Specific Detection ofPseudomonas syringaepv.phaseolicolaDNA in Bean Seed by Polymerase Chain Reaction-Based Amplification of a Phaseolotoxin Gene Region

The polymerase chain reaction (PCR) is described for the specific detection of the bean halo blight pathogen Pseudomonas syringae pv. phaseolicola in bean seed. The method involves the amplification of a segment of the tox (phaseolotoxin) gene cluster (23). Two oligonucleotide primers, designed according to the sequence of this segment, allowed the exclusive amplification of a 1.9-kb fragment from total DNA extracted from pure cultures of the pathogen and from water extracts of soaked bean seed. The amplified segment was visually detected by ethidium bromide staining when the original samples contained approximately 10 3 colony-forming units (cfu) per milliliter and by Southern blot hybridization in DNA extracts from pure cultures of the pathogen that contained as few as 10 cfu/ml [...]