Identification of stem melanosis of wheat (Pseudomonas cichorii) by real-time PCR
Keywords:
RT-PCR, bacterioses of grain crops, phytosanitary requirements, plant quarantine, diagnosis of phytopathogens, molecular genetic identification methodsAbstract
The phytopathogenic bacterium, Pseudomonas cichorii, affects a wide range of crops in filed and greenhouse production and causes wheat stem melanosis, which has been regulated by importers of Russian grain products. It is crucial to update the identification method based on real-time polymerase chain reaction (PCR) (PCR-RT). This method helps confirming P. cichorii in wheat samples. A 90 base pair long section of the hrcRST pathogenicity gene cluster was used as the target. Positive results were obtained for reference strains, confirmed by amplicon sequencing. Nucleotide sequences were then compared to the typical strain, DSM 50259. As a result of comparative DNA analysis, the sequences of the direct primer and probe were modified. The possibility of using a modification of 6FAM/BHQ1 dye/quencher available on the territory of the Russian Federation was demonstrated. The specificity of the new PCR-RT primer system PscF/PscHrc751R/PscP1 was assessed using 107 bacterial strains of the genus Pseudomonas, including P. cichorii, P. fuscovaginae, P. syringae, P. trivialis, P. viridiflava, P. chlororaphis, P. lutea and P. orientalis. The DNA of 4 strains (P. poae, P. graminis and 2 strains of P. fluorescens) showed a non-specific reaction at the 35–37 cycle threshold, with an accumulation that did not appear exponential. The analytical sensitivity of the test allows for the detection of P. cichorii at a concentration of 10^1 CFU/mL. The PCR-RV PscF/PscHrc751R/PscP1 test can be used as a screening tool for the detection of P. cichorii in plant products and for the characterization of pure bacterial cultures.
