SPECIFIC OLIGONUCLEOTIDE PRIMERS FOR THE IDENTIFICATION OF PSEUDOMONAS-SYRINGAE PV PISI YIELD ONE OF 2 POSSIBLE DNA FRAGMENTS BY PCR AMPLIFICATION - EVIDENCE FOR PHYLOGENETIC DIVERGENCE

Two unique DNA fragments, generated by RAPD-PCR, were used as probes a gainst dot-blots of representative isolates of the seven races of Pseu domonas syringae pv. pisi. DNA from each isolate hybridized only to on e 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 whi ch when used in PCR reactions with P. syringae pv. pisi cells gave spe cific amplification products (either a 272 bp or a 132 bp fragment) co rresponding to the original cloned fragments. When all four primers we re used in combination with specific DNA amplification reactions in 51 isolates of P. syringae pv. pisi they produced one of the two PCR ban ds. No bands were detected in a range of closely related P. syringae p athovars following PCR amplification. These results suggest that P. sy ringae pv. pisi can be unambiguously identified using specific oligonu cleotide primers and that isolates can be classified into two phylogen etic groups, I and II. (C) 1996 Academic Press Limited.