Strain bacillus thuringiensis var darmstadiensis n25 as means of integrated effect on harmful coleopteran insects and phytopathogenic fungi

FIELD: biotechnology.

SUBSTANCE: strain Bacillus thuringiensis var. darmstadiensis №25 (BtH10 №25) has insecticidal activity against pests - coleopteran insects and a wide range of antifungal activity against pathogenic fungi. It is deposited in State Scientific Institution All-Russia Institute for Agricultural Microbiology under the registration number RCAM01490. It can be used in the manufacture of polyfunctional means of protection of plants against harmful coleopteran insects and phytopathogenic fungi.

EFFECT: invention also enables to improve the germinating capacity of plants.

7 tbl, 1 ex

 

The invention relates to the microbiological industry, and can be used to protect plants from pests and diseases.

Protection of agricultural plants, vegetables, grain and fruit crops from diseases and pests is a serious economic problem. Annual losses to agriculture amount to several billion rubles (Kandybin NV, Patyk TI, Yermolov VP, Patyk V.F. Microbiocidal insect and its dominant Bacillus thuringiensis. Saint-Petersburg, Pushkin, 2009, 245 S.).

Among the most dangerous pests are beetles, in particular the Colorado potato beetle, which attacks potatoes, eggplant, tomatoes. The population of the bushes solanaceous crops in different years can reach 50-70%, and yield losses of 25-35%. To control Colorado potato beetle commonly used chemical insecticides that do not have a selective action. They can accumulate in the environment and thereby degrade the environment. On the basis of a group of bacteria Thuringiensis created a number of insecticidal biologics: Production, Methoxylation, Dendrobatidis, Colorado, but they do not cover the whole group mass of harmful species of coleopterous insects.

Known invention "bacterial Strain Bacillus thuringiensis spp. thuringiensis to produce ecotoxicology is bioinsecticides" in patent No. 2061376, application 93034131 priority from 07.07.1993,, IPC A01N 63/00, C12N 1/20, published 10.06.1996,

The patent describes the bacterial strain Bacillus thuringiensis 16-T50 deposited in PMBC no In-6404, which is obtained from the strain 98 by multi-selection on the basis of ecotoxicologie, thermal stability of the dispute and fagoustoichivye. An example of obtaining the culture fluid based on a specified strain.

Known invention "Strain of Bacillus thuringiensis producing endotoxin against Coleoptera and the method of its cultivation" in patent No. 2108384, the application 95111008 priority from 27.06.1995,, IPC C12N 1/20, A01N 63/00, published 10.04.1998,

The patent describes the bacterial strain Bacillus thuringiensis 16931, which is allocated from the dead of winter moths and deposited in PMBC no In-6649; describes the method of its cultivation. It is shown that this strain is effective against Coleoptera, including against the Colorado potato beetle.

Known invention "bacterial Strain Bacillus thuringiensis H8 designed to combat beetles insects" in patent No. 2204598, the application 2001128487 priority from 23.10.2001,, IPC C12N 1/20, A01N 63/00, published 20.05.2003,

The patent describes the bacterial strain Bacillus thuringiensis H8 deposited in PMBC No-8057, which is obtained from a strain of Bacillus thuringiensis H8 PMBC No-6306 through a multi-stage selection for signs of increased synthesis of the toxin against harmful W is starrily insects, in particular, the Colorado potato beetle, and the low-level sporoobrazovanie to prevent environmental pollution disputes in the application of the biopesticide based on this strain. An example of obtaining the culture fluid on the basis of the strain VKPM B-8057.

To combat plant pathogens of fungal or bacterial origin are widely used chemicals: Fundazol, Alto, Folicur and other Efficiency not more than 50-60% and they can cause resistance among pathogens that can accumulate in the environment. Currently, as an alternative means of protection of plants intensively use bacteria as antagonists of phytopathogens, i.e. the use of biological drugs, which are based on the metabolites of bacterial origin, showing antagonistic activity against phytopathogenic fungi: Flavobacterium, Minorin, agate-25, Backoff and others (Smirnov, O., Grishechkina S.D. the Study of the effect of biological preparations based on Bacillus thuringiensis on phytopathogenic fungi. Bulletin of plant protection, No. 1, 2010. P.27-35).

It is also known invention " Consortium of strains antagonists to combat bacterial and fungal plant diseases" in patent No. 2149552, the application 98111359 priority from 15.06.1998,, IPC A01N 63/00, C12N 1/20, SR 3900, published 27.05.2000,

The consortium, consisting of four strains of soil bacteria-antagonists: Bacillus polymixa, Bacillus thuringiensis, Pantoea agglomerans, Pantoea chlororatis, is used for obtaining a drug against bacterial and fungal diseases of plants. Preparation on the basis of the specified consortium of bacterial strains can be used to effectively suppress the development of grey mould and powdery mildew of tomato and cucumber, bacterial cancer in clover, vascular bacteriosis the cabbage, angular leaf spot of cucumber Fusarium wilt of tomato, Helminthosporium in barley, root rot in cotton. Pesticide drug activity comparable to the activity of chemical agents.

