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Populações De Alfa E Beta-Rizóbios De Feijão-Caupi Naturalmente Estabelecidas Em Solos Do Semiárido Tropical

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Populações De Alfa E Beta-Rizóbios De Feijão-Caupi Naturalmente Estabelecidas Em Solos Do Semiárido Tropical UNIVERSIDADE FEDERAL RURAL DE PERNAMBUCO ALEKSANDRO FERREIRA DA SILVA POPULAÇÕES DE ALFA E BETA-RIZÓBIOS DE FEIJÃO-CAUPI NATURALMENTE ESTABELECIDAS EM SOLOS DO SEMIÁRIDO TROPICAL Recife 2019 Aleksandro Ferreira da Silva Engenheiro agrônomo Populações de alfa e beta-rizóbios de feijão-caupi naturalmente estabelecidas em solos do semiárido tropical Tese apresentada ao Programa de Pós- Graduação em Ciência do Solo, da Universidade Federal Rural de Pernambuco, como parte dos requisitos para obtenção do título de Doutor em Ciência do Solo Orientadora Dra. Ana Dolores Santiago de Freitas Coorientadores Dr. Paulo Ivan Fernandes Júnior Dra. Maria do Carmo Catanho Pereira de Lyra Recife 2019 Autorizo a reprodução e divulgação total ou parcial deste trabalho, por qualquer meio convencional ou eletrônico, para fins de estudo e pesquisa desde que citada a fonte. Dados Internacionais de Catalogação na Publicação (CIP) Sistema Integrado de Bibliotecas da UFRPE Biblioteca Centra, Recife-PE, Brasil S586p Silva, Aleksandro Ferreira da Populações de alfa e beta-rizóbios de feijão-caupi naturalmente estabelecidas em solos do semiárido tropical / Aleksandro Ferreira da Silva. – 2019. 123 f. : il. Orientadora: Ana Dolores Santiago de Freitas. Coorientadores: Paulo Ivan Fernandes Júnior e Maria do Carmo Catanho Pereira de Lyra. Tese (Doutorado) – Universidade Federal Rural de Pernambuco, Programa de Pós-Graduação em Ciências do Solo, Recife, BR-PE, 2019. Inclui referências e apêndice(s). 1. Nitrogênio – Fixação 2. Leguminosa 3. Simbiose 4. Vigna unguiculata 5. Caatinga I. Freitas, Ana Dolores Santiago de, orient. II. Fernandes Júnior, Paulo Ivan, coorient. III. Lyra, Maria do Carmo Catanho Pereira de, coorient. IV. Título CDD 631.4 ALEKSANDRO FERREIRA DA SILVA Populações de alfa e beta-rizóbios de feijão-caupi naturalmente estabelecidas em solos do semiárido tropical Tese apresentada ao Programa de Pós-graduação em Ciência do Solo, da Universidade Federal Rural de Pernambuco, como parte dos requisitos para obtenção do título de Doutor em Ciência do Solo. Aprovada em 26 de fevereiro de 2019 _______________________________________________________________ Dra. Ana Dolores Santiago de Freitas Orientadora Universidade Federal Rural de Pernambuco BANCA EXAMINADORA ___________________________________________________ Profa. Dra. Lindete Míria Vieira Martins Universidade do Estado da Bahia ___________________________________________________ Dra. Maria do Carmo Catanho Pereira de Lyra Instituto Agronômico de Pernambuco ___________________________________________________ Prof. Dr. Jakson Leite Universidade Federal de Alagoas ___________________________________________________ Prof. Dr. Mario de Andrade Lira Júnior Universidade Federal Rural de Pernambuco Aos meus pais (Floraci Ferreira e Izidorio Anacleto), irmãos (Fábio Ferreira e Douglas Ferreira) e minha amada namorada (Kelly Alexsandra), por me ensinarem o valor do verdadeiro amor. DEDICO AGRADECIMENTOS A Deus, pelo dom da vida, por tudo que tenho conseguido e por ter me lapidado a cada manhã, proporcionando inconformismo construtivo, mantendo os meus pés no chão e também por não ter me deixado fugir nenhuma vez do caminho por mim escolhido. Aos meus pais (Floraci Ferreira da Silva e Izidorio Anacleto da Silva) e irmãos (Fábio Ferreira da Silva e Douglas Ferreira da Silva), por toda educação, amor, carinho, respeito e apoio durante toda minha vida. A minha namorada, Kelly Alexsandra Souza Menezes, por sempre acreditar em mim, pela paciência, amor, companheirismo e exemplo de ser humano. Te amarei eternamente. A minha orientadora, Dra. Ana Dolores Santiago de Freitas pela orientação, amizade, apoio, incentivo, paciência e principalmente pelo exemplo de pessoa que é. Agradeço imensamente por todos os ensinamentos. Aos co-orientadores, Dr. Paulo Ivan Fernandes Júnior e Maria do Carmo Catanho Pereira de Lyra, pelas contribuições. A Dr. Paulo agradeço também por todas as oportunidades desde a graduação e valiosas contribuições nessa reta final. A professora Lindete Míria Vieira Martins por ter feito parte da minha vida acadêmica desde o início. Agradeço pela confiança, ensinamentos, conselhos, e por ser uma pessoa incrível. Aos bolsistas Andressa e Pablo pela amizade e suporte em várias etapas do trabalho, serei sempre grato. Aos amigos que fizeram parte da realização desse sonho: Vinícius Gomes, Juscélia Ferreira, Mayame Brito, Adriana Bezerra, Juliet Emília, Emanuelle Maria e Cícero Abraão. Ao Grupo de Fixação Biológica de Nitrogênio – GFBN da UFRPE: Augusto Cesar, Sueide Karina, Jéssica Rafaella, Leandro Reis, Layane Barbosa e Edilandia. Aos amigos que fazem