Le cas de l'interaction modèle entre le riz et le champignon pathogène Magnaporthe oryzae a été étudié afin de mieux comprendre les effets d'une pratique culturale, la fertilisation azotée, sur la résistance. Ce travail a permis de mettre au point un système simplifié permettant d'étudier au laboratoire l'augmentation de la sensibilité induite par l'apport azoté, un phénomène dénommé NIS (Nitrogen-Induced Susceptibility), d'analyser les effets de l'azote sur la croissance de M. oryzae et son pouvoir pathogène, d'explorer les effets de l'azote au travers de la diversité du riz et des différents types de résistance (complètes et partielles), et d'identifier par cartographie des zones du génome du riz importantes pour la NIS. L'expression des gènes de défenses ne semble pas altérée en cas de régime riche en azote. Au contraire, le suivi cytologique de l'infection par M. oryzae a mis évidence que la pénétration du champignon n'était pas modifiée par la présence d'azote alors que sa croissance dans la plante est accrue. Le niveau de maladie a pu être augmenté par apport d'acides aminés 24h après le début de l'infection, suggérant qu'une relation trophique est probablement à la base de la NIS. Une analyse de l'effet de l'azote sur la sensibilité à la pyriculariose à travers la diversité du riz nous a permis de mieux caractériser la diversité du phénomène de NIS. La NIS est très polymorphe mais n'est pas corrélée à la différence liée aux sous-groupes indica et japonica du riz. Par ailleurs, un fort apport en azote a réduit la résistance déclenchée par le gène de résistance Pi1, suggérant que la robustesse de ces gènes peut être affectée par la NIS Enfin, un locus qui contrôle la sensibilité du riz à la pyriculariose sous un régime riche en azote a été identifié sur le chromosome 1. Plusieurs éléments suggèrent un lien possible entre l'efficacité de l'utilisation de l'azote (NUE) et la NIS. / Nitrogen-Induced Susceptibility (NIS) to plant diseases is a widespread phenomenon. In this work, we set an experimental system in which nitrogen supply strongly affects rice blast susceptibility whereas it is only slightly perturbing plant growth. We show that fungal growth is affected before and after penetration in the plant but that the final penetration rate is not affected; thus a change in penetration is not responsible for increased susceptibility. Differences in total nitrogen amount and defense gene expression before infection are unlikely to be responsible for the observed increase in penetration. After penetration, small changes in plant growth, but not modifications of the transcriptional regulation of defense genes, could be responsible for nitrogen-induced susceptibility. On the other hand, the fungus seems to perceive small differences in nitrogen amount after penetration and this may explain enhanced growth under high nitrogen regime. Indeed, exogenous treatment with some free amino acids after inoculation mimicked Nitrogen-Induced Susceptibility, further arguing that this phenomenon is mostly due to a trophic relation between the plant and the fungus. We also used our experimental system that does not strongly affect plant development to address the question of NIS polymorphism across rice diversity. We show that the capacity of rice to display NIS is highly polymorphic and does not correlate with difference related to indica/japonica sub-groups. We also tested the robustness of three different major resistance genes under high nitrogen. Nitrogen partially breaks down resistance triggered by the Pi1 gene. Cytological examination indicates that penetration rate is not affected by high nitrogen whereas growth of the fungus is increased inside the plant. Using the CSSL mapping population between Nipponbare and Kasalath, we identified a Kasalath locus on chromosome 1, called NIS1, which dominantly increases susceptibility under high nitrogen. We discuss the possible relationships between Nitrogen Use Efficiency (NUE), disease resistance regulation and NIS. This work provides evidences that robust forms of partial resistance exist across diversity and can be genetically mapped. This work also suggests that under certain environmental circumstances, complete resistance may breakdown, irrelevantly of the capacity of the fungus to mutate. These aspects should be considered while breeding for robust forms of resistance to blast disease.