Known invention "bacterial Strain of Bacillus thuringiensis, exhibiting fungicidal activity, insecticidal activity and inhibitory virulent properties of phytopathogenic bacteria Ervina carotovorola" in patent No. 2347809, the application 2007132825 priority on 31.08.2007,, IPC C12N 1/20, A01N 63/00, published 27.02.2009,

The bacterial strain Bacillus thuringiensis obtained by multistage selection from a material selected from natural samples, and deposited under the number VKPM B-9790. This strain has multiple pesticide activities, namely: insecticidal and fungicidal activity against larvae of Coleoptera (Colorado potato beetle, a large flour beetle is ka, oxalic and guelder rose leaf beetle), as well as the ability to suppress the manifestations of the virulence properties of pathogenic bacteria Ervina carotovorola spp. carotovorola.

Known bacterial strain Bacillus thuringiensis H10 No. 109 (BtH10No. 109) - producer basicola combining insecticidal and antifungal properties (Kandybin NV, Smirnov O., Barbashova NM New insecticidal preparation with a specific effect on the beetles. // Materials of all-Russian scientific-production meetings, Krasnodar 21-24 August 1994 2 Hours. Pushchino, 1994. S-181) prototype.

The results of studying the influence of strain of the bacteria Bacillus thuringiensis H10 No. 109 (BtH10No. 109) - producer basicola on phytopathogenic fungi published articles:

- Grishechkina S.D., Smirnov O., Kandybin NV Fungistatic activity of different subspecies of Bacillus thuringiensis // Mycology and Phytopathology. Volume 36, issue 1, 2002, Mushrooms pathogens;

- Grishechkina S.D., Smirnov O. Vegetation and field evaluation of antifungal effect of Bacillus thuringiensis // Bulletin of plant protection. All-Russian Institute of plant protection, Russian Academy of agricultural Sciences. Saint-Petersburg, Pushkin, 2010, №3. With 44-49.

The task of the claimed invention to provide a strain-based environmentally safe bacteria from the group of Bacillus thuringiensis with multifunctional properties: high insecticidal and EN is evangelinou activity against a wide group of mass of harmful species of Coleoptera and the growth-stimulating effect on plants, suitable for a highly effective insecticidal drug.

The task is solved in that as a means to obtain insecticidal drug multifunctional actions with insecticidal and antifungal activity and growth promoting properties of the used strain of Bacillus thuringiensis var. darmstadiensis No. 25.

Strain of Bacillus thuringiensis var. darmstadiensis No. 25 (BtH10No. 25) selected from the corpses of the Colorado potato beetle in the Leningrad region and deposited in 12.09.2012 Departmental collection of useful microorganisms for agricultural purposes RAAS (BKSM) of wildebeest WNISM under registration number RCAM01490, as evidenced by a certificate of Deposit from 30.10.2012, (reference attached).

Strain BtH10No. 25 has pronounced insecticidal properties against Coleoptera - phytophages. Identified as antifungal and growth-promoting properties of the strain.

Strain BtH10No. 25 is characterized by the following properties.

Cultural and morphological properties.

Vegetative cells of gram-positive, rod-like shape with a size of 1.2 to 1.5×2,5-4,5 µm, single with peritrichous type jgutikova. Form along with sporos personally tetragonal crystal, bipyramidal or ovoid shape with a size of 0.2-0.6×0.3 to 0.8 mm (edge length). Spores oval, subterminal 0.6-0.8×1.4 to 1.6 m is m In the cell, there arises a dispute and one crystal of endotoxin. In fish broth (RB) growth good, forms a film. When growing strain on the fish agar (RA) after 48 h at 28-30°C forms colonies flat, rough grayish-white color and irregular rounded, opaque. The diameter of the colonies 8-10 mm. On slices of potato growth moderate grayish-green color. During cultivation in liquid nutrient media the strain grows at temperatures 20-36°C (optimum 28-32°C) and the measure of acidity pH of 6.2-8.0 (optimum of 6.8 to 7.2) with shaking on a shaker at 220 rpm the Formation of spores and crystals of endotoxin ends after 60-72 hours of growth.

Biochemical properties.

Optional gone anaerobic.

It grows well on media containing organic nitrogen: peptone, casein hydrolysate, fish broth, fodder yeast.

From glucose, maltose, cellobiose produces acid but not gas. Not metabolizes galactose, arabinose, mannose, lactose, rhamnose, xylose, raffinose, lures, sorbitol, dulcet, Inositol, inulin.

Hydrolyzes esculin and starch, salicin does not break, sodium citrate does not use as sole source of carbon. Forms of amylase, protease, catalase and nitroreductase. Does not form lecithinase, urease, isindeterminate, phenylalkylamines indole and hydrogen sulphide.

Pigment is not about what ashet.

The strain produces a water-soluble thermostable exotoxin.

Antigenic properties.

Gives a positive reaction flagellar antigen with standard serum of Bacillus thuringiensis var. darmstadiensis (identified from this reaction and classification of Bacillus thuringiensis (1962, 1967, 1990) H. de Barjack. On serological properties refers to 10 Bacillus thuringiensis serotype.

The strain is resistant to the main production phages: M, K, e-5, E2-D5.

In terms of virulence, toxicity and toxigenicity strain of Bacillus thuringiensis var. darmstadiensis No. 25 (BtH10No. 25) not pathogenic for warm-blooded animals, belongs to the 4th class of hazard (low hazard) and satisfies the requirements of industrial microorganisms.

Strain BtH10No. 25 grows on nutrient groupresearch environments based on soy flour, corn starch, feed yeast.

The strain is stored on the beveled fish agar in test tubes at 4-5°C with periodic reseeding 1 time in 6 months, in dried state in a sealed ampoule and method of cryopreservation in the Departmental collection of useful microorganisms for agricultural purposes RAAS (RCAM).

