Colony PCR-single strand confirmation polymorphism for the detection of Ralstonia solanacearum in tomato.
In India bacterial wilt caused by Ralstonia solanacearum is the most destructive disease of vegetable plants. In an attempt to identify R. solanacearum by direct colony Polymerase Chain Reaction-Single Strand Confirmation Polymorphism (PCR-SSCP) technique. R. solanacearum was isolated and confirmed by biochemical, physiological, hypersensitivity and pathogenicity tests. In addition, 16S rRNA and specific PCR primers were used to detect the pathogen in infected plant material/soil sample. In order to perform a DNA-based diagnostic test, availability of a rapid and easy-to-perform DNA extraction protocol is essential. In the present study we evaluated colony PCR-SSCP as the easiest way to amplify the target DNA and PCR-SSCP is a simple and powerful technique for identifying sequence changes in amplified DNA. We have developed colony PCR-SSCP technique to identify phytopathogenic bacteria by the PCR product without DNA isolation was denatured and separated on a non-denaturing polyacrylamide gel. SSCP banding patterns were detected by silver staining of nucleic acids and compared with the other phytopathogenic bacteria. The application of colony PCR-SSCP technique for the detection and diagnosis of R. solanacearum is discussed in the present paper.