Identification of Pseudomonas fuscovaginae, Pseudomonas syringae and Xanthomonas translucens in wheat seeds using PCR

The causative agents of grain crops bacteriosis viz. Pseudomonas fuscovaginae , Pseudomonas syringae and Xanthomonas translucens are regulated by phytosanitary requirements of the largest importers of Russian grain - Egypt, Turkey, Bangladesh, Nigeria and Pakistan. Therefore, it requires the development of rapid methods for their diagnosis. The PCR method, which is the fastest and most reliable in testing laboratories, needs optimal preparation of the test material. The aim of the study was to optimize the process of preparing seed samples for subsequent detection and identification of P. fuscovaginae, P. syringae and X. translucens by PCR. Wheat grain samples were soaked in phosphate-buffered saline (PBS) for 2 hours and infected with suspensions of P. fuscovaginae, P. syringae pv. coronafaciens and X. translucens at various concentrations. Then, the infected grain samples were crushed and subjected to two-stage centrifugation. DNA was isolated from the obtained analytical samples and species-specific PCR was performed for each bacterial species. It was found that a two-hour soaking of the seeds and their treatment with a homogenizer is sufficient to effectively destroy each grain in the sample and ensure the release of bacteria into the liquid part of the sample. The first low-speed centrifugation allowed the crushed grain to settle efficiently and remove excess starch from the supernatant. High-speed centrifugation of the supernatant made it possible to obtain a concentrated microbiota contained in the grain sample. To obtain DNA of sufficient quality for PCR test, the kit ‘Proba-GS’ (AgroDiagnostika, Russia) was used for DNA extraction. Using ‘Pseudomonas fuscovaginae-RT’ kit (Syntol, Russia) and PsyF/PsyR and 4F1/4R 1 primers, DNA of P. fuscovaginae P. syringae and X. translucens , respectively, was successfully detected in each of the samples infected with these bacteria at concentrations of 103 CFU/ml. The absence of PCR inhibition was noted. The method of removing starch from samples for molecular diagnostics of phytopathogens was used for the first time. Application of these methods will allow diagnosing pathogens of bacterioses within one day.