RU2422511C1 - Brevibacillus laterosporus BACTERIA STRAIN PRODUCING WIDE SPECTRUM OF BIOLOGICALLY ACTIVE COMPOUNDS - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: Brevibacillus laterosporus bacteria strain, Russian National Collection of Industrial Microorganisms No. B-10531 is produced by multistage selection from natural Brevibacillus laterosporus 16-336 strain. An algicide activity of the strain makes 10.5-52.5 % of residual optical density in 24-h incubation. A diameter of a zone of growth retardation of plant pathogenic fungi makes 3-14 mm. A diameter of a zone of growth retardation of pathogenic bacteria makes 5-12 mm. ^ EFFECT: strain produces a wide spectrum of biologically active compounds for control of microscopic algae of various taxonomic types, plant pathogenic fungi and pathogenic bacteria. ^ 5 tbl, 7 ex

Description

The invention relates to microbiology and biotechnology, in particular the production and use of biological agents against phytopathogenic fungi, pathogenic bacteria and microscopic algae. In recent years, biological pesticides have been widely used to provide environmentally friendly farming conditions. Microorganisms continue to be the most sought-after producers of various compounds with antibacterial effect. There is growing interest in biological agents that inhibit the development of microscopic algae. Among the microorganisms producing biologically active compounds of various types, spore-forming gram-positive bacteria Brevibacillus laterosporus have occupied a special place in recent years. Interest in these bacteria is due to their lack of toxicity to warm-blooded animals.

The B. laterosporus strains have a number of biotechnological advantages: they are easily cultivated on relatively cheap nutrient media, do not require special conditions for cultivation, bacillus strains can be maintained in the laboratory for a long time. Currently, B. laterosporus strains are described that synthesize a number of biologically active compounds, including antibiotics, pesticidal factors, enzymes, algaecides, nematicides, etc. The above indicates a high biotechnological potential of strains of Brevibacillus laterosporus as possible producers of a wide range of antibiotic factors.

A number of the described strains of B. laterosporus can be used in medicine. So, B. laterosporus strains produce a large number of antibiotics of various chemical structures and mechanisms of action. The water-soluble antibiotic of non-protein nature, laterosporamine, had a weak inhibitory activity against gram-positive and gram-negative bacteria [1]. The analogue of the spergualin antibiotic, 15-deoxyspergualin, had antitumor activity and a pronounced immunosuppressive effect on various transplantation models [2-3].

The group of cyclic decapeptides of the thyrocidic family — loloatins, which have high antibiotic activity against pathogenic gram-positive bacteria, was isolated from the marine B. laterosporus strain [4]. The cyclic decapeptide laterocidin was isolated in Russia [5]. Given the toxicological safety and the ability to quickly colonize the intestine, some B. laterosporus strains can be used as effective probiotics. So, the bacteria B. laterosporus BOD is part of the probiotic preparations in gastroenterology used to treat diseases associated with impaired human intestinal microflora, which indicates their safety. This strain had antifungal and antibacterial effects [6]. B. laterosporus strains having an insecticidal effect against house flies, mosquito larvae, and corn rootworm have been described [7–9]. An insecticide based on the B. laterosporus strain caused 100% death of larvae and adult domestic flies [10].

There is evidence of antagonistic activity of B. laterosporus against plant pathogens. Strain B. laterosporus was used to create a biological fungicide to protect potatoes from phytopathogenic fungi [11]. Based on a number of data, some researchers propose including the B. laterosporus culture, along with other soil bacteria, in the composition of bacterial mixtures used to potentiate the effectiveness of chemical herbicides [12].

It was shown that strains of B. laterosporus can inhibit the development of fungi that affect humans and animals. The introduction of B. laterosporus culture to the animal in 7 days reduced the growth of Candida albicans by 95-99% [13].

Peptide antibiotic with mol. mass 1608 Yes, produced by the strain, isolated from marine sediment. Along with fungicidal activity against Candida albicans and Aspergillus fumigatus, the antibiotic also had antibacterial activity against a number of gram-positive and gram-negative bacteria [14].