Orientador: Anete Pereira de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T01:20:36Z (GMT). No. of bitstreams: 1 Schneider_DilaineRoseSilva_D.pdf: 11263694 bytes, checksum: 808a3a965f6f47fb9c2108a4c971b122 (MD5) Previous issue date: 2010 / Resumo: A brusone do arroz (rice blast disease) causada pelo ascomiceto fitopatógeno Magnaporthe grisea continua a ter um enorme impacto nas culturas de arroz (Oryza sativa) no Brasil e no mundo. PWL2, uma proteína efetora, é um conhecido produto de um gene AVR (avirulência). O gene PWL2 impede que o fungo infecte weeping lovegrass (Eragrostis curvula). Neste trabalho nós identificamos em uma linhagem de M. grisea um gene que produz uma proteína diferente de PWL2, denominada PWL2D. A seqüência do gene PWL2D tem duas bases que diferem do gene PWL2, as quais produzem alterações nos resíduos de 90 e 142 da proteína. A alteração do resíduo 90 (de D90 para N90) é fundamental para a avirulência. Neste trabalho foram efetuadas a clonagem do gene PWL2D no vetor pET32-Xa/LIC, a expressão em Escherichia coli e a avaliação da estrutura de PWL2D por técnicas espectroscópicas. A proteína PWL2D fusionada à cauda TRX é propensa a agregação, e sua solubilidade é melhorada quando super-expressa sem o seu peptídeo-sinal original. Os resultados estruturais obtidos indicam que a proteína PWL2D possivelmente é intrinsecamente desordenada. Foi elaborado um modelo para a resistência/susceptibilidade do hospedeiro à M. grisea considerando a atuação de PWL2D como uma proteína intrinsecamente desordenada. Os resultados obtidos deverão facilitar a análise estrutural de PWL2D e podem contribuir para a compreensão da função do gene nas interações fungo / planta. Oito diferentes genes de M. grisea, além de PWL2D, foram também estudados neste trabalho. Dentre estes, destacam-se o gene que produz a xilanase XYL5 e seu domínio catalítico, o gene que codifica a chaperona ABC1 e seus dois domínios funcionais, e o gene que codifica a trealase PTH9, sendo todos estes relacionados à patogenicidade do fungo M. grisea. A xilanase XYL5 (EC 3.2.1.8) e seu domínio catalítico conservado (XYL5/DOM) foram fusionados à Maltose Binding Protein (MBP) ou à tiorredoxina (TRX) e expressas em E. coli. A produção de proteína solúvel e ativa foi influenciada pelo tipo de fusão. Os extratos solúveis contendo as proteínas de fusão MBP-XYL5 e MBP-XYL5/DOM apresentaram atividade xilanolítica em relação ao controle. Entretanto, durante o processo de purificação, a atividade foi perdida. Assim, obteve-se pela primeira vez o gene de patogenicidade XYL5 de M. grisea expresso com sucesso em E. coli e sua atividade enzimática xilanolítica foi demonstrada. Não foi possível expressar a chaperona ABC1 na forma solúvel nos sistemas de expressão utilizados, e a sequência gênica referente à trealase PTH9 - por mostrar a presença de introns após o seqüenciamento do gene amplificado na linhagem de M. grisea em estudo, mostrou-se inadequado para a sua expressão protéica no sistema de expressão procariótico utilizado durante a realização deste trabalho / Abstract: The rice blast disease caused by the ascomycete phytopathogen Magnaporthe grisea continues to have a huge impact on crops of rice (Oryza sativa) in Brazil and worldwide. PWL2, an effector protein, is a product of an AVR (avirulence) gene . The gene PWL2 prevents fungus from infecting weeping lovegrass (Eragrostis curvula). In this work we identified in a strain of M. grisea a gene that produces a protein different from PWL2, called PWL2D. The gene sequence PWL2D has two bases that differ from PWL2 gene, which produce changes in residues 90 and 142 of the protein. The change of residue 90 (from D90 to N90) is critical to avirulence. In this work it was realized the cloning of the gene in the vector PWL2D pET32-Xa/LIC, the expression in Escherichia coli and the assessment of PWL2D structure by spectroscopic techniques. The protein fused to the tag PWL2D TRX is prone to aggregation, and its solubility is improved when overexpressed without its original signal peptide. The structural results obtained indicate that possibly the protein PWL2D is intrinsically disordered. A model for the resistance/susceptibility of the host to M. grisea was developed considering the performance of PWL2D as an intrinsically disordered protein. The results