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DALTON VINICIO DORIGHELLO VERSATILIDADE DE Bacillus Spp

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DALTON VINICIO DORIGHELLO VERSATILIDADE DE Bacillus Spp UNIVERSIDADE ESTADUAL PAULISTA “JÚLIO DE MESQUITA FILHO” Câmpus de Botucatu DALTON VINICIO DORIGHELLO VERSATILIDADE DE Bacillus spp. NO CONTROLE BIOLÓGICO DE DOENÇAS DE PLANTAS E NA PROMOÇÃO DE CRESCIMENTO DE SOJA Tese apresentada à Faculdade de Ciências Agronômicas da Unesp – Câmpus de Botucatu, para obtenção do título de Doutor em Agronomia (Proteção de Plantas). BOTUCATU 2017 DALTON VINICIO DORIGHELLO VERSATILIDADE DE Bacillus spp. NO CONTROLE BIOLÓGICO DE DOENÇAS DE PLANTAS E NA PROMOÇÃO DE CRESCIMENTO DE SOJA Tese apresentada à Faculdade de Ciências Agronômicas da Unesp – Câmpus de Botucatu, para obtenção do título de Doutor em Agronomia (Proteção de Plantas). Orientador: Dr. Wagner Bettiol BOTUCATU 2017 DEDICATORIA Aos meus pais, Vinicio Dorighello e Ivani Luvizotto Dorighello, pela dedicação, pelo incentivo e por tudo que fizeram por mim durante toda minha vida. Às minhas irmãs, Cristiane e Tatiane, pelo carinho. Aos meus cunhados, Júlio e Márcio, pelos bons momentos. À Mariana Modanez, minha noiva e futura esposa (09-09-2017), por estar ao meu lado durante toda a minha caminhada acadêmica e profissional, pela paciência, pelo incentivo, pela força e, principalmente, pelo companheirismo e amor. Aos meus avós, Pedro Dorighello e Ondina Dorighello (in memorian). Ao Campi (in memorian), muitas saudades! Ao Vicente Ferreira, meu tutor nos serviços rurais que, como um pai, foi também meu conselheiro e amigo. Aos momentos de dificuldade que me tornaram mais forte e maduro. AGRADECIMENTOS A Deus, pois sem Ele nada é possível. Ao meu orientador Dr. Wagner Bettiol que, apesar de todas as suas responsabilidades profissionais e familiares, se manteve presente durante esses anos e me possibilitou aprendizados que levarei para toda a vida. Agradeço também pelas conversas, conselhos, merecidos puxões de orelhas e por me mostrar, mesmo que involuntariamente, com que dedicação e gentileza devo tratar minha futura esposa e filhos. À UNESP/FCA, pela oportunidade de realização do curso. À Embrapa Meio Ambiente e à Embrapa Soja, por cederem a estrutura e materiais para a condução dos ensaios. À Dra. Regina Maria Villas Bôas de Campos Leite, pela ajuda e incentivo. À CAPES, pela concessão da bolsa de auxílio aos estudos. Ao Fabio Brandi da Bayer®, pelos materiais cedidos. Ao Dr. Nilson Molina Maia do Instituto Agronômico de Campinas, pela extração e fornecimento dos óleos de café. Às Dra. Fabiana Garboggini e Dra. Nataly Quiñones, por me possibilitarem os primeiros passos em biologia molecular. Aos professores do Programa de Pós-Graduação em Agronomia (Proteção de Plantas): Dr. Antônio Carlos Maringoni, Dra. Renate Krause Sakate, Dra. Silvia Renata Siciliano Wilcken, Dra. Raquel Ghini, Dr. Edson Luiz Furtado, Dr. Wagner Bettiol e Dr. Carlos Gilberto Raetano, pelo aprendizado nas disciplinas. Ao Dr. Cristiano Almeida, pelas esclarecedoras conversas sobre métodos estatísticos. Aos companheiros de república “das antigas”: Carlos Eduardo, Daniel, Lúcio, Renato, André e, especialmente, Cassiano Forner, pelo companheirismo, pelas conversas e ajuda nos trabalhos. Aos companheiros de república: Thiago, Carlos Albuquerque, Sr. Wilson e Matheus. Aos amigos de curso e trabalho: Mércia, Regiane, Michelle e Larissa. Às técnicas do LMA da Embrapa Meio Ambiente: Anamaria, Elke, Roseli e Márcia, pela ajuda e pelos bons momentos que passamos juntos; e aos funcionários: Henrique, Rafa, Celinha e Julius, pela colaboração neste trabalho. À equipe da Fitopatologia da Embrapa Soja: Allan, Ângelo e Tia Alda, pelos bons momentos e ajuda na condução do trabalho a campo. A todos meus companheiros de trabalho da Faculdade de Ensino Superior Santa Bárbara (FAESB), em especial à Carla Barreto, Thiago Mezette, Lilian Faustino e Odair Ferreira. A todos meus companheiros de trabalho da Itatijuca Biotech®. A todos meus companheiros de trabalho da MODO Topografia-Geo-Ambiental. A todos que direta ou indiretamente colaboraram para a realização deste trabalho. RESUMO O mercado de produtos de controle biológico, principalmente os que possuem isolados de Bacillus spp. em sua formulação, tem aumentado significativamente, devido às exigências de diminuição do uso de agrotóxicos e maior sustentabilidade do segmento agrícola. Assim, o presente trabalho teve por objetivos: 1- Avaliar o potencial de bactérias do gênero Bacillus no controle de diferentes doenças de plantas; 2- Selecionar isolados de Bacillus spp. promotores de crescimento da soja e antagonistas a Sclerotinia sclerotiorium; 3- Avaliar o potencial de um produto comercial à base de B. subtilis QST 713 e outro à base de óleo de café torrado para compor um programa de manejo da ferrugem-asiática da soja (Phakopsora pachyrhizi) em