Abstract

The aim of the present study is to develop a scheme for identification of Ralstonia solanacearum with high specificity based on conserved genomic regions. Short Tandem Repeats (STRs) in R. solanacearum genome were searched using Tandem Repeat Finder software. A total of 189 and 74 STRs were found in chromosomal and megaplasmid DNA respectively. Sequence homology of these STRs analyzed using BLAST showed that out of total 273 STRs only nine were found unique for R. solanacearum. Correspondingly nine pairs of primers were synthesized for the flanking regions of these STRs. Sequence homology of these primer pairs carried using BLAST revealed that out of the nine pairs, one pair uniquely matched only at a single locus in the Ralstonia chromosomal DNA. Polymerase chain reaction (PCR) amplification using templates from 44 different isolates of R. solanacearum yielding single sized amplicon ascertains the versatility and unambiguousness of the designed primers. The fact that the primer pair did not amplify the genomic DNA of 12 soil bacteria establishes the specificity to R. solanacearum. Thus the novel and specific primers designed for R.solanacearum would enable fast and definitive identification of the lethal pathogen. The designed primers would be of great importance for detection of R. solanacearum in seed tubers, soil and water streams thus helping in establishing preventive measures for checking pathogen spread. This would also allow facilitating epidemiological studies allowing better surveillance of this pathogen.

Key words: Ralstonia solanacearum, identification, specific primers, differentiation, polymerase chain reaction.