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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

Physiological and Molecular Plant Pathology (Impact Factor: 1.99). 10/1996; 49(4):233-245. DOI: 10.1006/pmpp.1996.0051

ABSTRACT Two unique DNA fragments, generated by RAPD-PCR, were used as probes against dot-blots of representative isolates of the seven races ofPseudomonas syringaepv.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 withP. syringaepv.pisicells 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 ofP. syringaepv.pisithey produced one of the two PCR bands. No bands were detected in a range of closely relatedP. syringaepathovars following PCR amplification. These results suggest thatP. syringaepv.pisican be unambiguously identified using specific oligonucleotide primers and that isolates can be classified into two phylogenetic groups, I and II.

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    • "), P. corrugata (Catara et al., 2000), P. syringae pv. pisi (Arnold et al., 1996). This method could be used to verify the efficacy of sanitation of an area and also to monitor the occurrence of the disease. "
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    • "Blots were hybridized ( 65 °C, 16 h) using the labelled probes in hybridization solution (Sambrook et al., 1989), and washed to low (2% SSC, 0.1% SDS) or high (0.1% SSC, 0.1% SDS) stringency, as described by Arnold et al. (1996). "
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    • ") and then washed to high stringency as described by Arnold et al. (1996). "
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