Studies on the detection methods of bacterial ring rot (Clavibacter michiganensis subsp. sepedonicus), bacterial wilt, and brown rot (Ralstonia solanacearum) pathogens on potato

Objective: The study's first aim is to detect two quarantine bacterial pathogens (Rsol and Cms) having zero tolerance in potato seed certification with the recommended methods by the European Community from various sources (potato plant, tuber, irrigation water, and soil) under current laboratory conditions. In addition, developing a real-time multiplex PCR protocol with newly designed primer-probe sets for the determination of Rsol and Cms from the same sources and evaluating the possibilities of its use in practice has been the ultimate goal of the study. Materials and Methods: To determine Rsol and Cms from different sources, IF, classical PCR, and real-time PCR) were used. Then, the newly designed primary probe sets of two bacteria (Rsol-1F/Rsol-1R, Rsol-Pr; Cms-1F/Cms-1R, Cms-Pr) and the Real-time PCR protocols proposed in these methods were compared. For this purpose, detection limits and PCR primer efficiencies were calculated in pure bacterial and DNA suspensions of both pathogens, artificially contaminated potato plant and tuber extracts, potato DNA and the presence of an appropriate primer probe. In addition, the presence of the agents in naturally latent infected tubers was examined, and the detection limits of Rsol were determined in the soil and irrigation water taken from 3 different potato-growing areas in Turkey. Results: The detection limit of Rsol was 1.9x103 cells/ml (3 cells), and the limit of detection of Cms was 1.9x102 cells/ml (5 cells) in the IF tests performed. With the classical PCR test using two different DNA extraction methods, the detection limit for Rsol was determined as six ng/µl for both methods, 0.419 ng/µl for Cms after the DNA extraction method, and 1.79 ng/µl after the boiling process. The determination of Rsol in soil and irrigation water samples by using a real-time PCR was performed with the new Rsol primer pair and probe set. The detection limit was 1.2x103 cells/ml (1.15x103 bacteria/gram) in Balikesir and Ödemiş and 1.2x105 cells (1.2x104 bacteria/gram) in soil samples of Afyonkarahisar for Rsol DNA obtained from the commercial kit. After DNA extraction by the boiling method, Rsol 1.2x103 cells/ml (1.2x102 bacteria/gram) could be detected only in the soil samples from Balıkesir and Ödemiş but not in the soil samples from Afyonkarahisar. Rsol was detected up to 1.2x103 cells/ml in the irrigation water samples from three provinces. Real-time PCR studies with new Rsol and Cms primer pairs and probe sets using potato plant extracts showed that the presence of potato DNA and COX primer pair and probe did not affect the detection limit. The detection limits were determined as Rsol 0.2 pg/µl (PCR efficiency between 77.80-86.33%), Cms 0.2 pg/µl (PCR efficiency between 74.42-83.08%) in these tests. In the samples prepared by mixing the bacterial suspension with tissue extracts consisting of two hundred potato tuber samples, with the new primer pair and probe set, the detection limit of Rsol was 1.08x103 cells/ml (PCR efficiency 97.39%), and with the primer pair and probe set of Weller (2000) was 1.08x102 cells/ml (PCR efficiency 259.75%). Likewise, the detection limit of Cms was 1.4x102 cells/ml (PCR efficiency 105.62%) with the new primer pair and probe set and 1.4x103 cells/ml (PCR efficiency 121.62%) with the sets of Schaad (1999). In multiplex PCR studies with the designed primer pair and probe set, Rsol was detected up to 0.2 pg/µl (PCR efficiency 90.29%, 90.94%, 90,07%, and 94.60%) in the presence of only Rsol, both bacteria, and potato DNA. For the Cms, the detection limit was calculated as 0.2 pg/µl in the presence of only Cms and potato DNA (PCR efficiency 90.98% and 88.59%). But the detection limit increased to 2 pg/µl (PCR efficiency 87.19% and 88.99%) with the existence of two bacteria and potato DNA. In the real-time PCR testing of naturally infected potato tuber samples, similar results were obtained as singleplex and multiplex use of the newly designed primer-probe sets of Rsol and Cms compared to primer pairs and probes of the recommended methods by the European Community. Conclusion: Rsol and Cms are bacterial pathogens subject to quarantine worldwide and have zero tolerance in seed potato certification. Potato tubers produced, imported, and exported legally must be tested. With the newly designed primer and probe sets, the presence of Rsol and Cms was successfully detected from potato plants and tubers, and the presence of Rsol from soil and irrigation waters. As a result, a multiplex real-time PCR protocol was created with the newly designed primer pair and probe set, allowing simultaneous detection of Rsol and Cms in potato tubers. Its efficiency and specificity were confirmed. Keywords: Ralstonia solanacearum, Clavibacter sepedonicus, Real-time PCR, Multiplex, soil, irrigation waters