Strain of bacteria bacillus thuringihnsis bios-1, possessing insectoacaricidal activity

FIELD: medicine.

SUBSTANCE: strain of bacteria Bacillus thuringiensis BIOS-1 VKPM B-10709, possessing insectoacaricidal activity against representatives of leaf-eating and sucking pests, such as representatives of orders Lepidoptera, Coleoptera, Homoptera, Thysanoptera and Acariformes, doing harm to crops, is deposited in All-Russian collection of industrial microorganisms (VKPM), Federal State Unitary Enterprise GosNIIgenetika under number B-10709.

EFFECT: invention makes it possible to increase mortality of representatives of leaf-eating and sucking pests, doing harm to crops.

6 tbl, 2 ex

 

The invention relates to the microbiological industry, Microbiology and biotechnology, namely the production of bacterial biological products intended for the control of pests of agricultural crops, which are the representatives of the orders Lepidoptera, Coleoptera, Homoptera, Thysanoptera and Acariformes.

Effective pest control plays an important role in maintaining and increasing yield, because agricultural production losses associated with the activity of insect pests, up to 30% of the expected harvest.

At the present time to control pests of agricultural crops is widely used chemicals. Prolonged use of the latter led to the formation of resistant to these drugs pest populations [1]. In addition, chemicals toxic to warm-blooded animals and beneficial insects, as well as able to penetrate into plants, resulting in poor sanitary and hygienic conditions of agricultural production. The most widely used of chemicals is karbofos.

An increasingly important role in protecting plants from pests play a biological control with the use of products derived from viral, bacterial and fungal pathogens or their products synthesized. The advantage of b is listwa known microbiological plant protection products compared to chemical - they are environmentally friendly.

The most widely as microbiological plant protection products on the basis of different strains of the entomopathogenic bacteria Bacillus thuringiensis. These bacteria during sporulation synthesize crystalline proteins, or, as they are called Delta-endotoxins, which differ among themselves on the spectrum of entomopathogenic actions [2]. Delta-endotoxins have a selective toxic effect only in relation to insects.

Some strains of Bacillus thuringiensis produce more and thermostable exotoxin, which is in contrast to endotoxin does not have the selectivity and toxicity manifests in relation to a very wide range of invertebrates and vertebrates, causing allergic reactions in individuals working with these drugs [3].

Known strains of Bacillus thuringiensis, which possess insecticidal activity against representatives of the order of Coleoptera and Lepidoptera [4, 5]. However, they are not toxic to the representatives of the orders Homoptera, Thysanoptera (insecticidal activity) and Acariformes (acaricide activity).

Known bacterial strain Bacillus thuringiensis ssp.kurstaki VKPM B-8715 with insecticide and acaricide activity against the representatives of the orders Lepidoptera, Coleoptera, Homoptera, Thysanoptera and Acariformes [6]. The strain chosen as a prototype.

The problem is obtained of the invention - is to teach the bacterial strain of Bacillus thuringiensis, with increased microbiological productivity and insecticide and acaricide activity.

The problem is solved by breeding strains of bacteria Bacillus thuringiensis ssp.kurstaki VKPM B-8715 on laboratory and industrial nutrient media. When mnogochislennyh the subcultures and rasiah to individual colonies was obtained strain of Bacillus thuringiensis BIOS-1, with increased insecticide and acaricide activity against listography and sucking pests.

The resulting strain deposited in Russian national collection of industrial microorganisms (VKPM) how Bacillus thuringiensis BIOS-1 VKPM B-10709.

The strain VKPM B-10709 has the following chacteristic:

Cultural and morphological characteristics.

Gram-negative motile Bacillus size of 4.1-1.6 ám. In the stationary growth phase to form crystals of endotoxins and oval spores.

Grows well in hydrolysates of casein (pancreatic hydrolysate of casein - 1, yeast extract and 0.2, MnSO4, - 0.5, and MgSO4- 3, KH2PO4- 0,1, glucose - 0,6, water - the rest, the pH of the medium to 7.4 and 7.5, medium composition in wt.%) at 27-29°C and a pH of 6.8 to 7.5. On agar medium casein (pancreatic hydrolysate of casein - 1, yeast extract - 0.5, and sodium chloride - 0.05, the agar - 1,5, water - the rest, the pH of the medium to 7.4 and 7.5, medium composition in wt.%) 10 days at 27-29°C forms a homogeneous colonies 3-5 mm in diameter, light gray in color with a slightly jagged edge. The structure of the ur fine.

