Strains of bacteria bacillus amyloliquefaciens, having fungicidal and bactericidal action, and biological product on its basis for protection of grain plants against diseases caused by phytopathogenic fungi

FIELD: biotechnology.

SUBSTANCE: bacterial strain Bacillus amyloliquefaciens of All-Russian collection of industrial microorganisms B-11475, which has fungicidal and bactericidal action, is proposed. Also the biological product for protection of grain plants from diseases caused by phytopathogenic fungi is proposed. The biological product is obtained by mixing the active ingredient in the form of culture liquid of the above mentioned strain with a titre of 2-3×109 CFU/ml, and the carrier in the form of fine granules of diatomite in a ratio by volume of 1: 3, followed by drying.

EFFECT: invention enables to increase yield of grain plants and reduce the percentage of infestation by phytopathogenic fungi.

2 cl, 7 tbl, 9 ex

 

The invention relates to agricultural biotechnology, in particular to the production and use of biological means of combating plant pathogens of fungal and bacterial origin. Protection of agricultural plants and vegetable crops, potatoes, grain and fruit from disease is a serious economic problem. Annual losses to agriculture amount to several billion rubles. Among the economically important plant diseases include diseases caused by microscopic fungi. Cereals (wheat, rye, barley, oats) are prone to fungal diseases etiology, which not only reduce by 20-30% level of agricultural production, but also reduce the quality of seeds (germination, growth rate). Phytopathogenic fungi produce toxic and carcinogenic metabolites that contaminate grain and products of its processing. Used chemical means of combating fungal diseases ("Alto", "Raxil", "Fundazol, Folicur") is not efficiently (efficiency is not more than 50-60%), therefore, requires the development of new environmentally safe and effective fungicides to protect them. Chemical fungicides induce resistance in pathogens, have a number of side effects on the environment, cause fit the toxic effect can accumulate in the environment. After their application has called the waiting period, i.e. regulated the time of appearance in the treated areas of people and technology.

There is a serious danger of chemical fungicides used for the processing of finished agricultural products, because we know the negative impact of chemicals on humans and animals. As an alternative plant protection products can be used for biological drugs, which are based on the metabolites of bacterial origin, providing antagonistic activity against various pathogens. Currently described fungicides peptide produced by bacteria of the genus Bacillus - furini, fungicide, surfactin [1-6]. The mechanism of antifungal action of the peptides caused either by lysis of the cells, either by suppression of the synthesis of components of cell walls. A number of strains .cereus produce antibiotic zwittermicin providing an inhibiting effect on phytopathogenic fungi [7].

Biological products based on microorganisms, producing antagonistic factors, can also be used for combating phytopathogenic fungi infecting seed. Known biological drug "Backoff", based on the strain ofBacillus subtilisPI is 215 [8]. The drug is recommended for the protection of plants from diseases. The active principle of the drug "Backoff", which is a strain of.subtilisIPM 215, considered as the closest analogue of the claimed strain.

The objective of the claimed invention:

- expand your Arsenal of strains of bacteria with antagonistic activity against a number of pathogens of cereal plants.

to develop on its basis the drug for crops protection of plants from diseases caused by phytopathogenic fungi.

Problem solved by:

- get a strain of bacteriaBacillus amyloliquefaciensVKPM B-11475 possessing fungicidal and bactericidal action and

development of a biological product for protection of cereal plants, caused by phytopathogenic fungi, obtained by mixing the active principle in the form of cultural liquid fungicidal strain of bacteriaBacillus amyloliquefaciensVKPM B-11475 with the title of 2-3×109CFU/ml and a carrier in the form of fine granules of diatomaceous earth in the ratio by volume of 1:3 with subsequent drying.

The inventive strain obtained in a multistage selection from natural strains ofBacillus amyloliquefaciensisolated from soil, and deposited in Russian national collection of industrial microorganisms under the number VKPM B-11475.

The inventive strain of Bacillus amyloliquefaciens In PMBC-11475 has the following characteristics:

1. Cultural and morphological characteristics.

Gram-negative motile Bacillus, peritricha size of 0.5-0.7×2.5 to 3.0 mm, the chains do not form. Strain Bac. amyloliquefaciens VKPM B-11475 hydrolyzes gelatin and starch, utilize citrate, reduces nitrate, forms catalase destroys casein, produces acid from glucose, car stereo and arabinose, lecithinase does not produce, has by and amylase activity and has no lipase activity.

