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Carolinarivieraduarte.Pdf Universidade de São Paulo Escola Superior de Agricultura “Luiz de Queiroz” Diversidade microbiana em solos sob florestas de Araucaria angustifolia Carolina Riviera Duarte Maluche Baretta Tese apresentada para obtenção do título de Doutor em Agronomia. Área de concentração: Solos e Nutrição de Plantas Piracicaba 2007 Carolina Riviera Duarte Maluche Baretta Engenheiro Agrônomo Diversidade microbiana em solos sob florestas de Araucaria angustifolia Orientador: Prof. Dr. MÁRCIO RODRIGUES LAMBAIS Tese apresentada para obtenção do título de Doutor em Agronomia. Área de concentração: Solos e Nutrição de Plantas Piracicaba 2007 Dados Internacionais de Catalogação na Publicação (CIP) DIVISÃO DE BIBLIOTECA E DOCUMENTAÇÃO - ESALQ/USP Maluche-Baretta, Carolina Riviera Duarte Diversidade microbiana em solos sob florestas de Araucaria angustifolia / Carolina Riviera Duarte Maluche-Baretta. - - Piracicaba, 2007. 184 p. : il. Tese (Doutorado) - - Escola Superior de Agricultura Luiz de Queiroz, 2007. Bibliografia. 1. Análise multivariada 2. Biologia 3. Microbiologia do solo 4. Pinheiro 5. Química do solo 6. Seqüências do solo 7. Solo florestal I. Título CDD 634.975 “Permitida a cópia total ou parcial deste documento, desde que citada a fonte – O autor” 3 OFEREÇO À Deus nosso Pai e criador. Família e amigos Aos meus avós queridos Ulisses e Ires, pelo amor incondicional e apoio em todos os momentos. À minha mãe Cristina, pelo amor e carinho, exemplo de luta e garra. Ao meu marido Dilmar, pelo companheirismo nas horas mais difíceis. A minha tia Luciana, que mesmo longe sempre esteve muito presente. AMO MUITO TODOS VOCES!!!!! DEDICO 4 AGRADECIMENTOS À Deus, por guiar meus caminhos, pela minha vida e oportunidade de crescimento diante dos obstáculos e, principalmente, por ter me dado neste plano terrestre a minha estrutura familiar. Ao meu orientador, Prof. Dr. Márcio Rodrigues Lambais, pelo estímulo e apoio em todas as fases deste trabalho, orientação, confiança, amizade e agradável convivência. À Prof. Dra. Elke J.B.N. Cardoso pela oportunidade de integrar a equipe do projeto temático BIOTA/FAPESP intitulado “Biodiversidade vegetal e de organismos edáficos em ecossistemas de Araucaria angustifolia naturais e impactados no Estado de São Paulo” Ao CNPq, pela concessão da bolsa de estudo e a Fapesp, pelo suporte financeiro na condução deste trabalho. Aos meus familiares, em especial agradeço aos meus avós maternos, Ulisses e Ires Duarte, e minha mãe Cristina, pela educação moral, apoio e amor incondicional. Se não fosse por vocês talvez esta jornada não tivesse chegado ao fim. Devo tudo a vocês! Aos meus tios Luciana e Gilmar, meus irmãos de coração, pelo carinho e apoio nesta e em muitas caminhadas. Obrigado pela presença em minha vida, sei que posso contar sempre com vocês !!! Ao meu marido, pelo amor, carinho, companheirismo e paciência dedicados. Agradeço também aos seus familiares, por torcerem sempre por nós. Aos professores doutores, José Paulo de Sousa e Paula Morais, e a Dra. Fernanda da Universidade de Coimbra, Portugal, pelo apoio e orientação durante o período de estágio no exterior. Aos técnicos dos laboratórios de Microbiologia e Microbiologia Molecular da ESALQ, Wladimir, Denise e Fernando, pessoas muito especiais, pela ajuda e amizade, e a funcionária Odete do laboratório de Microbiologia e Bioquímica da Universidade de Coimbra, Portugal. Aos colegas do projeto temático Biota/FAPESP, em especial Camila, Daniel, Ézio e Rafaela, pela amizade, apoio e importantes discussões sobre o trabalho. Aos colegas e amigos de laboratório, pelos momentos de trabalho, companheirismo e diversão: Adriano Lucheta, Gisele Lopes, Giselle, Lucas Azevedo, Márcio Morais, Rafael Armas, Robinson Moresca, Soraya Kiriachek, Winston e todos estagiários. Aos colegas que já se foram: Ana, Daniele Takahashi, Denise Santos, Juliano Cury, Karina Cenciani, Pablo Hardoim e Simão Lindoso. 5 Em especial gostaria de agradecer aos colegas: Adriano Lucheta, Daniele Takahashi, Gisele Lopes, Juliano Cury, Lucas Azevedo e Rafael Armas, pessoas maravilhosas as quais sempre pude contar durante toda esta minha jornada. As discussões, ajudas práticas e trocas de conhecimento sempre foram muito úteis. Aos amigos conquistados durante estes anos: Alessandra, Alexandre, André, Fabiana, Fernanda, Leandro, Maria Elda, Mylenne, Rafael, Simone, Pilar, Priscila, Paulo e Pereira. Ao programa de Solos e Nutrição de Plantas e a todos os funcionários do Departamento de Ciência do Solo da ESALQ/USP, em especial, à Flávia, Marta, Martinha e Nancy, pelo apoio nesse período. Aos colegas do Laboratório de Solos da Universidade de Coimbra (Portugal), em especial ao Alexandre, Carla, Cristina, Dalila, Julia, Paula Chung, Rui, Sara, Sonia, Thiago, pela amizade e acolhida em sua terra. A todos que de alguma forma colaboraram para a realização deste trabalho. MUITO OBRIGADO!