Strain B. laterosporus BOD, which is part of the widely used probiotic Flora Balance, in 28 days reduced the proportion of Candida albicans in the human intestinal tract from 100% to 19% [6]. Thus, the bacteria B. laterosporus BOD proved to be environmentally friendly transit organisms that ensure the development of a healthy or beneficial microflora of the digestive system.

Tillage with B. laterosporus bacteria increased plant growth and increased cereal yields by almost 30% compared to untreated soil [9]. Pre-treatment of the soil with B. laterosporus bacteria reduced the number of aerobic bacteria of the intestinal group, which ensured the fixation of the alkaline soil environment. Processing target plants with bacteria of this species inhibited the development of phytopathogenic bacteria and microscopic fungi, which increased the yield of plants [15].

Very interesting biologically active compounds include alkaline proteinase with a molecular mass of about 30 kDa, which is a pathogenicity factor for nematodes [16]. The wide variety of biologically active compounds produced by B. laterosporus is evidenced by antagonistic activity against white worms [17]. In Russia, the cyclic decapeptide laterocidin is described, which exhibits antagonistic activity against various species of organisms, including protozoa [5].

An intensive search is currently underway for new strains of bacilli, including among B. laterosporus bacteria, producers of lipopeptides and cyclopeptides, as potential producers of broad-spectrum antibiotics. In this regard, the ability to inhibit the growth of microscopic algae, an algipid effect, discovered in some B. laterosporus strains is of interest [18-19]. The problem of combating microalgae in recent years has become urgent. The inflow of industrial, domestic, and agricultural wastewater leads to water pollution, expressed in the "bloom" of water due to the development of various types of microscopic algae, and, above all, blue-green algae (cyanobacteria). The growth of microalgae leads to the “blooming” of water bodies and, in the future, is accompanied by the death of their excess biomass, the release of toxins, a violation of the oxygen regime, and organoleptic manifestations of decay. “Blooming water” leads to a deterioration in water quality, undesirable for domestic, recreational, fishery, energy use, causes a number of serious social and economic consequences, which determines the need to combat blue-green bacteria and their metabolic products. Given that microalgae also develop in rice fields, it seems relevant to search for strains with an algicidal effect.

The objective of the invention:

to obtain a strain of bacteria with a wide spectrum of antagonistic activity and combining algicidal, fungicidal and antibacterial activity.

The problem is solved by obtaining a strain of bacteria Brevibacillus laterosporus VKPM B-10531.

The inventive strain was obtained as a result of multi-stage selection from a natural strain of Brevibacillus laterosporus 16-336, isolated from natural sources, and deposited in the All-Russian collection of industrial microorganisms under the number VKPM B-10531.

As the closest analogue of the claimed object, the strain Brevibacillus laterosporus VKPM B-9405, which has an algicidal effect, is considered. The strain Brevibacillus laterosporus VKPM B-10531 differs from the closest analogue of Brevibacillus laterosporus VKPM B-9405 with a wide spectrum of antagonistic activity - algicidal, fungicidal and antibacterial.

1. The cultivation of the strain.

As nutrient media used NBY, ASC, SG, YM of the following composition (wt.%):

NBY environment:

nourishing broth "Difco" - 0.8; Difco Yeast Extract - 0.3; water - the rest.

ASC environment:

Difco Yeast Extract - 0.5; sucrose - 2.0; lemon acid - 1.17; Na ₂ SO ₄ - 0.4; (NH ₄ ) ₂ HPO ₄ - 0.42; Kcl - 0,076; MgCl ₂ × 6H ₂ O - 0.042; water - the rest

SG environment:

sodium succinate - 0.5; glycerol - 1.5; MgSO ₄ - 0.02; (NH ₄ ) ₂ HPO ₄ - 0.5; Kcl - 0.2; water - the rest.

YM environment:

hydrolysis yeast - 4.5; water - the rest.

