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Actinobactérias De Biomas Brasileiros: Biodiversidade E Potencial De Uso Na Agricultura

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Actinobactérias De Biomas Brasileiros: Biodiversidade E Potencial De Uso Na Agricultura Universidade de São Paulo Escola Superior de Agricultura “Luiz de Queiroz” Actinobactérias de biomas brasileiros: biodiversidade e potencial de uso na agricultura Harold Alexander Vargas Hoyos Tese apresentada para obtenção do título de Doutor em Ciências. Área de concentração: Microbiologia Agrícola Piracicaba 2018 Harold Alexander Vargas Hoyos Microbiologista Actinobactérias de biomas brasileiros: biodiversidade e potencial de uso na agricultura Versão revisada de acordo com a resolução CoPGr 6018 de 2011. Orientador: Prof. Dr. ITAMAR SOARES DE MELO Tese apresentada para obtenção do título de Doutor em Ciências. Área de concentração: Microbiologia Agrícola Piracicaba 2018 2 Dados Internacionais de Catalogação na Publicação DIVISÃO DE BIBLIOTECA – DIBD/ESALQ/USP Vargas Hoyos, Harold Alexander Actinobactérias de biomas brasileiros: biodiversidade e potencial de uso na agricultura / Harold Alexander Vargas Hoyos. - - versão revisada de acordo com a resolução CoPGr 6018 de 2011. - - Piracicaba, 2018. 130 p. Tese (Doutorado) - - USP / Escola Superior de Agricultura “Luiz de Queiroz”. 1. Actinobacterias 2. Sclerotinia sclerotiorum 3. Enzimas 4. Metabolitos secundários 5.Solubilização de fosforo 6. Diversidade Taxonômica 7. Biocontrole 8.Streptomyces I. Título 3 Dedico A minha família Nora e Ulises e Karen por ser a força para conseguir todo e pelo apoio infinito. A minha avó Ana Teresa, a definição de nobreza em pessoa. A minha companheira Mariana, pela companhia e carinho e a todos meus amigos que fizeram este caminho mais leve. 4 AGRADECIMENTOS A mis padres, Nora y Ulises, por ser el ejemplo y motivación para conseguir mis objetivos. Los Amo; A mi hermana Karen, por todo el amor y el cariño; A mi abuela Ana Teresa, la persona más noble que ya conocí en la vida. Mil gracias “mita” por todas las bendiciones; A mia tias Doris, Jenny, Sor Angela, Lili, sus familias, a todos mis primos por el apoyo en todo momento; A mis tios David, Balmes, Eunice, Arbey, Arlex y Bladimir, a todos mis primos por el apoyo y la compañia siempre. A minha companheira Mariana pelo amor, carinho em todo momento; Aos meus amigos ¨pa´las que sea, Diego, David, Janny, Jorge, Miguel, Jairo, Felipe, Federico, Victor, Ricardo; Ao meu orientador, Dr. Itamar Soares de Melo, muito obrigado pelo apoio, conselhos, confiança, paciência e ensinamentos; A minha amiga e colega a Dra. Suikinai, primeira pessoa que me acolheu no Brasil, infinitas gracias!!! Muito agradecido pela ajuda em todo momento, pelas conversas, paciência; A Dra. Carmen Fernandez Mendez, pela oportunidade de trabalhar no seu laboratório e as brilhantes sugestões ao meu trabalho; Ao Dr. Fernando Andreote, pelo apoio e ajuda na adaptação a vida acadêmica no Brasil, grande admiração; Aos pesquisadores de Embrapa Meio ambiente pelos conselhos e apoio: Dr. Rodrigo Mendes, Dra. Sonia de Queiroz, Dr. Miguel Dita, Dra. Katia de Lina Nechet; 5 A minha amiga Josiane Barros, pela ajuda em todo momento e as incontáveis conversas acadêmicas sempre acompanhadas de um bom café !!! Aos colegas e grandes amigos que tive o privilégio de conhecer durante esses últimos anos de vida acadêmica: Ana Paula, Rose, Rafael Silva, Stalin, Camila, Rafael Vasconcellos, Danilo, Vanessa, Lilian, Laura, Marta, Maria Carolina, Tiago, Leonardo, Diego, Fabio e Jorge; Ao Wallace, pelas atualizações do meu país, e como já comentei: ¨O laboratório precisa de mais pessoas como você A juanita, por las conversaciones en español e los buenos recuerdos de la patria querida; A Ana Gabriele, pelo apoio em todo momento e as conversas acadêmico/laborais; A la colonia colombiana, que fueron lo más próximo a una família todo este tiempo: Carlos, Walter, Felipe, Juan, Edgar, Jhon, Jhon Jairo, Andres, Pedro, Andrea, Juliana; Aos técnicos dos diferentes laboratórios, obrigado pela ajuda e boa disposição João Luiz, Ana Maria, Elke, Roselly, Marcia Parma, Tatiana, Gabriel, Juliane, Marley, Debora, Marcia; A Universidade de São Paulo especialmente ao Programa de Pós-Graduação em Microbiologia Agrícola da Escola Superior de Agricultura „Luiz de Queiroz‟ por todo apoio para meu crescimento profissional; A Embrapa Meio Ambiente pelo uso do Laboratório de Microbiologia Ambiental, Laboratório de Extratos Orgânicos, Laboratório de Resíduos e Contaminantes e demais para realização do meu trabalho; A FAPESP pela bolsa de doutorado concedida no processo 2013/25076-0 que viabilizou a elaboração desta tese; 6 SUMÁRIO RESUMO ........................................................................................................................................................................... 