As a solid nutrient media used agarized medium NBY, DPS, YM.

The composition of the medium NBY, wt.%:

Nutrient broth "Difco" (nutrient broth) - 0,8;

Yeast extract Difco" (yeast extract) to 0.3;

water - the rest.

pH of 6.8 to 7.2

The composition of the medium DPS, wt.%:

Hydrolytic yeast - 3,0;

corn flour - 1,5;

water - the rest.

pH of 6.8 to 7.2

The composition of the medium YM, wt.%:

hydrolytic yeast - 4,5;

water - the rest.

pH of 6.8 to 7.2

To prepare agar nutrient media in liquid medium was additionally added to the agar-agar (2.0 wt.%).

Under cultivation on agar media NBY, DPS and YM after 24-30 hours at a temperature of 28-30 With the strain forms a white opaque colonies with a diameter of 3-5 mm After 72 hours of growth on these nutrient medium strain produces spores. Free spores are elliptical in shape. The size of the spores of 0.5-0.6×0.9 to 1.5 mm.

On the basis of morphological, physiological and b is khimicheskikh signs, as well as the results of analysis of the nucleotide sequence of the variable regions of 16S rRNA of the claimed strain VKPM B-11475 identified as Bacillus amyloliquefaciens.

2. Fungicidal properties of the strain.

The strain is characterized by a broad spectrum of fungicidal activity and suppresses the development of the following phytopathogenic fungi: Alternaria tenius, Aspergillus niger, Fusarium frost, Fusarium solani, Fusarium culmorum, Fusarium avenaceum, Fusarium sporotrichioides, Microdochium nivale, Phomopsis helianthi, Phoma solanicola, Phytophtora infestans, Rhizoctonia solani, Sclerotinia sclerotiorum.

3. Antagonistic activity of the strain.

Strain of Bacillus amyloliquefaciens In PMBC-11475 synthesize biologically active substances and exhibits antagonistic activity with respect to:

- gram-positive bacteria: Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, B. megaterium, Bacillus, Staphylococcus ssp., Streptococcus ssp.

- gram-negative bacteria: Pseudomonas aureofaciens, Pseudomonas corrugata, Pseudomonas marginata, Erwinia cnrysanthemi, Xanthomonas campestris.

The closest analogue of the claimed strain a strain of B. subtilis IPM 215 and developed on the basis of the drug Backfit have a much narrower spectrum of fungicidal action. "Backoff" is recommended for treatment of potato tubers before putting it into storage for protection only from Fusarium wilt, late blight and stem canker. The inventive strain of Bacillus amyloliquefaciens In-11475, in addition to the pathogens of these diseases, inhibits the development of phytopathogen what's mushrooms-causative agents of superficial necrosis (Phoma solanicola), Alternaria (Alternaria tenius) and scab blight (Fusarium frost).

As the advantages of the proposed strain of Bacillus amyloliquefaciens In-11475 compared with the nearest equivalent strain of B. subtilis IPM 215 it should be noted its ability to produce surface-active substances (surfactants) in the process of cultivation, which is an important characteristic of the proposed strain, as in the future when creating the preparative form there is no need to introduce additional surfactants. In agriculture for plant protection surfactants are widely used for the formation of emulsions and as adhesives. Surfactants are used to improve the efficiency of transportation fungicidal components to plants, as they affect the permeability of cell membranes, in addition, surfactants have an inhibitory effect on the enzymatic system of phytopathogenic fungi.

Maintaining a strain of Bacillus amyloliquefaciens In PMBC-11475 spend on the shoals in standard test tubes with agar medium NBY. The deposited strain is carried out in a dried state. To obtain lyophilized vegetative material culture of cells grown on agar medium NBY to spore formation. Disputes and the remaining cells are washed with a protective medium containing sterilized skim milk. Ampoules with a suspension of cells and spores incubated 15 min at -70°C, and then rapidly the ro is transferred into the drying chamber, connected with the vacuum system lyophilization (model 75150 firm "Labkonko"). The drying time of 4 hours. Lyophilized samples stored in a refrigerator at a temperature of +4°C.