To prepare agarized culture media, agar-agar (2.0 wt.%) Was additionally added to liquid media.

2. Cultural and morphological characteristics.

Gram-positive movable sticks, peritrichi, 0.5-0.7 × 3.3-5.0 microns in size, do not form chains. Well sporulates in NBY liquid and solid nutrient media.

When cultured on NBY agar medium, after 48 hours at a temperature of 28-30 ° C, the strain forms rounded colonies with a diameter of 3-5 mm of light cream color with a smooth surface and an uneven edge. After 72-96 hours of growth on NBY, the strain forms spores. In the process of sporulation, a characteristic canoe-like structure is formed adjacent to the dispute from one of the sides. Free spores are elliptical in shape. The spore size is 0.8-1.2 × 1.4-1.7 microns.

3. Physiological and biochemical properties.

The cultivation of the strain of the claimed strain is carried out in the temperature range of 28-30 ° C at a pH of 6.8-7.2. The optimum temperature for the cultivation of strain B-10531 is 30 ° C and a pH of 7.0.

The strain forms catalase. Hydrolyzes casein and gelatin, does not hydrolyze starch. Urea does not break down. Does not ferment sucrose, arabinose, xylose, lactose. Ferments glucose, maltose, mannitol, fructose. Glucose is fermented without gas. Does not form acetone. Forms lecithinase, tween esterase. Grows in the presence of lysozyme.

Based on morphological (the presence of a canoe-like structure attached to the spore) and physiological and biochemical characteristics, the VKPM strain V-10531 is assigned to Brevibacillus laterosporus [20].

4. Antagonistic activity.

The strain exhibits antagonistic activity against microalgae.

The strain is characterized by a wide spectrum of algicidal activity and inhibits the development of the following microscopic algae (table 1).

Table 1 Taxonomy of used microalgae Type of microalgae Taxonomic type Anabaena sp. 5781 Cyanophyta Nostoc sp. A-10 Cyanophyta Microcystis aeroginosa 562 Cyanophyta Microcystis aeroginosa 905 Cyanophyta Cosmarium sp. Chlorophyta Chlorella vulgaris Chlorophyta Chlamydomonas reinhardi Chlorophyta Thalassiosira weissflogii Dinoflagellata Prorocentrum micans Dinoflagellata Amphidinium carterae Dinophyta

The strain also exhibits antagonistic activity against phytopathogenic fungi: Fusarium solani, Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum, Phomopsis helianthi, Phoma solanicola, Alternaria tenuis, Botrytis cinerea;

gram-positive bacteria: Staphylococcus aureus, Enterococcus faecalis;

Gram-negative bacteria: Salmonella enteritidis, Escherichia coli, Pseudomonas aeruginosa.

The invention is illustrated by the following examples.

Example 1

Cultivation of a strain of Brevibacillus laterosporus VKPM B-10531 in ASC and SG.

ASC and SG media were prepared in distilled water, the pH was adjusted to 7.0, poured into 5-10 ml tubes and autoclaved at a pressure of 0.8 atm at a temperature of 120 ° C for 30 min. Sterility and pH were monitored by taking appropriate samples. Then, in a test tube with ASC or SG medium, 1 ml of inoculum was prepared, prepared as follows: in a standard test tube with 5 ml of sterile NBY culture medium, a bacterial culture collected by loop from NBY solid agar medium was placed and incubated for 16 hours at a temperature of 30 ° C with shaking 250 rpm The contents of the tube, after light-optical control for the absence of extraneous microflora, were used as seed.

The inventive strain was cultured in standard 5 ml tubes in ASC or SG liquid culture media for 72-96 hours at 28-30 ° C with shaking at 250 rpm. The presence of bacterial cells and spores and the absence of extraneous microflora were evaluated by light microscopy and plating on nutrient agar. By 96 hours of cultivation, a slight spore formation was observed - 5-10% of spores, mainly 90-95% of vegetative cells. The microbiological method was used to evaluate the titer of the colony of the forming units (CFU) of the VKPM B-10531 strain, which was ~ 1-2 × 10 ⁹ / ml.

