Detection ofPseudomonas syringaepv.actinidiaeusing polymerase chain reaction (PCR) primers based on the 16S–23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions

Abstract

Several published polymerase chain reaction (PCR) primers to identifyPseudomonas syringaepv.actinidiae, the causal organism of bacterial canker of kiwifruit, were found not to be specific. Two new sets of PCR primers, PsaF1/R2 and PsaF3/R4, were designed to be complementary to a portion of the 16S–23S rDNA intertranscribed spacer (ITS) regions. These primers amplified a DNA fragment from strains ofP. syringaepv.actinidiae, but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen,P. syringaepv.theae. When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections asP. syringaepv.actinidiae, all except six cultures produced the expected product of 280 bp with PsaF1/R2 and 175 bp with PsaF3/R4. Results of multilocus sequence analysis using five housekeeping genes (gyrB,acnB,rpoD,pgiandcts) showed that none of these six strains was phylogenetically similar toP. syringaepv.actinidiae.In contrast to theP. syringaepv.actinidiaetype strain, these strains were positive in the determinative tests for ice nucleation and syringomycin production. It is suggested that these six strains were incorrectly identified asP. syringaepv.actinidiae. It was not possible to distinguishP. syringaepv.actinidiaefrom the phylogenetically similarP. syringaepv.theaeusing the ITS,gyrB,acnB,rpoD,pgiorctsgene regions to design PCR primers. BecauseP. syringaepv.theaeis unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue.