Detection of <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> using polymerase chain reaction (PCR) primers based on the 16S–23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions

<jats:p> Several published polymerase chain reaction (PCR) primers to identify <jats:italic>Pseudomonas syringae</jats:italic> pv. <jats:italic>actinidiae</jats:italic> , 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 of <jats:italic>P. syringae</jats:italic> pv. <jats:italic>actinidiae</jats:italic> , but not from 56 strains of bacteria from six genera and 17 species, except for a strain of the tea pathogen, <jats:italic>P. syringae</jats:italic> pv. <jats:italic>theae</jats:italic> . When tested against DNA extracted from a further 20 strains from Japan, Korea, Italy and the USA deposited in culture collections as <jats:italic>P. syringae</jats:italic> pv. <jats:italic>actinidiae</jats:italic> , 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 ( <jats:italic>gyrB</jats:italic> , <jats:italic>acnB</jats:italic> , <jats:italic>rpoD</jats:italic> , <jats:italic>pgi</jats:italic> and <jats:italic>cts</jats:italic> ) showed that none of these six strains was phylogenetically similar to <jats:italic>P. syringae</jats:italic> pv. <jats:italic>actinidiae.</jats:italic> In contrast to the <jats:italic>P. syringae</jats:italic> pv. <jats:italic>actinidiae</jats:italic> type 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 as <jats:italic>P. syringae</jats:italic> pv. <jats:italic>actinidiae</jats:italic> . It was not possible to distinguish <jats:italic>P. syringae</jats:italic> pv. <jats:italic>actinidiae</jats:italic> from the phylogenetically similar <jats:italic>P. syringae</jats:italic> pv. <jats:italic>theae</jats:italic> using the ITS, <jats:italic>gyrB</jats:italic> , <jats:italic>acnB</jats:italic> , <jats:italic>rpoD</jats:italic> , <jats:italic>pgi</jats:italic> or <jats:italic>cts</jats:italic> gene regions to design PCR primers. Because <jats:italic>P. syringae</jats:italic> pv. <jats:italic>theae</jats:italic> is unlikely to be found on kiwifruit, primers PsaF1/R2 and PsaF3/R4 are recommended for screening bacteria isolated from kiwifruit tissue. </jats:p>