POLYMERASE CHAIN-REACTION FOR VERIFICATION OF FLUORESCENT COLONIES OFERWINIA-CHRYSANTHEMI AND PSEUDOMONAS-PUTIDA WCS358 IN IMMUNOFLUORESCENCE COLONY STAINING

The potential of polymerase chain reaction (PCR) for verifying the ide ntity of colonies stained by the immunofluorescence colony-staining (I FC) procedure was investigated. Using primers directed against conserv ed sequences of the pectate lyase-genes coding for isozymes PLa, PLd a nd PLe of Erwinia chrysanthemi, the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained sample s with pure cultures. In pour plates with mixtures of Erw. chrysanthem i and non-target colonies from potato peel extracts, the identity of 9 0% of 113 target colonies was confirmed. Using primers directed agains t sequences of the ferric-pseudobactin receptor gene pupA of Pseudomon as putida WCS358, the identity of 96% of 22 target colonies was confir med in IFC-stained samples with pure cultures. In pour plates with mix tures of Ps. putida WCS358 and non-target bacteria from compost extrac ts, the identity of 59% of 108 fluorescent colonies was confirmed by P CR. It was shown that components from non-target bacteria lowered the threshold level of PCR for Ps. putida WCS358 100-fold.