Example 2

Cultivation of a strain of Brevibacillus laterosporus VKPM B-10531 in NBY medium.

For cultivation of the inventive strain B-10531 use NBY medium [21]. The medium is prepared in distilled water, the pH is adjusted to 7.0; poured into standard glass conical flat-bottomed Erlenmeyer flasks with a volume of 750 cm ³ of 50 ml each and sterilized at a pressure of 0.8 atm, a temperature of 120 ° C for 30 minutes. After sterilization of the flask, with the medium contained in them, it is cooled to +20 C. Sterility and pH of the medium are controlled by taking appropriate samples. 5 ml of VKPM B-10531 culture, obtained by culturing in NBY medium for 16 hours at 30 ° C with shaking at 250 rpm, is used as seed. The absence of extraneous microflora is controlled microscopically. The strain productivity is determined by the method of serial dilutions with subsequent seeding on an agar medium. A uniform growth of culture is observed throughout the entire volume of the medium. By 96 hours of cultivation, mainly 90-95% of spores and 5-10% of vegetative cells are observed.

The microbiological method was used to evaluate the CFU titer of VKPM strain B-10531, which was ~ 1 × 10 ⁹ / ml.

The VKPM B-10531 strain was maintained on jambs in standard tubes with NBY agar medium.

The strain was stored after lyophilization. Vegetative cell culture was grown on NBY agar medium until spores formed. Spores and remaining cells were washed away with a protective medium containing sterilized skim milk. Ampoules with a suspension of cells and spores were kept for 15 min at -70 ° C, and then quickly transferred to a drying chamber connected to a vacuum lyophilization system (model 75150 from Labkonko). Drying time was 4 hours. Lyophilized samples were stored in a refrigerator at a temperature of + 4 ° C.

Example 3

Cultivation of microscopic algae used to identify the algicidal properties of the strain.

Used strains of nitrogen-fixing filamentous species of microscopic algae of cyanobacteria: Anabaena sp. 5781 - received from the Department of Microbiology, Leningrad State University; Nostoc sp. A-10 - obtained from the collection of IMET, Jena, East Germany; Microcystis aeroginosa 905 and 562 non-fixing cyanobacterial nitrogen strains were obtained from the Institute of Hydrobiology of the Chinese Academy of Sciences (Hubei Province, Wuhan); strains of green (Cosmarium, Chlorella, Chlamydomonas) and seaweeds (Amphidinium, Prorocentrum, Thalassiosira) were obtained from the Department of Hydrobiology of the Biological Faculty of Moscow State University.

Strains of cyanobacteria Anabaena, Nostoc, and Cosmarium green algae were grown in modified BG-11 medium [22]. The strain Chlorella vulgaris was grown in Tamiya environment [23]. The strain Chlamydomonas reinhardy was grown in TAPs [24]. Microcystis aeruginosa strains were grown in B-12 medium [25]. Culture media for seaweeds: diatoms - Thalassiosira - weissflogii and dinofacies - Amphidinium carterae and Prorocentrum micans were prepared on artificial sea water with a salinity of 30 ° / _oo , which was pasteurized three times before adding additives. Then additives were added to this water according to the formulation of the medium f / 2 [26].

Microalgae strains were grown in the respective media of 50-100 ml in flat-bottomed glass flasks with a capacity of 250 ml at 25-30 ° С without aeration at room temperature and round-the-clock illumination. We used CBE LBU-30 fluorescent white fluorescent lamps, which provided illumination of 40 μmol quanta m ^-2 s ^-1 in the PAR area. Seaweed cultures: diatoms - Thalassiosira weissflogii and dinophytic - Amphidinium carterae and Prorocentrum micans were grown in 1 L flasks placed on a shaker (cell suspension volume 0.5 L) at a temperature of 20 ° C and 30 μmol quanta · m ^-2 · s ^{- 1} in the field of HEADLIGHTS created by fluorescent lamps. The duration of the light period was 14 hours per day.

