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Programa De Pós-Graduação Em Fitossanidade Tese 0 UNIVERSIDADE FEDERAL DE PELOTAS Programa de Pós-Graduação em Fitossanidade Tese Etiologia da mancha ocular na cultura do arroz causada por Bipolaris gigantea e alterações metabólicas desencadeadas nas plantas Priscila Rossatto Meneses Pelotas, 2017 1 PRISCILA ROSSATTO MENESES Etiologia da mancha ocular na cultura do arroz causada por Bipolaris gigantea e alterações metabólicas desencadeadas nas plantas Tese apresentada ao programa de Pós- Graduação em Fitossanidade da Universidade Federal de Pelotas, como requisito parcial à obtenção do título de Doutor em Fitossanidade (área de conhecimento: Fitopatologia) Orientador(a): Profa. Dra. Cândida Renata Jacobsen de Farias Coorientador: Prof. Dr. Leandro José Dallagnol Coorientador (a): Profa. Dra. Danielle Ribeiro de Barros Pelotas, 2017 Universidade Federal de Pelotas / Sistema de Bibliotecas Catalogação na Publicação M111e Meneses, Priscila Rossatto Etiologia da mancha ocular na cultura do arroz causada por Bipolaris gigantea e alterações metabólicas desencadeadas nas plantas / Priscila Rossatto Meneses ; Cândida Renata Jacobsen de Farias, orientadora ; Leandro José Dallagnol, Danielle Ribeiro de Barros, coorientadores. — Pelotas, 2017. 88 f. Tese (Doutorado) — Programa de Pós-Graduação em Fitossanidade, Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas, 2017. 1. Atividade bioquímica. 2. Drechslera gigantea. 3. Mancha ocular. 4. Oryza sativa. 5. Taxonomia. I. Farias, Cândida Renata Jacobsen de, orient. II. Dallagnol, Leandro José, coorient. III. Barros, Danielle Ribeiro de, coorient. IV. Título. CDD : 633.18 2 Banca examinadora: ______________________________________________ Profa. Dra. Cândida R. Jacobsen de Farias (Orientadora) (Departamento de Fitossanidade, FAEM-UFPel) ______________________________________________ Dr. Cley Donizeti Martins Nunes (Embrapa Clima Temperado) ______________________________________________ Profa. Dra. Danielle Ribeiro de Barros (Departamento de Fitossanidade, FAEM-UFPel) ______________________________________________ Dr. Eduardo Guatimosim (Departamento de Fitossanidade, FURG) ______________________________________________ Prof. Dr. Sidnei Deuner (Departamento de Botânica/Fisiologia Vegetal, UFPel) 3 Aos meus pais Verani Rossatto; Domingos Sávio R. Meneses e Jose Pedro C. Morales À minha avó Maria Salla Rossatto (in memorian) Aos meus irmãos (ãs) e sobrinhos (ãs) Dedico e Ofereço 4 Primeiramente agradeço a Deus por ter me privilegiado com a vida e por estar ao meu lado em todos os momentos me pegando no colo e dando forças para seguir em frente À Dra. Cândida R. Jacbsen de Farias, pela orientação, pelos ensinamentos, pelo apoio, pela confiança depositada durante toda pós-graduação e pela amizade. Por quem sempre guardarei carinho e respeito de uma eterna orientadora. Aos meus coorientadores, Dra. Danielle Ribeiro de Barros e Dr. Leandro José Dallagnol, pela orientação e por terem possibilitado parte da realização deste trabalho. À Universidade Federal de Pelotas, Faculdade de Agronomia Eliseu Maciel, em especial, ao Programa de Pós Graduação em Fitossanidade (PPGFS), extensivo a todos os professores e funcionários. Aos funcionários Serginho e Rosaria incansavelmente disponíveis, colaborando com os conhecimentos, bom funcionamento da estrutura e dos laboratórios, sempre de maneira gentil e prestativa. Ao Dr. Eduardo Guatimosim que se disponibilizou em contribuir enormemente para a execução deste projeto. Aos estudantes e futuros colegas Jessica U. Tavares, Alexandre M. Neumann, Guilherme Longaray e Ligiani Simões pela amizade, carinho e auxílio prestado. Aos colegas e amigos Viviana Gaviria, Victoria Moreira, Keilor Dorneles, Caliandra Bernardi, Vanessa Gonçalves, Cristiano Bellé, Wellington Rodrigues, Renata Moccelin e Silvia Maich que foram literalmente “os anjos” que tornaram todos os momentos muito mais doces ao longo dessa jornada. À todos os colegas do PPGFS, que dividiram comigo todos os momentos (difíceis, de descontração, de apuro) vivenciados ao longo do curso; Á colega e grande amiga Elen Bonilha, por estar ao meu lado em todos os momentos possíveis da minha jornada na pós graduação e na minha vida, pelo auxílio, apoio, amor, carinho e companheirismo. Ao meu amigo e namorado Elton Dalke, por estar sempre ao meu lado me motivando e em todos os momentos da minha vida, pela paciência, carinho, atenção e amor. À minha amada família e amigos, meus maiores incentivadores, por todo suporte, amor e carinho. E por fim, meu mais profundo e especial agradecimento à minha mãe, Verani Rossatto, meu maior exemplo de vida, de mulher, a pessoa que sempre esteve e está incondicionalmente ao meu lado. A todos meu carinhoso, sincero e eterno, MUITO OBRIGADA! 