Korotaieva N. Development of the biological formulation against crown gall disease in plants based on lactic acid bacteria

The dissertation is devoted to the study of endophytic microbiota of vessels and tissues of the crown gall of grapes, the search for active antagonists among lactic acid bacteria and to the development of the biological formulation based on lactic acid bacteria for prevention of the crown-gall disease in plants. The quantitative and qualitative composition of the endophytic microbiota of grapes and crown gall tissues was determined. PCR analysis of pathogenicity genes demonstrated that avirulent bacteria Rhizobium vitis and Rhizobium radiobacter predominated in crown galls. Pathogenic strains R. vitis ONU388, R. vitis ONU389 and R. vitis ONU390 were isolated. In the search of antagonistic strains against bacteria causing crown-gall disease in plants, all of the studied strains of Lactobacillus plantarum were found to contain from 2 to 6 genes which code for the synthesis of plantaricins. This correlates with the level of their antagonistic potential. An in vitro study of the antagonistic activity of lactobacilli against pathogenic bacteria causing the crown-gall disease demonstrated that 100% of rhizobia growth was inhibited by the lactobacilli synthesising organic acids (pH 3.5-4.0). However, only L. plantarum ONU87 produced a bacteriocin active against gram-negative rhizobia. The in vivo study based on the carrot discs model co-inoculation with rhizobia and the studied strains of L. plantarum demonstrated that among seven tested strains of L. plantarum the best results for tumor growth inhibition were shown by six strains of lactobacilli, suggesting high antagonistic activity of the mentioned lactobacteria. According to the degree of antagonistic activity, inhibition of tumor formation and the present spectrum of genes which code for bacteriocins, a promising strain of L. plantarum ONU87 was selected for further studies. The method of mathematical analysis based on the partial regression model was used to develop the optimal culture medium composition. With this medium the concentration of lactic acid bacteria in the stationary phase of growth increased to 3,51±0,103 ×1010 CFU/ml. A biotechnological production plan for the biological formulation was developed and the recommendations for storage were provided. It was established that treatment of grape shoots artificially infected by R. radiobacter C58 with the L. plantarum ONU87 strain reduced the amount of infected samples by 76,8% and stimulated growth and development of grapes.