Strain BtH10No. 25 exhibits insecticidal activity against a number of dangerous pests of agricultural crops - beetles (Coleoptera), such as: Colorado is the cue beetle, mustard and bread flea beetle, plavica, strawberry-raspberry weevil, buckwheat weevil, rape pollen beetle, leaf beetles (mustard, rape, horseradish, cabbage and elm) and other

Strain BtH10No. 25 is characterized by a wide spectrum of antifungal activity and inhibits the development of a wide range of phytopathogenic fungi: Botrytis cinerea Pers., Pythium spp., Bipolaris sorokiniana (Sacc.) Shoemaker, Verticillum dahliae Kleb., Rhizoctonia solani Kuhn, Alternaria alternata (Fries), Keissl, Sclerotinia sclerotiorum (Lib) de Bary, Fusarium avenaceum (Fr.) Sacc., F.oxysporum (Schlecht.), Snyd. et AZ., F. solani App. et Wr.

On the basis of strain BtH10No. 25 technology for producing new insecticidal biological product.

Example of getting a drug on the basis of strain BtH10No. 25.

First, in flasks (1 l) cooked groupresearch nutrient medium of the following composition, %: soy flour - 2,5; corn starch to 2.5; yeast - 0.5; K2HPO4to 0.3; MgSO4×7H2O - 0,02; CaCl2×2H2O - 0,01; rest (100) - water. Prior to sterilization pH is between 7.4 and 7.8.

The prepared environment is poured into the Erlenmeyer flask 40-50 ml and sterilized in an autoclave at a temperature of +(120-125°C. for 30 minutes. After sterilization, the pH of 6.8 to 7.2. Sterile nutrient medium is cooled to 30-35°C and seeded with a culture of the strain of the test tubes in a test tube culture on beveled RA contribute 5-8 ml of sterile water or saline solution. This is e a pipette gently rubbed culture, shaken until a homogeneous suspension and make the bulb of 0.2-0.5 ml of Culture grown in flasks on a shaker at 220-250 rpm and a temperature of 28-30°C for 56-72 hours until rash spores and crystal formation endotoxins (check is carried mikroskopirovanii). Grown in flasks culture fluid of strain BtH10No. 25 is used for planting medium in fermenters.

For growing the culture in the fermenter can be used any groupresearch nutrient medium, preferably of the same composition that was used to obtain the culture liquid in the Erlenmeyer flasks (composition specified above). In the fermenter any container filled with 50% water, were first dissolved salts, then brewed the estimated amount of corn starch at 80-85°C., cooled to 40°C, was added the calculated amount of soy flour, fodder yeast and mixed with water for 20 minutes. Drove pH to 8.0 using 40% sodium hydroxide (caustic soda). Then culture medium was heated to 80-85°C, kept the temperature for 30 minutes and sterilized at 1 ATM for 1 hour, then cooled to a temperature of 32-35°C. Prior to depositing the seed material from the fermenter took the test environment for the control of sterility. When viewed under the microscope field of view should not be the availability of microorganism is.

After that, in a nutrient medium in the fermenter was made 1-2% of the culture fluid from the Erlenmeyer flasks.

The process of growing the culture in the fermenter was conducted at 28-30°C With continuous stirring environment by means of stirrers, the pressure in the fermenter 0.2-0.4 atmospheres and appropriate aeration:

with 0-3 hour - only mixing;

from 3-12 hours - 0.5 V air/V environments/min;

with 12-42 h - 1,0 V air/V environments/min;

with 42 hour before the end of the fermentation - 0.5 V air/V environment/min

That is, the fermentation process was performed under conditions of high aeration and finished when reaching the content of free spores and endotoxin crystals up to 80-90% of the total number of inclusions at a ratio of 1:1. The titer of spores was determined by generally accepted by the serial dilution method. At the same titer dispute strain BtH10No. 25 received in the preparation of at least (3,8-4,0)·109spores/ml in the absence of extraneous microflora and virulent phage.

Thus was obtained Biopreparat-based strain BtH10No. 25, which represents a fluid, easily diluted with water to the desired working concentration, easy to use serial spraying equipment. The technology of its production does not require high energy consumption required for drying when receiving drugs dry form.

Performed laboratory tests of insecticides effectively the tee of the drug on the basis of strain BtH 10No. 25 with respect to mass insect phytophagous crops.

Laboratory determination of insecticidal activity of the drug on the basis of strain BtH10No. 25 was carried out on larvae of the Colorado potato beetle in the 2nd age. While forage (leaves of potato) was treated with an aqueous suspension of strain at concentrations of 5, 1 and 0.2%. In each variant of the experiment was planted on 25 Colorado potato beetle larvae of the 2nd age (in quadruplicate). For comparison in the experiments under the same conditions used drug on the basis of the strain-prototype BtH10No. 109 (Basical) and BtH1800 (Methoxylation liquid without drying), at the same concentrations. The account of the death of the larvae of the Colorado potato beetle was performed on day 7.

The content of exotoxin in the drug was determined on the larvae of house flies by the following method.

Drug-based strain BtH10No. 25 in 20 ml were centrifuged for 15 min at 8000 rpm the resulting centrate autoclaved at a pressure of 0.1 MPa and a temperature of 105°C for 20 minutes Then was prepared by dilution of the drug: 1:2; 1:4; 1:8; 1:16; 1:32, which corresponds to a concentration of 50; 25; 12,5; 6,25; 3,125 µl/g of feed. Each concentration was tested in four parallel definitions.

