We identified a synthetic hexapeptide that blocks Magnaporthe grisea appressorium formation, in artificial hydrophobic surface. The results suggest that peptides interfere with surface recognition. M. grisea non pathogenic pth1 mutants were complemented by N. crassa orthologous gene suggesting that the biochemical function of pth1 has not evolved specifically to play a role in appressorium development.

Made available in DSpace on 2014-06-11T19:28:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-10Bitstream added on 2014-06-13T20:18:27Z : No. of bitstreams: 1 goria_mm_me_botfca.pdf: 385248 bytes, checksum: aedd73597ba4895b9c4451da9ed36770 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O impacto da elevação da concentração de CO2 do ar sobre a brusone do arroz foi avaliado em estufas de topo aberto (OTCs) na Embrapa Meio Ambiente, Jaguariúna/SP, por dois anos. Foram realizados ensaios com cultivares de arroz em estufas com injeção de CO2, estufas sem injeção de CO2, e campo aberto, sem injeção de CO2 e sem estufa. Avaliaram-se as características de desenvolvimento das plantas, a incidência e a severidade da brusone do arroz, a caracterização química e microbiológica da rizosfera de plantas de arroz, e o teor de silício acumulado na parte aérea das plantas. No primeiro ensaio foi avaliada também a ocorrência de bactérias diazotróficas endofíticas nas raízes das plantas. A concentração média de CO2 atmosférico do tratamento em campo aberto foi 459,4 e 447,4 μmol mol-1 na safra 2007/08 e safra 2008/09, respectivamente. Por outro lado, as concentrações médias de CO2 foram 490,1 e 480,4 μmol mol-1 para o tratamento em estufa sem injeção de CO2 e 531,9 e 608,6 μmol mol-1 para o tratamento com estufa com injeção de CO2 na safra 2007/08 e safra 2008/09, respectivamente. Nos resultados obtidos, verificou-se o aumento significativo na altura de plantas das cultivares Agulha Precoce e Shao Tiao Tsao, na safra 2008/09, no tratamento com injeção do gás. Nas cultivares Caloro e Agulha Precoce, nas safras 2007/08 e 2008/09, respectivamente, o ambiente com a concentração de CO2 do ar elevada aumentou a severidade da brusone nas folhas das plantas. A análise química e microbiológica da rizosfera não apresentou diferenças entre os ambientes com e sem injeção do gás. A massa seca da parte aérea das plantas, a massa das panículas e a massa dos grãos não sofreram alteração devido à elevação do CO2 atmosférico. O aumento da concentração de CO2 do ar pode alterar o crescimento das plantas e a severidade da brusone, acarretando... / The impact of elevated atmospheric CO2 concentration on rice blast disease was evaluated in open-top chambers (OTCs) in Embrapa Meio Ambiente, Jaguariúna /SP, for two years. Trials were developed under OTCs with injection of CO2, OTCs without injection of CO2, and field, without injection of CO2 and without OTC. The characteristics of rice plants growth, the incidence and severity of rice blast, chemical and microbial characterization of rizosphere of rice plants, and leaf silicon content were evaluated. On the first trial it was also examined the occurrence of diazotrophic bacteria in rice plant´s root. Actual season-long average CO2 concentration in field without injection of CO2 and without OTC were 459,4 e 447,4 μmol mol-1 in 2007/08 and 2008/09, respectively. For the other hand, actual season-long average CO2 concentration were 490,1 and 480,4 μmol mol-1 in OTCs without injection of CO2 and 531,9 and 608,6 μmol mol-1 for the treatment under OTCs with CO2 enrichment in 2007/08 and 2008/09, respectively. As results, Agulha Precoce and Shao Tiao Tsao, in 2008/09, it was found a significant increase on rice growth, on treatment with CO2 injection. On Caloro and Agulha Precoce, in 2007/08 and 2008/09, respectively, the atmosphere with elevated CO2 increased the severity of leaf blast. No significant difference was detected on rizhosphere chemical and microbiological analysis in the atmosphere with injection of the gas. CO2 enrichment resulted in a non-significant increase in grain weight, plant dry weight and the panicles weight. The increase of CO2 atmospheric concentration may alter the rice plant´s growth and the severity of rice blast, and consequently, the strategies of disease management.

