Applying Molecular Methods in Diagnosing the Pathogen of Bacterial Leaf Spot in Cauliflower Pseudomonas syringae pv. maculicola (McCulloch) Young et al.

Abstract

Studies were performed to evaluate the applicability and validation of the PCR test according to Inoue and Takikawa (2021) in the diagnosis of Pseudomonas syringae pv. maculicola (McCulloch) Young et al. (Psm). The test was optimized for real-time application. For this purpose, the SYBR Green I intercalating dye with a fluorescence emission wavelength of 520 nm was added to the reaction mixture. Method performance criteria were determined according to EPPO standard PM 7/098 (4). Evaluation the applicability of the test in real-time mode showed sufficient analytical sensitivity, which was 10³–10⁴ CFU/mL, which corresponded to a low and medium degree of infection. Determination of analytical specificity showed false positive results with P. syringae pv. tomato. There is also in silico data on a 100% match with P. syringae pv. avii, pv. persicae, and pv. spinaciae. These pathovars affect other crop species; there is also no information on their distribution in Russia; therefore, they were not considered in these studies. Determining selectivity showed the effect of the matrix on the sensitivity of the test. Thus, the detection threshold decreased from 10³ to 10⁴ CFU/mL when studying extracts of vegetative parts of rapeseed as compared to seeds. The repeatability and reproducibility of the test was 100% when the concentration of the target object in the matrix was 10⁴ CFU/mL. The obtained values of the test efficiency criteria indicate the possibility of using PCR according to Inoue and Takikawa (2021) for direct diagnosis of the phytopathogen from the plant extract of seeds and vegetative parts of Psm host plants. The method was tested on rapeseed samples from Tula and Lipetsk oblasts and Zabaykalsky krai. Psm genetic material was found in one rape sample from Tula oblast.