SENSITIVE AND SPECIFIC DETECTION OF XANTHOMONAS-CAMPESTRIS PV PELARGONII WITH DNA PRIMERS AND PROBES IDENTIFIED BY RANDOM AMPLIFIED POLYMORPHIC DNA ANALYSIS

The random amplified polymorphic DNA method was used to distinguish st rains of Xanthomonas campestris pv. pelargonii from 21 other Xanthomon as species and/or pathovars. Among the 42 arbitrarily chosen primers e valuated, 3 were found to reveal diagnostic polymorphisms when purifie d DNAs from compared strains were amplified by the PCR. The three prim ers revealed DNA amplification patterns which were conserved among all 53 strains tested of X. campestris pv. pelargonii isolated from vario us locations worldwide. The distinctive X. campestris pv. pelargonii p atterns were clearly different from those obtained with any of 46 othe r Xanthomonas strains tested. An amplified 1.2-kb DNA fragment, appare ntly unique to X. campestris pv. pelargonii by these random amplified polymorphic DNA tests, was cloned and evaluated as a diagnostic DNA pr obe. It hybridized with total DNA from all 53 X. campestris pv. pelarg onii strains tested and not with any of the 46 other Xanthomonas strai ns tested. The DNA sequence of the terminal ends of this 1.2-kb fragme nt was obtained and used to design a pair of 18-mer oligonucleotide pr imers specific for X. campestris pv. pelargonii. The custom-synthesize d primers amplified the same 1.2 kb DNA fragment from all 53 X. campes tris pv. pelargonii strains tested and failed to amplify DNA from any of the 46 other Xanthomonas strains tested. DNA isolated from saprophy tes associated with the geranium plant also did not produce amplified DNA with these primers. The sensitivity of the PCR assay using the cus tom-synthesized primers was between 10 and 50 cells. The techniques us ed could provide a general method to identify pathovar-specific DNA pr imers for rapid, sensitive, and specific detection and identification of plant-pathogenic bacteria.