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Proteobacteria from Brazilian Soils

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Proteobacteria from Brazilian Soils IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF DIAZOTROPHIC β- PROTEOBACTERIA FROM BRAZILIAN SOILS KRISLE DA SILVA 2009 KRISLE DA SILVA IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF DIAZOTROPHIC β-PROTEOBACTERIA FROM BRAZILIAN SOILS Thesis presented to the Federal University of Lavras as part of the requirementes of Agricultural Microbiology Program to the obtainment of the title of “Doctor”. Major Professor Prof. Fátima Maria de Souza Moreira LAVRAS MINAS GERAIS - BRASIL 2009 Ficha Catalográfica Preparada pela Divisão de Processos Técnicos da Biblioteca Central da UFLA Silva, Krisle da. Identification and functional characterization of diazotrophic β- proteobacteria from Brazilian soils / Krisle da Silva. – Lavras : UFLA, 2009. 124 p. : il. Tese (doutorado) – Universidade Federal de Lavras, 2009. Orientador: Fátima Maria de Souza Moreira. Bibliografia. 1. Fixação biológica de nitrogênio. 2. Betaproteobacteria. 3. Leguminosas. 4. Floresta Amazônica. 5. Microbiologia do solo. I. Universidade Federal de Lavras. II. Título. CDD – 631.46 KRISLE DA SILVA IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF DIAZOTROPHIC β-PROTEOBACTERIA FROM BRAZILIAN SOILS Thesis presented to the Federal University of Lavras as part of the requirementes of Agricultural Microbiology Program to the obtainment of the title of “Doctor”. APROVED on 26th August 2009 Prof. Peter Vandamme UGhent Prof. Emanuel de Souza Maltempi UFPR Prof. Fábio Lopes Olivares UENF Prof. Ricardo Magela de Souza UFLA Profa. Patrícia Gomes Cardoso UFLA Prof. Fátima Maria de Souza Moreira UFLA (Major Professor) LAVRAS MINAS GERAIS – BRASIL A Deus, pela presença, amor incondicional, pela força para enfrentar as dificuldades e por preencher minha vida com pessoas maravilhosas. OFEREÇO A Ires Aurora da Silva, pelo amor, dedicação e companherismo, presentes durante toda minha vida. DEDICO AGRADECIMENTOS Primeiramente, a Deus, pois cada conquista, seja ela grande ou pequena, não seria possível sem sua presença em minha vida. Essa é, sem dúvida, mais uma conquista com as bênçãos de Deus. À Universidade Federal de Lavras, em especial ao Programa de Pós- Graduação em Microbiologia Agrícola, pela oportunidade de realização do curso. À Fundação de Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), pela concessão da bolsa de estudos. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), pela concessão da bolsa de estudos para a realização do doutorado sanduíche. Ao projeto TSBF-CIAT/GEF-UNEP, “Conservation and Sustainable Management of Below Ground Biodiversity”, pelo financiamento para execução deste trabalho. À professora Fátima Maria de Souza Moreira, pela orientação durante o mestrado e doutorado, pela confiança, pelo exemplo e amizade. Ao Prof. Peter Vandamme, pela excelente acolhida e pela coorientação, que foi muito positiva para a realização deste trabalho. A todos os professores do Programa de Pós-Graduação em Microbiologia Agrícola. Um agradecimento especial à pesquisadora Diva de Souza Andrade, pela amizade e iniciação em Microbiologia do Solo. Aos funcionários Marlene Aparecida de Souza e Manuel Aparecido da Silva, pela contribuição na realização deste trabalho e pela amizade. À funcionária do Laboratório de Microbiologia da Universidade de Ghent Evie de Brandt, pela paciência e contribuição a este trabalho. A Magda Aparecida Melo, por toda a dedicação durante o doutorado e pela grande disposição. A todos os amigos do Laboratório de Microbiologia do Solo, em especial Ligiane, Adrianinha, Rogério, Pedro e Michele Rocha. Aos membros da banca examinadora, professores Peter Vandamme, Emanuel Maltempi de Souza, Fábio Lopes Olivares, Ricardo Magela e Patrícia, pelas sugestões e críticas construtivas dadas a este trabalho. Às amigas de república durante mestrado e doutorado: Patrícia, Taís, Gláucia, Sílvia, Fabiana e Ludmila. Vocês foram minha nova família durante o perído em que vivemos juntas. Às amigas que fiz na Bélgica, que fizeram com que os dias nublados e frios se tornassem mais alegres: Inês, Tatiana, Luana, Jéssica, Tânia, Margo e Luciane. Aos meus grandes amigos e/ou irmãos: Meire, Amanda, Gláucia, Cândido e Sandro. Agradeço por todos os momentos que dividimos e que, sem dúvida, foram inesquecíveis. Tenho certeza de a nossa amizade não há distância ou tempo que desgate. Amo vocês. As minhas queridas amigas e/ou irmãs de Londrina, Daiane e Cintia. Apesar da distância, vocês estão sempre perto, seja no meu coração ou na minha mente. Amos vocês! A minha querida mãe, Ires Aurora da Silva, obrigada, pelo amor, exemplo de ser humano, educação e confiança. Amo você demais! Aos meus queridos familiares, Cleonice, Kátia, Diego, Ivani, Ladislau, Pedro e Murilo, pelo grande carinho. APRESENTAÇÃO Nesta tese