Molecular Detection Of Two Plant Quarantine Pseudomonas Syringae Pathovars

Pseudomonas syringae pv. pisi and Pseudomonas syringae pv. maculicola, causing bacterial blight of pea and bacterial spot of Brassicaceae, respectively, are both listed in the Plant Quarantine Pest of China. Little information is available for detection of these two bacteria. At present study, specific primers were designed for detecting the two pathogens and a reliable, sensitive and rapid detection method was established. It provides an important way for plant quarantine and disease management.One hundred and fifty bacterial strains were used for validating specific primer sets, including a collection of target bacterialstrains as well as closely related strains with economically or environmentally important ones. The two pairs of primers were very specific to the target bacteria without false positive or false negative reaction. The minimum detection concentration for P. syringae pv. pisi by conventional PCR was103fg/μl of genomic DNA or102CFU/ml bacteria cells and102fg/μl or10CFU/ml by Real-time PCR assay. Moreover, the specific primer was able to detect the existence of target pathogen in102and103fold diluted plant extracts from artificially infected plant sample by conventional PCR and Real-time PCR, respectively.The minimum detection concentrations for P. syringae pv. maculicolaby conventional PCR and Real-time PCR were both102fg/μl and10CFU/ml. In the stimulation of the seed detection of P. syringae pv. maculicola, the minimum detection concentrationof seed suspension was105CFU/ml and103CFU/ml by conventional PCR and Real-time PCR, respectively. In the stimulation detection of partially infected seed lot, conventional PCR could detect5%infected seed lot, while Real-time PCR was able to detect2.5%infected seed lot. Morever, the sensitivity of detection could be further increased by combining enrichment of the target bacteria with conventional or Real-time PCR assay,0.1%infected seeds could be detected using this method.