On the H-antigen refers to serovariants III (IV).

Under cultivation of strain both in liquid and on solid nutrient media output phage does not occur.

Physiological and biochemical characteristics.

The sources of carbon assimilates with the formation of acid, glucose, maltose, corn and potato starch, soy and corn flour. Not absorb lactose, xylose, arabinose, lecithin. Peptonized milk, hydrolyzes starch, gelatine not thins.

The sources of nitrogen: restores nitrates, assimilates ammonium forms of nitrogen.

Relationship to temperature: grows well at 27-29°C, the optimum temperature of 28°C.

Relation to the pH of the medium: grows well when 6,8-7,5, optimum pH of 7.2. Relation to oxygen: aerobe.

Relationship to phage: sustainable for the main industrial phages P., 22, s/t, And-77, 25-16.

Beta-exotoxin not synthesize.

Not pathogenic for humans, domestic and wild animals, beneficial insects, birds and fish.

Not a genetically modified strain.

Claimed properties: synthesizes components with insecticide and acaricide activity against the representatives of the orders Lepidoptera, Coleoptera, Homoptera, Thysanoptera and Acariformes.

Marker signs: activity against the representatives of the orders Lepidoptera, Coleoptera, Homoptera, Thysanoptera and Acariformes, the lack of ability to synthesize beta-Exo is oxen.

Storage of strain: strain is stored in the vials after lyophilization paracrystalline mixture in the usual way.

The invention is illustrated by the following examples.

EXAMPLE 1. Microbiological yield was obtained strain.

Assessment of microbiological productivity of the obtained strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 on laboratory culture media was carried out according to two main parameters, namely the title dispute and concentration of the main insecticidal protein (Delta-endotoxin) in comparison with the prototype strain of Bacillus thuringiensis ssp.kurstaki VKPM B-8715.

For this purpose, the strains of Bacillus thuringiensis were grown in flasks with a capacity of 750 ml in volume environment growing 200 ml to stage full of sporulation on a circular shaker at 28°C and shaking 250 rpm/min For growing used medium of the following composition, wt.%:

1. Wednesday 1:

Pancreatic hydrolysate of casein1
Yeast extract0,2
MnSO4,0,5
MgSO43,
KH2PO40,1
Glucose the 0.6
Waterrest
the pH of the medium7,4-7,5

2. Wednesday 2:

Trypticase casein hydrolysate1
Yeast extract0,2
MnSO40,5
MgSO43,
KH2PO40,1
Glucosethe 0.6
Waterrest
the pH of the medium7,4-7,5

3. Wednesday 3:

Peptone1
Yeast extract0,2
MnSO4,0,5
MgSO43
KH2PO40,1
Glucose0,,
Waterrest
the pH of the medium7,4-7,5

The grown culture was separated by centrifugation and washed 1M NaCl in the presence M EDTA. Crystal proteins from paracrystalline mixture was dissolved in carbonate buffer pH 11.0 in an hour, undissolved precipitate was separated by centrifugation, the pH of the supernatant was made up to 10.0. The protein concentration was determined by Bradford method [7], using bovine serum albumin as standard. Complete dissolution was controlled by re-extraction in the same buffer. If re-extraction was dissolved not more than 7% protein. Determination of the titer of viable spores was carried out on Petri dishes with agar medium of the following composition, wt.%:

Pancreatic hydrolysate of casein1
Yeast extract0,5
Sodium chloride0,05
Agar1,5
Waterrest
the pH of the medium7,47,5

The research results are summarized in table 1:

Table 1
Nutrient mediumWednesday 1Wednesday 2Wednesday 3
The strains of Bacillus thuringiensisPMBC
In-8715
PMBC
In-10709
PMBC
In-8715
PMBC
In-10709
PMBC
In-8715
PMBC
In-10709
The title dispute, ×108per ml4,561,82,63,55,0
Protein concentration (mg/ml)0,250,40,350,50,250,4

As follows from table 1, the resulting strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 on the three studied laboratory nutrient medium is superior in microbial productivity (titer dispute, conc is ntrace main insecticidal protein, Delta-endotoxin) strain of Bacillus thuringiensis ssp.kurstaki VKMV-8715 (prototype) by 25-30%.

