Bioprospecção De Actinobactérias Associadas À Esponja Marinha Aplysina Fulva: Isolamento, Caracterização E Produção De Compostos Bioativos

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Bioprospecção De Actinobactérias Associadas À Esponja Marinha Aplysina Fulva: Isolamento, Caracterização E Produção De Compostos Bioativos Universidade de São Paulo Escola Superior de Agricultura “Luiz de Queiroz” Bioprospecção de actinobactérias associadas à esponja marinha Aplysina fulva: isolamento, caracterização e produção de compostos bioativos Fábio Sérgio Paulino da Silva Tese apresentada para obtenção do título de Doutor em Ciências. Área de concentração: Microbiologia Agrícola Piracicaba 2015 Fábio Sérgio Paulino da Silva Licenciado em Ciências Biológicas Bioprospeção de actinobactérias associadas à esponja marinha Aplysina fulva: isolamento, caracterização e produção de compostos bioativos Orientador: Prof. Dr. ITAMAR SOARES DE MELO Tese apresentada para obtenção do título de Doutor em Ciências. Área de concentração: Microbiologia Agrícola Piracicaba 2015 Dados Internacionais de Catalogação na Publicação DIVISÃO DE BIBLIOTECA - DIBD/ESALQ/USP Silva, Fábio Sérgio Paulino da Bioprospecção de actinobactérias associadas à esponja marinha Aplysina fulva: isolamento, caracterização e produção de compostos bioativos / Fábio Sérgio Paulino da Silva. - - Piracicaba, 2015. 159 p. : il. Tese (Doutorado) - - Escola Superior de Agricultura “Luiz de Queiroz”. 1. Metabólitos secundários 2. Fungicidas 3. Pythium aphanidermatum 4. Phytophthora capsici 5. Magnaporthe grisea 6. Algicida 7. Selenastrum capricornutum 8. Herbicida 9. Pré- emergência 10. Agrostis stolonifera 11. Taxonomia polifásica 12. Bioprospecção I. Título CDD 589.92 S586B “Permitida a cópia total ou parcial deste documento, desde que citada a fonte – O autor” 3 Dedico Aos meus pais, aos meus irmãos, a minha companheira Fabiana, e a todos os amigos que sempre influenciaram positivamente meu galgar nesta breve, porém feliz existência. 4 5 AGRADECIMENTOS A DEUS. Palavras não bastam para descrever a imensa gratidão; Aos meus pais, Flávio e Áurea, por todo amor, dedicação e exemplo de educação, que serviram de pilares para minha existência; Aos meus irmãos, Bruno e Cássio por todo amor e carinho; A minha eterna companheira Fabiana pelo amor, carinho e compreensão nesta fase de minha jornada; Aos meus amigos do peito, George, Marcelo, Jean, Deyvit, Anderson, Tiago, Andre, Gabriel, Franco, Solano e Samuel, por todo o companheirismo, que desde a infância só trazem boas lembranças; Ao orientador e amigo, Dr. Itamar Soares de Melo pela oportunidade, sugestões, confiança, paciência e ensinamentos; Aos colegas e grandes amigos que tive o privilégio de conher durante os anos de vida acadêmica: Élcio, Ismael, Victor, Marcelo, Robson, Antover, Tirone, Daniela, Sidney, Karine, Éder, Adriano, Rafael Valadares, Elisa, Bruna, Alice, Silvia, Rafael Vasconcellos, Danilo, Camila, Ana Gabriele, Suikinai, Vanessa, Taketani, Tiago, Tatiana, João Luiz, Rafael Silva, Wallace, Rose, Joseane, Ana Paula, Stalin, Mendes, Jorge e Harold; Aos Professores e amigos que me orientaram desde o início da graduação: Prof. José Antônio Peters, Prof. Marco Antônio Bacarin, Profª. Andrea Bittencourt Moura e Prof. Marcos Rogério Tótola; A Embrapa Meio Ambiente pelo fornecimento do espaço no Laboratório de Microbiologia Ambiental e por todas as condições para realização dos trabalhos; 6 A Universidade de São Paulo em especial ao Programa de Pós-Graduação em Microbiologia Agrícola da Escola Superior de Agricultura ‘Luiz de Queiroz’ pela oportunidade do doutoramento e por todo apoio que contribuiu para meu crescimento profissional; Ao CNPq pela bolsa de doutorado concedida e pelo apoio financeiro atravézs do projeto Arquipélago e Ilhas Oceânicas – Chamada 39/2012 que viabilizou a elaboração desta tese; A Marinha do Brasil em especial ao Comandante Carvalho e a Secretaria da Comissão Interministerial para os Recursos do Mar (SECIRM), que junto ao CNPq, viabilizaram o projeto e a expedição ao Arquipélago de São Pedro e São Paulo; Á Tripulação “safa” do TRANSMAR pela logística e por toda a “faina” de travessia de Natal- RN até o Arquipélago de São Pedro e São Paulo; Ao apoio do grande amigo Fernando Rodrigues que com sua experiência, auxiliou e conduziu da melhor forma possível todos os mergulhos no Arquipélago; Aos parceiros do projeto sem os quais seria impossível a realização deste trabalho: Msc. Danilo Tosta Souza, Camila Pansa, Roseli de Cássia de Almeida Pamplona, Jorge Fernandes Albano de Morais, Dr. Rodrigo Taketani, Drª. Sonia Queiroz, Dr. Eduardo Crevelin, Dr. Luiz Alberto Beraldo de Moraes, Drª. Suikinai Nobre Santos, Dr. Tiago Domingues Zucchi e Dr. Fernando Coreixas de Moraes. 7 "A percepção do desconhecido é a mais fascinante das experiências. O homem que não tem os olhos abertos para o misterioso passará pela vida sem ver nada." Albert Einstein 8 9 “Educação não transforma o mundo. Educação muda as pessoas. Pessoas mudam o mundo” Paulo Freire 10 11 SUMÁRIO RESUMO ................................................................................................................... 