5 Resumo Meneses, Priscila Rossatto. Etiologia da mancha ocular na cultura do arroz causada por Bipolaris gigantea e alterações metabólicas desencadeadas nas plantas. 2017. 88f. Tese (Doutorado) – Programa de Pós-Graduação em Fitossanidade. Universidade Federal de Pelotas. Pelotas, RS, Brasil. A Região Sul do Brasil apresenta-se em posição de destaque no cenário orizícola brasileiro, com grande potencial de crescimento. A ocorrência de doenças está entre os fatores que podem restringir a expressão do seu potencial produtivo, reduzindo a área fotossintética, acarretando perdas na produção de grãos e queda na produtividade. Manchas foliares, relacionadas à doença conhecida como mancha ocular, venham sendo observadas desde a safra 2006/07 no estado do Rio Grande do Sul (RS). O objetivo do presente trabalho foi identificar e caracterizar a etiologia do patógeno causador da doença, bem como, informações referentes aos testes de patogenicidade e avaliar os componentes de resistência, variáveis bioquímicas de mecanismos de defesa e respostas citológicas envolvidas no processo de infecção. Para isso o patógeno foi isolado de plantas sintomáticas e analisado quanto as suas estruturas reprodutivas e colônias, comparando-as com a literatura padrão. A extração de DNA, amplificação por PCR e sequenciamento e análise de regiões conservadas do genoma, consistiram nas etapas para obter a descrição molecular do isolado. Componentes de resistência, atividade enzimática, peroxidação lipídica e quantificação de lesões foram analisados em diferentes períodos de inoculação e estádios fenológicos, para as cultivares BRS Querência e Puitá Inta CL. O presente estudo conclui, satisfatoriamente, que o agente causal da manha ocular do arroz é o fungo Bipolaris gigantea. Ainda, independente do estádio, a cv BRS Querência mostrou maior suscetibilidade à doença, causando danos celulares, a nível de membrana, devido a ação desencadeada pelo fungo, através do acúmulo de espécies reativas de oxigênio. Puitá Inta CL apresentou resposta de defesa através da ação de enzimas antioxidantes. Os resultados esclareceram à correta taxonomia do agente causal da mancha ocular da cultura do arroz e demostraram as primeiras evidências das respostas de defesa de cultivares ao patógeno. Palavras-chave: atividade bioquímica, Drechslera gigantea, mancha ocular, Oryza sativa, taxonomia. 6 Abstract Meneses, Priscila Rossatto. Etiology of eyespot in the rice crop culture caused by Bipolaris gigantea and metabolic alterations unleashed on the plants. 2017. 87f. Tese (Doutorado) – Programa de Pós-Graduação em Fitossanidade. Universidade Federal de Pelotas. Pelotas, RS, Brasil. The lands in southern Brazil are in a highlighted position of the rice cropping area, with great potential to increase. The occurrence of diseases is among the factors that can restrict its potential, reducing the photosynthetic area, causing losses on the grains’ production and reduction of yield. Foliar leaf spots related to the eyespot disease are being observed since 2006/07’s rice harvest, in the state of Rio Grande do Sul (RS).The objective of the study is identify and characterize the etiology of the pathogen causing the disease, as well, acquire information regarding the pathogenicity tests and evaluate the resistance components, biochemical variables of defense mechanisms and cytological responses involved in the infection process. In order to accomplish that, the pathogen was isolated from symptomatic plants and its reproductive structures and its colonies were analyzed and compared to the literature. The DNA extraction, amplification by PCR and sequencing and analysis of genome consisted on the steps to obtain the molecular description of the isolate. Components of resistance, enzymatic activity, lipid peroxidation and lesion quantification were analyzed in different periods of inoculation and phenological stages, for cultivars BRS Querência and Puitá Inta CL. The present study concludes that the agent which is causing the eyespot on rice is the Bipolaris gigantea fungus. Moreover, independently of stage the cv BRS Querência showed major susceptibility to the disease, causing cellular damages at membrane level, due to the action triggered by the fungus, through the accumulation of reactive oxygen species. Puitá Inta CL showed defense response by action of antioxidant enzymes. The results showed the correct taxonomy of the causal agent of zonate eyespot on rice crop culture and demonstrate the first evidence of defense responses of cultivars pathogen. Keyword: activity biochemical, Drechslera gigantea, Oryza sativa, taxonomy, zonate eyespot. 7 Lista de Tabelas Artigo 1 Tabela 1 - Lista de pares de primers, utilizados para amplificação das regiões genômicas estudadas, suas respectivas sequências, tamanho de fragmento esperado e referências bibliográficas.
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