MOLECULAR-GENETIC IDENTIFICATION OF STRAINS OF BACTERIA-ANTAGONISTS OF PHYTOPATOGENS
The article is devoted to application of molecular genetic approach (DDSL - double digest and selective label) for fast and inexpensive identification of bacterial strains which are used as antagonists suppressing phytopathogenic microorganisms. Results demonstrate a potential for routine usage of this technique. It based on generation of genetic profile specific for each bacterial strain certifying its origin. Introduction. Microbiological methods of plant protection from phytopathogens and insect pests have recently acquired particular importance due to the global trend towards the use of environmentally friendly food products. Among such microorganisms, strains of Xenorhabdus nematophila and Bacillus thuringiensis can be underlined. The industrial use of these bacteria requires regular confirmation of their authenticity to the original patented originals from the established collections of beneficial microorganisms. Object. The object of research are isolates of bacteria belonging to two different species - Xenorhabdus nematophila and Bacillus thuringiensis. The former being a part of intestine microbiota in soil nematodes is able to produce secondary metabolites deadly for harmful insects. The latter possesses valuable feature - under appropriate conditions it produces spores which are highly stable and constitute a functionally active part of biopreparations. Materials and methods. The previously developed method for genetic certification of bacterial strains, based on double digest and selective label of DNA fragments (DDSL), was used for molecular-genetic identification of strains of these two species. The method uses two restriction endonucleases simultaneously, one produces “sticky” ends that can be labeled with biotinylated deoxycytosine triphosphate, and the other produces blunt ends that are not capable of incorporating the label. As a result, out of thousands of DNA fragments, only a limited number will carry the tag; the number and distribution of such labeled fragments after electrophoresis in a conventional agarose gel is easily visualized on a nylon filter. The set of fragments is characteristic for each bacterial strain and is a sort of genetic certificate. The aim of the work was to apply this method to identify strains of two bacterial species. Results and conclusion. Using Bacillus thuringiensis as an example, it was demonstrated that the resolution of the method depends on the number of detected DNA fragments. For example, Sgr AI/ Eco 24I enzyme pair generates more than 25 fragments whereas Sgs I/ Eco 32I - just a few bands. The higher number of bands the better resolving power of any technique. In case of application of Sgr AI/ Eco 24I all isolates were discriminated into distinct genotypes except pair EП-Е isolates. In contrast, Sgs I/ Eco 32I generating a small number of bands did not resolve most strains. As for entomopathogenic bacteria Xenorhabdus nematophila the optimal pair of enzymes was a combination of restriction endonucleases SgsI/Eco 32I. Application of Sgs I/ Eco 32I enzymes in Xenorhabdus nematophila revealed the identity of the C/s and P strains, while the J strain differed significantly from them. DDSL method makes it possible to identify rearrangements in the genomic DNA for the certification of strains of beneficial bacteria producing bioinsecticides and having antagonistic activity. Thus, this typing technique can be a valuable tool in identification of bacterial strains having antagonistic activity and nowadays used commercially.