Development of a quantitative PCR method to detect the bacterial gall pathogen Pseudomonas amygdali pv. loropetali from loropetalum plant materials.

Abstract

Bacterial gall caused by Pseudomonas amygdali pv. loropetali (PAL) is a prevalent problem on Loropetalum chinense 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 Pseudomonas species, Agrobacterium tumefaciens, or Burkholderia contaminans. Melting curve analysis demonstrated that all PAL PCR products shared the same melting temperature of 79°C. TaqMan-based qPCR analysis of the serially diluted genomic DNA from PAL strain AAC exhibited a strong linear response for regressed cycle threshold (Ct) and logarithm copy values (Adjusted R2 = 0.9944) with a high amplification efficiency (E = 1.96), while the linear response (Adjusted R2 = 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, while restrictive, still allow a practical detection of PAL strains associated with plant tissue that can be utilized in epidemiological studies to develop disease management options.