8 ABSTRACT .................................................................................................................................................................... 10 1. INTRODUÇÃO........................................................................................................................................................... 11 2. OBJETIVOS ............................................................................................................................................................... 16 2.1. OBJETIVO GERAL ................................................................................................................................................... 16 2.2. OBJETIVOS ESPECÍFICOS ......................................................................................................................................... 16 REFERÊNCIAS .............................................................................................................................................................. 17 3. DIVERSIDADE DE ACTINOBACTÉRIAS ISOLADAS DE TRÊS BIOMAS BRASILEIROS E SUA ATIVIDADE ANTAGÔNICA CONTRA SCLEROTINIA SCLEROTIORUM ......................................................... 23 RESUMO ......................................................................................................................................................................... 23 ABSTRACT .................................................................................................................................................................... 23 3.1 INTRODUÇÃO .......................................................................................................................................................... 25 3.1. MATERIAL E MÉTODOS .......................................................................................................................................... 26 3.1.1. Obtenção dos isolados .................................................................................................................................. 26 3.1.2. Avaliação de antagonismo contra Sclerotinia sclerotiorum ......................................................................... 26 3.1.3. Inibição do crescimento de micielial e da germinação de escleródios de Sclerotinia sclerotiorum ............. 26 3.1.4. Compatibilidade com bactérias diazotróficas ............................................................................................... 27 3.1.5. Capacidade de solubilizar in vitro duas fontes de fósforo ............................................................................ 27 3.1.6. Identificação molecular ................................................................................................................................. 28 3.2. RESULTADOS E DISCUSSÃO .................................................................................................................................... 29 3.2.1. Avaliação da capacidade antagônica de actinobactérias contra Sclerotinia sclerotiorum .......................... 29 3.2.2. Inibição do crescimento de micielial e da germinação de escleródios de Sclerotinia sclerotiorum ............. 29 3.2.3. Compatibilidade com rizobactérias .............................................................................................................. 30 3.2.4. Caracterização Molecular dos isolados de Actinobactérias ......................................................................... 31 3.3. CONCLUSÕES ......................................................................................................................................................... 51 REFERÊNCIAS .............................................................................................................................................................. 52 4. SOLUBILIZAÇÃO DE FÓSFORO E PROMOÇÃO DE CRESCIMENTO EM SOJA POR ACTINOBACTÉRIA DO CERRADO ......................................................................................................................... 59 RESUMO ......................................................................................................................................................................... 59 ABSTRACT .................................................................................................................................................................... 59 4.1. INTRODUÇÃO ......................................................................................................................................................... 61 4.2. MATERIAL E MÉTODOS .......................................................................................................................................... 62 4.2.1. Locais de Amostragem .................................................................................................................................. 62 4.2.2. Isolamento e purificação
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