For testing in a glass jar with a capacity of 200 ml was made: 2 ml of the drug on the basis of strain BtH10No. 25 of the respective cultivation; 11 ml of 2.5%aq is th suspension of milk powder; 7 g of sterilized wheat bran. In the control experiment in banks instead of the dissolved drug was introduced 2 ml of sterile water. Feed with appropriate dilutions of the drug in each pot was thoroughly mixed and placed there by 25 larvae of house flies, holes cans tied-calico and kept in a thermostat at a temperature (28±1)°C and relative humidity of 70-85%. On the 5th day was chosen puparia and left in the same conditions until the flies.

For comparison in the experiments under the same conditions used drug on the basis of the strain-prototype BtH10No. 109 (Basical) and BtH1800 (Methoxylation liquid without drying), at the same concentrations.

The exotoxin activity was assessed by the number of deaths on day 8 of the flies-adjusted mortality in the control was determined by the formula:

X=(K-B)/A×100, where

K - the number of flies coming in control;

In the number of flies coming into the experience.

According to the results of the above laboratory experiments for each case was determined indicator LC50the dose of the drug (in %, in ál/g of feed, kg, ml, etc)that causes 50% death of the insects.

The value of LC50was calculated by the formula Cerberus (see, for example, the monograph "the ECOLOGY of BACILLUS THURINGIENSIS" authors Vratya, TI, Patyk, Kiev, ed. PGAA, 2007, 217 pages):

Lg LC50=lgm-δ(ΣL1-0,5),

where Cm- the maximum is th of the tested concentrations;

σ is the logarithm of the relationship of each previous concentration subsequent to (the logarithm of the ratio of dilution);

ΣL1- the sum of the relations of the number of insects that have died from this concentration, the total number of insects that were exposed to this concentration.

The results of insecticidal activity and education of exotoxin (values LC50)obtained in the above experiments, preparations based on strain BtH10No. 25, strain-prototype BtH10No. 109 (Basical) and analogue BtH1800 (Methoxylation liquid without drying), are presented in table 1.

Table 1
The biological activity of the strains based on the BtH
Drug-based strainInsecticidal activity: LC50for L2the Colorado potato beetle, %The content of the exotoxin: LC50for L2house flies, µl/g feed
BtH10No. 250,272,98
BtH10No. 1090,384,79
BtH1No. 8000,29 3,05

From the data presented in table 1, it follows that a drug based on the strain BtH1025 on insecticidal activity of larvae of the Colorado potato beetle 1.4 times more effective than drug-based strain-prototype BtH10109, and the content of exotoxin he surpasses it in 1.6 times. Comparison of strain BtH1025 strain BtH1800 shows that insecticidal activity and content of exotoxin they are close analogs.

Performed laboratory experiments on determination of insecticidal activity of the strain VTN10No. 25 in respect of leaf beetles (Chrysomelidae): rapeseed, cabbage, horseradish, Oriental mustard, elm.

First method described above in Example, received medication on the basis of strain VTN10No. 25, which was then diluted with distilled water to concentrations: 1,0; of 0.2 and 0.04%. The leaves of rape, horseradish, mustard, cabbage, elm dipped in the drug in Petri dishes in appropriate dilutions. After drying the cuttings leaves wrapped with a wet swab was placed in penicillin bottles with water and transferred into molds. On leaves of each variant plants were planted on 25 larvae of the corresponding leaf, the molds were covered with gauze or baseway cloth. The experiments were set at room temperature, each version of the experiment in triplicate. DL the comparison in the experiments under the same conditions used drug on the basis of the strain-prototype BtH 10No. 109 (Basical) at the same concentrations. Insecticides strains was assessed by the amount (dose) of drug causing 50% death of the larvae (LC50on the 5 th day experience. The results of the experiments are shown in table 2.

Table 2
Insecticidal activity of strains BtH10in respect of the leaf
Drug-based strainThe dose that causes 50% loss of larvae, (LC50), %
Oriental mustard leafRapeseed leafCabbage leafShitty leafElm leaf
BtH10No. 250,0620,0560,050,060,07
BtH10No. 1090.0860,0770,0640,080,09

The results presented in table 2, indicate a high Antonien the th activity of the strain BtH 10No. 25 against the larvae of leaf beetles on different cultures. The activity of strain BtH10No. 25 1.3-1.4 times higher than the activity of the strain prototype BtH10No. 109.

The effectiveness of the drug on the basis of strain BtH10No. 25 was also tested under field conditions on potato against the Colorado potato beetle in the farms of the Leningrad region: JSC "upstream"OPH "Suyda"and Stavropol Krai (statr) on an area of 1 ha 0.5 ha and 3 ha, respectively.

Potato crops, inhabited the Colorado potato beetle with a ratio of larvae in ages: L1=58,6%; L2=36.5 per cent; L3=4.9%of treated liquid preparation obtained as described in Example a method based on strain VTN10No. 25. For comparison in the experiments under the same conditions used drugs on the basis of the strain-prototype BtH10No. 109 (Basical) and strain-analogue BtH1800 (Methoxylation liquid without drying), at the same concentrations. Average larvae per 25 bushes considered before treatment and at the 5th and 10th days after treatment. The processing performed tractor sprayer, the dose of 15-20 l/ha, the fluid flow 400 l/ha the Results of field experiments in the farms of the Leningrad region and the Stavropol territory are shown in table 3.