aplicação sequencial ou alternada com um fungicida. Inicialmente foi avaliado o efeito de B. subtilis, B. firmus e B. amyloliquefaciens no controle dos patógenos Erysiphe diffusa, Podosphaera xanthii, Sclerotinia sclerotiorum, P. pachyrhizi, Fusarium solani, Fusarium oxysporum f. sp. phaseoli, Colletotrichum lindemuthianum e Corynespora cassiicola em condições controladas. Neste estudo, nenhum dos isolados controlou Erysiphe difusa e Podosphaera xanthii. Em folhas destacadas, todos os isolados reduziram o diâmetro da lesão de S. sclerotiorum e a severidade de P. pachyrhizi, sendo mais efetivos com aplicação preventiva. B. subtilis, B. firmus e B. amyloliquefaciens também inibiram o desenvolvimento de F. oxysporum f. sp. phaseoli, C. cassiicola e C. lindemuthianum. No entanto, apenas B. amyloliquefaciens inibiu o desenvolvimento de F. solani. Todos os isolados de Bacillus diminuíram a germinação dos esporos destes patógenos, exceto os de C. lindemutianum. Os isolados B. subtilis, B. firmus e B. amyloliquefaciens possuem potencial para serem explorados como agentes de biocontrole para as doenças testadas, exceto para os oídios. Porém, novos testes devem ser realizados em condições reais de ocorrência das doenças. Também foi realizada seleção de bactérias do gênero Bacillus para promoção de crescimento de plantas de soja e antagonismo a S. sclerotiorium. Foram selecionados oito isolados promissores em promover crescimento in vivo, os quais foram, posteriormente, avaliados em casa-de-vegetação em relação a capacidade de promoverem crescimento e sobre a atividade das enzimas relacionadas à defesa das plantas, por meio da inoculação de sementes, com ou sem a co-inoculação do Bradyrhizobium japonicum. Os resultados demonstraram que a seleção realizada in vivo para promotores de crescimento foi eficiente, pois todos os isolados selecionados diferiram da testemunha em pelo menos um dos componentes avaliados. Os isolados de Bacillus AP-3, AP-25, AP-51, AP-106, AP-117, AP-40, QST-713 e QST-2808 promoveram crescimento de plantas de soja e inibiram, significativamente, S. sclerotiorum. O isolado de Bacillus AP-25 (Bacillus subtilis subsp. inaquosorum) aumentou a atividade da peroxidase. Em condições de campo, o isolado de B. subtilis QST 713 e óleo de café torrado foram utilizados em aplicações sequencias e alternadas com o fungicida Piraclostrobina + Epoxiconazol para controle da ferrugem asiática da soja. Os resultados dos dois ensaios nos campos experimentais mostraram que as aplicações sequenciais do óleo de café, do produto à base de Bacillus e do fungicida reduziram, em média, 31%, 45% e 68% respectivamente, a área abaixo da curva de progresso da doença. Apesar disso, apenas o tratamento com a aplicação sequencial do produto contendo B. subtilis apresentou a mesma produtividade e peso de 100 sementes do tratamento com a aplicação sequencial de fungicida. O isolado de B. subtilis QST 713 pode ser indicado para compor um programa de manejo da ferrugem asiática da soja em condições de baixa intensidade da doença. Palavras-chave: Bacillus spp., doenças de plantas, promoção de crescimento, Glycine max ABSTRACT The market of products for biological control, especially formulations using Bacillus spp., has increased significantly, due to the requirements for the reduction of the use of agrochemicals in crop protection and sustainability of the agricultural sector. The objectives of this thesis were: 1- To evaluate the potential of Bacillus in plant disease control; 2- To select isolates of Bacillus spp. for growth promotion of soybean and antagonists to Sclerotinia sclerotiorium; 3- To evaluate the potential of a commercial product based on B. subtilis QST 713 and another product based on roasted coffee oil in order to compose a management program for asian soybean rust (Phakopsora pachyrhizi) in sequential or alternating application with a fungicide. Initially, the effect of B. subtilis, B. firmus and B. amyloliquefaciens on the control of pathogens Erysiphe diffusa, Podosphaera xanthii, Sclerotinia sclerotiorum, P. pachyrhizi, Fusarium solani, Fusarium oxysporum f. sp. phaseoli, Colletotrichum lindemuthianum and Corynespora cassiicola was evaluated under controlled conditions. In this study, Bacillus isolates did not controlled Erysiphe diffusa and Podosphaera xanthii. All isolates were effective in reducing the diameter of the S. sclerotiorum lesion and the severity of P. pachyrhizi, being more effective by preventive application in detached leaf. B. subtilis, B. firmus and B. amyloliquefaciens inhibited the mycelial growth of F. oxysporum f. sp. phaseoli, C. cassiicola and C. lindemuthianum. However, only B. amyloliquefaciens inhibited F. solani. All Bacillus isolates inhibited spore germination of these pathogens, except for C. lindemutianum. The isolates of B. subtilis, B. firmus and B. amyloliquefaciens
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