Bacterial strain bacillus thuringiensis varthuringiensis n800/15 as agent for preparing entomocidal biopreparation

FIELD: medicine.

SUBSTANCE: invention refers to microbiology, particularly to the protection of agricultural, medicinal and forest crops. The strain Bacillus thuringiensis var. thuringiensis No.800/15 (BtH1 800/15) possesses the entomocidal activity on depredators - phytophage insects. The strain is deposited in State Scientific All-Russia Research Institute for Agricultural Microbiology (GNU VNIISHM), No. 611.

EFFECT: strain may be used in preparing the high-efficacy entomocidal biopreparation.

3 tbl, 1 ex

 

The invention relates to biotechnology, namely microbiological remedies agricultural, medicinal and forest crops from harmful insects, i.e. possessing insecticidal action.

It is known that as entomopathogens used strains-producers bitoxibacillina, namely Bacillus thuringiensis subsp. thuringiensis 202, 98 and 620, are capable of forming two insecticidal toxin: protein crystals endotoxin released from cells during lysis of their shells, and water-soluble exotoxin nucleotide nature produced during vegetative growth of the culture (Nvention, Nimbusbase, Ass, Lincoserve. Comparative effectiveness of three strains of Bacillus thuringiensis subsp. thuringiensis /bul. ARRIAM No. 53, L., 1990, p.44-46). As a result of comparison of biological and insecticidal activity of these three strains revealed that the strains 98 and 620 superior in insecticidal activity strain 202.

Strain of Bacillus thuringiensis subsp. thuringiensis 98 (BtH198) isolated from dead dolls cabbage moths, is a producer of insecticidal endotoxin, is stored in the collection "Vniigenetika" No. CMPL IN-2452.

Strain BtH198 has a sufficiently high insecticidal activity against Colorado potato beetle and other insect pests (prototype).

Methoxylation is produced by cultivation of the strain BtH198 on fateley environment, containing ammonium phosphate, magnesium sulfate, calcium chloride, starch, corn, and fodder yeast, green molasses, with aeration for 48 h, the title dispute in the culture fluid of 3.5-3.75 billion/ml, the content of the exotoxin 0.5 mg/ml of the obtained culture liquid contribute 1% of sodium chloride and dried. Get Methoxylation, which contains spores 65-72 billion/year and 1% of the exotoxin.

The lack of strain of Bacillus thuringiensis subsp. thuringiensis 98 (BtH198) is that it quickly loses the insecticidal activity when stored.

Methoxylation-based strain BtH198 get in the dry form, and therefore, its production is energy-intensive and costly because of the operation of drying. In addition, the high temperatures have a detrimental effect on the endotoxin crystals, destroying them, which leads to lower insecticidal activity of Methoxylation.

The task of the invention is the identification of strain-based environmentally safe bacteria from the group of Bacillus thuringiensis, suitable for a highly effective insecticidal drug in liquid form against the massive insects phytophagous pests of agricultural and forest crops and retain its insecticidal activity during prolonged storage.

The task is solved in that as a means for the floor is to be placed insecticidal drug use strain of Bacillus thuringiensis var. thuringiensis No. 800/15.

Strain of Bacillus thuringiensis var. thuringiensis No. 800/15 (BtH1800/15) isolated from dead larvae of the Colorado potato beetle in the Leningrad region and deposited 20.03.2006, Departmental collection of useful microorganisms for agricultural purposes RAAS (BKSM) of wildebeest WNISM under registration No. 611 in the group of spore-forming microorganisms, as evidenced by a certificate of Deposit from 02.07.2010, (reference attached). The strain is characterized by the following properties.

Cultural and morphological properties.

Gram-negative motile Bacillus, peritricha, vegetative cells solitary or in short chains of 2-4 cells in the chain. The size of cells one-day culture fish agar (RA) is(2,5-3,0) × (0,8-0,9) µm. 3-4 days of growth on RA in cells formed spores oval subterminale size(1,1-1,3)×(0,8-0,9) μm and parasporal enable endoxana (crystals) correct bobbidee shape with a size of 1.1-1.3 μm.