The preparation of the proposed drug

1. Getting active start - culture liquid strain of Bacillus amyloliquefaciens In-11475

Strain of Bacillus amyloliquefaciens In-11475 cultivated in clarified environment YM of the following composition (wt.%): hydrolytic yeast - 4,5; water - the rest for 72 hours at 30°C with aeration in a rocking chair at 260 rpm as a result, the culture fluid, which is the active beginning of the proposed drug.

2. The carrier for immobilization of active factor as a carrier for immobilization of active start - culture liquid bacteria Bacillus amyloliquefaciens B-11475, selected fine diatomite, which is a natural sedimentary rock, composed mainly of the remains of diatoms. Natural diatomite is usually loose or poorly cemented, light gray with yellowish or pinkish tinge breed. Chemically diatomite more than 80% consists of water silica. Easily sterilized (withstands temperatures up to 1400°C), is a light porous smallest granules with a particle size fractions from 0.1 to 10.0 mm; effective pore diameter of 50 to 100 nm, with a bulk density not bol is e 620 kg/m 3[9]. Is an inert material that does not interact with the most aggressive environments. Diatomaceous earth is a natural material with the requisite consumer features to create a biological product: sterile, chemically inert, lightweight, free-flowing, non-caking, non-flammable, biologically stable and environmentally friendly, not prone processes of decay, air and water-permeable, has a high adsorption properties and can absorb up to 120% water by weight. Due to the highly porous microstructure granules of diatomaceous earth save and prolong the action of insertion biofungicide not only in the processing of grain, but capable, when introduced into the soil to prevent contamination of crops during the whole vegetative growth.

3. The composition and benefits of the proposed drug

The drug consists of the culture fluid of the strain Bacillus amyloliquefaciens B-11475 containing complex spores, vegetative cells with a title CFU/ml (colony forming units) from 8×108up to 2-3×109and the products of metabolism, immobilized on highly porous fine granules of diatomaceous earth.

The advantage of the proposed drug is that it is well kept, convenient for transportation, provides a stable volume, non-caking, and fungicidal properties manifests as during the processing of the seeds of cereal plants, and is ocve during vegetative growth.

The invention is illustrated by the following examples:

Example 1. Cultivation of a strain of Bacillus amyloliquefaciens B-11475

For cultivation of strain B-11475 use standard glass conical flat-bottomed Erlenmeyer flask with a volume of 750 cm3that fill the 50 ml sterile liquid medium YM or NBY.

Clarified environment YM prepared with tap water. Prepared environment boiled for 10 min, cooled to room temperature and decanted and poured adosados, and the residue discarded; bring the pH of the clarified medium (nadeshiko) to 7.2. In the Erlenmeyer flask with a volume of 750 ml poured into 50 ml of the clarified medium and sterilized in an autoclave under a pressure of 0.8 ATM at a temperature of 121°C for 30 minutes After sterilization, the flasks are cooled to a temperature of +20°C and monitored by sampling for sterility and pH environment.

In 50 ml of sterile clarified environment YM add 2 ml of inoculum grown in the medium NBY on the rocking chair (260 rpm) at 28°C for 14 hours with a title - 1-2×109CFU/ml of Bacterial culture to obtain biomass grown in the same conditions as the seed material within 72 hours. Every day (after 24 hours of growth) take samples for microbiological control, which includes checking the status of culture and the absence of extraneous microflora. To 72 hours cultivation circulatory strain of Bacillus amyloliquefaciens VKMV-P is 2-3×10 9CFU/ml

Example 2. Evaluation of fungicidal activity of the inventive strain under laboratory conditions

The strain Bacillus amyloliquefaciens VKPM B-11475 grown as in example 1. For the analysis of antifungal activity using the culture fluid containing 2-3×109CFU/ml Testing antifungal activity carried out by the method of the hole. For this purpose, the strains of phytopathogenic fungi (table 1) were seeded on Petri dishes with agar and potato-sucrose medium. Potato-sucrose agar-agar is prepared by adding to the strained broth potato sucrose and agar-agar.

Potato-sucrose agar, wt.%:

Sucrose2,0
agar-agar2,0
potato brothrest
pH6,8-7,2

In the agar-agar with borax cut out holes with a diameter of 8 mm, which bring in 150 μl of culture fluid of the proposed strain. The effectiveness of antifungal effect of the proposed strain is determined by the diameter of the zone of no growth of the test cultures. The results are presented in table 1.