Evaluation of the obtained strain on industrial nutrient medium was performed according to the setting of microbiological productivity as a title dispute.

For preparation of inoculum the resulting strain and the prototype were grown in 2 ml of medium 1 for 12 hours at 28°C as described above. Then 1 ml of microbial cultures seeded in flasks with a capacity of 750 ml volume of medium growing 50 ml-driven polysaccharide environment (DPS) of the following composition, wt.%: yeast - 36, corn flour and 26.8, soy flour to 5.0, chalk - 3,0, water - the rest, the pH of the medium was 7.2-7.5 and cultivated on a pie rocking chair to the stage full of sporulation. Determination of the titer of viable spores was carried out on Petri dishes with agar medium as described above. The results of the experiment are shown in table 2.

Table 2
StrainsPMBCPMBC.
Bacillus thuringiensisIn-8715In-10709
Microbiological production(billion JV./ml)4,36,0

From the results shown in the tables is 2, it follows that the productivity of the obtained strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 industrial environment, higher productivity of the strain Bacillus thuringiensis ssp.kurstaki VKPM B-8715 (prototype).

Microbial productivity (title dispute, the concentration of the main insecticidal protein, Delta-endotoxin) of the obtained strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 on laboratory culture media and industrial nutrient medium is higher than that of strain-prototype (Bacillus thuringiensis ssp.kurstaki VKPM B-8715).

EXAMPLE 2. Evaluation of insecticide and acaricide activity of the obtained strain.

Determination of insecticide and acaricide activity of the obtained strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 and prototype Bacillus thwingiensis ssp.kurstaki VKPM B-8715, Baloo conducted by representatives of the orders Lepidoptera, Coleoptera, Homoptera, Thysanoptera and Acariformes in the laboratory.

The resulting strain VKPM B-10709 and prototype VKPM B-8715 grew to the stage full of sporulation as in example 1 in environment 1.

Evaluation of insecticidal activity for representatives of Lepidoptera were conducted at two sites: the caterpillars of the Gypsy moth Lymantria dispar 2 age and caterpillars mill the liquidation Galleria mellonella age 7.

Caterpillars of the moth grew stored in the refrigerator at a temperature of +4°C egg in a medium of the following composition, wt.%:

Haricot beans are swollen72
The dry yeastof 5.4
Agarof 5.4
Paper filter finely chopped2
Sucrose4
Potassium phosphoric acid disubstituted1,4
Ascorbic acid1,4
Linseed oil0,7
Benzoic acid + alcohol3,5
Formalin (40%)1,8
KON1,4
The oak leaf dry (powdered)1

When biotesting 3 g medium was mixed with 1 ml of the test suspension of spore-crystal mixture (twice washed with saline) and well rubbed the mixture in a porcelain mortar with a pestle and mortar. The resulting mixture with a spatula transfer on the cover of the Petri dishes and placed in the form of a ring with a diameter of about 3, see Inside of this ring was planted on ten tracks. At the bottom of the Petri dishes placed is whether a disk of filter paper. Each sample suspension was tested in 3 parallel replications. The experiment was carried out at room temperature and natural light. The number of dead insects was counted on day 6. The results of the experiment are shown in table 3.

Table 3
The strains of Bacillus thuringiensisMortality (%) Gypsy moth (Lepidoptera, moth Lymantria dispar)
VKPM B-871560
VKPM B-1070975

The resulting strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 on insecticidal activity against moth exceeds the prototype of Bacillus thuringiensis ssp.kurstaki VKPM B-8715 25%.

Evaluation of insecticidal activity against the mill the liquidation (Galleria mellonella) 7 age was performed by means of grafting larvae mill the liquidation in a Petri dish filled with food (Merv + wheat bran).

Food was Packed up in Petri dishes (9 g each), which were introduced into 3 ml of the drug.