On RA after 48 h at 26-28°C, the culture forms a flat colony grayish-white color round shape with rough edges, the surface of the grain diameter of the colony 22-24 mm. Strain grows at a temperature of 20 to 34°C (opt 26-32°C) and the measure of acidity pH 6.4 to 8.0 (opt of 6.8 to 7.2). Fish broth (RB) after 24 h grows with the formation of the film (the veil).

Biochemical his is TBA.

Optional gone anaerobic. Gelatino roszival on the 5th day, casein breaks down, peptonizing milk with alkalinization after 24-28 h, then acidify and discolor the litmus paper. In the medium Clarke forms acetylserotonin (AMK).

Gidrolizuet starch, uses citrates, pigment and urease does not form. Indole and hydrogen sulfide does not use, reduces nitrate to nitrite.

Breaks down glucose, mannose, levulose, sucrose, maltose, cellobiose, glycerin and salicin with the formation of acid. Does not use galactose, arabinose, xylose, rhamnose, lactose, raffinose, mannitol, sorbitol, dulcet, Inositol, inulin.

Antigenic properties.

Gives a positive reaction flagellar antigen with standard serum of Bacillus thuringiensis var. thuringiensis H-1 at a dilution of 1:6400.

The strain is identified according to this reaction, as well as on the classification of Bacillus thuringiensis (1962, 1967, 1990) H.de Barjack.

The strain is resistant to phage production: K, M, H, e-5, And-77, D2-D5.

Strain low toxicity to warm-blooded animals.

The strain grows on nutrient groupresearch environments; stored in a lyophilized state or on the shoals of RA in test tubes at 4-5°C with periodic re-seeding once in 6 months.

Strain BtH1800/15 possesses insecticidal properties and are promising as a means for the production of a biological product to protect selskohozyaistvennyh cultures from mass insect phytophages.

Based on this strain BtH1800/15 developed technology of production of new insecticidal biological product, named by the authors Biaxin.

An example of retrieving Biotoxin.

First, in flasks (1 l) was prepared groupresearch nutrient medium of the following composition, %:

Corn flour1,5-2,4
Fodder yeastof 3.0-3.5
(NH4)2HPO40,5-0,6
MgSO4×7H2O0,02-0,04
CaCl2×2H2O0,15-0,20
Water waterup to 100.

Using alkali (for example, 10% NaOH) bringing the pH to 7.4-7.8 for. Then this medium is poured 30-50 ml Erlenmeyer flask with a capacity of 750 ml and sterilized for 40 minutes at 120-122°C. After sterilization, the pH was 6.8 to 7.2. Prepared nutrient medium had cooled and seeded culture of strain BtH1800/15.

For the planting medium in the flasks is spore culture of strain BtH1800/15 obtained by flushing 5-10 ml of physiologic saline in a test tube culture on the sloped agar. The resulting usues the culture of the strain which was to obtain a homogeneous suspension and added into the flask with a prepared nutrient medium at the rate of 0.3-0.5 ml of suspension in 30-50 ml of medium. Seeded flasks were placed on a rocking chair at 200-220 rpm and 28-30°C in 56-72 hours to complete rash spores and crystals endoxana (validation was performed with mikroskopirovanii). Grown in flasks culture fluid of strain BtH1800/15 used for the planting medium in fermenters.

As a nutrient medium for fermenters can be used any groupresearch environment, preferably of the same composition that was used to obtain the culture liquid in the flasks, i.e. on the basis of fodder yeast and corn flour (listed above).

In this first in tap water, heated to 40°C, peppered with constant stirring all the other ingredients, the obtained suspension was heated to 80°C and kept for 30 min, after which the pH was brought to 7.8-8.0 and the medium was sterilized for 1 h at 140°C.

Presterilizing nutrient medium to the fermenter was cooled to 30-35°C and made it the culture fluid of strain BtH1800/15, pre-grown in flasks, at the rate of 0.7-1.0% of the culture fluid of the volume of the nutrient medium in the fermenter.