e fizeram parte do Laboratório de Microbiologia do Solo da Embrapa Semiárido: Pamella Sena, Valéria Borges, Viviane, Claudia, Tailane, Thaise, Lucas e Reginaldo. Agradeço pela amizade, acolhimento e ensinamentos. A professora Carolina Etienne pelo carinho e convivência desde o mestrado. Obrigado pela amizade e ensinamentos. Ao amigo Jakson Leite por sempre se dispor a tirar minhas dúvidas inerentes ao trabalho. Agradeço pela oportunidade de ter me apresentado a professora Lindete e, consequentemente, por ter despertado em mim o interesse em fazer pesquisa. É um exemplo de profissional. A professora Giselle Fracetto e ao professor Felipe Fracetto pela amizade, ensinamentos e confiança. A Universidade Federal Rural de Pernambuco, em especial ao PPGCS, pela oportunidade, acolhimento e condições concedidas para a realização do curso. A Empresa Brasileira de Pesquisa Agropecuária – EMBRAPA, pela parceria na realização da pesquisa, sob supervisão de Dr. Paulo Ivan Fernandes Júnior. A Sra. Maria do Socorro Santana, secretária do Programa de Pós-Graduação em Ciência do Solo pela cordialidade e presteza. Aos professores da banca examinadora, pela participação e valorosas sugestões atribuídas a este trabalho. A todos os professores do PPGCS, pelos ensinamentos transmitidos. A todos que, direta ou indiretamente, contribuíram para a realização deste sonho. MUITO OBRIGADO Populações de alfa e beta-rizóbios de feijão-caupi naturalmente estabelecidas em solos do semiárido tropical RESUMO Recentemente, importantes contribuições para o avanço do conhecimento da ecologia e diversidade de micro-organismos em diferentes ecossistemas brasileiros tem levado ao enriquecimento da taxonomia de rizóbios, inclusive com a descrição de novas espécies de microssimbiontes. A grande diversidade de condições edafoclimáticas (tipos de solo, precipitação média anual, etc.) e de características da vegetação da caatinga, tornam esse bioma um campo vasto para estudos de ecologia de rizóbios e para buscas de novas estirpes elites para recomendação como inoculantes. Objetivou-se com este trabalho, avaliar as características das populações de alfa e beta-rizóbios naturalmente estabelecidas em solos do Semiárido de Pernambuco cobertos com vegetação nativa. Para a realização de uma amostragem representativa das diferentes condições edafoclimáticas, foram selecionados pontos em áreas com cobertura de caatinga densa (com pouca interferência antrópica) e nas principais classes de solos ocorrentes no bioma: Argissolo, Neossolo Litólico, Neossolo Quartzarênico, Neossolo Regolítico, Latossolo, Luvissolo e Planossolo. As comunidades rizobianas nativas das amostras de solo coletadas foram acessadas em experimento em casa de vegetação utilizando o feijão- caupi como planta-isca. Essa espécie foi escolhida por sua conhecida habilidade de estabelecer simbiose com comunidades de alfa e beta-rizóbios. Dos nódulos coletados no experimento de planta-isca foram obtidos 434 isolados, dos quais 338 foram positivos para a amplificação do gene nodC (nodC+). Em sua maioria são isolados de crescimento rápido, com metabolismo que acidifica o meio de cultura e produtores de muito muco. Os 338 isolados foram submetidos à Análise de Restrição do DNA Ribossomal Amplificado (ARDRA), para avaliar a variabilidade genética da coleção nodC+. A análise conjunta dos perfis de restrição do 16S rRNA, obtida a partir das endonucleases HaeIII, MspI e Hin6I, permitiu a formação de 209 grupos a 100 % de similaridade, evidenciando grande variabilidade genética entre os isolados. A partir do dendrograma do ARDRA, determinou-se um “threshold” de 75 % de similaridade e, a partir desse agrupamento, selecionou-se representantes dos grupos (um total de 60) para serem submetidos à amplificação e sequenciamento do gene 16S rRNA. Dos 60 isolados, selecionou- se, aleatoriamente, 30 para serem sequenciados os genes nodC, recA e gyrB. Levando em consideração o sequenciamento do 16S rRNA, Paraburkholderia (18) foi o gênero com maior número de isolados, seguido de Rhizobium (13) e Bradyrhizobium (10). Analisando a árvore com base nos 24 isolados com sequência de boa qualidade do gene recA, verificou-se que oito isolados fazem parte da classe das betaproteobactéria pertencentes ao gênero Paraburkholderia. Para os alfa-rizóbios, apenas 2 isolados agruparam com o gênero Rhizobium e 12 ficaram agrupados com Bradyrhizobium. A maioria dos isolados agrupados com o gênero Paraburkholderia na árvore do gene nodC apresentaram alta similaridade com as estirpes referência. Por apresentarem sequências de recA e gyrB divergentes, na árvore concatenada, os isolados C88-4 e C59-4 formaram um grupo com baixa similaridade com as estirpes de referência de beta-rizóbios. Em alfa-rizóbios o comportamento foi semelhante. Este padrão de agrupamento demonstra a elevada variabilidade genética dos isolados e aponta para a existencia de novos grupos taxonômicos dentre
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