Doctor of Philosophy / Department of Plant Pathology / Scot H. Hulbert / The rice cultivar Oryzica Llanos 5 (OL5) possesses a high level of resistance to the fungus Magnaporthe grisea. The number and chromosomal location of quantitative trait loci (QTL) conferring resistance against eight isolates of the blast fungus were tested in two different populations of recombinant inbred lines from the cross Fanny x OL5. Twenty one QTL were detected and associated with the resistance traits, disease leaf area and lesion type, on 9 rice chromosomes. Eight of these 21 resistance loci had significant resistance effects in both experiments, while the others had effects that were only statistically significant in one experiment. Most, but not all, of the QTL occurred in the same genomic regions as either genes with major race-specific effects or other resistance QTL that had been described in previous experiments. Most of the QTL appeared to be race-specific in their effects but it is possible some of the QTL with smaller effects were nonspecific. One of the blast isolates used was FL440, which causes limited disease on OL5 and was probably virulent on most or all of the major genes from OL5. Three QTL affected resistance to FL440 in both experiments, one of which mapped to a region on chromosome 9 where no blast resistance genes have yet been mapped. An advanced backcross strategy with marker-assisted selection for OL5 alleles in QTL regions was used to generate five BC2F3 populations carrying five different target regions associated with partial resistance to rice blast disease. Three of five of these populations were analyzed for segregation for resistance to the M. grisea isolate FL440. One QTL designated qrbr-11.3 near the bottom of rice chromosome 11 was found to be significantly associated with partial blast resistance in 120 lines of a BC2F3 population (P< 0.01). This QTL accounted for 12.4% and 8.0% of the phenotypic variation in diseased leaf area and lesion type observed under greenhouse inoculation. Examination of the genomic sequence at the qrbr-11.3 locus showed that twenty-nine candidate resistance genes are present at that locus (~1.8 Mb), twenty-seven of which are predicted NBS-LRR genes. Ultimately, the information from this study can be integrated into the development of improved lines with OL5-derived QTL for resistance.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice (Oryza sativa L.) production worldwide is constrained by rice blast disease caused by the ascomycetous fungus Magnaporthe oryzae. Rice blast has become a model system for the study of fungal plant diseases based on its global relevance to agriculture and on our ability to apply molecular genetic and genomic analyses to both the pathogen and the plant. We have applied molecular and cellular analyses to understand critical processes in the M. oryzae disease cycle. The dark melanin pigment produced by the fungus is critical for the function of its specialized appressorial cell, which punches the leaf surface by generating the highest pressure known in any biological system, estimated at 80 times the atmospheric pressure. Without melanin, the fungus can neither generate this pressure nor puncture the plant surface and disease does not occur. M. oryzae genome sequencing identified a cluster of melanin biosynthesis genes that included an attractive candidate for the transcription factor that regulates melanin biosynthesis in appressoria. We report the structural and functional characterization of this putative transcription factor, although its role remains elusive. Host cellular responses after appressorial penetration are equally important in determining if disease will occur. We have characterized the cellular response of one rice variety to a compatible fungal strain (causes disease), an incompatible strain (fails to cause disease due to specific triggering of rice defenses) and a non-host strain (causes disease in barley but not in rice). Distinctive fungal and rice cellular responses correlated with the outcome of each particular pathogen-strain rice interaction. We report contrasting responses in two rice leaf sheath assays that are amenable to live cell microscopy, as well as a novel cellular response of crystalline aggregations deposited inside the host cell under appressoria on the leaf surface. Our studies have important implications for future analyses of pathogenicity and host specificity in rice blast disease.

