticides commonly used in soybean production systems. This research aims to provide management recommendations for <em>S. sclerotiorum </em>in soybean using <em>C. minitans </em>and <em>B. amyloliquefaciens </em>and to develop guidelines for how to incorporate the biofungicides into an established soybean pest management program. To assess the effectiveness of <em>C. minitans </em>and <em>B. amyloliquefaciens </em>as biocontrol agents of <em>S. sclerotiorum </em>dual culture, amended media, and soil plate assays were conducted along with experiments in the growth chamber and field. The presence of a distinct inhibition zone surrounding the <em>B. amyloliquefaciens </em>colony in the dual culture assay and the absence of mycelial growth on the media plates amended with <em>B. amyloliquefaciens </em>confirmed that the bacteria can control the mycelial growth of <em>S. sclerotiorum </em>through antibiosis. The absence of an inhibition zone surrounding the <em>C. minitans </em>isolate in the dual culture assay along with the degradation of sclerotia following treatment with <em>C. minitans </em>in the soil plate assay indicates an inability to limit the mycelial growth of <em>S. sclerotiorum </em>and confirms that the primary mode of action is mycoparasitism. In the growth chamber, <em>B. amyloliquefaciens</em> at 14.03 L/ha applied using the dip method significantly reduced Sclerotinia stem rot lesion length when compared to the non-treated control and resulted in the lowest lesion area under the disease progress curve (lAUDPC). When <em>B. amyloliquefaciens </em>and <em>C. minitans </em>were applied in the field, no differences were observed between treatments for soybean moisture, test weight, or yield. To evaluate the sensitivity of <em>B. amyloliquefaciens </em>and <em>C. minitans</em> biological activity on <em>S. sclerotiorum </em>to pesticides commonly used in soybean production systems a poison plate assay as well as soil plate, growth chamber, and field experiments were conducted. In the poison plate assay <em>C. minitans </em>was most sensitive to the preemergence herbicide flumioxazin and the synthetic fungicides boscalid and fluazinam, while <em>B. amyloliquefaciens </em>was sensitive only to the synthetic fungicide fluazinam. In the soil plate assay the mycoparasitic activity of <em>C. minitans </em>on sclerotia of <em>S. sclerotiorum </em>was sensitive to flumioxazin, metribuzin, glyphosate, picoxystrobin, and boscalid. In the controlled environment experiments, none of the pesticides tested decreased the efficacy of <em>B. amyloliquefaciens</em>. There were no significant interactions between <em>C. minitans </em>and <em>B. amyloliquefaciens </em>with preemergence herbicides, postemergence herbicides, and synthetic fungicides for soybean moisture, test weight, and yield. This research demonstrates that <em>B. amyloliquefaciens </em>and <em>C. minitans </em>are effective biocontrol agents of <em>S. sclerotiorum </em>in soybean. However, antagonistic relationships exist between the biofungicides and certain preemergence, postemergence, and synthetic fungicides used in soybean production systems.</p>

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Soybean stem white rot, caused by Sclerotinia sclerotiorum, occurs in more than one million hectares in Brazil. This disease should be managed by integrated control methods, including genetic resistance. This study evaluated and developed a reliable method for the selection of soybean genotypes resistant to stem white rot. The work was based on rating inoculations on the stem. The methods evaluated consisted of different inoculation sites in the plant, wounding or not or cutting the stem of the plants, and inoculating PDA disks containing fungal mycelium. The tests were conducted in two different environments, a controlled one, where plants were grown in a greenhouse and brought into the laboratory to be inoculated and incubated in a growth chamber where the temperature is favorable for fungal growth, and a field, where the pathogen is subjected to adverse environment conditions. Spearman´s coefficients of rank correlation was used to measure the correspondence between ranks of cultivar and ranks of the same cultivars in field experiments to establish the best inoculation method. It was observed that temperature growth conditions, and inoculation method may affect the selection of resistant soybean genotypes against stem rot. The best method to evaluate soybean resistance was by wound inoculating the middle third of the plant when the plants were at the end of flowering and early grain filling, with PDA disks and evaluating 14 days later. This method was rs = 0.86, and the variety that can be considered as resistance standard in other studies was Emgopa 316, while the susceptible standards were BRSMG Garantia and BRSMG68 Vencedora. / A podridão branca da haste da soja causada por Sclerotinia sclerotiorum ocorre mais de um milhão de hectares no Brasil. Esta doença deve ser manejada por métodos de controle integrado, entre eles a resistência genética que deve ser pesquisa. O objetivo do trabalho foi avaliar e definir um método confiável na seleção de genótipos de soja. Quanto ao método de inoculação, o trabalho se baseou em avaliar inoculações realizadas no caule. As metodologias diferiram quanto ao local de inoculação na planta, uso ou não de ferimentos, ou o uso do corte do ápice das plantas na inoculação, utilizando o disco de BDA contendo micélio do fungo. Os testes foram conduzidos em dois ambientes distintos, em ambiente controlado, onde plantas foram cultivadas em casa-de-vegetação e levadas para o laboratório para serem inoculadas e incubadas em câmara de crescimento onde a temperatura é extremamente favorável ao desenvolvimento do fungo e no campo onde a doença estará sujeito às condições ambientais adversas. Para definir qual o melhor método de inoculação, como também os genótipos que possuem maior resistência parcial, calculou correlações pelo coeficiente de Spearman entre os métodos avaliados e métodos com o ranking geral das cultivares. Observou-se que a temperatura, condições de cultivo, e o método de inoculação podem influenciar na seleção dos genótipos quanto à resistência à podridão da haste da soja. Definiu que o melhor método de avaliação à resistência de genótipos de soja foi a inoculação realizada com ferimento no terço médio da planta onde as plantas se encontravam no final do florescimento e início de enchimento dos grãos, com discos de BDA e avaliadas 14 dias após a inoculação, este método obteve rs=0,86, e a cultivar que pode ser considerado com padrão de resistência em outros estudos foi a Emgopa 316, e o padrão de suscetibilidade a BRSMG Garantia e BRSMG 68 Vencedora. / Mestre em Agronomia