Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes.
During the past few years, Ralstonia solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, a method was developed based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of 5 R. solanacearum strains and a R. pickettii strain were PCR amplified, sequenced, and analysed by sequence alignment. This resulted in the construction of an unrooted tree, and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into 2 clusters. Based on the alignments, 2 specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related R. syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, 2 independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissues of the weed host Solanum dulcamara (bittersweet) in contaminated areas.