Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2009. / Submitted by Luis Felipe Souza (luis_felas@globo.com) on 2008-11-27T19:02:17Z No. of bitstreams: 1 Dissertacao_2007_ DanielAgustinho.pdf: 633554 bytes, checksum: 7fdafd2cfb150d3fac3b9cba5430e0e3 (MD5) / Approved for entry into archive by Georgia Fernandes(georgia@bce.unb.br) on 2009-02-11T15:57:22Z (GMT) No. of bitstreams: 1 Dissertacao_2007_ DanielAgustinho.pdf: 633554 bytes, checksum: 7fdafd2cfb150d3fac3b9cba5430e0e3 (MD5) / Made available in DSpace on 2009-02-11T15:57:23Z (GMT). No. of bitstreams: 1 Dissertacao_2007_ DanielAgustinho.pdf: 633554 bytes, checksum: 7fdafd2cfb150d3fac3b9cba5430e0e3 (MD5) / A seringueira (Hevea spp.) é uma planta nativa da região amazônica, da qual se extrai o látex para a fabricação da borracha natural. O Brasil já foi o maior produtor mundial de látex, e este já foi responsável por 40% da exportações brasileiras. Esta planta é suscetível ao ataque do fitopatógeno Microcyclus ulei, causador do mal das folhas da seringueira, que é também endêmico da região amazônica e o principal responsável pela queda de produção de látex na América Latina e perda do mercado para países asiáticos como Sri Lanka e Malásia. Entretanto, surgiu dentro deste contexto o fungo micopatogênico Dicyma pulvinata que apresenta alta capacidade de controle do fitopatógeno M. ulei. Considerando esse fato, D. pulvinata pode ser utilizado como uma ferramenta para o controle biológico do mal das folhas da seringueira. Neste trabalho foi analisada a capacidade de produção de glicosil hidrolases (β−1,3-, β−1,3-1-4- e β−1,4-glucanase e quitinase ) por dois isolados CEN62 e CEN93, que haviam demonstrado, em estudos anteriores, serem bons antagonistas do fitopatógeno M. ulei. Assim, foi iniciado um projeto de avaliação da capacidade de produção dessas enzimas por estes dois isolados, e a caracterização de suas propriedades bioquímico-moleculares. Os dois isolados demonstraram capacidades de produção das enzimas β−1,3-, β−1,3-1,4-glucanase e quitinase. No entanto, apenas o isolado CEN62 apresentou atividade de β-glucanase no sobrenadante do meio de cultura. Nenhum dos dois isolados secretou atividade de quitinase. Entretanto, a atividade de quitinase significativa foi encontrada associada ao micélio. Não foi possível, no entanto, a identificação de moléculas relacionadas a essa atividade, quer seja procedendo a lise do micélio na presença de detergente, quer procedendo a lise por sonicação Assim, foi escolhido o sobrenadante do meio de cultura do isolado CEN62 para etapas posteriores de purificação, por apresentar uma atividade de β−1,3-glucanase. O sobrenadante do meio de cultura do isolado CEN62 foi então caracterizado bioquimicamente, observando-se que a atividade de β−1,3-glucanase teve um máximo a 60ºC e pH 5,5, e a atividade de β 1,3-1,4-glucanase o máximo foi de 50ºC e pH 5,5. A β−1,3-glucanase apresentou atividade correspondente a uma meia vida de 10 e 1 minutos a 70ºC e 80ºC respectivamente. O sobrenadante do meio de cultura do isolado CEN62 de D. pulvinata , crescido por dez dias em meio contendo quitina como fonte de carbono, apresentando atividade de β-1,3-glucanase, foi ultrafiltrado com uma membrana de exclusão de 30kDa, concentrado e submetido a uma cromatografia de troca iônica em resina S-Sepharose. Essa cromatografia resultou em um