Indirect immunofluorescence colony staining method for detecting bacterial pathogens of tomato

An indirect immunoflouorescence colony staining method was developed for the detection of important seed-borne bacterial pathogens of tomato. The method involves the use of specific antiserum for initial binding of target bacteria and visualization of positive colonies with a commercially available secondary antiserum conjugated with FITC and observed under a fluorescence microscope. The indirect method is especially suitable for laboratories, seed companies, and quarantine stations which have no facilities for conjugation of primary antiserum. It is more economical and overcomes the problems generally encountered with variable conjugate quality in new batches of conjugates prepared from the same stock of primary antiserum. The assay is easy to perform and results can be easily assessed by visual scoring or image analyser. Results are available in 4–5 days as compared to 30–45 days in traditional methods. The resulting bacterial culture can be tested by PCR or host infectivity and a culture can be stored for future reference. Used in combination with highly specific antibodies (commercially available monoclonal and recombinant antibodies) it can be used as a very sensitive detection tool and has application potential in localization studies as well. Choosing the right secondary conjugate is however necessary to get best results in the assay

Indirect immunofluorescence colony staining method for detecting bacterial pathogens of tomato

An indirect immunoflouorescence colony staining method was developed for the detection of important seed-borne bacterial pathogens of tomato. The method involves the use of specific antiserum for initial binding of target bacteria and visualization of positive colonies with a commercially available secondary antiserum conjugated with FITC and observed under a fluorescence microscope. The indirect method is especially suitable for laboratories, seed companies, and quarantine stations which have no facilities for conjugation of primary antiserum. It is more economical and overcomes the problems generally encountered with variable conjugate quality in new batches of conjugates prepared from the same stock of primary antiserum. The assay is easy to perform and results can be easily assessed by visual scoring or image analyser. Results are available in 4–5 days as compared to 30–45 days in traditional methods. The resulting bacterial culture can be tested by PCR or host infectivity and a culture can be stored for future reference. Used in combination with highly specific antibodies (commercially available monoclonal and recombinant antibodies) it can be used as a very sensitive detection tool and has application potential in localization studies as well. Choosing the right secondary conjugate is however necessary to get best results in the assay

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 specie