In the fermenter cultivation process was performed under the following conditions:

the cultivation temperature 28-30°C;

- the mixer rotation speed of 200-220 rpm;

- pressure in the apparatus of 0.4-0.5 kgf/cm2;

- air:

from 0 to 12 is - 0,2 Vd/ Venvironment/min;

from 12 to 24 h to 0.5 Vd/ Venvironment/min;

from 24 to 42 h of 0.8-1.0 Vd/ Venvironment/min;

42 h before the end of the fermentation - 0.5 Vd/ Venvironment/min

That is, the fermentation process was performed under conditions of high aeration and ended with the achievement of the contents in the received drug 80-85% free spores and crystals of endotoxin at a ratio of 1:1. At the same titer strain BtH1800/15 in the obtained liquid product was not less than 3,8-4,0 mlrdsr/ml in the absence of extraneous microflora and virulent phage.

So? was obtained Biopreparat-based strain BtH1800/15 (Biaxin), which is a liquid, easily diluted with water to the desired working concentration, easy to use serial spraying equipment. Production of the drug factories, biofactories in the regions consumer will not require the cost of transport and delivery over long distances. As Biaxin is liquid, the preparation does not require large energy consumption for the operation of drying.

Performed laboratory testing of the effectiveness of drug-based strain BtH1800/15 - Biaxina with respect to mass insect phytophagous crops.

For example, the laboratory is haunted determine the insecticidal activity of the drug on the basis of strain BtH 1800/15 (Biotoxin) were performed on larvae of the Colorado potato beetle in the 2nd age. While forage (leaves of potato) were treated with Biaxina in dilutions 1:20; 1:100; 1:500, which corresponds to a concentration of 5.0; 1.0; 0.2%of ensuring the destruction of the test object from 30 to 95%. In each variant of the experiment was planted on 25 Colorado potato beetle larvae of the 2nd age (in triplicate). For comparison in the experiments under the same conditions used drug on the basis of the strain-prototype BtH198 (Methoxylation liquid without drying), at the same concentrations. The control feed was treated with water. The account of the death of the larvae of the Colorado potato beetle was performed on day 7.

We also conducted laboratory tests insecticidal activity Biotoxin (preparation based on strain BtH1800/15) on tracks 2-instar Gypsy moth. The experiments were carried out in glass jars with a volume of 0.8 liters of this food (the branch of an Apple tree with leaves) were treated with Biaxina five concentrations: 2,0; 1,0; 0,5; 0,25; 0,125%. In each variant of the experiment was planted on 25 caterpillars Gypsy moth 2nd age (in triplicate). For comparison in the experiments under the same conditions used drug on the basis of the strain-prototype BtH198 (Methoxylation liquid without drying) in the same concentrations. The control feed was treated with water. The account of the death of the moth caterpillars were performed on day 7.

Experience is by definition the content of exotoxin in drug Biaxin was evaluated on the larvae of house flies by the following method.

Drug-based strain BtH1800/15 (Biaxin) 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 three times).

For testing in a glass jar with a capacity of 200 ml was made:

2 ml of the drug Biaxin appropriate cultivation;

- 11 ml of 2.5%aqueous suspension of powdered milk;

- 7 g sterilized wheat bran.

In control instead of Biotoxin used the drug on the basis of the strain - prototype BtH198 (Methoxylation liquid without drying) in the same concentrations. Feed with appropriate dilutions of the drug in each pot was thoroughly mixed and placed there by 25 larvae of house flies. The exotoxin activity was assessed by the number of dead larvae of house flies on the 8th day of the experience.

The results of the experiments for each of them was determined indicator LC50the dose of the drug (in %, in ál/g of feed, ...)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, Teatime, Kiev, ed. PGAA, 2007, 217 pages):

lg50=lgCm-σ(ΣL1-0,5),

where Cm- m is ximala tested concentration;

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.

Obtained in these experiments the values LK50for drug-based strain BtH1800/15 (Biotoxin) and drug-based strain - prototype BtH198 (Methoxylation), characterizing their insecticidal activity, are presented in table 1.