pico de proteínas com atividade de β-1,3-glucanase o qual foi em seguida, recromatografado em uma coluna de troca iônica (DEAE-Sepharose). Um novo pico de proteínas com três espécies protéicas foi obtido, conforme mostrado em gel de poliacrilamida em condições desnaturantes. O rendimento deste processo de purificação parcial desta β-1,3-glucanase foi de 0,023%, e o fator de purificação de 345,3 vezes. _______________________________________________________________________________ ABSTRACT / The rubber tree (Hevea spp.) is a native plant from the amazonic region, from which the latex is extracted in order to make the natural rubber. Brazil once was the mundial leader in latex production, and it was the responsible for 40% of brazilian exportations. However, this plant is vulnerable to the attacks from the phytopathogen Microcyclus ulei, agent of the leaf blight of the rubber tree, and is also endemic from the amazonic region and the main responsible for the fall in production in latex in latin America, and loss of market shares for the asian countries of Sri Lanka and Malasia. Nonetheless, in this context, the mycopathogen fungus Dicyma pulvinata came forth, and shows high capacities in the biological control of M. ulei. Base don this, there is a new alternative for the biological control for the leaf blight of the rubber tree. In this work the capacity of production of glicosyl hidrolases (β−1,3-, β−1,3-1-4- e β−1,4-glucanase e quitinase) of two isolates CEN62 e CEN93, which had demonstrated in anterior studies to be good antagonists of the phytopatogen M. ulei, were analyzed. Then, a project started to evaluate the capacity of production of these enzymes by these two isolates, and the characterization of their biochemical and molecular properties. The two isolates have demonstrated capacity to product the enzymes β−1,3-, β−1,3-1,4-glucanase and quitinase. Nevertheless, just the isolate CEN62 demonstrated activity of β-glucanase in the medium filtrate. None of the isolates secreted quitinase activity. However, a significant activity of quitinase was found associated to the mycelium. It was not possible, however, to extract this activity nor by cell lysis using detergent, nor by lysing by sonication.Then, the CEN62 medium filtrate was chosen for further purification steps, by showing β−1,3-glucanase activity. The isolate CEN62 medium filtrate was then biochemically characterized, being possible to evaluate that the β−1,3-glucanase activity had a maximum at 60ºC and pH 5,5, and the β 1,3-1,4-glucanase activity had its maximum at 50ºC and pH 5,5. The β−1,3-glucanase activity showed a half-life period of tem and one minutes at 70ºC e 80ºC respectively. The CEN62 medium filtrate of D. pulvinata after growing for tem days in medium containing chitin as carbon source, and containing β-1,3-glucanase activity was ultrafiltrated with an exclusion membrane of 30kDa, and the concentrated was submitted to ion exchange chromatography in S-Sepharose resin. This chromatography resulted in a protein peak with β-1,3-glucanase activity, which was, thereafter, rechromatographed in a ion exchange column (DEAE-Sepharose), resulting in a new protein peak, with three protein species as shown in SDS-PAGE. The yield of this process of β-1,3-glucanase partial purification was of 0,023%, and purification factor of 345,3 times.

