Specific oligonucleotide primers for the identification of Pseudomonas syringae pv pisi yield one of two possible DNA fragments by PCR amplification: Evidence for phylogenetic divergence

Two unique DNA fragments, generated by RAPD-PCR, were used as probes against dot-blots of representative isolates of the seven races of Pseudomonas syringae pv. pisi. DNA from each isolate hybridized only to one of the two probes. Fragments identified from isolates 1691 (race 7) and 203 (race 2), were cloned into pUC18 and sequenced. The resulting sequences were used to design two pairs of oligonucleotide primers which when used in PCR reactions with P. syringae pv. pisi cells gave specific amplification products (either a 272 bp or a 132 bp fragment) corresponding to the original cloned fragments. When all four primers were used in combination with specific DNA amplification reactions in 51 isolates of P. syringae pv. pisi they produced one of the two PCR bands. No bands were detected in a range of closely related P. syringae pathovars following PCR amplification. These results suggest that P. syringae pv. pisi can be unambiguously identified using specific oligonucleotide primers and that isolates can be classified into two phylogenetic groups, I and II. (C) 1996 Academic Press Limited.