Table 1.
The biological activity of drugs on the basis of BtH1
LC50, %, for L2LC50, %, for L2The content of the exotoxin:
MedicationColorado potato beetlemothLC50, ál/g of feed for L2
house flies
Drug-based
strain BtH1980,510,426,82
(Methoxylation -
the prototype)
A new medication
based0,240,23,05
strain BtH1800/15
(Biaxin)

From the data in table 1 it follows that a drug based on the strain BtH1800/15 (Biaxin) insecticidal activity for Colorado potato beetle larvae and caterpillars Gypsy moth 2.1 times more of the drug on the basis of the strain - prototype BtH198 (Methoxylation), but by the content of exotoxin it exceeds the th 2.24 times.

In addition, they conducted laboratory experiments on the preservation of insecticidal activity of strains BtH1800/15 and BtH198 after their long-term storage. The results of the experiments are shown in table 2.

Table 2.
Biological characteristics of strains BtH1after long term storage
IndicatorsThe original valuesAfter 1 yearAfter 2 years
Strain
BtH198BtH1800/15BtH198BtH1800/15BtH198BtH1800/15
The title dispute billion/ml2,114,02,053,921,98the 3.8
The content of the exotoxin: LC50, ál/g of feed for L2house flies 5,042,9of 5.43,06,43,05
Insecticidal activity: LC50, %, for L2Colorado potato beetle0,540,280,580,290,620,3

From the data presented in table 2, it is seen that after 2 years of storage the insecticidal activity of the strain prototype BtH198 against the Colorado potato beetle has decreased by 15%, while the strain BtH1800/15 - only 7%, which confirms the possibility of long-term storage. After 2 years of storage strain BtH1800/15 92% more strain - prototype BtH198, but by the content of exotoxin and insecticidal activity against the Colorado potato beetle 2.1 times higher.

The effectiveness of the drug on the basis of strain BtH1800/15 (Biotoxin) was also tested in field conditions in the farms of the Leningrad region: SE OPH "Kaleici"OPH "Suyda" and OPH "upstream"against Colorado potato beetle. The results of field experiments in the farms of the Leningrad region are shown in table 3.

Table 3.
The effectiveness of the drug on the basis of strain BtH1800/15 on potato against the Colorado potato beetle in farms lenoble
Treatment optionThe treatment area, haThe consumption rate of the product, l/haThe rate of fluid flowThe average number of pest (ind. per 1 plant)Processing efficiency, % at day accounting
l/haBefore processingDay accounting714
714
SE OPH "Kaleici"
Biaxin31530026,7 1,01,696,495,9
Control (without treatment)0,25--19,330,425,1*The increase in num. pest 30%
OPH "Suyda"
Biaxin21240028,41,50,994,796,8
Control (without treatment0,25--26,436,734,6*The increase in num. pest 31%
OPH "upstream"
Biaxin31540030,23,0 0,890,097,4
Control (without treatment)0,25--23,428,130,9The increase in num. pest 37%
Note: * - part of the larvae left in the soil.

In table 3 the data confirm the high insecticidal activity Biotoxin - drug-based strain BtH1800/15.

Drug-based strain BtH1800/15 (Biaxin) safe for warm-blooded animals.

Virulence, dissemination, toxicity and toxigenicity of strain BtH1800/15 were studied in accordance with the Methodological guidelines of the Ministry of health of the USSR №4263-87, No. 2620-82 and taking into account the recommendations of the who (bull. Who, 1981, No. 6, p.20-27) on outbred white rats and outbred white mice.

Virulence and dissemination of strain BtH1800/15 studied after a single dose daily agar culture in physiological solution in the stomach, white mice and white rats in doses of 106, 107, 108and 109and intraperitoneally to 106, 107, 108and 109microbial cells is to the animal. In the experiment used 12 animals per dose (6 males and 6 females). During the observation period, clinical symptoms of the animals was not observed, death was absent. Within 30 days after the injection of cultures of microorganisms, animals were killed by inhalation of CO2and method prints did the seeding from the blood and internal organs (lungs, liver, kidneys and spleen) on Petri dishes with agar medium. The growth of culture in the sowing of the bodies of animals using both methods of introduction was not found.

Toxicity strain BtH1800/15 was assessed by intraperitoneal administration to mice agar suspension culture of microorganisms, prepared in sterile saline and inactivated by heating at 60°C for 30 minutes at concentrations of 107, 108and 109microbial cells per animal (6 animals per dose).