Example 4

Determination of algicidal activity of strain VKPM B-10531.

Algicidal activity of the strain was detected by co-cultivation. To quantify the algicidal effect, a 96-hour culture of bacilli in a 5: 1 ratio was added to the liquid culture of blue-green (Nostoc, Anabaena, Microcystis) and green algae (Cosmarium, Chlorella, Chlamydomonas) and the residual optical density was determined by the formula: (OD _О ), as (OD _t / OD _n ) × 100, where OD _N is the initial OD, OD _T is the OD after incubation for T hours, respectively.

The optical density (at a wavelength of 590 nm) of the mixture was measured at time zero and after joint incubation.

The algicidal activity of the claimed strain was evaluated by the decrease in optical density after 24 hours of incubation. The decrease in the optical density of the mixture with Nostoc and Anabaena was 10 times, and Microcystis, Cosmarium, Chlorella was one and a half to two times, which indicated its lytic effect on microalgae (table 2). Microalgae cell lysis was also detected by optical microscopy.

table 2 Algicidal activity of strain VKPM B-9844 grown in ASC medium The residual optical density after 24 hours of incubation,% Nostoc Anabaena Microcystis Cosmarium Chlorella Chlamydomonas No processing 100.0 100.0 100.0 100.0 100.0 100.0 Processing strain B-10531 10.5 12.5 45.0 50,5 52,5 65.5 Processing strain B-9405 20.1 22.2 63.1 65.5 66.8 Not determined

It should be noted the significant turbidity of the incubation mixtures of Microcystis, Cosmarium, Chlorella, Chlamydomonas with the strain VKPM B-10531. However, discoloration of the resulting mixture occurs. It should be noted that the cyanobacteria Anabaena and Nostoc are more sensitive to the influence of the culture fluid of the studied strain and the discoloration of algae cells begins already at 1-2 hours of joint incubation. Lysis of vegetative cells of all types of microalgae was revealed microscopically.

Example 5

Determination of the spectrum of action of algicidal factor strain VKPM B-10531.

To assess the antagonistic (algicidal) effect, the color change of incubation mixtures of planktonic forms of microalgae and VKPM strain V-10531 was determined. Microalgae synthesize various pigments (chlorophyll, phycobilins, carotenoids), which determine their color. In all experiments, discoloration of incubation mixtures of microalgae with VKPM strain B-10531 was observed. Measurement of the optical density of a mixture of seaweed Amphidinium, Prorocentrum, Thalassiosira with VKPM strain B-10531 was not possible due to the physicochemical properties of the high turbidity of the resulting suspensions.

Table 3 The spectrum of algicidal action of the culture fluid of the strain VKPM B-10531 Taxonomic type of algae Preferred habitat Initial color of the mixture Color measurement level through 24 hours 96 hours * Anabaena 5781 fresh water blue green +++ +++ Nostoc a-10 fresh water blue green +++ +++ Microcystis aeruginosa 562 fresh water yellow green ++ +++ Microcystis aeruginosa 905 fresh water yellow green ++ +++ Cosmarium sp. fresh water green + +++ Chlorella vulgaris fresh water green + ++ Chlamydomonas reinhardy fresh water green + ++ Prorocentrum micans sea water light green + ++ Prorocentrum micans sea water light green + ++ Amphidinium carterae sea water brown + ++ * (+) - color change of the mixture to light green (or yellow), (++) - lightening the mixture to yellow, (+++) - complete discoloration of the mixture of microalgae and strain B-10531

As follows from table 3, all strains of microalgae are sensitive to the algicidal effect of strain B-10531, but the level of their sensitivity varies depending on the exposure time. Optical microscopy revealed the lysis of vegetative microalgal cells of the studied taxonomic types.

Example 6

Determination of the fungicidal activity of the strain Brevibacitlus laterosporus VKPM B-10531.