Made available in DSpace on 2014-06-11T19:34:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-06Bitstream added on 2014-06-13T21:06:54Z : No. of bitstreams: 1 sambugaro_r_dr_botfca.pdf: 1143633 bytes, checksum: 25f9ce4aa41c21b2302d4b0e2e19e340 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / The rubber tree is a tropical species that loses its leaves periodically. This deciduifolio habit is an important character for the rubber tree, that is, clones uniform defoliation provides a reduction in the pathogens inocule whose reproductive structures are located in the old leaves. After defoliation, it occurs the foliar renewal and it is at this stage, also known as refoliation, that the rubber tree presents susceptible leaflets in which occur most part of the foliar deseases, such as leaf blight, where the cycle of the pathogen (Microcyclus ulei (P. Henn.)v. Arx) is straightly related with the foliar stages (B, C and D). Therefore, the knowledge on these stages, clones phenologic behavior, factors that influence this character and local climate conditions is important for their selection. The objectives of this work were: characterize through quantitative and descriptive foliar morphology the stages B, C and D; characterize the phenologic behavior and the incidence of South American Leaf Blight; correlate the beginning of the defoliation period with climatic parameters, and leaf blight incidence, during the foliar renewal period, with climatic parameters. For the stages characterization, it was used the clone PB 235, from which the leaves central leaflets were collected, at different stages. It was measured the foliolar limbus lenght, width and area, and the petiole lenght and diameter. It was also determined the form of the limbus, apex and base of the foliolar lamina, as well the leaf coloration. For the other objectives, the clones Fx 3844, Fx 3864, Fx 2261, IAN 873, IAN 717 and RRIM 600 of rubber trees from Vale do Ribeira region, São Paulo state were used, being the fallen leaflets collected weekly in four baskets by clone, randomly distributed. The leaflets were totalized, separated by age and analyzed as for the incidence of the fungus Microcyclus ulei... (Complete abstract click electronic access below)

Orientador: Edson Luiz Furtado / Banca: Nilton Luiz de Souza / Banca: Roberto Antonio Rodella / Banca: José Otavio Machado Menten / Banca: Adriana Martins / Abstract: The rubber tree is a tropical species that loses its leaves periodically. This deciduifolio habit is an important character for the rubber tree, that is, clones uniform defoliation provides a reduction in the pathogens inocule whose reproductive structures are located in the old leaves. After defoliation, it occurs the foliar renewal and it is at this stage, also known as refoliation, that the rubber tree presents susceptible leaflets in which occur most part of the foliar deseases, such as leaf blight, where the cycle of the pathogen (Microcyclus ulei (P. Henn.)v. Arx) is straightly related with the foliar stages (B, C and D). Therefore, the knowledge on these stages, clones phenologic behavior, factors that influence this character and local climate conditions is important for their selection. The objectives of this work were: characterize through quantitative and descriptive foliar morphology the stages B, C and D; characterize the phenologic behavior and the incidence of South American Leaf Blight; correlate the beginning of the defoliation period with climatic parameters, and leaf blight incidence, during the foliar renewal period, with climatic parameters. For the stages characterization, it was used the clone PB 235, from which the leaves central leaflets were collected, at different stages. It was measured the foliolar limbus lenght, width and area, and the petiole lenght and diameter. It was also determined the form of the limbus, apex and base of the foliolar lamina, as well the leaf coloration. For the other objectives, the clones Fx 3844, Fx 3864, Fx 2261, IAN 873, IAN 717 and RRIM 600 of rubber trees from Vale do Ribeira region, São Paulo state were used, being the fallen leaflets collected weekly in four baskets by clone, randomly distributed. The leaflets were totalized, separated by age and analyzed as for the incidence of the fungus Microcyclus ulei... (Complete abstract click electronic access below) / Doutor

