Detecção de Ralstonia solanacearum em plantas infectadas de eucalipto via PCR em tempo real

• Ano de defesa: 2011
• Autor(a) principal: Pereira, Camila Santana
• Orientador(a): Alfenas, Acelino Couto
• Banca de defesa: Guimarães, Lúcio Mauro da Silva
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Genética e Melhoramento
• Departamento: Genética animal; Genética molecular e de microrganismos; Genética quantitativa; Genética vegetal; Me
• País: BR
• Palavras-chave em Português: Ralstonia solanacearum; Eucalipto; PCR em tempo real
• Palavras-chave em Inglês: Ralstonia solanacearum; Eucalyptus, real-time PCR
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::GENETICA::GENETICA VEGETAL
• Link de acesso: http://locus.ufv.br/handle/123456789/4789

Bacterial wilt caused by Ralstonia solanacearum is currently one of the most important bacterial diseases in eucalyptus. The use of healthy eucalyptus cuttings is the most effective method to prevent the introduction and spread of the pathogen in disease free areas. Infected plants can be identified visually bybacterial cell exudation from the host tissue and confirmed by analysis of PCR. However, in asymptomatic minicuttings with latent infections, in which the xylem shows low concentrations of bacterial cellsit is not possible to visualize wilting symptoms bacterial exudation. Thus, for certification thateucalyptus cuttings are pathogen free, it is necessary to employ a sensitive method able to detect the bacteria, even when present in low concentrations in the host tissue.This study aimed to evaluate the real-time PCR for detection of bacteria in the latent period of infection. Among six primers previously tested, only the oligo224R/224F was specific to R. solanancearum, that amplifies the r16S ribosomal gene region. Subsequently,it was developed a protocol to extract bacterial DNA from infected plant tissue consisting of tissue maceration in liquid nitrogen, addition of saline solution, centrifugation, discard of the supernatant and DNA extraction. Real-time PCR of fourinfected eucalyptus cuttings with R. solanacearum(UFV32)evaluated at 20 days after inoculationshowed 105 to 106 colony forming units (cfu) of R. solanacearum / g of host tissue. The detection limit of real-time PCR was 103cfu / g, copared with 107cfu / mL for the conventional PCR. The method proved to be efficient for detection and quantification of R. solanacearum directly from infected eucalyptus plant tissue.