Development of immunofluorescence colony staining (IFC) for detection of Xanthomonas campestris pv. vesicatoria and Clavibacter michiganensis subsp. michiganensis in tomato seeds.

Immunofluorescence colony-staining (IFC) is based on sample pour plating in combination with immunofluorescence staining for recognition of the target colony. IFC was optimised for detecting Xanthomonas campestris pv. vesicatoria (Xcv) and Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato seed lots. Optimum incubation periods for colony growth were 2 d for Xcv and 3 d for Cmm. For both targets, the fluorescence intensity of the target colonies was positively correlated with the incubation temperature in the range tested and the period of colony growth. In general a lower brilliance was observed in IFC preparations of pure cultures than when seed extracts was added. The maximum density of bacterial colonies in the pour plates for both targets was about a thousand times higher per square cm as for surface plating. In comparison with IF (immunofluorescent cell staining) and isolation, IFC was 10 times more sensitive than IF. In samples with a target to saprophyte ratio higher than 1/100, 10 to 100 times higher numbers of positive colonies of the target were found in IFC using 16 mm diameter wells in miniaturized routine system, than by dilution plating on trypticase soy agar (TSA) and selective media. The recovery of Xcv in pour plates at various density of the target was still 46-68% for samples with a high microbial background (105 or more colonies per cm). For the slow growing Clavibacter, recovery was reduced to ca. 12-17% for samples with a high microbial background. A series of 23 commercial seed lots were tested for Xcv with IF and IFC. Using IF seven samples were positive based on IF-positive cells in the preparation. In IFC no typical fluorescent positive Xcv colonies were observed. From the weak cross-reacting colonies, viable cells were collected for pure culturing. Verification confirmed that the colonies of the weakly cross-reacting species belonged to other species