RU2182172C1 - Strain of bacterium bacillus subtilis with wide spectrum of antagonistic activity - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: invention relates to the strain of bacterium of genus Bacillus subtilis that is an antagonist of with respect to the strains of pathogenic microorganisms causing diseases in plants, humans and agriculture animals. The strain is isolated from the health wheat plant in Ufa region and deposited at number VKM B-2218D in all-Russian collection of microorganisms of IBFM RAN. EFFECT: new strain indicated above, valuable properties of strain. 6 tbl

Description

 The invention relates to microbiology and biotechnology and is a new strain of bacteria of the genus Bacillus, which is an antagonist to strains of pathogens that cause disease in humans, farm animals and plants.

 Currently, probiotic preparations from bacteria of the genus Bacillus, which suppress the development of pathogenic microorganisms, are widely used in agriculture for the suppression of phytopathogenic bacteria and fungi (bacispecin-BM, phytosporin) (1, 2), in medicine (biosporin, bactisporin, sporobacterin) (3-5), veterinary medicine for the treatment of various diseases of birds and farm animals (BCL bactocellolactin, subaline, sporolact) (6-8). The probiotic preparations listed above are based on different strains of bacteria of the genus Bacillus, which are antagonistic to pathogenic bacteria and fungi that cause diseases in either plants or animals and people. However, there are no strains with a spectrum of antagonistic activity sufficient to simultaneously protect against pathogenic microorganisms that cause diseases in plants, people and animals.

 The objective of the invention is the selection of a strain of bacteria of the genus Bacillus, which has antagonistic activity against pathogenic strains of bacteria and fungi that cause diseases of plants, humans and farm animals and which can be used to protect against a complex of diseases caused by fungi and bacteria.

 Closest to the invention is a strain of B. subtilis 10 VIZR (9), which has a wide spectrum of antagonistic activity to phytopathogenic fungi, on the basis of which the biological product Alirin B was obtained. However, the strain does not inhibit the growth of pathogenic bacteria and its antagonistic activity against strains of fungi causing diseases of humans and animals.

 The aim of the invention is a new strain of bacteria of the genus Bacillus, with antagonistic activity against pathogenic fungi and bacteria that cause diseases of plants, humans and farm animals.

 The strain Bacillus subtilis 11 B was isolated from a healthy wheat plant in the Ufa region. Identification of the strain was carried out according to the "Determinator of bacteria Burgee" M. Mir, 1997.

 The strain was deposited under the number VKM V-2218D in the All-Russian collection of microorganisms of IBPM RAS 08/19/1999

 The strain culture is characterized by the following properties: B. subtilis VKM B-2218 D - gram-positive aerobic spore-forming bacilli producing catalase. On meat-peptone agar (MPA), wort agar give abundant growth. On the MPA it forms grayish-white small-wrinkled colonies with wavy edges, slightly growing into agar, of a viscous consistency. Growth in a liquid medium is accompanied by turbidity of the medium, the formation of a film and sediment. After 18 hours of growth, straight rod-shaped cells, 2-3 × 0.6 μm in size, located singly, in pairs or in a chain are detected in culture smears. The cells are motile. When spore formation, the cell does not swell. The spores are oval, size 0.9x0.6 microns, located in the cell eccentrically.

 The culture ferments glucose, b-galactose, lactose, sucrose and maltose. Does not decompose mannitol, inositol, sorbitol and arabinose. It gives a positive Voges-Proskauer reaction, hydrolyzes starch, gelatin, does not hydrolyze urea, utilizes sodium citrate with glucose, does not use propionate and sodium malonate, and grows weakly under anaerobic conditions. It is characterized by the presence of arginine dehydrolase, lysine decarboxylase, the absence of phenylalanyl deamylase, ornithine decarboxylase. Forms acetylmethylcarbinol.

 Reduces nitrates, does not form gas on a medium with nitrates under anaerobic conditions. Does not form indole and hydrogen sulfide.

 The culture of the strain was tested for harmlessness (virulence, toxicity and toxigenicity).

To study virulence, the strain was grown on meat-peptone agar for 24 hours at a temperature of 37 ^o C. The grown biomass was washed with 0.9% sodium chloride solution and bacterial suspensions were prepared with cell concentrations from 0.5 to 5.0 billion / v ml The resulting suspensions were administered to white mongrel mice weighing (16 ± 1) g in two ways: once intraperitoneally and five times orally (one dose daily for 5 days). The test results are presented in table 1 (see the end of the description).