Tab the Itza 1
The spectrum of fungicidal activity of the strain Bacillus amyloliquefaciens VKPM B-11475
The test-cultureThe culture fluid
Alternaria tenuis+
Aspergillus niger+
Fusarium frost+
Fusarium solani+
Fusarium culmorum+
Fusarium avenaceum+
Fusarium sporotrichioides+
Microdochium nivale+
Phoma solanicola+
Phytophtora infestans+
Rhizoctonia solani+
Sclerotinia sclerotiorum+
* Sign "+" marked growth inhibition of the fungus

As follows from table 1, the inventive strain has a broad spectrum of fungicidal activity, which is localized in the culture fluid.

Example 3. OC the NCA bactericidal activity of the inventive strain

Evaluation of bactericidal activity of the inventive strain in the laboratory carried out as follows. The strain is grown as in example 1. For analysis of bactericidal activity using the culture fluid containing 2-3×109CFU/ml Testing of bactericidal activity carried out by the method of the hole. For this bacterial culture test (table 2) were seeded on Petri dishes with agar medium NBY, then in the wells of 5 mm diameter contribute 50 μl of culture fluid and incubated at 30°C for 24-48 hours. The effectiveness of the bactericidal action of the inventive strain is determined by the diameter of the zone of no growth of the test cultures. The results are presented in table 2.

Table 2
Spectrum of bactericidal activity of the strain Bacillus amyloliquefaciens VKPM B-11475
The test-cultureThe culture fluid
Erwinia chrysanthemii+
Pseudomonas corrugata+
Pseudomonas aureofaciens+
Pseudomonas marrginata+
Bacillus cereusBacillus thuringiensis+
Bacillus sphaericus-
Bacillus subtilis+
B. megaterium Bacillus+
Staphylococcus aureus+
Xanthomonas campestris+
* Sign "+" marked growth inhibition of test bacteria; "-" denotes the absence of inhibition of growth of test bacteria

As follows from table 2, the inventive strain has a broad spectrum of bactericidal activity, which is localized in the culture fluid.

Example 4. Determination of surface-active properties of the proposed strain

Surface-active properties of the strain (the ability to form surfactants evaluated on the following criteria - foaming activity, the ability to emulsification and surface tension. Strain of Bacillus amyloliquefaciens In PMBC-11475 grown on clarified environment YM on a pie rocking chair (260 rpm) at 30°C for 120 hours.

Foaming was determined after removal from rocking the ratio of volume of foam to the volume of the culture fluid, expressed in %.

To determine the emulsifying activity (Es) adosados obtained after separation of the biomass by centrifugation of the culture fluid of strain at 10,000 rpm, mixed with kerosene in the ratio 2:3, shaken at 300 rpm on a shaker for 3 min and left at room temperature for 24 hours.

Emulsifying activity index (emulsification) estimated as the ratio of the formed emulsion to the volume of the culture fluid in %.

Surface tension is measured stalagmometer method [10] in a volume of 5 ml at 20°C.

Table 3
Characteristics of the claimed ability of the strain to form surface-active substances (surfactants)
StrainFoaming, %The formation of emulsions at 20°C E24%Surface tension, mn/m
The strain Bacillus amyloliquefaciens VKMV-11475387827

From the results shown in table 3, it follows that the claimed strain capable of producing surface-active substances (surfactants), allowing the cooking is on the basis of the drug do not add additional adhesives, necessary for immobilization of the active agent in the granules of the media.

Example 5.

Evaluation of fungicidal activity of the culture liquid of the claimed strain under artificial infection of wheat seeds by phytopathogenic fungi Fusarium frost and Microdochium nivale

In model experiments seeds of wheat, pre-decontaminated by treatment with 0.1%solution of AgNO3, soaked in the culture fluid of strain for two hours. Then the seeds are dried in air for 3 hours and infect spore suspension (105-106spores/ml) of phytopathogenic fungi Fusarium frost and Microdochium nivale.

After infection, the seeds are spread out in a sterile moist chamber (Petri dishes with a layer of cotton wool and filter paper, moistened in accordance with standard sterile water). In each Cup put 20 seeds; repetition - 5x. Cup of seeds incubated at 25-27°C for 10 days. Evaluate the germination, early germination and infection of seeds by the fungi (table 4).

