Cloning of a specific DNA region of white top pathogen of pea and its detection by polymerase chain reaction

摘要

White top strain (WT strain) of Pseudomonas syringae pv. pisi (Ppi) is a variant strain causing white top disease of peas. The WT strain is distinguishable from common Ppi strains only by symptom expression chlorosis and whitening of apical shoots. To develop a specific detection method for the WT strain, we cloned a specific DNA region of the WT strain using transposon tagging. Five mutants defective in white top symptom expression were obtained. A part of the Tn5-flanking region was cloned and labeled as a hybridization probe. One clone, pAY3, gave two signal bands, one of which was detected from the genomic DNA of all the WT and the common Ppi strains; another was specific to WT strains. A restriction map of pAY3 showed that it contains two BamHI fragments; one is 5.0kb in length involving a part of Tn5, and the other is 1.5kb, did not carry Tn5, and may have been accidentally ligated into pAY3. The 1.5-kb band was subcloned as pAY13 and was used as a probe. It hybridized specifically to WT strains. These results suggested that the WT strains have a specific DNA region and that part of the region was successfully cloned. Sequence analysis of pAY13 showed that it is similar to part of nonribosomal peptide synthetases (NRPSs) genes. The deduced amino acid sequence of pAY13 suggested the existence of eight conserved motifs of NRPSs. WT strain-specific PCR primers, PS1 and PS2, were designed from the DNA sequence. These primers gave a specific amplification product of 981bp from both the genomic DNA and a direct cell preparation of WT strains. No specific amplicon was produced from Ppi strains that caused only water-soaked lesions or from strains of other P. syringae pathovars. A specific amplicon was not produced from four strains of the pea pathogen: P. marginalis pv. marginalis, P. viridiflava, Erwinia carotovora ssp. carotovora, Xanthomonas campestris pv. pisi. Using the primers, WT strain was detected from water-soaked lesions and green and white tissues without water soaking.