Orientador: Edson Luiz Furtado / Coorientador: José Raimundo De Souza Passos / Banca: Juliana Cristina Sodario Cruz / Banca: Ana Carolina Firmino / Resumo: O maior entrave à expansão da heveicultura no Brasil é a doença conhecida por mal das folhas causada pelo fungo Microcyclus ulei, cujos sintomas ocorrem nas folhas jovens, causando intensas desfolhas, diminuindo a produção de látex e até mesmo a morte em plantas suscetíveis. Consiste numa ameaça constante aos plantios do Oriente. Com a evolução dos programas de melhoramento genético tradicional, atualmente são utilizados clones híbridos interespecíficos, os quais são mais produtivos e apresentam melhor resistência aos patógenos. Através dos programas de melhoramento genético tradicional, não houve progresso significativo quanto à resistência ao mal das folhas, devido à seleção serem dirigida para clones com resistência completa. Neste patossistema a resistência horizontal (RH) e resistência vertical (RV) podem agir simultaneamente, sendo complexa e de difícil quantificação. Este trabalho teve como objetivo caracterizar o tipo de lesão foliar do patossistema Hevea sp. x M. ulei, verificar a suscetibilidade clonal aos patógenos, analisar a freqüência de infecção visando quantificar a resistência no patossistema Hevea sp. x M. ulei e sugerir clones diferenciadores de seringueira além de quantificar as raças de M. ulei no Brasil. Os parâmetros monocíclicos (período latente e diâmetro de lesão) são aplicáveis na quantificação de resistência no patossistema Hevea sp. x M. ulei. O período de latência teve pouca variação entre os clones. O diâmetro de lesão teve variação ampla entre os clones, sendo um parâmetro discriminante para a resistência horizontal e resistência vertical. Várias raças deste patógeno foram descritas no Brasil, baseada em uma série de clones diferenciadores. Diante disso foi utilizado testes com 11 clones diferenciadores já conhecidos na literatura: MDF 180, Fx 3844, Fx 985, Fx 4098, Fx 2261, Fx 2804, Fx 3899, IAN 6158, IAN 3087, ... / Abstract: The biggest barrier to expansion of rubber cultivation in Brazil is the disease known as leaf blight caused by the fungus Microcyclus ulei whose symptoms occur in young leaves, causing severe defoliation, reducing the production of latex and even death in susceptible plants. Is a constant threat to the plantations of the East. With the evolution of traditional breeding programs currently interspecific hybrid clones are used, which are more productive and have better resistance to pathogens. Through traditional breeding programs, there was no significant progress for resistance to leaf blight, due to selection be directed to clones with complete resistance. This pathosystem horizontal resistance (HR) and vertical resistance (VR) can act simultaneously, being complex and difficult to quantify. This study aimed to characterize the type of leaf injury pathossystem Hevea sp. x M. ulei verify clonal susceptibility to pathogens, to analyze the frequency of infection to quantify the resistance in the pathosystem Hevea sp. x M. ulei and suggest different clones of rubber and quantifies the races of M. ulei in Brazil. Monocyclic parameters (latent period and lesion diameter) are applicable in the quantification of resistance in the pathosystem Hevea sp. x M. ulei. The latency period had little variation among clones. The diameter of the lesion was wide variation between the clones, being a discriminant parameter for horizontal resistance and vertical resistance. Several races of this pathogen have been described in Brazil, based on a number of clones' differentiators. Grounded on the reactions of 11 different clones already known in the literature: MDF 180, Fx 3844, Fx 985, Fx 4098, Fx 2261, Fx 2804, Fx 3899, IAN 6158, IAN 3087, IAN 717 and PA 31 containing the species Hevea brasiliensis, Hevea benthamiana and Hevea pauciflora, we performed an analysis grouping for the binary data referring to virulence ... / Mestre