Table 4
Characterization of wheat seeds treated with the culture fluid of the proposed strain, with subsequent infection of these seeds with a mixture of phytopathogenic fungi Fusarium frost and Microdochium nivale
OptionSeed germination (%)The beginning of germination (days)Contamination mushrooms 10 day (%)
Control (without treatment)93,02-318,0
Seed treatment of wheat culture fluid of strain VKMV-1147598,02-32,0

As follows from table 4, the seed treatment of wheat culture fluid of the proposed strain leads to an increase in seed germination, reduction of infection by phytopathogenic fungi without affecting the start time of germination.

Example 6.

Determine the effectiveness of the proposed strain on disputes of various phytopathogenic fungi.

Stage dispute is one of the phases (that rests phase) life cycle of microscopic fungi. Phytopathogenic fungi in the resting stage pose a serious threat to plants as they germinate plants severely damaged. For evaluating the effect of the strain Bacillus amyloliquefaciens VKPM B-11475 spores of pathogenic fungi treated with culture filtrate liquid and analyze the effect after 24 hours of incubation. The United States is m VKPM B-11475 effect on resting cells of fungi in all cases, but in a different way, there is no formation of growth tubes mushrooms and mycelium is not formed (table 5).

Table 5
The influence of the culture fluid of the proposed strain on the germination of spores of pathogenic fungi after 24 hours incubation
Phytopathogenic fungiThe length of the growth tubes, mcmThe nature of the damage caused by the action of the filtrate of the culture fluid of the strain Bacillus amyloliquefaciens VKPM B-11475
controlthe filtrate of the culture fluid
Fusarium frost29,700,0Lysis of conidia
Fusarium solani67,000,0Protoplasmatologia conidia
Phytophthora infestans36,300,0The lack of germination of zoospores

Example 7. The preparation of the proposed drug

For preparation of drug crops the optimum fluid strain VKPM B-11475, grown as in example 1, is mixed with fine granules of diatomaceous earth in the ratio by volume of 1:3 and dried at 50°C for 15-20 hours. The obtained powder preparation should be stored at room temperature in a container with a tightly closed tube to prevent moisture. Fungicidal properties of the drug are evaluated directly after drying.

Example 8.

Evaluation of biological activity of the claimed preparation in the laboratory.

The fungicidal activity of the claimed drug determine method holes on the cups, sown by phytopathogenic fungiFusarium solani, Fusarium frost, Fusarium sporotrichioides, Fusarium avenaceum, Fusarium culmorum. Dry powder, diluted as follows: 100 mg of the dry powder is dissolved in 9 ml of sterile distilled water (dilution 10 times). A detergent solution is left on the table at room temperature for 30 minutes and Then by serial dilution, prepare working solutions with a ratio of 10:1/100 and 1/1000, as in example 2. As a positive control, use the same dilution of the original culture liquid, and as a negative control of sterile distilled water.

The fungicidal activity of dilutions of the dry powder of the proposed drug with the fungicidal activity of the corresponding dilution of the original cult of the Central fluid (table 6).

Table 6
Comparison of the fungicidal activity of the proposed drug with the original culture fluid of strain Bacillus amyloliquefaciens VKPM B-11475
OptionCultivationPhytopathogenic fungi (zone of inhibition of growth in mm)
Fusarium solaniFusarium frostFusarium sporotrichioidesFusarium avenaceumFusarium culmorum
The culture fluid of strain VKPM B-114751/106106118
1/10026474
1/100013242
the claimed preparation 1/107117128
1/10036475
1/100013232

Example 9.

Evaluation of biological activity of the claimed preparation in the field.

Evaluation of biological activity of the claimed preparation in the field is carried out by spraying the infected phytopathogenic fungi winter wheat cultivar Bezostaya-1 flowering 1% aqueous suspension of the proposed drug. As controls use not processed grain wheat grain wheat, treated with a chemical fungicide "Palikur" (Bayer, Germany). The biological activity of the proposed drug is evaluated for yield and infestation by pathogenic fungi of the harvest of winter wheat.

Table 7
The biological activity of the inventive p is eparate field
OptionWheat yieldContamination of grains by phytopathogenic fungi, %
kg/ha%
Control (without treatment)16,010032

Processing wheat declare medication19,01198
Processing wheat preparation "Palikur"17,410928

As follows from Table 7, the processing of wheat grain of the claimed preparation in the field leads to increased productivity and reduction in the percentage of infection obtained harvest phytopathogenic fungi, and fungicidal activity of the inventive preparation is not only not inferior to the reference chemical fungicide "Palikur", but exceeds it by 4%.