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Este estudo descreve a diversidade de actinobactérias isoladas da esponja marinha Aplysina fulva e o potencial destes microorganismos como produtores de metabólitos bioativos com propriedades fungicidas e herbicidas. Actinobactérias são prolíficas produtoras de compostos farmacologicamente importantes, pois cerca de 70% dos antibióticos naturalmente derivados que estão atualmente em uso clínico são produzidos por estes microorganismos. Entretanto este valor é ainda inexpressivo na indústria agrícola. Agroquímicos sintéticos ainda são dominantes no mercado apesar de estarem menos efetivos contra plantas daninhas e patógenos cada vez mais resistentes. Neste trabalho, um total de 21 actinobactérias foram isoladas com a utilização de meios seletivos. Análises filogenéticas baseadas no sequenciamento parcial do gene que codifica para o rRNA 16S mostrou que estes microorganismos pertencem a oito gêneros do filo Actinobacteria: Kocuria; Citricoccus; Terrabacter; Gordonia; Agrococcus; Tsukamurella; Brevibacterium e Streptomyces. Os extratos de todos os isolados foram testados para verificar a produção de metabólitos secundários com propriedades fungicidas contra os fungos fitopagênicos de importância agrícola: Pythium aphanidermatum; Phytophthora capsici e Magnaporthe grisea. O extrato bruto de 43% dos isolados mostrou atividade fungicida para ao menos um dos patógenos. O perfil químico do extrato dos isolados com bioatividade positiva foram similares mesmo entre gêneros diferentes. Os metabólitos do Streptomyces ASPSP 103 foram mais eficientes devido à forte inibição contra todos os patógenos testados. Portanto este isolado foi selecionado e testado para atividade herbicida por meio de screening que teve início com testes de atividade algicida contra a microalga Selenastrum capricornutum. Acreditamos que actinobactérias associadas a esponjas marinhas desempenham um papel de defesa química contra microalgas que possam obstruir os porócitos asfixiando o animal, e que estes compostos algicidas possivelmente tenham ação herbicida. Foi verificada atividade do extrato bruto do Streptomyces ASPSP 103 contra S. capricornutum, e a atividade herbicida pré-emergência com um efeito fraco em Lactuca sativa (dicotiledônea) e uma forte inibição em Agrostis stolonifera (monocotiledônea). A purificação do extrato bruto para isolamento do composto bioativo foi guiado por bioensaio contra Pythium aphanidermatum, um oomiceto de rápido crescimento e sensível aos metabólitos de ASPSP 103 previamente testados. Foi identificado o composto da classe butenolida com atividade herbicida préemergência contra Agrostis stolonifera (IC50 33.43 &mu;g/mL). Este é o primeiro relato da atividade de butenolida para atividade herbicida. Estudos aprofundados em taxonomia mostraram que as características filogenéticas, morfológicas e químicas do isolado ASPSP 103 são consistentes com o gênero Streptomyces. Portanto devido algumas diferenças em parâmetros taxonômicos, ASPSP 103T foi proposto como linhagem tipo para uma nova espécie de Streptomyces, para qual o nome Streptomyces atlanticus sp. nov. foi sugerido. Estes resultados enfatizam o potencial de Streptomyces marinhos para produzir compostos bioativos com potencial de aplicação em agrobiotecnologia. / Actinobacteria are producers of important pharmacological compounds. About 70% of natural antibiotics are derived from these microorganisms. However, the use of natural compounds are still limited in the agricultural industry, even considering that synthetic pesticides are less effective against pathogens and weed plants. This study describes the diversity of actinobacteria associated with the marine sponge Aplysina fulva and their potential as producers of bioactive compounds with fungicidal and herbicidal properties. In this study, a total of 21 actinomycetes were isolated with the use of selective media. Phylogenetic analyzes based on partial sequencing of the gene encoding for 16S rRNA showed that these microorganisms belong to eight Actinobacteria genera, including Kocuria, Citricoccus, Terrabacter, Gordonia, Agrococcus, Tsukamurella, Brevibacterium and Streptomyces. The extracts of all isolates were tested for the production of secondary metabolites with fungicidal properties against the following phytopathogenic fungi: of Pythium aphanidermatum, Phytophthora capsici and Magnaporthe grisea. The crude extract of 43% of the isolates showed fungicidal activity for at least one of the pathogens. The chemical profiles of the actinobacteria extracts with positive bioactivity were similar even among different genus. The metabolites of Streptomyces ASPSP 103 were more efficient because of the strong inhibition against all tested pathogens. So, the isolate ASPSP 103 was selected and tested for herbicide activity through screening for algaecide activity towards microalgae Selenastrum capricornutum. We believe that actinobacteria associated with marine sponges play a role in chemical defense against algae that can obstruct the pores, choking the animal. These algaecides compounds possibly have herbicide action. Activity of the Streptomyces ASPSP 103 crude extract against S. capricornutum was observed. In addition, it was observed a weak pre-emergence herbicide activity on Lactuca sativa (dicot) and a strong inhibition in Agrostis stolonifera (monocot). The purification of the crude extract to isolate the bioactive compound was guided by bioassay against Pythium aphanidermatum, a fast growing oomycete and sensitive to metabolites from ASPSP 103 previously tested. The butenolide compound was identified with pre-emergence herbicidal activity against Agrostis stolonifera (IC50 33.43 &mu;g/mL). This is the first report of butenolide activity with herbicide activity. Taxonomy studies showed that the phylogenetic, morphological and chemical characteristics of the isolated ASPSP 103 are consistent with the Streptomyces genus. Then, considering some differences in taxonomic parameters, ASPSP 103T was proposed as line type for a new species of Streptomyces, for which the name Streptomyces atlanticus sp. nov. was suggested. These results emphasize the potential of marine Streptomyces to produce bioactive compounds with potential biotechnological application in agricultural industry.

