SPECIFIC DETECTION OF PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA DNA 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. DNAs from 57 strains of other bacteria, including the bean pathogens P. s. pv. syringae and Xanthomonas campestris pv. phaseoli, and presumptive saprophytes associated with bean seed did not contain sequences homologous to the 1.9-kb fragment and did not produce this or other discrete bands upon amplification. The detection threshold in water extracts from soaked bean seeds was 10-20 cfu/ml. Presumptive epiphytes present at concentrations greater-than-or-equal-to 10(4) cfu/ml did not interfere with detection even when the pathogen was present at concentrations of less-than-or-equal-to 30 cfu/ml. In control experiments, heterologous DNA increased the intensity of the 1.9-kb band. The method was used to detect the pathogen in a commercial seed lot that failed to yield the pathogen by conventional methods.