References

1. Akra E., Jacques P., Wathelet B., Paquot M., Fuchs R., H. Budzikiewicz, Thonart P. 2001.

2. Influence of culture conditions on lipopeptide production by Bacillus subtilis. Appl. Biochem. Bioctechnol. 91-93: 551-561.

3. M.A. Klich, A.R. Lax, Bland J.M. 1991. Inhibiton of some mycotoxigenic fungi by iturin A, a peptidolipid produced by Bacillus subtilis. Mycopathology. 116:77-80.

4. F. Ahimou, Jacques P., Dejeu M. 2000. Surfactin and iturin A effect on Bacillus subtilis surface hydrophobility. Enzyme Microb. Technol. 27:749-754.

5. Volpon L., F. Besson, Lancelin, J. 2000. NMR structure plipastatins A and In from Bacillus subtilis inhibitors ofphospholipase A2. FEMS Lett. 485: 76-80.

6. Volpon L., F. Besson, Lancelin, J. 1999. NMR structure of active and forms of the sterol-dependet antifungal antibiotic bacillomycin L. Eur. J. Biocem. 263:1-12.

7. Milner J.L., Raffel S.J., Lethbridge B.J., Handelsman J. 1995. Culture condition that influence accumulation ofzwittermicin A by Bacillus cereus UW85. Appl. Environ. Biotechnol. 43: 685-691.

8. Patent RU 2019966.

9. THE 9692-0003-59266087-05 "Materials sorption and filtration".

10. Ostroumov S.A., Lazareva E.V. Surface tension of aqueous solutions of sodium dodecyl sulfate in the presence of aquatic plants. - Water: technology and ecology. 2008, No. 3,p.57-60.

1. The strain of bacteriaBacillus amyloliquefaciensVKPM B-11475 possessing fungicidal and bactericidal action.

2. Biological preparation for crops protection of plants from diseases caused by phytopathogenic fungi, obtained by mixing the active principle in the form of cultural liquid of the strain according to claim 1 with a titer of 2-3 x 109CFU/ml and a carrier in the form of fine granules of diatomaceous earth in the ratio by volume of 1:3 with subsequent drying.



 

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3 tbl

FIELD: biotechnology.

SUBSTANCE: invention can be used for development of antitoxic preparations and feed additives for prevention of mycotoxicosis in farm animals and poultry. The bacterial strain Lactobacillus acidophilus is deposited in the Russian Collection of Microorganisms under the registration number RCM B-2794D. The strain has resistance to T-2 toxin and capacity to destroy it metabolically in the habitat of bacteria. The resistance to the strain T-2 toxin is up to 77%, and the ability to destroy T-2 toxin is 1.8Ч10-12 nmol/CFU.

EFFECT: increased resistance of the strain.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns a method for identifying lactic acid bacilli. The presented method is based on a combination and a polymorphism of toxin-antitoxin RelBE and MazEF gene superfamilies and characterised by the fact that identifying is ensured by genome DNA amplification with using a set of oligonucleotides of certain structure; PCR products are analysed in agarose gel, while a size of the produced fragment is determined by means of a DNA marker. The analysed strain is referred to a specific group, species or strain in case of producing fragments when using certain oligonucleotides.

EFFECT: presented invention can be used for identifying the lactic acid bacilli strains in human microbiota, food stuff, as well as for detecting the analysed strain in clinical samples, or molecular targeting in commercial preparations.

3 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention concerns biotechnology and nanotechnology. The method includes transforming archaeal cells with a recombinant plasmid, growing cells, selecting flagella and modifying the surface of the flagella. The plasmid structure contains recombinant genes for synthesis of flagellins A1 and A2 which form flagella, wherein the sequence of flagellin A1 or flagellin A2 or sequences of flagellin A and flagellin A2 contain at least one peptide insert for selective binding of metal ions or nanoparticles. The point of the peptide insert in flagellin A1 is defined in the region between first and second glycosylation sites located between position 86 and position 96 of SEQ ID NO:2, and the point of the peptide insert in flagellin A2 is defined in the region between first and second glycosylation sites located between position 82 and position 92 of SEQ ID NO:3, where the length of the peptide insert is 5 to 60 amino acids. The method includes selecting archaeal flagella containing peptide inserts for non-covalent bonding with metal ions, performing fragmentation of flagella into fragments and modifying the surface of flagella by binding peptide inserts with metal ions and oxidising metals, washing, drying and packing the obtained nano-structured material.