To identify fungicidal activity used the culture fluid of strain B-10531. Testing fungicidal activity was carried out by the method of wells. To do this, the tested phytopathogenic fungi were sown in the center of the Petri dish with potato agar (wt.%: Bacto-agar - 2.0; glucose - 1.5; potato broth - up to 1 l, pH - at 25 ° С - 7.2) . 200 μl of 72-hour culture fluid of the strain was added to wells cut in agar with a diameter of 10 mm. The plates were incubated in the dark at room temperature. After 48 hours, the presence or absence of fungal growth inhibition zones was assessed. The effectiveness of fungicidal action was determined by the diameter of the zone of absence of growth of phytopathogenic fungi.

Table 4 Phytopathogenic fungi Zones of growth inhibition of phytopathogenic fungi by the culture fluid of strain B-10531 grown on media (mm) Ym SG NBY Fusarium solani fourteen* 12 10 Fusarium graminearum 5 3 3 Rhizoctonia solani eleven 12 - Sclerotinia sclerotiorum fourteen 13 13 Phoma solanicola 10 10 12 Alternaria tenuis 13 10 four Botrytis cinerea 12 10 12 (*) is the diameter of the zone of growth inhibition; (-) - absence of a zone of growth inhibition.

As follows from table 4, the inventive strain inhibited the growth of the mycelium of the studied phytopathogenic fungi.

Example 7

Determination of the spectrum of antibacterial action of the strain VKPM B-10531.

The antibacterial effect of the culture of strain B-10531 was detected on gram-positive and gram-negative test bacteria. Test microorganisms were cultured at 37 ° C and 220 rpm for 24 hours in Mueller Hinton Broth (HIMEDIA, India), wt.%: Casein acid hydrolyzate - 30.0; beef extract - 1.75; soluble starch - 0.15; pH - (at 25 ° C) - 7.4 ± 0.2. Testing of antibacterial activity was carried out by the method of wells in a dense agar medium. For this, bacterial test cultures with an optical density of 0.5 were plated on Petri dishes with 20 ml of Muller-Hinton agar medium. The cups were left at room temperature for 30 minutes, after which 4 mm diameter wells were cut out in agar, into which 25 μl of a culture fluid (QL) sample of strain B-10531, cultured on NBY and ASC media for 72 hours, as well as fractions, were added. sediment and supernatant. The precipitate was separated by centrifugation on an Eppendorf high speed centrifuge at a speed of 10,000 rpm for 15 min at a temperature of 4 ° C. The precipitate was resuspended in an equal volume of sterile distilled water. Petri dishes with samples of QL fractions, sediment and supernatant were incubated at 37 ° C. The antagonistic effect of the strain was evaluated by the presence of a zone of growth inhibition of test bacteria after 24-48 hours and the measurement of zones of inhibition of growth of test microorganisms.

Table 5 The spectrum of antibacterial activity of fractions of strain B-10531 cultivated on NBY and ASC media, on gram-positive and gram-negative test bacteria Fractions of the culture fluid of strain B-10531 Zones of growth inhibition of test bacteria (mm) Staphylococcus aureus Enterococcus faecalis Salmonella enteritidis Escherichia coli Pseudomonas aeruginosa 36 / NBY / KZh 10* 10 9 10 10 36 / NBY / but 8 7 - - - 36 / NBY / oc 10 9 7 6 8 36 / ASC / KZL eleven 12 10 7 10 36 / ASC / but 10 10 6 7 - 36 / ASC / oc 9 9 5 6 - (*) is the diameter of the zone of growth inhibition; (-) - absence of a zone of growth inhibition.

As follows from table 6, the antibacterial activity of the fractions of strain B-10531 varies depending on the culture medium both in spectrum and in diameter of the growth inhibition zone of the test bacteria.

Bibliography

1. Shoji, J., Sakazaki, R., Wakisaka, Y., Koizumi, K., Mayama, M., Matsuura, S. and K. Matsumoto. J. Antibiotics. 1976, V.29, No.4, p. 390-393.

