Immunofluorescence colony-staining (IFC) for detection and quantification of Ralstonia (Pseudomonas) solanacearum biovar 2 (Race 3) in soil and verification of positive results by PCR and dilution plating

A procedure was developed for specific and sensitive quantitative detection of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil. It is b ased on immunofluorescence colony-staining (IFC) followed by confirmation o f the identity of fluorescent colonies by PCR-amplification or dilution pla ting on a semi-selective medium, SMSA. Addition of sucrose and the antibiot ics cycloheximide and crystal violet to the non-selective trypticase soy br oth agar resulted in increased colony size and staining intensity of R. sol anacearum in IFC. Verification of IFC-results by picking cells from IFC-pos itive colonies followed by dilution plating of the suspended cells on SMSA was highly efficient. The success rate was 92% and 96% with 'spiked' and na turally contaminated soils respectively. Several other bacterial species wh ich cross-reacted with polyclonal antibodies in IFC also grew on SMSA and w ere difficult to distinguish from R. solanacearum, thereby necessitating co nfirmation of the results. Rapid verification of IFC-positive results direc tly by PCR-amplification with primers D2/B specific to division 2 of R. sol anacearum had a success rate of 86% and 96% with 'spiked' and naturally con taminated soil samples, respectively. Primers D2/B reacted with all R. sola nacearum division 2 strains, and strains of R. syzygii and the banana blood disease bacterium, but not with saprophytic bacteria cross-reacting in IFC with R. solanacearum antibodies. In comparative tests, IFC was able to det ect consistently ca. 100 cfu g(-)1 of soil, a detection level similar to th at found with direct plating on SMSA, but less laboriously, whereas detecti on level with a bioassay on tomato plants was only 10(4)-10(5) cfu g(-1) of soil.