EFFECT: method enables to obtain a coating for forming active surfaces on flexible and solid substrates or capsules using archaeal flagella, which enable non-covalent bonding of a wide range of substances such as metal ions, metal nanoparticles, semiconductors and other ligands.

6 cl, 11 dwg, 1 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention concerns a method for Staphylococcus aureus genotyping. The presented method involves preparing a pure culture on a solid nutrient medium with DNA purification and amplification by multiplex polymerase chain reaction (PCR) and result detection by agarose gel electrophoresis. The multiplex PCR involves using four pairs of primers complementary to extracellular thermonuclease (nuc) gene, Panton-Valentine leukocidin (pvl) gene, toxic shock protein (tst) gene and methicillin resistance (mecA) gene sites. Staphylococcus aureus is genotyped by the presence or absence of pvl and tst virulence genes and mecA methicillin resistance genes or combinations thereof.

EFFECT: invention enables Stafylococcus aureus genotyping in a combination with the multiplex reaction, as well as provides eliminating the preliminary stage of microorganism identification.

1 dwg, 2 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: strain Rhodotorula sp. 51-18-2P is deposited in the Russian National Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms n.a. GK Scriabin of RAS under the registration number RNCM Y-2993D. The strain is capable to destroy crude oil and petroleum products in contaminated water or soil.

EFFECT: invention enables to improve the efficiency of cleaning of soil, water, waste water and sludge from crude oil and petroleum products.

3 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: bacterial strain Bacillus vallismortis RNCIM B-11017 is grown and a suspension is made of it, which is applied in cryogenic soil and the water environment. It is exposed under the specified parameters from 7 to 60 days and the quantitative content of crude oil and petroleum products in the cryogenic soil and water environment is determined.

EFFECT: invention enables to reduce the time of denaturation of crude oil and petroleum products and to reduce their concentration in the cryogenic soil and water environment.

3 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The method is intended for the identification of nosocomial strains of microorganisms in carrying out epidemiological monitoring. Water-alcohol solutions of 3-4 aniline dyes are prepared. A standardised suspension of the studied microbial culture is added, incubated; the obtained results are estimated and the identity of strains is determined. In case of the identity of sensitivity indices of the separated and studied microbial cultures to each corresponding aniline dye the identification of nosocomial strain is stated.

EFFECT: invention makes it possible to increase the method accuracy.

4 tbl, 3 ex

FIELD: biotechnology.

SUBSTANCE: new strain of hybrid cultured animal cells Mus musculus 2F9 - producer of monoclonal antibodies specific for lysostaphin inhibiting its lytic activity and suitable for its quantitation is proposed. High-affinity monoclonal antibodies (mAb) of hybridoma 2F9 enables to determine lysostaphin in the samples to the concentrations of 0.8 ng/ml. The mAb of hybridoma 2F9 can be used to study the structural and functional properties of lysostaphin and quality control of preparations based on lysostaphin.

EFFECT: improved properties.

3 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, in particular to obtaining nutrient media for cultivation of a listeriosis causative agent. A nutrient medium contains fermentative hydrolysate of soya beans, fermentative hydrolysate from an activated embryo-egg mass of quails, sodium chloride, potassium phosphate 2-substituted 3-aqueous, sodium phosphate 2-substituted 12-aqueous, microbiological agar and distilled water in a specified component ratio.

EFFECT: invention makes it possible to simplify the nutrient solution for cultivation of the listeriosis causative agent.

3 ex

FIELD: agriculture.

SUBSTANCE: group of inventions, including a strain of unicellular green algae Parachlorella nurekis and its application for destruction of cyanobacteria relates to biotechnology. The strain Parachlorella nurekis 1904 KIEG is deposited in the Culture Collection of Algae and Protozoa (Culture Collection of Algae and Protozoa, CCAP), Scottish Marine Institute, Dunbeg, OBAN, Argyll, PA37 1QA, Scotland, UK under the registration number CCAP No.259/1 and can be used for destruction of cyanobacteria.

EFFECT: inventions enable to reduce the amount of cyanobacteria in water reservoirs.

2 cl, 6 dwg, 1 tbl, 2 ex

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