Specific oligonucleotide primers for the rapid identification and detection of the agent of tomato pith necrosis, Pseudomonas corrugata, by PCR amplification : evidence for two distinct genomic groups

Abstract

Unique DNA bands from strains representative of two groups of Pseudomonas corrugata , as shown by amplification of their genomic DNA by polymerase chain reaction using short random sequence oligonucleotide primers (RAPDPCR), were isolated, cloned and sequenced. Two pairs of specific primer sequences, based on the ends of the cloned unique DNA bands from strains IPVCT10.3 and IPVCT8.1, were used in multiplex PCR with a range of P. corrugatastrains. All strains produced one of the two specific bands, 1100 bp (from the IPVCT10.3-based primers) and 600 bp (from the IPVCT8.1-based primers), representing groups designated I and II, respectively. The primers were also tested on a wider range of Pseudomonasspecies, including the closely-related fluorescent Pseudomonas genomospecies FP1, FP2 and FP3: none of these bacteria produced any bands following amplification by PCR with these primers. The primer sets detected P. corrugata in tomato pith necrosis-infected plants providing a useful tool for rapid identification and epidemiological studies.