Made available in DSpace on 2014-06-11T19:30:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-28Bitstream added on 2014-06-13T19:00:18Z : No. of bitstreams: 1 000758939.pdf: 1540122 bytes, checksum: d22f9c87e9c7253bc25e906de41e8311 (MD5) / O maior entrave à expansão da heveicultura no Brasil é a doença conhecida por mal das folhas causada pelo fungo Microcyclus ulei, cujos sintomas ocorrem nas folhas jovens, causando intensas desfolhas, diminuindo a produção de látex e até mesmo a morte em plantas suscetíveis. Consiste numa ameaça constante aos plantios do Oriente. Com a evolução dos programas de melhoramento genético tradicional, atualmente são utilizados clones híbridos interespecíficos, os quais são mais produtivos e apresentam melhor resistência aos patógenos. Através dos programas de melhoramento genético tradicional, não houve progresso significativo quanto à resistência ao mal das folhas, devido à seleção serem dirigida para clones com resistência completa. Neste patossistema a resistência horizontal (RH) e resistência vertical (RV) podem agir simultaneamente, sendo complexa e de difícil quantificação. Este trabalho teve como objetivo caracterizar o tipo de lesão foliar do patossistema Hevea sp. x M. ulei, verificar a suscetibilidade clonal aos patógenos, analisar a freqüência de infecção visando quantificar a resistência no patossistema Hevea sp. x M. ulei e sugerir clones diferenciadores de seringueira além de quantificar as raças de M. ulei no Brasil. Os parâmetros monocíclicos (período latente e diâmetro de lesão) são aplicáveis na quantificação de resistência no patossistema Hevea sp. x M. ulei. O período de latência teve pouca variação entre os clones. O diâmetro de lesão teve variação ampla entre os clones, sendo um parâmetro discriminante para a resistência horizontal e resistência vertical. Várias raças deste patógeno foram descritas no Brasil, baseada em uma série de clones diferenciadores. Diante disso foi utilizado testes com 11 clones diferenciadores já conhecidos na literatura: MDF 180, Fx 3844, Fx 985, Fx 4098, Fx 2261, Fx 2804, Fx 3899, IAN 6158, IAN 3087,... / The biggest barrier to expansion of rubber cultivation in Brazil is the disease known as leaf blight caused by the fungus Microcyclus ulei whose symptoms occur in young leaves, causing severe defoliation, reducing the production of latex and even death in susceptible plants. Is a constant threat to the plantations of the East. With the evolution of traditional breeding programs currently interspecific hybrid clones are used, which are more productive and have better resistance to pathogens. Through traditional breeding programs, there was no significant progress for resistance to leaf blight, due to selection be directed to clones with complete resistance. This pathosystem horizontal resistance (HR) and vertical resistance (VR) can act simultaneously, being complex and difficult to quantify. This study aimed to characterize the type of leaf injury pathossystem Hevea sp. x M. ulei verify clonal susceptibility to pathogens, to analyze the frequency of infection to quantify the resistance in the pathosystem Hevea sp. x M. ulei and suggest different clones of rubber and quantifies the races of M. ulei in Brazil. Monocyclic parameters (latent period and lesion diameter) are applicable in the quantification of resistance in the pathosystem Hevea sp. x M. ulei. The latency period had little variation among clones. The diameter of the lesion was wide variation between the clones, being a discriminant parameter for horizontal resistance and vertical resistance. Several races of this pathogen have been described in Brazil, based on a number of clones’ differentiators. Grounded on the reactions of 11 different clones already known in the literature: MDF 180, Fx 3844, Fx 985, Fx 4098, Fx 2261, Fx 2804, Fx 3899, IAN 6158, IAN 3087, IAN 717 and PA 31 containing the species Hevea brasiliensis, Hevea benthamiana and Hevea pauciflora, we performed an analysis grouping for the binary data referring to virulence ...

Submitted by MARCOS LEANDRO TEIXEIRA DE OLIVEIRA (marcosteixeira@ufv.br) on 2018-11-01T11:22:07Z No. of bitstreams: 1 texto completo.pdf: 993827 bytes, checksum: 8498e005f6e2efc67c76122e44845c7f (MD5) / Made available in DSpace on 2018-11-01T11:22:07Z (GMT). No. of bitstreams: 1 texto completo.pdf: 993827 bytes, checksum: 8498e005f6e2efc67c76122e44845c7f (MD5) Previous issue date: 2018-07-06 / O mal das folhas da seringueira (MDF) é uma doença destrutiva causada pelo fungo Pseudocercospora ulei que pode afetar severamente a seringueira em monocultura na América tropical. O agente causal da doença é um patógeno hemibiotrófico que apresenta crescimento lento in vitro. O isolamento de DNA de boa qualidade em quantidades adequadas não é uma tarefa fácil devido à falta de um protocolo padrão e a dificuldade de cultivar P. ulei em condições de laboratório. Embora o MDF seja conhecido por mais de um século, informações sobre a estrutura genética na região amazônica, seu centro de origem, permanecem desconhecidas. Neste