Este estudo descreve a diversidade de actinobactérias isoladas da esponja marinha Aplysina fulva e o potencial destes microorganismos como produtores de metabólitos bioativos com propriedades fungicidas e herbicidas. Actinobactérias são prolíficas produtoras de compostos farmacologicamente importantes, pois cerca de 70% dos antibióticos naturalmente derivados que estão atualmente em uso clínico são produzidos por estes microorganismos. Entretanto este valor é ainda inexpressivo na indústria agrícola. Agroquímicos sintéticos ainda são dominantes no mercado apesar de estarem menos efetivos contra plantas daninhas e patógenos cada vez mais resistentes. Neste trabalho, um total de 21 actinobactérias foram isoladas com a utilização de meios seletivos. Análises filogenéticas baseadas no sequenciamento parcial do gene que codifica para o rRNA 16S mostrou que estes microorganismos pertencem a oito gêneros do filo Actinobacteria: Kocuria; Citricoccus; Terrabacter; Gordonia; Agrococcus; Tsukamurella; Brevibacterium e Streptomyces. Os extratos de todos os isolados foram testados para verificar a produção de metabólitos secundários com propriedades fungicidas contra os fungos fitopagênicos de importância agrícola: Pythium aphanidermatum; Phytophthora capsici e Magnaporthe grisea. O extrato bruto de 43% dos isolados mostrou atividade fungicida para ao menos um dos patógenos. O perfil químico do extrato dos isolados com bioatividade positiva foram similares mesmo entre gêneros diferentes. Os metabólitos do Streptomyces ASPSP 103 foram mais eficientes devido à forte inibição contra todos os patógenos testados. Portanto este isolado foi selecionado e testado para atividade herbicida por meio de screening que teve início com testes de atividade algicida contra a microalga Selenastrum capricornutum. Acreditamos que actinobactérias associadas a esponjas marinhas desempenham um papel de defesa química contra microalgas que possam obstruir os porócitos asfixiando o animal, e que estes compostos algicidas possivelmente tenham ação herbicida. Foi verificada atividade do extrato bruto do Streptomyces ASPSP 103 contra S. capricornutum, e a atividade herbicida pré-emergência com um efeito fraco em Lactuca sativa (dicotiledônea) e uma forte inibição em Agrostis stolonifera (monocotiledônea). A purificação do extrato bruto para isolamento do composto bioativo foi guiado por bioensaio contra Pythium aphanidermatum, um oomiceto de rápido crescimento e sensível aos metabólitos de ASPSP 103 previamente testados. Foi identificado o composto da classe butenolida com atividade herbicida préemergência contra Agrostis stolonifera (IC50 33.43 &mu;g/mL). Este é o primeiro relato da atividade de butenolida para atividade herbicida. Estudos aprofundados em taxonomia mostraram que as características filogenéticas, morfológicas e químicas do isolado ASPSP 103 são consistentes com o gênero Streptomyces. Portanto devido algumas diferenças em parâmetros taxonômicos, ASPSP 103T foi proposto como linhagem tipo para uma nova espécie de Streptomyces, para qual o nome Streptomyces atlanticus sp. nov. foi sugerido. Estes resultados enfatizam o potencial de Streptomyces marinhos para produzir compostos bioativos com potencial de aplicação em agrobiotecnologia. / Actinobacteria are producers of important pharmacological compounds. About 70% of natural antibiotics are derived from these microorganisms. However, the use of natural compounds are still limited in the agricultural industry, even considering that synthetic pesticides are less effective against pathogens and weed plants. This study describes the diversity of actinobacteria associated with the marine sponge Aplysina fulva and their potential as producers of bioactive compounds with fungicidal and herbicidal properties. In this study, a total of 21 actinomycetes were isolated with the use of selective media. Phylogenetic analyzes based on partial sequencing of the gene encoding for 16S rRNA showed that these microorganisms belong to eight Actinobacteria genera, including Kocuria, Citricoccus, Terrabacter, Gordonia, Agrococcus, Tsukamurella, Brevibacterium and Streptomyces. The extracts of all isolates were tested for the production of secondary metabolites with fungicidal properties against the following phytopathogenic fungi: of Pythium aphanidermatum, Phytophthora capsici and Magnaporthe grisea. The crude extract of 43% of the isolates showed fungicidal activity for at least one of the pathogens. The chemical profiles of the actinobacteria extracts with positive bioactivity were similar even among different genus. The metabolites of Streptomyces ASPSP 103 were more efficient because of the strong inhibition against all tested pathogens. So, the