The article presents the results of research on the development of parameters for practical application of phage biopreparation Pseudomonas syringae in order to indicate the data of phytopathogenic bacteria in sanitary control objects. The introduction of the article describes pathogens of Pseudomonas syringae and plants that they affect, which proves research actuality aimed at developing speed-up methods of indication and identification of bacteria-pathogens, which will allow specialists to develop measures to combat above-mentioned phytopathogens in a short time. It was established that cultivation of the "bacteriophage-test material" system at a temperature of 28 ± 1 0C during 3.5 hours allows detecting Pseudomonas syringae bacteria in soil samples, river water, and cucumber seeds by the RSF method at a concentration of 103 MK/ml., increase in the exposure time does not improve the quality of the reaction. The bacteriophages Ps. s-7 UlGAU and Ps. s-27 UlGAU in monoculture were used in experiments at a concentration of 103 BFU/ml. When testing a polyphage biological product in RSF, including all the above-mentioned bacteriophages, it was found that the results obtained in the studies of the river water sample were similar to the experimental data on cucumber seed sample. Concentration of detected Pseudomonas syringae bacteria was 103 m.k./ml. It was experimentally established that reducing the initial titer of polyphage biopreparation to a concentration of 102 BFU / ml allowed for the indication of Pseudomonas syringae bacteria in a soil sample at a concentration of 103 mk/g.
Phytopathogenic bacteria Pseudomonas syringae cause diseases of many cultivated plants, causing tumor neoformation, rot, chlorosis, necrosis, etc . The advanced biological mean to control bacteriosis in crop science is bacteriophages. In this work full biological characteristic of 8 bacteriophages is shown, active according to Pseudomonas syringae. The studied phages formed similar negative colonies- clear, rounded, in diameter of 5-9 mm. Lytic actifity of phages Pseudomonas syringae by Appelman from 10-4 to 10-8; by Gratia from 1,0±0,1×106 to 2,0±0,1×109 (BFU/ml). Bacteriophages Ps.s-7 UlGAU, Ps.s-13 UlGAU and Ps.s-27 UlGAU did not change lytic activity when storing in fridge during 12 months. Lytic activity of phages Ps.s-1 UlGAU, Ps.s-8, Ps.s-15 UlGAU, Ps.s-30 UlGAU, Ps.s-77 UlGAU in the same conditions fell within 1-2 orders. Spectrum of lytic activity of phages varied from 21,4% (Ps.s-13 UlGAU) to 85,7% (Ps.s-7 UlGAU, Ps.s-27 UlGAU). The study of phage specificity on 15 species og heterologous cultures showed that phages are specific for Pseudomonas syringae. Phages are moderately stable to heating and lose activity during 30 –minute temperature effect above 62°С. The optimal way to relieve phage lysate from living cells of Pseudomonas syringae was thrichloromethane at a ratio of 10:1and temporal exposition 45 minutes. On the basis of obtained data we determined capacity of each bacteriophage for the use as biocontrol agent. For further research for the study of molecular genetic characteristics we selected advanced strains of phages Pseudomonas syringae - Ps.s-7 UlGAU и Ps.s-27 UlGAU.
The article presents the results of studies on isolation and study of biological properties of bacteriophages specific for phytopathogenic bacteria Pseudomonas syringae. 8 new virulent bacteriophages were isolated and selected from environmental objects. The main biological properties of isolated phages, including lytic activity, were studied (according to Appelman, the indicator was equal to from 10-5 to 10-8; by diffusion method in «soft agar» - from 1.0±0.1*106 to 2.0±0.1*109 (PFU/ml), «spot test» - spot test confirmed the results obtained by diffusion method in «soft agar» - n6 – n9) and its diapason, which ranged from 21.4% (Ps.s-13 UlGAU) to 85.7% (Ps.s-7 UlGAU, Ps.s-27 UlGAU), morphology of plaque-forming units (morphology of negative colonies), specificity within genus and species, changes in lytic activity index during storage, features of impact of physical and chemical factors (trichloromethane at the ratio of 1:10, the impact of which during 5-85 minutes does not influence on biological activity of phages when determining by indirect method; urea, whose 30% solution reduces the yield of plaque-forming units of each bacteriophage studied by about 77.6-85.5 %; temperature in the range of -5 to 10°C and from 50 to 100°C). The study of biological properties allowed us to systematize biological features of each of isolated clones of virulent bacteriophages and select the 4 most promising bacteriophages for further research. The research is carried out with the support of Russian Foundation for Basic Research, the project “Fundamental foundations for the development of phage preparation specific to Pseudomonas syringae, and applied aspects of its application for phagoidentification and bioprocessing of food and agricultural raw materials” No 19-44-730014.
The article presents results of studies on development of isolation scheme and bacteriological identification of Pseudomonas syringae bacteria and its approbation. The introduction of the article describes the objects of Pseudomonas syringae contamination - fruit trees and shrubs, agricultural plants, which proves the relevance of the research in the field of expanding of laboratory methods for identifying phytopathogenic microorganisms. The author’s bacteriological scheme includes the use of King B Medium (Pseudomonas F Agar; Pronadisa 1532) as a selective medium. Initially, the isolated bacteria are differentiated to Pseudomonas genus, the following paramters are studied: anaerobic fermentation, production of enzymes catalase, lecithinase, lipase; hydrolysis of starch and gelatin; fermentation of glucose and lactose, also, a test for maceration is put. The second stage of the research includes the study of the growth of bacterial cultures on meat-and-peptone agar at 41 ° C and at 5% of NaCl; oxidase production, arginine hydrolase; fermentation of mannose and sorbitol; formation of levan, hydrogen sulfide and indole, esculin, a hypersensitivity reaction is set. The determined parametres allow to type the representatives of the genus to Pseudomonas syringae species within 192 hours. During the research, a collection of 12 strains of Pseudomonas syringae bacteria was formed, isolated from 97 objects of phytosanitary supervision and identified according to the developed technique. The proposed bacteriological scheme allows to differentiate the above microorganisms on the basis of the analysis of 25 parameters. The application of a phage biological product as a diagnosticum (according to the Otto method) expands the spectrum of the analyzed biological properties of the isolated and identified Pseudomonas syringae bacteria.
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