15 ABSTRACT ............................................................................................................... 17 LISTA DE FIGURAS ................................................................................................. 19 LISTA DE TABELAS ................................................................................................. 23 1 INTRODUÇÃO ....................................................................................................... 25 2 OBJETIVOS ........................................................................................................... 27 2.1 Objetivo principal ................................................................................................. 27 2.2 Objetivos específicos........................................................................................... 27 3 REVISÃO BIBLIOGRÁFICA ................................................................................... 29 3.1 A demanda por novos agroquímicos e a importância das Actinobactérias para a agrobiotecnologia ...................................................................................................... 29 3.2 Actinobacterias em ambientes marinhos ............................................................. 32 3.3 Estratégias de isolamento de actinobactérias marinhas...................................... 33 3.4 Taxonomia de microorganismos ......................................................................... 36 3.5 Esponjas marinhas .............................................................................................. 37 3.6 Associação entre esponjas e microorganismos e o potencial destes na produção de compostos bioativos ............................................................................................. 40 3.7 Aplysina fulva: distribuição, descrição taxonômica e potencial biotecnológico .... 43 Referências ............................................................................................................... 45 4 DIVERSIDADE DE ACTINOBACTÉRIAS PRODUTORAS DE COMPOSTOS FUNGICIDAS ISOLADAS DE APLYSINA FULVA DO ARQUIPÉLAGO DE SÃO PEDRO E SÃO PAULO............................................................................................. 53 Resumo ..................................................................................................................... 53 Abstract ..................................................................................................................... 53 4.1 Introdução ........................................................................................................... 54 4.2 Material e Métodos .............................................................................................. 55 4.2.1 Local de amostragem ....................................................................................... 55 4.2.2 Isolamento Seletivo para actinobactérias ......................................................... 58 4.2.3 Identificação molecular de actinobactérias ....................................................... 60 4.2.4 Extração dos metabólitos secundários ............................................................. 61 4.2.5 Atividade antifúngica dos extratos de actinobactérias ...................................... 62 4.2.6 Perfil químico dos extratos brutos .................................................................... 62 12 4.3 Resultados e Discussão ..................................................................................... 63 4.3.1 Identificação da esponja marinha .................................................................... 63 4.3.2 Isolamento Seletivo ......................................................................................... 63 4.3.3 Identificação molecular de actinobactérias ...................................................... 64 4.3.4 Atividade antifúngica de extratos bruto de actinobactérias .............................. 70 4.3.5 Perfil químico dos extratos brutos .................................................................... 74 Referências............................................................................................................... 86 5 ATIVIDADE HERBICIDA DO COMPOSTO BUTENOLIDA PRODUZIDO PELO STREPTOMYCES SP. ASPSP 103 ......................................................................... 93 Resumo .................................................................................................................... 93 Abstract .................................................................................................................... 93 5.1 Introdução ..........................................................................................................
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