In order to more fully study the safety of the strain when administered orally, high doses of the strain were tested with a single oral administration to mice (Table 2). It was established that a single administration of per os high doses of the strain - (10-20) • 10 ⁹ cells / ml does not lead to a decrease in the weight of animals on the first day after administration and on the following days the weight of the animals increases.

To study the toxicity (Table 3), the strain was grown on meat and peptone agar for 24 hours at 37 ^° C, washed with 0.9% sodium chloride solution, and suspensions containing 1.0 to 5.0 billion cells per 1 were prepared ml, which were then inactivated by heating at 100 ^o C for 1 hour. Suspensions were administered by the methods described in the study of virulence.

To study toxigenicity (Table 4), the strain was grown for 10 days on meat-peptone broth at a temperature of 37 ^o C. After the expiration of the term, the culture fluid was centrifuged at 8000 rpm for 30 minutes. A supernatant was used for administration to mice. The control was a group of mice that received sterile meat-peptone broth.

 Observations of animals were carried out for 5 days after the end of the course of injections or oral administration. During this period, all animals remained active, ate well-fed diets, gained weight, physiological functions and behavioral reactions did not change. The data obtained indicate the safety of the strain.

To determine the specific activity, the antagonistic activity of the strain was studied in relation to test cultures of pathogenic microorganisms causing plant diseases (Table 5), humans and animals (Table 6). The study was carried out by the method of delayed antagonism. For this, the strain culture was seeded with a loop in the center of a Petri dish with agarized Gause N2 medium. Crops were incubated in an incubator at 37 ^o C for 72 hours. Then, test microorganisms (500 millionth suspension of diurnal cultures in physiological saline) were seeded to the grown culture. Analysis was carried out after 18 hours of incubation at 37 ^o With the size of the zones of lack of growth of test cultures. The growth of test cultures was controlled by their parallel growth on plates with agarized Gause N2 medium without culture of the studied strain. The obtained data of the test results are presented in table. 5, 6.

 From these tables it follows that the B. subtilis 11 B strain has a wide spectrum of antagonistic activity against pathogenic bacteria and fungi that cause diseases of humans, animals, and plants, which makes it possible to create a probiotic drug based on it that can treat a wide range of diseases and can be used in medical, veterinary and agricultural practice.

Used sources of information
1. Vyalykh L.K., Melentyev L.I. Induction of rust resistance in barley and wheat caused by the drug Bacispecin BM / Microbiol. protection of the biosphere in the regions of the Urals and North. Caspian Sea: Abstract. doc. All symp -Orenburg, 1991. 23-24.

 2. Menlikeev M.Ya., Smirnov VV, Baiguzina F.A. et al. Phytosporin biological product for protecting plants from diseases / Patent N 2099947, RU, C 12 N / 20, publ. 12/25/97, bull. N 12.

 3. Smirnov V.V., Reznik S.R., Sorokulova I.B. et al. Biosporin preparation for the prevention and treatment of human gastrointestinal diseases // A. p. N 1722502. - SU - A 61 K 39/02, publ. 03/30/92.

 4. Mikhailova N.A., Kuznetsova T.N., Kunyagina O.V. / Therapeutic and prophylactic drug Baktisporin // Patent N 2130316, prior. 01/23/97.

 5. Nikitenko V.I. Bacterial drug for the prevention and treatment of inflammatory processes and allergic diseases // International application. N 89/09607, WO, publ. 10/19/1989.

 6. Ivanovsky A. A., Vepreva N. S., Zimireva V. V., Lagunova O. P. A method of obtaining a probiotic for veterinary medicine / RU Patent N 2084233, publ. 07/20/97. - Bull. N 20.

 7. Smirnov V.V., Reznik S.R., Sorokulova I.B. and other Preventive biological product subalin / RU Patent N2035185, publ. 01/31/92. - Bull. N 14.

 8. Reznik S. R., Sorokulova I. B., Vyunitskaya V. A. and other Preventive biological product sporolact // Patent N 2035186. - RU. - A 61 K 35/66, publ. 05/20/95, bull. N 14.

 9. Novikova I.I., Litvinenko A.I., Nugumanova T.A., Kalko G.V. The bacterial strain Bacillus subtilis to obtain a drug against phytopathogenic fungi // Patent N 2081167 RU. - C 12 N 1/20, publ. 06/10/97. Bull. N 16.

Claims (1)

 The bacterial strain Bacillus subtilis 11 B, deposited under the number VKM B-2218D in the collection of microorganisms of the IBPM RAS, has a wide antagonistic activity against strains of pathogens that cause diseases in plants, people and farm animals.

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