apresenta-se parte dos resultados do projeto internacional “Conservação e Manejo Sustentado da Biodiversidade do Solo”, implementado em sete países tropicais - Brasil, Costa do Marfim, India, Indonesia, Quênia, Mexico e Uganda. Este projeto é coordenado pelo Tropical Soil Biology and Fertility Institute do CIAT (TSBF-CIAT) com cofinanciamento do "Global Environmental Facility" (GEF) e suporte para implementação do "United Nations Environment Program" (UNEP). O componente brasileiro do projeto é denominado BiosBrasil e é coordenado pela Universidade Federal de Lavras (UFLA) (www.biosbrasil.ufla.br). As opiniões expressas nesta publicação são de responsabilidade de seus autores e não necessariamente refletem aquelas das instituições dos autores, do "United Nations Environment Programme" ou do "Global Environmental Facility". SUMMARY Page TABLE LISTS.................................................................................................. i FIGURE LISTS................................................................................................ iii GENERAL ABSTRACT.................................................................................. v RESUMO GERAL........................................................................................... vi CHAPTER 1..………………………………………………………………... 01 1 Introdução Geral…………………………………………………………… 02 2 Referencial Teórico........................................................................................ 04 2.1 O gênero Burkholderia............................................................................... 04 2.2 Burkholderia spp. como bactérias promotoras do crescimento vegetal (PGPB).............................................................................................................. 05 2.3 Estirpes de Burkholderia e fixação biológica de nitrogênio...................... 08 2.4 Complexo Burkholderia cepacia (Bcc)………………………………….. 13 2.5 Complexo B. cepacia X ambiente.............................................................. 14 2.6 Estudos de identificação de Burkholderia spp............................................ 18 2.7 O gênero Cupriavidus................................................................................. 21 2.8 O gênero Cupriavidus e a fixação biológica de nitrogênio........................ 23 3 Referências Bibliográficas............................................................................. 31 CHAPTER 2: Diazotrophic Burkholderia spp. (B. fungorum, B. contaminans and B. lata) isolated from the Amazon region exhibit phenotypical, functional and genetic diversity………………………………. 53 1 Resumo.......................................................................................................... 54 2 Abstract.......................................................................................................... 55 3 Introduction……………………………………………………………….... 56 4 Material and Mhetods……………………………………………................ 58 4.1 Origin of isolates…………………………………………………………. 58 4.2 Preliminary identification………………………………………………... 62 4.3 Phenotypic characterization….................................................................... 62 4.3.1 Cultural characterization………………………………………………. 62 4.3.2 Biochemical characterization………………………………………….. 63 4.3.3 SDS-PAGE of whole-cell proteins…………………………………….. 63 4.4 Plant growth promotion traits…………………………………………... 63 4.4.1 Phosphate solubilization assay…………………………………………. 63 4.4.2 Nitrogenase activity……………………………………………………. 64 4.4.3 Antifungal-activity of Burkholderia isolates…………………………... 64 4.5 Genetic characterization…………………………………………………. 65 4.5.1 16S rRNA sequencing…………………………………………………. 65 4.5.2 gyrB gene sequencing………………………………………………….. 66 4.5.3 Bcc-specifc recA gene and MLST……………………………………... 67 5 Results……………………………………………………………………… 69 5.1 Phenotypic characterization……………………………………………… 69 5.1.1 Cultural characterization……………………………………………….. 69 5.1.2 Biochemical characterization…………………………………………... 69 5.1.3 SDS-PAGE of whole-cell proteins…………………………………….. 69 5.2 Plant growth promotion traits……………………………………………. 73 5.2.1 Phosphate solubilization assay…………………………………………. 73 5.2.2 Nitrogenase activity…………………………………………………… 73 5.2.3 Antifungal-activity of Burkholderia isolates…………………………... 74 5.3 Genetic characterization…………………………………………………. 75 5.3.1 16S rRNA sequencing…………………………………………………. 75 5.3.2 gyrB genes amplification and sequencing……………………………... 76 5.3.3 Bcc-specifc recA gene and MLST……………………………………... 78 6 Discussion………………………………………………………………….. 80 6.1 Phenotypic characteristics and Plant growth promotion traits .…………. 80 6.2 Genotipic characterization……………………………………………….. 81 7 Conclusions.................................................................................................... 83 8 References.....................................................................................................
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