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During the past few years,Ralstonia (Pseudomonas )solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of fiveR. solanacearum strains and oneRalstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in whichR. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed forR. solanacearum and the closely relatedRalstonia 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, two independent targets for identification ofR. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed hostSolanum dulcamara (bittersweet) in contaminated areas.

Detection of Ralstonia solanacearum , Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes