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Pseudomonas Fluorescentes Provenientes Da Rizosfera De Solanáceas No UNIVERSIDADE DE BRASÍLIA INSTITUTO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE FITOPATOLOGIA PROGRAMA DE PÓS-GRADUAÇÃO EM FITOPATOLOGIA Pseudomonas fluorescentes provenientes da rizosfera de solanáceas no controle de Ralstonia solanacearum em tomateiro JOSEFA NEIANE GOULART BATISTA Brasília - DF 2015 JOSEFA NEIANE GOULART BATISTA Pseudomonas fluorescentes provenientes da rizosfera de solanáceas no controle de Ralstonia solanacearum em tomateiro Dissertação apresentada à Universidade de Brasília como requisito parcial para a obtenção do título de Mestre em Fitopatologia pelo Programa de Pós Graduação em Fitopatologia. Orientador Carlos Hidemi Uesugi, Dr. BRASÍLIA DISTRITO FEDERAL – BRASIL 2015 FICHA CATALOGRÁFICA Batista, G. N. J. Pseudomonas fluorescentes provenientes da rizosfera de solanáceas no controle de Ralstonia solanacearum em tomateiro. Josefa Neiane Goulart Batista. Brasília, 2015 Número de páginas p.: 49. Dissertação de mestrado - Programa de Pós-Graduação em Fitopatologia, Universidade de Brasília, Brasília. Murcha Bacteriana, controle biológico, Pseudomonas, tomate. I. Universidade de Brasília. PPG/FIT. II. Título. Pseudomonas fluorescentes provenientes da rizosfera de solanáceas no controle de Ralstonia solanacearum em tomateiro. Dedico a minha filha Júlia, ao meu pai Josvaldo, a minha mãe Nelma, a minha irmã Josiane e minha sobrinha Giovanna. Agradecimentos A Deus. Aos meus pais Josvaldo e Nelma. A minha filha Júlia, minha irmã Josiane e minha sobrinha Giovanna. Ao meu orientador Professor Carlos Hidemi Uesugi. Aos colegas do mestrado e doutorado Elenice, João Gilberto, Pedro Victor, Ricardo, Cleia, Maurício, Rafaela, Maria Geane, Karina, Carina. Aos funcionários do departamento de fitopatologia José Ribamar, José Cezar, Arlindo, Arenildo, Maria e Mariza Sanchez (in memoriam). Aos professores: Juvenil Cares, Cléber Furlanetto, Helson Mário, Adalberto Café, Rita de Cássia Pereira Carvalho, Renato Resende, Alice Nagata, Robert Miller, José Carmine Dianese e Luíz Eduardo Blum. Aos funcionários da Estação Experimental de Biologia, Fábio Fonseca, Evandro Calixto, Aldo Mostardi, Francisco e Olinda. Ao Programa de Pós Graduação em Fitopatologia da Universidade de Brasília. A Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) pela bolsa. Trabalho realizado junto ao Departamento de Fitopatologia do Instituto de Ciências Biológicas da Universidade de Brasília, sob orientação do Dr. Carlos Hidemi Uesugi. Apoio Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes). Pseudomonas fluorescentes provenientes da rizosfera de solanáceas no controle de Ralstonia solanacearum em tomateiro. JOSEFA NEIANE GOULART BATISTA DISSERTAÇÃO APROVADA em __/__/____por: ___________________________________________ Dr. Luiz Eduardo Bassay Blum (Examinador Interno) ___________________________________________ Dr. Reinaldo José de Miranda Filho (Examinador Externo) ___________________________________________ Dr. Carlos Hidemi Uesugi (Orientador Presidente) BRASÍLIA – DISTRITO FEDERAL BRASIL 2015 Sumário Índice de Figuras ....................................................................................................................... vi Índice de Tabelas ...................................................................................................................... vii Resumo .................................................................................................................................... viii Abstract ..................................................................................................................................... ix 1. Introdução ........................................................................................................................... 1 2. Objetivo Geral ..................................................................................................................... 3 2.1 Objetivos específicos ....................................................................................................... 3 3. Revisão bibliográfica .......................................................................................................... 4 3.1 A cultura do tomateiro ................................................................................................. 4 3.2 Murcha bacteriana do tomateiro .................................................................................. 6 3.3 Controle biológico ..................................................................................................... 12 3.4 Pseudomonas ............................................................................................................. 16 4. Material e Métodos ........................................................................................................... 