Development of a Quantitative PCR Method to Detect the Bacterial Gall Pathogen <i>Pseudomonas amygdali</i> pv <i>. loropetali</i> from Loropetalum Plant Materials

Bacterial gall caused by <i>Pseudomonas amygdali</i> pv. <i>loropetali</i> (PAL) is a prevalent problem on <i>Loropetalum chinense</i> shrubs in commercial plant nurseries. A method was developed to reliably detect PAL on the surface of loropetalum twigs. A whole-genome analysis resulted in the identification of a locus encoding an AraC regulator that is specific to PAL. A pair of primers and a TaqMan probe were designed based on a 71-base-pair sequence in this locus. Positive results of PCR amplification were obtained with genomic DNA samples from all PAL strains but not from those of other <i>Pseudomonas</i> species, <i>Agrobacterium tumefaciens</i>, or <i>Burkholderia contaminans</i>. Melting curve analysis demonstrated that all PAL PCR products shared the same melting temperature of 79°C. TaqMan-based quantitative PCR (qPCR) analysis of the serially diluted genomic DNA from PAL strain AAC exhibited a strong linear response for regressed cycle threshold and logarithm copy values (adjusted <i>R</i>^<i>2</i> = 0.9944) with a high amplification efficiency (E = 1.96), whereas the linear response (adjusted <i>R</i>^<i>2</i> = 0.8885) for PAL genomic DNA extracted from serially diluted bacterial cell suspension had a reduction in detection sensitivity. The limits of detection and quantification of PAL from the spiked plant twigs (diameter × length = ∼0.45 × 2.45 cm) were 873 and 14,724 cells, respectively, using a modified Promega Wizard extraction protocol. These limits of the qPCR method, although restrictive, still allow a practical detection of PAL strains associated with plant tissue that can be used in epidemiological studies to develop disease management options.