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 6 SUMÁRIO RESUMO………………………………….………………………………………………... 9 ABSTRACT………………………….……………………………………………………... 10 1 INTRODUÇÃO………………….……………………………………….………………. 11 2 DESENVOLVIMENTO …………………………………………………………………. 13 2.1 Revisão bibliográfica …………………………………………………………………... 13 2.1.1 Araucaria angustifolia (Bertoloni) O. Ktze .................................................................. 13 2.1.2 Impacto da ação antrópica em Florestas de Araucária .................................................. 15 2.1.3 Fogo em Florestas de Araucária …………………........................................................ 16 2.1.4 Efeito do reflorestamento e do fogo sobre atributos microbiológicos do solo.............. 17 2.1.5 Diversidade microbiana do solo ……………………………………………………… 19 2.1.6 Métodos de avaliação de comunidades microbianas dos solo ...................................... 23 2.1.6.1 Biomassa microbiana do solo, atividade respiratória e qüociente metabólico ........... 24 2.1.6.2 Capacidade de utilização de substratos de carbono (Biolog) ..................................... 24 2.1.6.3 Perfis de ácidos graxos ligados a ésteres de fosfolipídios (PLFAs) ……………….. 25 2.1.6.4 Medida da diversidade microbiana através de técnicas moleculares ………………. 26 2.2 Objetivos .......................................................................................................................... 30 2.2.1 Objetivo geral ………………………………………………………………………… 30 2.2.2 Objetivos específicos ………………………………………………………………… 30 2.2 Material e métodos ........................................................................................................... 31 2.2.1 Descrição da área de estudo .......................................................................................... 31 2.2.2 Esquema experimental .................................................................................................. 37 2.2.3 Amostragem .................................................................................................................. 38 2.2.4 Processamento das amostras ......................................................................................... 38 2.2.5 Avaliação dos atributos químicos ................................................................................. 39 2.2.6 Avaliação dos atributos microbiológicos ...................................................................... 39 2.2.7 Análises da estrutura das comunidades de Bacteria e Archaea por DGGE.................. 40 2.2.7.1 Extração do DNA total do solo .................................................................................. 40 2.2.7.2 PCR ………………………………………………………………………………… 41 2.2.7.3 DGGE ......................................................................................................................... 42 7 2.2.8 Análise de comunidades de Bacteria através do seqüenciamento de clones do gene rRNA 16S ..................................................................................................................... 43 2.2.8.1 Seleção das amostras .................................................................................................. 43 2.2.8.2 Amplificação de fragmentos do gene rRNA 16S ....................................................... 43 2.2.8.3 Purificação dos amplicons .......................………………………………………….. 43 2.2.8.4 Clonagem dos amplicons do gene rRNA 16S .................…………………………... 44 2.2.8.5 Extração do DNA plasmidial ………………………………………………………. 45 2.2.8.6 Seqüenciamento dos clones do gene rRNA 16S ……………………………….…... 46 2.2.9 Capacidade de utilização de substratos de carbono (Biolog) ......……………...……... 46 2.2.10 Perfis de ácidos graxos ligados a ésteres de fosfolipídios (PLFAs) ………..………. 47 2.2.11 Análise dos dados ........................................................................................................ 49 2.2.11.1 Análise estatística de dados químicos e microbiológicos ........................................ 49 2.2.11.2 Análise da diversidade genética …………………………………………………... 49 2.2.11.3 Análises das seqüências do gene rRNA 16S ............................................................ 50 2.2.11.4 Análise estatística da capacidade de utilização de substratos de carbono (Biolog) 51 2.2.11.5 Análise estatística dos perfis de ácidos graxos ligados a ésteres de fosfolipídios (PLFAs) …………………………………………………………………………… 54 2.3 Resultados e Discussão ………………………………………………………………… 56 2.3.1 Atributos químicos do solo …………………………………………………………... 56 2.3.2 Atributos microbiológicos do solo …………………………………………………… 60 2.3.3 Análise canônica discriminante (ACD) de atributos químicos e microbiológicos do solo ..............................................................................................................................
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