Sensitive and specific detection of phaseolotoxigenic and nontoxigenic strains of Pseudomonas syringae pv. phaseolicola by TaqMan real-time PCR using site-specific recombinase gene sequences

Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight, is the most important bacterial pathogen of bean. Both nontoxigenic (Tox") and toxigenic (Tox*) strains of this pathogen cause halo blight in beans. However, nontoxigenic strains cannot be detected by currently available molecular and serological tools. In this study, a TaqMan probe and primer set were designed based on the phage integrase family site-specific recombinase of P s. pv. phaseolicola 1448A because it is known that most site-specific recombinases are structurally and functionally diverse. The specificity of the probe and primers was evaluated using purified DNA from 29 isolates of 3 different pathovars of P syringae. The probe and primer set were able to detect Tox" and Tox* isolates of P s. pv. Phaseolicola, but no other phytopathogenic bacteria. The assay was also able to detect at least 5 genome equivalents of cloned amplified target DNA, using purified DNA, or 7 colony forming unit (CFU) per reaction when using calibrated cell suspensions. Thus, the TaqMan real-time PCR-based method can be used for the rapid detection of both types of P s. pv. Phaseolicola, and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen, and guide plant disease management. (C) 2009 Elsevier GmbH. All rights reserved.