On Genotyping Bacterial Strains of the Genera Pectobacterium and Pseudomonas: Pathogens of Bacterioses in Potatoes

Abstract

Molecular-genetic research methods make it possible to identify phytopathogens with comprehensive characteristics of their hereditary material. We suggest a genotyping method based on the use of two restriction endonucleases for the cleavage of bacteria’s genomic DNA. Taq DNA polymerase added to the reaction mixture provides labeling of DNA fragments with biotinylated deoxycytosinetriphosphate (Bio-dCTP). The label is only incorporated into DNA having 3-prime recessed ends formed by the first enzyme. The second restriction endonuclease produces only blunt ends of fragments that are unable to incorporate the label. As a result of the DDSL (double digest selective label) reaction, 20–50 distinct DNA fragments are visualized on the filter, the amount and distribution of which are specific for each bacterial strain. This research was carried out using two pairs of restriction enzymes (XbaI/DraI and XbaI/Eco24I). The results indicate the same discriminatory capacity of these enzymes. Some advantage was noted for the XbaI/DraI combination, which makes it possible to detect differences in the region of longer DNA fragments in genetic profiles. The genetic profile generated by the BcuI/Eco32I pair of enzymes in Ps. fluorescens 894 was significantly different from that of Ps. xanthochlora, which could be anticipated while comparing these two species. In general, the results of genotyping agreed with the data on the origin of bacterial strains. In particular, microorganisms with the same genetic profile, D822 and D828, G399 and G400, as well as C960 and C966, were obtained from affected tubers of the same experimental field. This suggests the possibility of the contact spread of the pathogen. In some cases, the identity of strains was observed, despite their different geographic origin. This fact confirms contamination that occurred in the past during the use of the foreign seed potato.