A SIMPLE AND EFFICIENT PCR METHOD FOR THE SPECIFIC DETECTION OF PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA IN BEAN-SEEDS

Using Southern blot hybridizations, it was found that the gene encodin g the phaseolotoxin-insensitive ornithyl carbamoyl transferase (argK) was specific for Pseudomonas syringae pv. phaseolicola, the causal age nt of the halo-blight disease. Based on these findings, a PCR protocol was developed for the specific detection of P.syringae. pv. phaseolic ola in water-extracts of soaked bean seed. For this PCR protocol, two oligonucleotide primers were designed, based on the sequence of argK, which allowed the detection of a specific 1 kb fragment. The protocol is simple since PCR was directly applied to bacterial suspensions, thu s avoiding DNA extraction. The sensitivity of detection was increased by allowing the bacteria present in seed extracts to multiply in semi- selective media for 18 h prior to PCR amplification. The detection thr eshold by visual detection using ethidium bromide staining was one nat urally infected seed in lots of 400 to 600 seeds.