Rapid detection and quantification of viable Pseudomonas syringae pv. lachrymans cells in contaminated cucumber seeds using propidium monoazide and a real-time PCR assay

Abstract

Cucumber angular leaf spot, caused by Pseudomonas syringae pv. lachrymans (Psl), is one of the most devastating bacterial diseases in cucumber worldwide. Seedborne transmission of the pathogen is one of the principal modes for disease spread. To date, the detection of Psl is mainly achieved by culture methods, which are time and labour-consuming. In the present work, propidium monoazide (PMA) treatment combined with a quantitative real-time PCR (PMA-qPCR) assay was developed for quantifying viable Psl cells in contaminated cucumber seeds. PMA selectively penetrates compromised membranes of dead cells and inhibits DNA amplification during real-time PCR. The primers, based on a 162-bp amplicon from gap1 gene, were highly specific for Psl at the species level. PMA at 60 μmol L−1 was suitable for selective quantification of viable cells. The limit of detection (LOD) of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated cucumber seeds was 3.25 × 102 CFU·mL−1 and 47.73 CFU·g−1, respectively. For naturally contaminated seeds, quantifiable levels of viable cells were observed in eight out of the 37 samples, and ranged from 103 to 104 CFU·g−1. This assay has been confirmed to be a rapid and selective method to quantify the viable cells of Psl in bacterial suspension and cucumber seeds. Application of the assay may potentially improve pathogen control and disease management.