isolate ASPSP 103 was selected and tested for herbicide activity through screening for algaecide activity towards microalgae Selenastrum capricornutum. We believe that actinobacteria associated with marine sponges play a role in chemical defense against algae that can obstruct the pores, choking the animal. These algaecides compounds possibly have herbicide action. Activity of the Streptomyces ASPSP 103 crude extract against S. capricornutum was observed. In addition, it was observed a weak pre-emergence herbicide activity on Lactuca sativa (dicot) and a strong inhibition in Agrostis stolonifera (monocot). The purification of the crude extract to isolate the bioactive compound was guided by bioassay against Pythium aphanidermatum, a fast growing oomycete and sensitive to metabolites from ASPSP 103 previously tested. The butenolide compound was identified with pre-emergence herbicidal activity against Agrostis stolonifera (IC50 33.43 &mu;g/mL). This is the first report of butenolide activity with herbicide activity. Taxonomy studies showed that the phylogenetic, morphological and chemical characteristics of the isolated ASPSP 103 are consistent with the Streptomyces genus. Then, considering some differences in taxonomic parameters, ASPSP 103T was proposed as line type for a new species of Streptomyces, for which the name Streptomyces atlanticus sp. nov. was suggested. These results emphasize the potential of marine Streptomyces to produce bioactive compounds with potential biotechnological application in agricultural industry.

La première partie du travail présenté ici se concentre sur l’optimisation d’une cathode à oxygène. Tout d’abord, l’utilisation d’une nouvelle enzyme (la BOD de Magnaporthe oryzae) permet de multiplier le courant de réduction de l’oxygène en eau jusqu’à neuf fois. Ensuite la synthèse d’un polymère rédox adapté a permis d’améliorer le coefficient de diffusion des électrons dans l’hydrogel résultant en l’augmentation de la densité de courant générée. Enfin nous sommes passés d’uneélectrode de carbone en 2 dimensions à une fibre d’or poreuse tridimensionnelle. Après modification de cette fibre avec l’hydrogel rédox à base de BOD de M. oryzaenous avons évalué sa biocompatibilité : in vitro les tests ont montré l’absence totale de cytotoxicité et seule une très faible réponse inflammatoire ; in vivo aucune infection ne s’est déclarée pendant les 8 semaines d’implantation dans les souris etla formation d’une capsule fibrotique autour de l’électrode traduit sa bonne intégration dans les tissus de l’animal. La seconde partie concerne la biopile dans son intégralité, construite à partir de la cathode optimisée et d’une anode adaptée à base de GDH. Elle permet de générer jusqu’à 240 μW.cm-2 dans du tampon Pipes/CaCl2 à 5mM de glucose. La biopile a ensuite été testée dans du sang humain total. Un maximum de 129 μW.cm-2 a été obtenu dans un échantillon avec une glycémie de 8,2 mM sous air. De plus nous avons constaté que la densité de puissance délivrée augmente proportionnellement avec la glycémie des différents échantillons de sang testés, faisant de la biopile à la fois une source d’électricité et un biocapteur à glucose ce qui n’avait jamais été démontré auparavant. / The first part of the work presented here focuses on the optimization of an oxygen cathode. First, the use of a new enzyme (BOD from Magnaporthe oryzae) permit to increase the current of reduction of oxygen into water by a factor nine. Then the synthesis of a suitable redox polymer greatly improved the diffusion coefficient of electrons in the hydrogel, resulting in an increase of the current density. Finally we switched from a two-dimensional carbon electrode to a three-dimensional porous gold fiber. After modification of the fiber with the redox hydrogel based on BOD from M. oryzae, we assessed its biocompatibility: in vitro the tests showed the total absence of cytotoxicity and only a very low inflammatory response; in vivo noinfection appeared during the 8 weeks of implantation in mice and the formation of afibrotic capsule around the device reflects its successful integration into the animal tissues.The second part concerns the full biofuel cell, elaborated from the optimized cathode and an adapted GDH-based anode. It could generate up to 240 μW.cm-2 at 5mMglucose in Pipes/CaCl2 buffer. The biofuel cell was then tested in whole human blood. A maximum of 129 μW.cm-2 was obtained in a sample with 8,2 mM glycaemiaunder air. In addition we observed that the delivered power density increased proportionally with the glycaemia of the different blood samples tested, making the biofuel cell both a power source and a glucose biosensor at the same time which had never been shown before.