A Quantitative PCR Assay for Specific Detection of Pseudomonas cannabina pv. alisalensis

Pseudomonas cannabina pv. alisalensis is a gram-negative bacterium that causes bacterial leaf blight in Brassica crops, an important disease that could bring severe damage to the host plants. The aim of this study was to develop a tool that can reliably and accurately quantify P. cannabina pv. alisalensis and distinguish it from other closely related bacterial pathogens. Two species and six pathovars of Pseudomonas were tested: three pathovars, P. syringae pv. coriandricola, P. syringae pv. philadelphi, and P. syringae strains from Vicia faba, were found or confirmed to be members of P. cannabina based on the multilocus sequence analysis and repetitive element sequence-based PCR results. The quantitative PCR (qPCR) assay was evaluated for specificity and examined for detection limit in pure bacterial cells and bacteria-spiked plant samples. The assay was applied in monitoring the quantities of the P. cannabina pv. alisalensis DNA over time in inoculated turnip green leaves. The newly developed qPCR assay detected the target DNA in P. cannabina pv. alisalensis suspension as low as 100 CFU/ml and did not detect any of the nontarget bacteria. The qPCR assay detected P. cannabina pv. alisalensis in all the inoculated samples at least 5 days before the symptoms became visible; bacterial quantity increased significantly in the first 3 days after inoculation but slowed down afterward. The new qPCR assay for P. cannabina pv. alisalensis detection will facilitate early detection and disease diagnosis, assist research to provide epidemiological insights for the pathogen, and guide implementation of strategies to manage disease and prevent its spread.