A Rapid and Sensitive PCR-Based Assay for Concurrent Detection of Bacteria Causing Common and Halo Blights in Bean Seed

Abstract

A quick polymerase chain reaction (PCR)-based procedure was developed for concurrent detection of the causal agents of bean common blight, Xanthomonas campestris pv. phaseoli, and bean halo blight, Pseudomonas syringae pv. phaseolicola, in bean seed. A rapid DNA extraction procedure, consisting of briefly wetting intact or crushed seeds with a solution of sodium hydroxide, was used to extract bacterial DNA from externally and internally blight-contaminated seeds. G+C-rich oligonucleotide primers were designed from the phaseolotoxin gene cluster of P syringae pv. phaseolicola and tested under high-stringency conditions in PCR assays. The HB14 primers specifically directed the amplification of a 1.4-kb fragment from DNA of 19 P syringae pv. phaseolicola isolates, whereas templates from 62 other bacterial strains, including the bean pathogens P syringae pv. syringae and X. campestris pv. phaseoli, and plant-pathogenic species of Agrobacterium, Clavibacter, Erwinia, and Xanthomonas did not produce any discrete bands upon amplification. The HB14 primers for P. syringae pv. phaseolicola, and the previously reported G+C-rich X4 primers, specific for X. campestris pv. phaseoli, amplified discrete DNA fragments by soaking extracts of white bean seeds contaminated with halo and common blights, respectively. DNA extracts from blight-infested colored seeds were recalcitrant to PCR-amplification unless polyvinylpyrrolidone was added to the extraction buffer. In combination, X4 and HB14 primers successfully detected individual and mixed infections of bean common and halo blights and yielded distinctive DNA fragments from batches containing as few as 1 infected seed in 10,000 seeds.