should facilitate structural analysis of PWL2D and may contribute to the understanding of gene function in the interactions fungus/plant. Eight different genes of M. grisea, besides PWL2D, were also studied in this work. Among these, stands out the gene that produces xylanase XYL5 and its catalytic domain, the gene that codify the chaperone ABC1 and its two functional domains, and the gene that codify the trehalase PTH9, all them being related to the pathogenicity of the fungus M. grisea. The xylanase XYL5 (EC 3.2.1.8) and its retained catalytic domain (XYL5/DOM) were fused to the solubilizing proteins (MBP) or thioredoxin (TRX) and expressed into E. coli. The production of soluble and active protein was influenced by the type of fusion. The soluble extracts containing the fusion proteins MBP- XYL5 and MBP-XYL5/DOM showed xylanolytic activity compared to the control. However, during the purification process, the activity was lost. Thus, we obtained for the first time the gene pathogenicity XYL5 M. grisea expressed successfully in E. coli and its enzymatic xylanolytic activity was demonstrated. It was not possible to express the chaperone ABC1 in soluble form in the expression systems used, and the gene sequence related to trehalase PTH9 - by showing the presence of introns after the sequencing of the gene amplified in the strain of M. grisea under study, rendered inadequate for its protein expression in the prokaryotic system used during the realization of this work / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Magnaporthe grisea is a notorious pathogenic fungus that causes rice blast disease worldwide. Proteins secreted by the fungus are likely candidates for being effectors that are potentially recognized by determinants of resistance or susceptibility in host plants. However, knowledge of the role of secreted proteins of M. grisea is still limited. In this study, I identified 29 proteins that were secreted into culture filtrates from M. grisea strains expressing candidate proteins. I confirmed secretion of these proteins and tested them for elicitor activity on plants. Among them, I studied two groups: cell wall degrading enzymes (CWDEs) and small cysteine-rich proteins. Cysteine-rich proteins have been shown in other systems to function as elicitors. Initially, I expressed and purified proteins in M. grisea to obtain proteins by a homologous expression system. Although this was effective for a number of proteins, the need for greater amounts of protein led me to express several proteins in the Pichia pastoris system. Several candidate proteins were purified and found to induce symptoms on rice and maize. Hypothetical proteins MG10424.4 and MG09998.4 were both found to have elicitor activity. Lipase MG07016.4 did not induce response of plants and we concluded that the lipase activity of MG07016.4 does not function as an elicitor. I also purified a small cysteine-rich protein, which belongs to the group of cluster 180 proteins in M. grisea, MG10732.4 from P. pastoris. It is able to cause yellowing symptoms and hydrogen peroxide production in plants and it might contain elicitor activity.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-20T18:33:23Z No. of bitstreams: 1 texto completo.pdf: 20005358 bytes, checksum: 1121af69167ca6262a2059e1892c3134 (MD5) / Made available in DSpace on 2017-04-20T18:33:23Z (GMT). No. of bitstreams: 1 texto completo.pdf: 20005358 bytes, checksum: 1121af69167ca6262a2059e1892c3134 (MD5) Previous issue date: 2003-12-12 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Em Magnaporthe grisea, agente causal da brusone, mutagênese insercional mediada por transformação tem constituído estratégia para a identificação de genes essenciais para a patogenicidade em arroz. A técnica REMI, integração mediada por enzima de restrição, merece destaque em virtude da eficiência de transformação e da predominância de integrações simples. Visando a implantação de programa de mutagênese insercional em M. grisea, os objetivos deste trabalho incluíram: (1) adequar as condições para a obtenção e regeneração de protoplastos do ascomiceto, (2) estabelecer sistema de transformação REMI em M. grisea, avaliando o potencial dos protoplastos e do vetor pAN7-1 e (3) selecionar e caracterizar mutantes com patogenicidade alterada em plantas de arroz. Produção