15,0
Table 3
The effectiveness of drugs on the basis of strains BtH on potato against the Colorado potato beetle in field experiments
Drug-based strainArea, haDose, l/haMaggots, %:
after 5 daysafter 10 days
CJSC "upstream" of the Leningrad oblast
BtH10No. 251,020,0an 80.2the 98.9
BtH10No. 1091,020,060,488,4
BtH1No. 8001,020,063,589,2
OPH "Suyda" Leningrad oblast
BtH10No. 250,515,075,9of 99.1
BtH10No. 1090,568,586,5
BtH1No. 8000,515,064,7of 87.3
Stavropol Krai (statr)
BtH10No. 253,015,0to 83.597,9
BtH10No. 1093,015,070,186,3
BtH1No. 8003,015,072,388,0
The control without treatmentThe increase in the number of 1.52 times

The data of table 3 indicate a high efficiency of the drug on the basis of strain BtH10No. 25 against the Colorado potato beetle, exceeding the effectiveness of drugs on the basis of strain-analogues.

There were also growing field trials on the basis of strain BtH10No. 25, poluchennogo the above method, strain the prototype BtH10No. 109 and strain-analogue BtH1No. 800 strawberries before flowering against strawberry-raspberry weevil on a plot of 100 m2. Used a hand sprayer, consumption of the drug is 10 l/ha of working liquid - 400 l/ha Defeat strawberry plants weevil considered before treatment and on the 10th, 20th days after spraying.

The results of the experiment are shown in table 4.

Table 4
The effectiveness of drugs on the basis of strains BtH against strawberry-raspberry weevil
Drug-based strainDefeat strawberries, %
Before processing10th dayDay 20
BtH10No. 256,16,89,4
BtH10No. 1095,97,914.4V
BtH1No. 8005,814,723,5
Control (without treatment)5,318,928,2

The results presented in table 4, indicate a high activity of the drug on the basis of strain BtH10No. 25 against strawberry-raspberry weevil. The defeat of the strawberry weevil when it is processed by the agent on the basis of strain BtH10No. 25 1.5 times lower than in the processing of drug-based strain-prototype BtH10No. 109, 2.5 times lower than in the processing of drug-based strain BtH1No. 800, and 3 times lower compared to control.

For evaluation of insecticidal activity of the drug, derived from strain BtH10No. 25 above in the Example method, against different species of coleopterous insects on agricultural crops have been conducted field experiments. Testing against crucifer flea beetles, cabbage leaf and cabbage moths were carried out on plots of 20 m2with turnip, rape, cabbage, horseradish and horseradish against shitty of a leaf. Used to process the hand sprayer, consumption of the drug - 8-12 l/ha of working liquid - 400 l/ha Efficacy strain was assessed by % the death of insects on the 5th and 10th days after treatment of plants with the drug. The results of the experiment are given in table 5.

Table 5
The effectiveness of the drug on the basis of strain BtH10No. 25 against various species of insects on crops
InsectsCultureDose preparation consumption, l/haInsects, %
on the 5th dayon the 10th day
Crucifer flea beetle (genus Phyllotreata)Rutabaga1273,495,7
rape1271,694,5
Cabbage noctuid moth (Mamestra brassicae L.)Cabbage1089,3100
Cabbage leaf (Phaedon cochleariae F.)Cabbage882,5the 98.9
horseradish1079,9 99,6

The data of table 5 indicate a high insecticidal activity of the drug on the basis of strain BtH10No. 25 against different insect species in different crops.

Antifungal activity of strain BtH10No. 25 against phyto-pathogenic fungi was determined by the method of agar blocks in Petri dishes (Methods of experimental Mycology / edited Wearily. Kiev, 1982.). Drug-based strain BtH10No. 25 obtained in the above way, was made in the melted and cooled to 40°C environment of čapek. On the surface of the solidified agar was placed blocks cut from a 10-day culture of the tested fungi. Control was medium without the addition of the drug. For comparison in the experiments under the same conditions used drugs on the basis of the strain-prototype BtH10No. 109 (Basical) and BtH1800 (Methoxylation liquid without drying) at the same concentrations.

The diameter of the colonies of the fungus was measured after 3 days. Inhibitory activity was calculated by the formula:

X=(Dto-Dabout)/Dto×100, where

X is the degree of inhibition of colony growth of the fungus, cm;

Dtoand Dabout- the diameter of the colonies of the fungus in control and experience, respectively.

The results of the experiment are shown in table 6.

Table 6
Antifungal activity of preparations on the basis of strains BtH
View phytopathogenic fungusInhibition of colony growth of the fungus after 3 days., %
BtH10No. 25BtH10No. 109BtH1No. 800
Botrytis cinerea Pers1008342
Pythium spp.867023
Bipolaris sorokiniana756111
Verticillum dahliae Kleb.52490
Rhizoctonia solani Kuhn43289
Alternaria alternata (Fries), Keissl36310
Fusarium avenaceum (Fr.) (Sacc)5144 0
Fusarium oxysporum (Schlecht.) Snyd. et Hans.53490
F.solani App. et Wr.26190

From the results shown in table 6, it follows that the antifungal effect of the drug on the basis of strain BtH10No. 25 14-17% higher than drug-based strain-prototype BtH10No. 109, and several times higher than the effect of the drug on the basis of a strain of 1 serotype BtH1No. 800 for all types of phytopathogens. 100% effect of inhibiting the growth of the fungus occurs when the effect of the drug on the basis of strain BtH10No. 25 on the fungus Botrytis cinerea is the causative agent of grey mould.