18 4.1 Localização da área e a coleta do material ................................................................ 18 4.2 Locais dos ensaios ..................................................................................................... 19 4.3 Isolamento, purificação e manutenção de Pseudomonas fluorescentes. ................... 19 4.4 Testes in vitro – Prospecção da antibiose por difusão em dupla camada .................. 21 4.5 Seleção dos isolados .................................................................................................. 22 4.6 Testes in vivo: Efeito dos isolados de Pseudomonas fluorescentes na supressão de Ralstonia solanacearum no tomateiro. ................................................................................. 22 5. Resultados ......................................................................................................................... 24 5.1 Isolamento de Pseudomonas fluorescentes ............................................................... 24 5.2 Testes in vitro – Prospecção da antibiose por difusão em dupla camada. ................. 24 5.3 Testes in vivo: Efeito dos isolados de Pseudomonas fluorescentes na supressão de Ralstonia solanacearum no tomateiro. ................................................................................. 26 6. Discussão .......................................................................................................................... 33 7. Conclusões ........................................................................................................................ 39 8. Referências bibliográficas ................................................................................................. 40 v Índice de Figuras Figura 1. Etapas do teste in vitro, de antibiose por difusão em dupla camada, para seleção de Pseudomonas fluorescentes antagonistas a Ralstonia solanacearum, segundo Romeiro (2007, com modificações) citado por Marques (2012). ....................................................................... 22 Figura 2. Isolados de Pseudomonas fluorescentes sob luz ultravioleta. .................................. 24 Figura 3. Teste de antibiose in vitro pelo método de difusão em dupla camada contra Ralstonia solanacearum isolado UnB 1173, halos de inibição formados pelos isolados JCP 8.1 e JCP 8.5. ............................................................................................................................ 25 Figura 4. Relação entre plantas com e sem sintoma de Murcha Bacteriana, no experimento com a estirpe de R. solanacearum UnB 1173. Plantas tratadas com isolados de Pseudomonas fluorescentes. ............................................................................................................................ 28 Figura 5. Relação entre plantas com e sem sintoma de Murcha Bacteriana, no experimento com a mistura das estirpes UnB 1033, UnB 1103 e UnB 1173 de R. solanacearum. Plantas tratadas com isolados de Pseudomonas fluorescentes. ............................................................ 29 Figura 6. A-Sintoma de murcha em tomate. B- teste do copo feito em planta com sintoma de murcha. ..................................................................................................................................... 33 vi Índice de Tabelas Tabela 1. Hospedeiras e local de ocorrência de Ralstonia solanacearum de acordo com raças e biovares (Buddenhagen et al., 1962; Hayward, 1991). .............................................................. 8 Tabela 2. Classificação para Ralstonia solanacearum baseado no genótipo (Fegan & Prior, 2005). .......................................................................................................................................... 9 Tabela 3. Nome popular, nome científico, número de amostras coletadas e número de isolados obtidos das espécies vegetais de onde foram obtidos os isolados de Pseudomonas fluorescentes. ............................................................................................................................ 19 Tabela 4. Isolados de Pseudomonas fluorescentes, nome científico das espécies de onde foram coletados e local de origem dos isolados. ................................................................................ 20 Tabela 5. Inibição de Ralstonia solanacearum por isolados de Pseudomonas fluorescentes provenientes de diferentes solanáceas. ..................................................................................... 25 Tabela 6. Efeitos dos isolados de Pseudomonas fluorescentes no controle da Murcha Bacteriana causada por R. solanacearum UnB 1173. .............................................................. 27 Tabela 7. Efeitos dos isolados de Pseudomonas fluorescentes no controle
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