Detection of Viable Cells of Ralstonia solanacearum in Soil Using a Semiselective Medium and a PCR Technique

A semiselective medium (PCCG) and a polymerase chain reaction (PCR) technique were combined to detect viable cells of Ralstonia (Pseudomonas) solanacearum E. F. Smith (synonym Burkholderia solanacearum) in soil. DNA was extracted from 92 strains of soil bacteria including R. solanacearum that grew on PCCG and then used as template for PCR with a pair of primers designed to amplify a single fragment (281 bp) of R. solanacearum DNA. The 281-bp fragment was amplified only from DNA of R. solanacearum (12 strains). For DNA from soil bacteria other than R. solanacearum, the PCR amplification generated no products for 66 strains, a single DNA band with different sizes from 281 bp for 2 strains, and several DNA bands for 12 strains. Southern analysis showed that any of those products other than the 281-bp fragment had no homology with the 281 -bp fragment of R. solanacearum, indicating the specificity of the primers to generate the 281-bp fragment from R. solanacearum. A simple method consisting of a plating step using PCCG and succeeding PCR for amplifying the 281-bp fragment from colonies on PCCG was described