In pot experiments on tomato (cultivar "Gribovskaya") on infectious background antifungal effect of strain BtH10No. 25 against the fungus Fusarium oxysporum was 70%, while in the control of the affected plants reached 100%.

Assessment photoregulatory activity of the strain BtH10No. 25, i.e. its ability to affect plant growth promoting action, conducted in the laboratory experiments. Seeds of agricultural plants: tomato, cabbage, lettuce, cucumber, zucchini before sowing were soaked for 3 hours in preparation on the basis of strain BtH10the 25, obtained as described in Example way to compare is in preparation on the basis of strain-prototype BtH10No. 109, and in control - in the water. After soaking, seeds were germinated in rolls of filter paper at a temperature of 26°C and humidity of 86%.

After 5 days, take into account the germination of seeds after 3 weeks was controlled by the length of shoots and roots. The results of the experiment are shown in table 7.

Table 7
Photoregulatory activity of preparations on the basis of strains BtH10when pre-soaking of seeds of agricultural crops
The cult-
RA
% of control
Seed germinationThe height of seedlingsLength of roots
BtH10No. 25BtH10No. 109BtH10No. 25BtH10No. 109BtH10No. 25BtH10No. 109
Tomato10494 156143112110
Cabbage9896149140158150
Salad110107123113136128
Zucchini135121159140162144
Beets1089810398114102
Cucumbers115110104101121106

The results of the tests of the drug on the basis of strain BtH10No. 25, shown in table 7, showed that this strain does not possess phytotoxic the stew. Moreover, it stimulates the germination of seeds and growth of seedlings and roots of plants, i.e. has a growth promoting property. This photoregulatory (growth-stimulating) the activity of the inventive strain BtH10No. 25 on 3-19% more than in strain-prototype BtH10No. 109.

Thus, the claimed bacterial strain of Bacillus thuringiensis var. darmstadiensis No. 25 (BtH10No. 25) has multifunctional properties: high insecticidal and antifungal activity against a wide group of mass of harmful species of Coleoptera and growth promoting effects on plants and suitable for the production of highly effective insecticidal biological product to protect crops from harmful mass insect phytophages and fungi pathogenic fungi.

The bacterial strain of Bacillus thuringiensis var. Darmstadiensis No. 25, deposited in the collection of the GSI ARRIAM RAAS (Departmental collection of useful microorganisms for agricultural purposes) under the registration number RCAM01490 as polyfunctional plant protection from harmful beetles insects and phytopathogenic fungi.



 

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FIELD: biotechnologies.

SUBSTANCE: strain of bacteria Bacillus atrophaeus VKPM V-10592 is grown, and suspension is prepared from it, which is introduced into permafrost soil and water environment. Maintained at the specified parameters from 7 to 60 days, and then they determine quantity content of oil and oil products in permafrost soil and water environment.

EFFECT: invention makes it possible to reduce time for oil and oil product denaturation and to reduce their concentration in permafrost soil and water environment.

5 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Claimed is method of obtaining androst-4,9(11)-dien-3,17-dione from phytosterol. Microbiological oxidative elimination of side chain at atom C17 with formation of 9α-hydroxyandrost-4-en-3,17-dione is performed. Biomass is separated. 9α-hydroxyandrost-4-en-3,17-dione is extracted from clarified cultural liquid with aprotic organic solvent, selected from aromatic hydrocarbons or organochlorine hydrocarbons. After that, reaction of 9α-hydroxygroup of 9α- hydroxyandrost-4-en-3,17-dione dehydration is carried out in obtained extract. As dehydration agent applied is mineral acid, which contains water and is selected from group, which includes orthophosphoric, pyrophosphoric and chloric acids. Mineral acid is applied in quantity from 1 to 10 mol per 1 mol of 9α- hydroxyandrost-4-en-3,17-dione. In the process of dehydration reaction removal of excessive water is carried out either in presence of effective quantity of pyrophosphoric acid or by azeotropic distillation.

EFFECT: invention makes it possible to intensify dehydration process with application of smaller quantity of mineral acid and exclude side product formation.

11 cl, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, microbiology, and concerns the recovery and identification of pseudotuberculosis and intestinal yersiniosis agents (Y. Pseudotuberculosis and Y. Enterocolytica). A nutrient medium contains microbiological agar (dry), lactose, glucose, urea, calcium chloride, 1% alcoholic phenol red, 1% alcoholic methylene blue and distilled water in specific proportions.

EFFECT: invention enables reducing the length of studies.

3 dwg, 2 tbl

FIELD: biotechnology.

SUBSTANCE: growing of Staphylococcus aureus is carried out in a nutrient medium containing yolk-salt agar. At a stage of preparation for analysis the growth stimulators of Staphylococcus aureus are introduced into the nutrient medium in the form of aqueous solutions at concentrations of 10-4-10-6 wt %. The following compounds are used as growth stimulators: tris(2-hydroxyethyl)ammonium 4-chlorophenyl-sulfanylacetate or tris(2-hydroxyethyl)ammonium 2-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 2-methyl-4-chlorophenyloxyacetate or tris(2-hydroxyethyl)ammonium 1-benzylindol-3-yl-sulfanylacetate.

EFFECT: invention enables to accelerate growing of Staphylococcus aureus for diagnostics of infections, by reducing the time of growing from 48 to 6-9 hours in comparison with the control in a standard nutrient medium.