In each Petri dish (after drying of feed) was infused for 10 caterpillars of the liquidation 7 age (weight 75±5 mg) in three replications. Petri dishes with the tracks contained on the lab tables at temperature 25±20°C and relative humidity of the air is 60-80%. Mortality of larvae was calculated for 6 days. The results of the experiment are shown in table 4.

Table 4
The strains of Bacillus thuringiensisMortality (%) mill the liquidation (Lepidoptera, Galleria mellonella)
VKPM B-871530
VKPM B-1070939

The resulting strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 on insecticidal activity against mill the liquidation exceeds the prototype of Bacillus thuringiensis ssp.kurstaki VKPM B-8715 30%.

Evaluation of the insecticidal activity of the obtained strain VKPM B-8715 for members of the order of Coleoptera were performed on larvae of the Colorado potato beetle Leptinotarsa decemlineata.

Colorado potato beetle larvae younger children (3rd and early 4th instar) were placed in Petri dishes with potato leaves treated by immersion in solutions of drugs. Table 5 shows the results of the death of the larvae of the Colorado potato beetle on the 5th day.

Table 5
The strains of Bacillus thuringiensisMortality (%) Colorado potato beetle (Coleoptera, Leptinotarsa decemlineata)
VKPM B-8715 72
VKPM B-1070988

The resulting strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 on insecticidal activity against larvae of the Colorado potato beetle exceeds the prototype of Bacillus thuringiensis ssp.kurstaki VKPM B-8715 25%.

A comparative assessment of insecticide and acaricide activity of the obtained strain against the representatives of the orders Homoptera, Thysanoptera and Acariformes conducted at three sites: bocheva aphid, Western flower thrips and spider mite.

For test objects in the laboratory used the method of immersion of host plants in solution culture fluid with spores and crystals of the investigated strains obtained after completion of sporulation as in example 1 on the environment of growing 1.

For melon aphid (Aphis gossypii Glov., Homoptera) cut the leaves of cucumber were immersed in solutions of the test samples for 6 seconds, after which they were placed on moist filter paper in a Petri dish and was inhabited by the female aphid.

For Western flower thrips (Frankliniella occidentalis Pergande, Thysanoptera) used dvuhlitrovye bean plant. 7 days prior experience on plants of the female thrips were laying eggs. After hatching the larvae of the plants were cut and the leaves were immersed in solutions of the test samples for 6 seconds, after which he set the glasses of water, substituting the petioles plastic mugs, almasan the e on the edges with petroleum jelly.

In the case of the common spider mite (Tetranychus urticae Koch, Acariformes) cut dvuhlitrovye bean plants the day before the experience was inhabited by the female mite. The plants were immersed in solutions of drugs for 6 seconds and was installed in vessels of water.

The results of the death of the test object on the 5th day are shown in table 6.

Table 6
The strains of Bacillus thuringiensisVKPM B-8715VKPM B-10709
Mortality (%) melon aphid (Homoptera, Aphis gossypii Glov.,)5569
Mortality (%) spider mites (Acariformes, Tetranychus urticae Koch,)7995
Mortality (%) Western flower thrips (Thysanoptera, Frankliniella occidentalis Pergande,)4961

As follows from the results presented in table 6, the resulting strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 on insecticide and acaricide activity against the representatives of the orders Homoptera, Thysanoptera and Acariformes exceeds the prototype of Bacillus thuringiensis ssp.kurstaki VKPM B-8715 20-25%.

Insecticide and acaricide activity of the obtained strain of Bacillus thuringiensis BIOS-1 VKPM B-10709 n is otiv of representatives of the orders Lepidoptera, Coleoptera, Homoptera, Thysanoptera and Acariformes exceeds the prototype of Bacillus thuringiensis ssp.kurstaki VKPM B-8715 20-30%.

Sources of information

1. National Research Council. 1986. Pesticide reistance: strategies and tactics for management. National Academy of Sciences, Washington, D.C.

2. Crickmore, N., Zeigler, D.R., Feitelson, J., Schenepf, E., van Rie, J., Lereclus, D., Baum, J. and Dean, D.H. 1998. Revision of the nomenclature for the Bacillus thuringienis pesticidal crystal proteins. Environ. Molec. Biol. Rev. 62:807-813.