eficiente de protoplastos foi alcançada com o uso simultâneo de 10 mg de Lysing Enzymes e 10 mg de Cellulase Onozuka R10 em 3 mL de MgSO 4 a 1,2 M / NaH 2 PO 4 a 0,01 M (pH = 5,8). Protoplastos de M. grisea I-22 liberados com 3 horas de hidrólise enzimática apresentaram maior capacidade de regeneração da parede celular. Quando expostos ao vetor pAN7-1, os protoplastos foram prontamente transformados para a resistência à higromicina. Quando pAN7-1- HindIII foi usado para transformar I-22 na presença de HindIII, a freqüência de transformantes foi 1,1 a 8,1 vezes superior ao tratamento sem a adição da endonuclease de restrição. No geral, a melhor concentração de HindIII foi 5 unidades/reação de transformação. A partir de testes de patogenicidade envolvendo 125 transformantes, principalmente gerados por REMI, foi possível selecionar cinco mutantes com alterações consistentes na patogênese. Dois desses mutantes, T108 e T93, causaram poucas lesões em folhas de arroz, enquanto o mutante T251 não foi patogênico. A alteração na patogenicidade de T108 foi acompanhada pela menor capacidade de desenvolvimento in vitro. Quando inoculado em plantas de arroz, o mutante T41 apresentou agressividade reduzida, caracterizada por lesões arredondadas de tamanho limitado. Por sua vez, o período de incubação para o mutante T72 foi mais longo do que o do isolado selvagem. Além disso, atrasos consideráveis na germinação de conídios e na formação de apressórios foram detectados em T72. Os mutantes T93 e T251 apresentaram fenótipos semelhantes quando em cultura, caracterizados pela pigmentação marrom. Análises Southern blots de quatro mutantes indicaram que em 50 % dos casos, T108 e T251, apenas uma cópia de pAN7-1 se integrou em um único sítio no genoma. No mutante T251 ocorreu evento REMI propriamente dito. Os mutantes T41 e T93 apresentaram padrões de integração mais complexos. / Transformation mediated-insertional mutagenesis of phytopathogenic fungi is an important tool to identify genes involved in pathogenicity. An improved version of this method is the restriction enzyme mediated integration, or REMI. We chose REMI to begin an insertional mutagenesis project in M. grisea. In this work, were reported the: (1) protoplasts production and regeneration of M. grisea, (2) transformation of protoplasts with pAN7-1 mediated by restriction enzyme and (3) identification and characterization of five transformants with pathogenicity defects in rice, at the phenotypic and molecular levels. The highest protoplasts production was obtained with Lysing Enzymes plus Cellulase Onozuka R-10 and the osmotic buffer MgSO 4 at 1.2 M / NaH 2 PO 4 at 0.01 M (pH = 5.8). The highest regeneration frequency was obtained with protoplasts produced after 3 hours of incubation. The I-22 protoplasts were readily transformed for hygromycin resistance. When pAN7-1-HindIII was used to transform fungal protoplasts in the presence of the HindIII, the transformation efficiency was increased 1.1 to 8.1-fold. The optimal HindIII concentration for enhanced transformation corresponded to 5 unit/transformation mix. Out of 125 transformants screened for the ability to infect rice plants, five showed changes in pathogenicity. The T108 and T93 mutants caused few lesions in rice leaves, while the T251 mutant was non-pathogenic. The alteration in pathogenicity of T108 was accompanied by reduced development in culture. The T41 mutant caused small and limited round lesions. The incubation period of the T72 mutant was longer than of wild type. Furthermore, late germination and appresorium formation was detected in the T72 mutant. The T93 and T251 mutants had similar phenotypes, characterized by a brown-pigmented colony. Four mutants (T41, T93, T108 and T251) were examined by Southern blots. The T108 and T251 mutants contained one copy of the vector integrated at a single site in the genome. REMI event occurred in the T251 mutant. More complex integration events were observed in the T41 and T93 mutants. / Tese importada do Alexandria