2 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: invention is production of the nutrient medium, which creates optimal conditions for growing legionella, comprising: enzymatic hydrolyzate of pig lung, enzymatic hydrolyzate of chicken egg yolk, potassium monophosphate, trihydrate disubstituted potassium phosphate, L-cysteine hydrochloride, activated carbon, microbiological agar and distilled water at a predetermined ratio of ingredients.

EFFECT: invention enables to produce the high-quality, easy-to-prepare nutrient medium, to reduce the time of growing legionella.

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology and deals with method of obtaining preparation based on vaccine strain of plague microbe. Claimed invention includes preparing inoculation native culture of plague microbe, concentration of microbe suspension, preparing vaccine suspension and obtaining dry form of preparation, with process of preparing inoculation culture including cultivation of microbes in liquid nutritional medium in flasks for 48 h at temperature 26…28°C and contibuous aeration with not less than 10 l min-1. with passaged stabilised starting culture, obtained as a result of three successive passages through organism of guinea pigs and mixed with glycerol-lactose-polyglucinum liquid in ratio 2:1; for preparation of vaccine suspension used is optimised in component composition protective drying medium, lyophilisation being carried out with observance of the specified regimen.

EFFECT: claimed solution makes it possible to obtain product with higher activity with reduced duration of process of its manufacturing.

3 dwg, 6 tbl

FIELD: biotechnologies.

SUBSTANCE: nutritive medium includes lactoserum, yeast autolysate, acetic acid 70%, agar and cabbage brew at the specified component ratio.

EFFECT: invention allows increasing selectivity of nutritive medium and simplifying its production.

8 ex

FIELD: biotechnologies.

SUBSTANCE: method for obtaining spore material of bacteria of Clostridium type provides for production of inoculum of bacteria in a full synthetic growth medium, seeding of inoculum and cultivation under the corresponding conditions in growth medium including potato, glucose, ammonium sulphate and chalk. During the main fermentation process for 18-28 parts of bacterial culture growth depending on time of occurrence in one field of view of at least 80-100 thickened forms of cells, n-butanol in the amount of 0.16-0.81 wt % is added to growth medium. Number of formed spores is 1.08-2.86·108 in one millilitre of the medium.

EFFECT: increased content of spores in spore material.

1 tbl, 15 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes an association of strains of bacteria-oil decomposers, which have been extracted from oil-contaminated soil, Acinetobacter species B-1037, Pseudomonas species B-989, Bacillus species B-1040, deposited at The State Research Centre of Virology and Biotechnology VECTOR. Besides, at least 30% bacteria of each strain is contained in the association. Remediation of oil-contaminated soils includes water suspension of lyophilic dried biomass of the strain association based on 109 cells per square metre. Strains of the association can utilise a wide range of oil components at the temperature of 10-15°C.

EFFECT: improving cleaning efficiency of oil-contaminated soils.

2 cl, 5 dwg, 2 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: bacillus subtilis subsp.subtilis BKM B-2711D strain has apparent antagonism in relation to Escherichia coli, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes, and resistance to streptomycin and tetracycline antibiotics. It is deposited in the All-Russia Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms Named after G.K. Skryabin of the Russian Academy of Science (IBFM RAN) and has the following registration number: BKM B-2711D, and can be used at production of probiotic bacterial preparations that can be used in veterinary medicine.

EFFECT: invention allows increasing protease and xylanase activities.

1 tbl, 3 ex

FIELD: agriculture.

SUBSTANCE: invention relates to the field of agriculture, namely to viticulture. The method comprises initially single seeding in a row of vines on 3-year agro-technological cycles in inter-row spacing unsown in the previous cycle of winter triticale alternating in varieties cycles of selection of Krasnodar Research Institute of Agriculture n.a. Lukyanenko. Yearly in the cycles the green mass is trimmed at the stem elongation of plants in the spring. In the summer in the inter-row spacing soil the matured plant mass is embedded with ears of corn of triticale and agrobiological stimulant of effective microorganisms (EM) "Baikal EM-1" based on the special nutrient medium "EM syrup". At that together with the biomass of triticale and the stimulant EM in the inter-row spacing soil the organic matter of vegetable pulp (OP) is embedded at a rate of 380 kg/ha.

EFFECT: method enables to increase the natural fertility of the soil, vineyard productivity, to improve quality of grapes grown and vine-winemaking products through the creation of agro-technical conditions of increased growth and development of micro-organisms of the soil biota by increasing their biological activity due to the greater number of the organic matter in the treated soil.

1 tbl, 1 ex

FIELD: agriculture.

SUBSTANCE: agent for control of plant diseases comprises: at least one compound chosen from tetrazolyl oxime derivatives represented by the formula , and their salts: in the formula (I) X is C1-6-alkyl group, C1-6-alkoxy group, halogen atom, nitro group, cyano group, C6-10-aryl group or C1-6-alkyl-sulfonyl group; n is an integer from 0 to 5; Y is C1-6 alkyl group; Z is a hydrogen atom, an amino group or a group represented by the formula NHC(-O)-Q; Q is a hydrogen atom, C1-8-alkyl group, C1-6-haloalkyl group, C3-6-cycloalkyl group, C1-8-alkoxy group, C3-6-cycloalkoxy group, C7-20-aralkoxy group, C1-4-alkylthio-C1-8 alkyl group, C1-4-alkoxy-C1-2-alkyl group, C1-4-acylamino-C1-6-alkyl group, C1-4-acylamino-C1-6-alkoxy group, C1-8-alkylamino group, C2-6-alkenyl group, C7-20-aralkyl group or C6-10-aryl group; R is an halogen atom; m is an integer from 0 to 3; and at least one compound selected from the group consisting of triflumizole, hydroxyisoxazole, acetamiprid and their salts.

