Final report, September 29, 1992-December 30, 1994 : a DNA-based assay to detect Xanthomonas campestris pv. campestris

The most effective means of control of black rot is the use of disease-free seed. However, seeds are currently assayed using techniques that are slow, expensive, and lack the sensitivity desired. This report summarizes the results of a project to develop a new assay method utilizing the Polymerase Chain Reaction (PCR) increasing the speed and sensitivity of assays at a reduced overall cost. In previous work, a region of the Xcc genome was identified with specificity for pathogenic strains of the bacterium and the DNA sequence was determined and utilized to generate oligonucleotide primers for use in PCR. Three primer pairs showed promise for use in PCR following tests of 21 primers in 32 pairwise combinations. The primer pairs reacted with 250 of 254 pathogenic strains from a geographically diverse collection, and did not react with 111 of 113 non-pathogenic strains. Using these primer pairs, a hemi-nested PCR assay was developed that is effective using either genomic DNA or intact bacterial cells. Preliminary tests using seed wash extracts from 20 cabbage seed lots showed 100% correspondence in detection of Xcc between traditional plating and PCR assays. (Author abstract, modified)