L'intégrité de paroi cellulaire est cruciale pour la survie du champignon pendant son cycle de développement ou en réaction à des conditions de stress. Chez la levure, les voies de signalisation de MAP kinase, Slt2, et calcineurin, régulent la réparation de paroi cellulaire. MPS1, l'orthologue de SLT2, chez Magnaporthe grisea est essentiel pour la réparation de la paroi cellulaire et pour la pénétration de l'appressorium (Xu, et al., 1998). Chez la levure, Slt2 active les facteurs de transcription Rlm1, Swi4 et Swi6, alors que le calcineurin active Crz1. Par ailleurs, PaIdc1 a un rôle dans la localisation nucléaire de PaMpk1 (orthologue de Slt2 et Mps1). (Corinne Jamet-Vierny, et al., 2007). MgIDC1, Le gène orthologue de PaIDC1 a été, de même, identifié chez M. grisea. ScAGS1 (α-glucan synthase), est un composant principal de la paroi principal des champignons. CaCAS5, est un régulateur transcriptionel, régule plusieurs gènes de la paroi cellulaire et ScKnr4 est impliqué dans la régulation de l'activité de 1,3--glucan synthase et la formation de la paroi cellulaire Ces gènes ont été identifiés chez M. grisea et des mutants de délétion ont été construits par le remplacement de gène chez M. grisea. Les mutants Guy11ΔKU80Δmps1 et Guy11ΔKU80Δidc1 montrent une forte réduction de mycélium aérien. Guy11ΔKU80Δmps1, Guy11ΔKU80Δrlm1 et Guy11ΔKU80Δswi4 ont une très forte réduction de sporulation. Guy11ΔKU80Δmps1, Guy11ΔKU80Δrlm1 et Guy11ΔKU80Δcrz1 ont une forte réduction de pouvoir pathogène. De plus, les mutants Guy11ΔKU80Δmps1 et Guy11ΔKU80Δswi4 sont inhibé, de 100% en présence d'un mélange de l'Aculéacine et Nikkomycine à faible dose (DI20). Les résultats montrent que chez M. grisea, il existe deux voies impliquées dans l'intégrité de la paroi cellulaire. La voie MAPK MPS1, qui régule les facteurs de transcription Rlm1, Swi4, Swi6 et la voie de Calcineurine, qui régule Crz1. D'après les analyses, SWI4 est impliquée dans la régulation des gènes de glucan synthases et chitine synthase, quand RLM1 n'est impliqué que dans la régulation des gènes de chitine synthase. Dans la voie de Calcineurine, CRZ1 contrôle les gènes de chitine synthase aussi. Ces études suggèrent que les facteurs de transcription qui sont régulés par Mps1 et Calcineurine sont spécialisés dans la régulation des gènes de cible spécifiques à l'intégrité et la réparation de la paroi cellulaire. / The cell wall integrity is crucial for the survival of fungi during its development or in reaction in stress conditions. In yeast, the MAP kinase pathway Slt2, and the calcineurin pathway are responsible of the cell wall repair. MPS1, the orthologous of SLT2, in Magnaporthe grisea is essential for the repair of the cell wall and the penetration of the appressorium (Xu, and Al, 1998). In yeast, Slt2 activates the transcription factors Rlm1, Swi4 and Swi6, whereas the calcineurin activates Crz1. In addition, PaIdc1 has a role in the nuclear localization of PaMpk1 (orthologous of Slt2 and Mps1). (Corinne Jamet-Vierny, and Al, 2007), in Podospora anserina. MgIDC1, the gene orthologous of PaIDC1 , likewise, was identified in M. grisea. ScAGS1 (α- glucan synthase), is the principal component of the wall of fungi. CaCAS5, is a transcriptional regulator, controls several genes of the cell wall and ScKnr4 is implied in the regulation of the activity of 1,3-- glucan synthase and the formation of the cell wall These genes was identified at M. grisea and deletion mutants were obtained by the gene replacement in M. grisea. Mutants Guy11ΔKU80Δmps1 and Guy11ΔKU80Δidc1 show a strong reduction of mycelium. Guy11ΔKU80Δmps1, Guy11ΔKU80Δrlm1 and Guy11ΔKU80Δswi4 have a very strong reduction of sporulation. Guy11ΔKU80Δmps1, Guy11ΔKU80Δrlm1 and Guy11ΔKU80Δcrz1 have a strong reduction of pathogenicity. Moreover, mutants Guy11ΔKU80Δmps1 and Guy11ΔKU80Δswi4 are 100% inhibited, in presence of a mixture of Aculéacine and Nikkomycine with low dose (DI20). The results show that in M. grisea, there are two pathways implied in the cell wall integrity. The MAPK pathway Mps1, which controls the tr anscription factors Rlm1, Swi4, Swi6 and the calcineurin pathway, which controls the transcription factor, Crz1. According to this study, SWI4 is implied in the regulation of glucan synthases and chitin synthase genes, when RLM1 is implied only in the regulation of chitin synthase genes. In the calcineurin pathway, CRZ1 controls chitin synthase genes. These studies suggest that the factors of transcription which are controlled by Mps1 and Calcineurin are specialized in the regulation of target genes, specific to the cell wall integrity and cell wall repair.

