On DDSL-based genotyping of potato bacteriosis agents, their antagonists and microbial biodestructors for plant protection and ecotechnologies

Intensification of agricultural and industrial production necessitates environmentally friendly technologies to prevent human habitat from chemical pollutions. Microbial producers of biologicals for biocontrol of plant pathogens and hydrocarbon destructors for bioremediation are characterized by high spontaneous genetic variability which can lead to a change in their activity. Therefore, in stabilizing selection, it is necessary to confirm strain affiliation. Here, we presents data on the application of the double digest and selective label (DDSL) technique developed by us to study the genetic profiles of plant pathogenic agents of the genera Pseudomonas , Pectobacterium , their antagonists Bacillus subtilis ( Bs ), and the hydrocarbon destructors of the genus Pseudomonas . The study confirmed high biological efficiency of two selected Bs strains, the M-22 and I5-12/23 against bacterial diseases of stored potato tubers. In addition, destructors from the genus Pseudomonas were genetically identified. The aim of the study was to evaluate genetic diversity among Pseudomonas , Pectobacterium , and Bs strains to select effective microbial antagonists and hydrocarbon destructors. The DDSL technique uses two restriction endonucleases for bacterial genomic DNA digestion. Taq DNA polymerase supplemented into reaction mix provides simultaneous labeling DNA fragments by biotinylated deoxycytidine triphosphate (Bio-dCTP). Only fragments digested with one of the restriction enzymes producing fragments with 3´-recessed ends are subjected to labeling. The second restriction enzyme produces only blunt ends which can not bind Bio-dCTP tag. As a result of DDSL reaction 20 to 50 clear DNA fragments are visualized on the filter, and their quantity and distribution are characteristic for each bacterial strain. Genotyping allows generating genetic profile for each bacterial strain, i.e., assigning a “bar-code” to the bacteria that identifies a given microbe with confidence. Genotyping P. atrosepticum D822 и G784 allows for identification of about 50 DNA fragments more than 20 % of which were specific for only one of the compared strains. We used two pairs of restriction enzymes - XbaI/DraI and XbaI/Eco24I. Our results indicate on equal discriminatory ability of these two enzyme combinations when compared P. atrosepticum strains D822 и G784. We noted some advantage of XbaI/DraI enzymes because of its ability to identify differences in genetic profiles in a range of longer DNA fragments. The optimal enzymes for Pseudomonas genus genotyping were restriction endonucleases BcuI/Eco32I, for Bs the first restriction enzyme was SgsI (39 cleavage sites), the second was Eco32I reducing the size of the obtained DNA fragments. High antagonistic activity of B. subtilis strain I5-12/23 which belongs to I genotypic group was shown in laboratory experiments with artificial contamination of potato tubers with P. atrosepticum 1944 и P. carotovorum subsp. carotovorum 481 . Index of development of bacterial soft rot disease after treatment of infected tubers by B. subtilis strain I5-12/23 was 0-0.02, in control the index was 4.04. Biological activity of this strain after treatment of infected tubers was up to 100 % whereas chemical fungicide Maxim KS gave rise to only 77.7 % value. Experiments conducted in potato storage houses confirmed high biological activity of two selected bacillus strains against bacterial diseases during potato tuber storage. B. subtilis strain I5-12/23 demonstrated highly expressed antagonistic activity against causal agents of bacterial soft rot, ring rot as well as fusarium dry rot potato diseases. Significant suppression of potato tuber diseases in comparison with control after treatment by selected antagonist strains was demonstrated, and this effect was comparable with that of chemical standard Maxim KS. Percentage of healthy tubers treated by bacillus strains was in the range of 30.4-35.5 % whereas in control this value did not exceed 13.3 %. Thus, yield of healthy products compared to control was 2.7 times higher. The most efficient B. subtilis strain I5-12/23 effectively suppressed causal agents for ring rot and fusarium dry rot potato diseases. Prevalence of ring rot disease was 2.6-2.9 %, fusarium dry rot - 1.5-3.0 %, the values which are significantly lower than those in samples treated by B. subtilis М-22 and Maxim KS (4.8 and 3.5 %; 9.0 and 4.1 %, respectively). After genotyping, destructor strains of the genus Pseudomonas utilizing difficult-to-oxidize compounds, including heavy oil fractions and polyaromatic hydrocarbons (benzopyrene, chrysene, phenanthrene, anthracene, chrysene, naphthalene) have been deposited in the VIZR collection. The range of their activity is enough to compose associations for utilizing specific pollutants. Thus, DREAM genotyping identifies bacterial strains to confirm their origin in the course of development and use of biological products for various purposes.