2. Fudaba, Y., Fukuda, Y., Yamamoto, H., Ohdan, H., Shintaku, S., Shibata, S., Miyata, Y., Marubayashi, S, Asahara, T., and Dohi, K 1998 - Transplant. Proc., 1998, V.30, No.7, p. 3582-3583.

3. Tanabe, K., Takahashi, K., Nemoto, K., Okada, M., Yasuo, M., Hayasaka, Y., Toma, H., and Ota, K.J.Urol. Vol., 1994, V.152, No.2, part 1, p. 562-566.

4. Gerard J., Haden P., Kelly M., Andersen R. J. Nat. Prod., 1999, V. 62 (1), p. 80-85.

5. RF patent No. 2229520.

6. Huynh H., Le Hong Due, Cutting S. FEMS Microbiology Rev., 2005, V.29, p. 813-835.

7. Kuznetsova N.I., Smirnova T.A., Shamshina T.N., Ganushkina L.A., Azizbekyan P.P. Biotechnology, 1995, vol. 3-4, p. 11-16.

8. Orlova M.V. Smirnova T.A., Ganushkina L.A., Yacubovich V.Y., Azizbekyan P.P. Appl. Environ. Microbiol., 1998, v. 64, N.7, p. 2723-2728.

9. Aronson A., Dunn P. U.S. Patent No.5055293, 1993.

10. Ruiu L., Satta A., Floris I. Environ Entomol., 2008, V.37 (2), p. 505-509.

11. RF patent 2242125.

12. Branly K., Atkins R. U.S. Patent No.6232270. 2001.

13. O′Donnell B. Patent U.S. No.5455028, 1995.

14. Ren Z.Z., Zheng Y., Sun M., Liu J.Z., Wang Y.J. Wei Sheng Wu Xue Bao., 2007, V. 47 (6), p. 997-1001.

15. O'Donnell B. Patent U.S. No.5702701. 1997.

16. Qiuhong N. .. Xiaowei H., Baoyu T., Jinkui Y., Jiang L., Lm Z., Keqin Z .. Appl Microbiol Biotechnol., 2007, V.74 (2), p. 372-380.

17. Chang S.F., Wan Y.L., Cui J.Y., Wang B.S. Acta Entomol. Sin., 1984, V.26, p. 419-425.

18. Kuznetsova N.I., Azizbekyan P.P., Konyukhov I.V., Pogosyan S.I., Rubin A.B. Reports of the Academy of Sciences, 2007, vol. 241, No. 2, pp. 262-266.

19. RF patent 2323968.

20. Shida O., Takagi H., Kadowaki K., Komagata K. 1996. Proposal for new genera, Brevibaciillus gen.nov. and Aneurinibacillus gen. nov. Int. J. Sysyt. Bacteriol., 45: 939-946.

21. Stanier, R. Y., Kunisawa, R., Mandel M., Cohen-Basire, G. Bacteriool. Rev. 1971, T.35 (2), 171-205.

22. Stanier, R. Y., Kunisawa, R., Mandel M., Cohen-Basire, G. Bacteriool. Rev. 1971, T.35 (2), 171-205.

23. Vonshak A. Microalgae: technology for laboratory cultivation of biomass in outdoor plants. In the book. Photosynthesis and bioproductivity: determination methods. M., Agropromizdat. 1989. S.310-328.

24. Harris E.H. 1989. The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego, p. 780.

25. Nakagawa, M., Y. Takamura, and 0. Yagi. 1987. Isolation of the slime from a cyano-bacterium, Microcystis aeruginosa K-3A. Agric. Biol. Chem. 51: 329-337.

26. Guillard R.R.L., Ryther J.H. // Can. J. Microbiol. 1962. V.8. 923-931.

Claims (1)

The bacterial strain Brevibacillus laterosporus VKPM B-10531, producing a wide range of biologically active compounds to combat microscopic algae, phytopathogenic fungi and pathogenic bacteria.