L'intégrité de paroi cellulaire est cruciale pour la survie du champignon pendant son cycle de développement ou en réaction à des conditions de stress. Chez la levure, les voies de signalisation de MAP kinase, Slt2, et calcineurin, régulent la réparation de paroi cellulaire. MPS1, l'orthologue de SLT2, chez Magnaporthe grisea est essentiel pour la réparation de la paroi cellulaire et pour la pénétration de l'appressorium (Xu, et al., 1998). Chez la levure, Slt2 active les facteurs de transcription Rlm1, Swi4 et Swi6, alors que le calcineurin active Crz1. Par ailleurs, PaIdc1 a un rôle dans la localisation nucléaire de PaMpk1 (orthologue de Slt2 et Mps1). (Corinne Jamet-Vierny, et al., 2007). MgIDC1, Le gène orthologue de PaIDC1 a été, de même, identifié chez M. grisea. ScAGS1 (α-glucan synthase), est un composant principal de la paroi principal des champignons. CaCAS5, est un régulateur transcriptionel, régule plusieurs gènes de la paroi cellulaire et ScKnr4 est impliqué dans la régulation de l'activité de 1,3--glucan synthase et la formation de la paroi cellulaire Ces gènes ont été identifiés chez M. grisea et des mutants de délétion ont été construits par le remplacement de gène chez M. grisea. Les mutants Guy11ΔKU80Δmps1 et Guy11ΔKU80Δidc1 montrent une forte réduction de mycélium aérien. Guy11ΔKU80Δmps1, Guy11ΔKU80Δrlm1 et Guy11ΔKU80Δswi4 ont une très forte réduction de sporulation. Guy11ΔKU80Δmps1, Guy11ΔKU80Δrlm1 et Guy11ΔKU80Δcrz1 ont une forte réduction de pouvoir pathogène. De plus, les mutants Guy11ΔKU80Δmps1 et Guy11ΔKU80Δswi4 sont inhibé, de 100% en présence d'un mélange de l'Aculéacine et Nikkomycine à faible dose (DI20). Les résultats montrent que chez M. grisea, il existe deux voies impliquées dans l'intégrité de la paroi cellulaire. La voie MAPK MPS1, qui régule les facteurs de transcription Rlm1, Swi4, Swi6 et la voie de Calcineurine, qui régule Crz1. D'après les analyses, SWI4 est impliquée dans la régulation des gènes de glucan synthases et chitine synthase, quand RLM1 n'est impliqué que dans la régulation des gènes de chitine synthase. Dans la voie de Calcineurine, CRZ1 contrôle les gènes de chitine synthase aussi. Ces études suggèrent que les facteurs de transcription qui sont régulés par Mps1 et Calcineurine sont spécialisés dans la régulation des gènes de cible spécifiques à l'intégrité et la réparation de la paroi cellulaire.

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.

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.