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

文献类型： 外文期刊

作者： Meng, Xianglong;Chai, Ali;Chen, Lu;Shi, Yanxia;Xie, Xuewen;Li, Baoju;Meng, Xianglong;Ma, Zhanhong

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关键词： cucumber angular leaf spot;cucumber seeds;propidium monoazide (PMA);Pseudomonas syringae pv. lachrymans;real-time PCR;viable cells

期刊名称： CANADIAN JOURNAL OF PLANT PATHOLOGY

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收录情况： SCI

摘要： 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 mu mol 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 x 10(2) 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 10(3) to 10(4) 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.

分类号： S4

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