EFFECT: invention enables to improve the efficiency of disease control.

2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, in particular to veterinary liquid external composition for local trasderamal treatment or prevention of parasite infections of animals, in particular, ruminant animals, such as cattle and sheep. Claimed veterinary composition includes effective quantity of clorsulon, macrocyclic lactone, selected from ivermectin or eprinomectin, glycol ether, propyleneglycol dicaprylate/dicaprinate, stearylstearate, palmitate and myristate, as well as stability enhancer, which represents glycerolformal or PEG.

EFFECT: invention makes it possible to obtain veterinary composition, which has increased stability and ensures improved bioavailability of active substances in case of local application.

5 cl, 9 dwg, 15 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and is used to preparing the cell cultures of Lactobacillus reuteri, containing reuterin to be kept inside the cells. The method involves the fermentation of cell cultures, the addition of 1,2-propanediol or glycerol to the reuterin-producing cell systems Lactobacillus reuteri in the beginning of the fermentation, the addition of glycerol to the cell cultures of Lactobacillus reuteri during the production and preservation of the cells Lactobacillus reuteri. The product prepared by the declared method contains the kept cells Lactobacillus reuteri with reuterin kept in the cells.

EFFECT: group of inventions allows producing the large amounts of reutrin-loaded Lactobacillus reuteri to be used for the purpose of both preventing and treating diseases, and as a food additive.

9 cl, 9 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to agriculture. The composition contains (a) at least one imidazole compound of formula : where R is a C1-6 alkyl group or a C1-6 alkoxy group and n is an integer from 1 to 5, and (b) polyoxins. The composition is applied onto plants.

EFFECT: invention increases treatment efficiency.

5 cl, 2 tbl, 2 ex

FIELD: agriculture.

SUBSTANCE: method of plant cultivation comprises includes treatment of plant seeds prior to their sowing and of plant in the growing season with a biological product in liquid form. As a biological product, a mixture of strains of bacteria of B. subtilis of RNCIM B-10641, B. amyloliquefaciens RNCIM B-10642 and B. amyloliquefaciens RNCIM B-10643 is used, with a titer of not less than 106 CFU/ml in the form of an aqueous suspension.

EFFECT: invention provides increase in productivity of plants and restoration of soil microbiocenosis.

3 cl, 4 dwg, 5 tbl, 9 ex

FIELD: agriculture.

SUBSTANCE: biological preparation for stimulation of growth and protection of plants from diseases, increase in productivity and soil fertility contains biomass Bacillus amyloliquefaciens RNCIM B-11008 and humates at the following ratio of components in vol. %: biomass Bacillus amyloliquefaciens RNCIM B-11008 - vegetative cells and spores of 1.24-1.30×1010 CFU/ml of culture fluid and the spore content 94% of the total amount of CFU - 99.0, humates - 1.0.

EFFECT: biological preparation enables to protect plants from fungal and bacterial diseases, to improve the phytosanitary condition of soil and improve its fertility, to increase crop yields and improve the quality of agricultural products.

5 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to agriculture. Pesticide combination contains at least two active ingredient components together with one or more conventional additives. Component (I) is imidacloprid and component (II) is one or more growth activators selected from a group comprising harpin and acibenzolar-S-methyl. To control or prevent damage by pests and/or pathogenic damage to plant propagation material, plants and/or plant organs which grow at a later point in time, said pesticide combination is applied onto a plant, part of a plant or the area surrounding the plant.

EFFECT: invention enables to increase biological activity of the combination.

12 cl

FIELD: agriculture.

SUBSTANCE: method relates to agriculture, namely, to protection of crops. The method is implemented by obtaining a salt extract of biologically active substances extracted from a biomass of soil oligochaetes Nicodrilus caliginosus after their treatment with potassium chloride in crystalline form, and pre-sowing treatment of seeds or spraying plants during the growing season with optimum dose of the extract of biologically active substances in a proportion of 1:30 parts of the biomaterial.

EFFECT: method enables to reduce the dosages of biologically active substances and to increase crop yields.

3 tbl, 4 ex

FIELD: food industry.

SUBSTANCE: microbicidal or microbiostatic composition comprises bacteriocin and extract containing phenolic diterpenes and obtained or extracted from plants of family Labiatae. The extract contains phenolic diterpenes in a quantity greater than 1.0 wt % in conversion to the composition weight. The plant of family Labiatae is rosemary. This composition is applied in method of preventing and/or suppression and/or destruction of microorganism in food product. The method is carried out by contacting food with the composition. Also a set for obtaining the composition and food products containing the composition are offered.

EFFECT: invention has a capacity to inhibit growth of pathogenic microorganisms in food products.

39 cl, 11 dwg, 7 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: strain of virus of nuclear polyhedrosis of cotton budworm Helicoverpa armigera Hbn has high antiviral activity in relation to cotton budworm. It is deposited in the State collection of the Federal Service for Consumer Rights Protection and Human Welfare of causative pathogens of viral infections, rickettsial diseases of Federal Budget Institution of Science of State Science Centre of virology and biotechnology "Vector" under the registration number V-607, and can be used in the production of biological insecticides for agriculture.

EFFECT: invention enables to improve the antiviral activity in relation to cotton budworm.

1 dwg, 3 tbl, 4 ex

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