3. McConnell, E. and Richards, A.G. 1959. The production by Bacilllus thuringiensis Berliner of a heat-stable exotoxin toxic to insects. Can. J. Environ. 5: 161-168.

4. USA patent 6,156,308.

5. RF patent №2125091.

6. RF patent №2278159.

7. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72: 248-254.

The bacterial strain Bacillus thuringiensis BIOS-1 VKPM B-10709 with insecticide and acaricide activity against members of the leaf-eating and sucking pests.



 

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1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves Aeromonas sobria vaccine strain seeding, cultivation and inactivation. Aeromonads are cultivated in a liquid nutrient medium of a Hottinger broth in a fermenter for 4-6 hours at temperature 26±1°C, immediately after seeding, an oxidation-reduction potential of the cultural liquid is reduced to (-40) - (-100) mV. Thereafter, before termination of a cultivation process, pO2 in the cultural fluid is kept at 20-30 % from air oxygen saturation, pH of the cultural fluid is regulated at 7.1-7.3 pH units, and fractional glucose supply is carried out in doses to the concentration 0.25-0.35 % with glucose limitation of aeromonad growth.

EFFECT: method allows higher yield of the vaccine with reduced time of preparation.

4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: according to the invention, versions of an immunobiological antiallergic agent contain Lactobacillus acidophilus 100 "аш" PA strain, collection No. 207, is stored in the State collection of microorganisms of normal microflora Federal State Research Institution Gabrichevskiy Moscow Research Institution of Epidemiology and Microbiology of Federal Service for Supervision of Consumer Rights and Human Welfare in a culture medium in amount 106-1010 CFU/ml. The immunobiological antiallergic agent additionally contains species-specific virulent bacteriophages and bacteriophages with induced virulence, a biomass of Lactobacillus acidophilus "КзШз4" strain in a culture medium in amount 106-1010 CFU/ml, a biomass of Lactobacillus acidophilus NKi strain in the culture medium in amount 106-1010 CFU/ml and a biomass of Bifidobacterium bacteria in the culture medium in amount 106-1010 CFU/ml. The immunobiological antiallergic agent additionally contains target additives in amount 0.01-95.0 wt % from mass of the agent. The additives are selected from a number of: glycine, cysteine, copper sulphate, copper gluconate, quinosol, chitosan, licorice root extract, hips extract, origanum extract, cranberry extract and a flavouring agent. The immunobiological antiallergic agent is presented in the form of a suspension. The Lactobacillus acidophilus 100 "аш" PA strain is produced by means of a sequence of multiple passages through a nutrient medium MPC with added sources of histamine, Cu1+, Cu2+, Co2+, Ni2+ ions and at culture pH 6.2-7.4. The agent is used for prevention and treatment of allergic or pseudo-allergic disease without an accompanying pathology or in a combination with an infectious disease, intestinal dysbacteriosis and psychological adaptation disorders.

EFFECT: more efficient antiallergic action of the immunobiological agent on the basis of lactobacilli.

10 cl, 3 tbl

FIELD: medicine.

SUBSTANCE: invention refers to a mutant bacterium producing higher levels of intracellular and/or extracellular folate as compared with a wild type which is sensitive to methotrexate, and has growth rate at least 0,1 µ/hour when grown on a medium which contains methotrexate 1.25 mg/l and no folate-dependent metabolites. Also, the invention refers to a composition for foodstuff or food additive enrichment containing said bacterium, and to a method of sampling said bacteria.

EFFECT: invention allows producing high-folate bacteria.

13 cl, 3 dwg, 2 tbl

FIELD: agriculture.

SUBSTANCE: agent is a strain of a ectomycorrhiza-forming fungus Suillus sibiricus (6)No.LE(BIN)2178. The strain is deposited in the Collection of basidium fungi cultures LE(BIN) of the Institution of the Russian Academy of Science of the Botanical Institute named after V.L. Komarov, RAS, with appointment of a registration number No.LE(BIN)2178.

EFFECT: invention makes it possible to eliminate difficulties related to heterovegetative reproduction and re-planting of precious breeds of coniferous trees, to increase establishment of conifers at woodless areas and urbanised lands, on depleted soils in landscape and gardening works, to strengthen dams and to arrange green spaces of coal quarries.

6 dwg, 5 tbl

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