A PCR diagnostic assay for rapid detection of plant pathogenic pseudomonads

Molecular detection of phytopathogens is increasingly being applied to identify regulated organisms at the border in many parts of the world. However, even with molecular tests, complete phenotyping and identification of a strain is often time consuming and sometimes inconclusive. In this study, a leaf-based pathogenicity test was used to separate pseudomonads into two groups, Group A containing pathogens, and Group B containing saprotrophs. Comparative genomics of 56 pseudomonad genomes from different plant hosts (including 29 strains from kiwifruit) agreed with kiwifruit pathogenicity test results, placing pathogens into Group A and saprotrophs into Group B. Sixteen loci were found unique to Group A. A PCR assay was developed for amplification of one of these loci, the trehalose phosphatase gene. The generation of this 655 bp amplicon was associated with production of water-soaked lesions on inoculated kiwifruit leaves by pseudomonads in Group A. This test was validated for further strains from all seven pathogenicPseudomonasphylogroups, non-pathogenic pseudomonads, and other bacterial genera. The sensitivity of the PCR was comparable to the limit of recovery of pseudomonads by culturing. This simple PCR assay could be used as part of a testing pipeline at the border and for general surveillance for screening plants with and without symptoms, offering the potential to detect uncharacterized pseudomonads that may pose a biosecurity risk. The method was shown to be able to rapidly identify pathogens cultured from plant material with symptoms, or, more importantly, to detect pathogens directly from plant tissue.