trabalho foram avaliados protocolos adequados para a extração de DNA e a estrutura genética da população de P. ulei na região amazônica. Foram comparados seis protocolos de extração em relação a pureza e ao rendimento do DNA. Um total de 61 isolados foram amostrados ao longo das bacias hidrográficas Madeira, Purus e Juruá localizadas na região amazônica e genotipados para 12 locos SSR. O protocolo de extração de DNA de Doyle e Doyle foi o único que apresentou concentração consistente e melhor qualidade do DNA extraído. Houve desequilíbrio de ligação entre os alelos e diferenciação genética entre populações geograficamente distantes foi detectada. A análise de componentes principais revelou agrupamento dos isolados de acordo com os limites das bacias hidrográficas. Foram formados dois grupos, um constituído pelos indivíduos da bacia do rio Madeira e o outro composto por indivíduos dos rios Purus e Juruá. / South American leaf blight (SALB) is a destructive disease caused by the fungus Pseudocercospora ulei that can severely affect rubber tree in monoculture in tropical America. The causal agent of the disease is a hemibiotrophic pathogen, that grows slowly under in vitro conditions. Isolation of good quality DNA in proper quantities is not an easy task due to the difficulties in the handling of P. ulei under laboratory conditions and the lack of a standard protocol. Although SALB has been known for nearly a century, information on the genetic structure of P. ulei in the Amazon region, its center of origin, remains unknown. In this work, adequate protocols for DNA extraction were assessed and the population genetic structure of P. ulei in the Amazon region was determined using 12 microsatellite loci (SSR). Six extraction protocols were compared regarding yield and purity of the DNA. A total of 61 isolates were sampled along the Madeira, Purus and Juruá watersheds located in the Amazon region and SSR genotyped. The Doyle and Doyle DNA extraction protocol was the one that presented consistent and better quality extracted DNA. There was linkage disequilibrium between alleles and genetic differentiation between geographically distant populations was detected. Principal component analysis revealed clustering of the isolates according to the watershed boundaries. Two groups were formed, one comprised by individuals from the Madeira river and the other comprised of individuals from the Purus and Juruá rivers.

Menace potentielle pour l'hévéaculture mondiale, la maladie sud-américaine des feuilles de l'hévéa provoquée par le champignon Microcyclus ulei est aussi responsable du faible développement de cette culture en Amérique latine. La sélection de variétés résistantes est privilégiée pour anticiper une apparition accidentelle de la maladie dans les pays encore épargnés et pour développer la culture de l'hévéa dans les zones infestées. La principale source de résistance décrite jusqu'à présent n'est pas fonctionnelle face à des isolats très agressifs du champignon. Une autre source de résistance identifiée chez un cultivar originaire du Pérou se maintient depuis plus de trente ans en conditions de forte infestation. La cartographie génétique réalisée sur la descendance de ce cultivar en croisement révèle l'existence de deux gènes majeurs de résistance, l'un situé sur le groupe de liaison g15 et efficace contre les isolats de Guyane et l'autre sur le groupe de liaison g13 efficace vis-à-vis des isolats de l'état de Bahia. L'analyse de la diversité des populations naturelles du sud-ouest Amazonien fait apparaître une structure en trois groupes principaux recouvrant les états brésiliens de l'Acre, du Rondônia et du Mato Grosso. La population Madre de Dios au Pérou est rattachée au groupe de l'Acre. La différentiation entre les populations est expliquée principalement par l'isolement par la distance et secondairement par l'existence de bassins hydrographiques. Le déséquilibre de liaison entre marqueurs liés est plus étendu dans le groupe Acre que dans le groupe Rondônia, mais reste faible dans les deux cas. Une association avec le caractère de résistance à M. ulei est détectée avec un marqueur microsatellite situé à proximité du gène majeur de résistance du groupe de liaison g15. Une autre association est détectée dans une portion du génome où aucun locus de résistance n'était identifié jusqu'à présent. L'importance de ces résultats pour la sélection de nouveaux cultivars résistants à M. ulei est discutée.