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 μg/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 μg/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.

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Made available in DSpace on 2016-06-02T18:55:25Z (GMT). No. of bitstreams: 1 5478.pdf: 1057989 bytes, checksum: fd5f9b601fb9dcd1e824044cd0b9b00b (MD5) Previous issue date: 2013-06-26 / Financiadora de Estudos e Projetos / Blast disease, caused by Magnaporthe grisea (anamorph Pyricularia grisea), is one of the main diseases of wheat and is disseminated throughout main producing states of the Country. It has been causing significant yield loss and is a limiting factor for the crop s expansion in central Brazil where the weather is highly favorable. The use of resistant cultivars can be influenced by weather conditions and concentration of inoculum. Chapter 1 of the present dissertation describes the influence of period of foliar wetness on the occurrence of blast in wheat seedlings in two experiments. The first one employed cultivars BH1146 (resistant) and BR40 (susceptible) inoculated with the Pyricularia isolate PR06- 03. In the second assay, the cultivars were BR18 (resistant) and Anahuac (susceptible) inoculated with the fungal isolate PR01-23. Five treatments were the period of wetness (6, 10, 14, 18 and 24h) in ten replications. Analysis was performed using a completely randomized design with a factorial plan of the treatments (five foliar wetness periods and 2 cultivars). Results showed that the minimum period needed for the development of the disease was 10 hours when inoculated with the isolate PR06-03. The longer the foliar wetness period the higher the incidence and severity of the disease regardless the resistance of cultivars. Chapter 2 aimed to determine the influence of the weather and concentration of P. grisea on the incidence and severity of blast. The experiment used totally randomized design with a factorial plan of the treatments: four concentrations (10³, 104, 105, 106 spores/ml) and two cultivars, ten repetitions. The wheat cultivars were BR18 (resistant) and Anahuac (susceptible) which were inoculated with 50ml of P. grisea suspension (isolate PR06-03) at the concentrations 10³, 104, 105, 106 spores/ml. The seedlings were submitted to temperatures of 22ºC and 28ºC for 18 hours of foliar wetness. The results showed that at 22ºC higher disease severity occurred on susceptible cultivar whereas the disease was the same for both cultivars at 28ºC. The resistant cultivar reacted like a susceptible when submitted to high temperature and high concentration of pathogen. / A brusone é uma das principais doenças do trigo e está disseminada nos principais estados produtores do país, causando danos significativos e sendo fator limitante para a expansão da cultura no Brasil central, principalmente por ser uma doença altamente influenciada pelo clima. Seu controle através de cultivares resistentes pode ser influenciado pelas condições climáticas e pela concentração de inóculo na lavoura. O Capítulo 1 desta dissertação descreve a influência do período de molhamento foliar na ocorrência da brusone, em plântulas de trigo, em dois experimentos. O primeiro com o isolado do fungo Pyricularia grisea (PR01-23) e duas cultivares de trigo no estádio de plântula, com reação diferenciada ao isolado (Anahuac suscetível e BR18 resistente) e o segundo com o isolado do fungo (PR06-03) e duas cultivares de trigo no estádio de plântula (BR40 suscetível e BH1146 resistente). Foram empregadas dez repetições de cinco tratamentos com os diferentes períodos de molhamento foliar (6, 10, 14, 18 e 24h). Avaliou-se a incidência e a severidade da brusone em porcentagem. A análise foi feita através de um delineamento estatístico inteiramente casualizado, com um esquema fatorial dos tratamentos (cinco períodos de molhamento e duas cultivares). Os resultados mostraram que o tempo mínimo requerido para o aparecimento da doença nas plântulas foi de 10 horas, quando inoculadas com o isolado PR01-23 e de 14 horas, quando inoculadas com o isolado PR06-03. Quanto maior o período de molhamento foliar, maior a incidência e a severidade da doença, independente da resistência da cultivar. O Capítulo 2 teve como objetivo determinar a influência da temperatura e concentração de inóculo de Pyricularia grisea na incidência e na severidade da brusone. O delineamento experimental utilizado foi inteiramente casualizado, com um esquema fatorial dos tratamentos: quatro concentrações de inóculo (10³, 104, 105 e 106 esporos/mL) e duas cultivares, dez repetições. Utilizou-se duas cultivares de trigo no estádio de plântula (BR18 resistente e Anahuac suscetível) que foram inoculadas com 50 mL de suspensão de P. grisea (isolado PR06-03) nas concentrações 10³, 104, 105 e 106 esporos/mL. As plantas foram submetidas às temperaturas de 22ºC e 28ºC por 18 horas de molhamento foliar. Avaliou-se a incidência e a severidade da brusone em porcentagem. Os resultados obtidos mostraram que à 22ºC a severidade foi maior na cultivar suscetível e igual para as cultivares a 28ºC. A cultivar resistente se comportou de forma semelhante a suscetível, quando submetida à alta temperatura e altas concentrações de inóculo.

Orientador: Raquel Ghini / Banca: Wagner Bettiol / Banca: Alfredo Seiti Urashima / Resumo: 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... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: 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. / Mestre

<p>Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).</p><p>Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus <i>Gaeumannomyces graminis</i>. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.</p><p>Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast <i>Pichia pastoris</i> with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.</p><p>Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).</p><p>Mn-lipoxygenase is an <i>R</i>-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other <i>R/S</i> lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.</p><p>The genome of the rice blast fungus, <i>Magnaporthe grisea</i>, contains putative genes of lipoxygenases and LDS. Mycelia of <i>M. grisea</i> were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from <i>G.graminis</i>. </p><p>Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.</p>

Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases). Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus Gaeumannomyces graminis. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones. Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast Pichia pastoris with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases. Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”). Mn-lipoxygenase is an R-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other R/S lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols. The genome of the rice blast fungus, Magnaporthe grisea, contains putative genes of lipoxygenases and LDS. Mycelia of M. grisea were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from G.graminis. Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.