Toxigenicity strain BtH1800/15 studied in mice by intraperitoneal and intragastric administration of sterile filtrate of cultural Noi fluid filtration through the Millipor filter with pore size 0.22 μm) 3 and 7 day cultures at doses of 0.3 ml, 0.6 ml and 1.0 ml (6 animals per dose). Animals of the control group was injected with sterile liquid nutrient medium in the same amounts. The death of the mice was not. LD50has not been installed. In both ways it exceeds 1.0 ml per animal is for 3 and 7 daily cultural liquids.

That is, in terms of virulence, dissemination, toxicity and current-sidenote strain of Bacillus thuringiensis var. thuringiensis No. 800/15 (BtH1800/15) is not pathogenic for warm-blooded animals and, in accordance with the Methodological guidelines of the Ministry of health No. 2620-82 and No. 4263-87, strain BtH1800/15 belongs to the 4th class of hazard (low hazard) and satisfies the requirements of industrial microorganisms.

The results of the biological product are consistent with international practice extensive use of bioinsecticides on the basis of strains of bacteria of the group of Bacillus thuringiensis.

Thus, based on the above, it can be argued that the drug liquid form on the basis of strain BtH1800/15 (Biaxin) has high insecticidal activity against dangerous mass pests - insects-phytophages, characterized safety for humans and warm-blooded animals, and that the strain BtH1800/15 retains its insecticidal activity during prolonged storage.

And this, in turn, characterizes the strain of Bacillus thuringiensis var. thuringiensis No. 800/15 (BtH1800/15) as promising as a means for the production of insecticidal biological product.

The bacterial strain of Bacillus thuringiensis var. thuringiensis No. 800/15 deposited in the GSI ARRIAM (Departmental collection of useful microorganisms for agricultural purposes - BKSM) the registration number 611 in the group of spore-forming microorganisms, as a means to obtain insecticidal biological product.



 

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59 cl, 4 dwg, 9 tbl, 5 ex

FIELD: agriculture.

SUBSTANCE: invention relates to the field of agriculture. The method of cultivation of soyabean using polycomponent fertiliser "Bioplant Flora", including presowing treatment of seeds with rizotorfin. The presowing treatment with rizotorfin is performed in the mixture with polycomponent fertiliser "Bioplant Flora" at a dose of 1 l/t. At that foliar fertilising of plants is carried out with the polycomponent fertiliser "Bioplant Flora" in the phase of 4-5 leaves and end of bean-formation at a dose of 0.25-1 l/ha.

EFFECT: method enables to improve the yield and quality of soyabean by increasing the amount of nitrogen-fixing nodules on soybean root system and improve the nutrient regime of plants, also it is more efficient to use the elements of mineral nutrition of fertiliser, thus reducing human-induced impact on the environment.

4 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, veterinary science and plant protection. The bacterial strain Bacillus subtilis IC-1435-1-1 is deposited in the Russian National Collection of Industrial Microorganisms, registration number B-10641. The bacterial strain Bacillus amyloliquefaciens IC-1436-1-23 is deposited in the Russian National Collection of Industrial Microorganisms, registration number B-10642. The bacterial strain Bacillus amyloliquefaciens IC-1437-1-23 is deposited in the Russian National Collection of Industrial Microorganisms, registration number B-10643. The strains are produced by selection and promote the recovery of microbiocenoses in the soil and animals gastrointestinal tract, possess bactericidal, fungicidal and virucidal activity. A preparation on the basis of the strains contains an excipient or water with a bacterial biomass in the spore form Bacillus subtilis, the Russian National Collection of Industrial Microorganisms B-10641, or Bacillus amyloliquefaciens, the Russian National Collection of Industrial Microorganisms B-10642, or Bacillus amyloliquefaciens, the Russian National Collection of Industrial Microorganisms B-10643, or a mixture thereof in ratio 1:1:1 with titre of each bacterial strain not less than 1·104 CFU/g or 1·104 CFU/ml.

EFFECT: preparation possess bactericidal and fungicidal activity.

7 cl, 9 dwg, 12 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbological industry, particularly production of biologically active substances and can be used in producing fusicoccins for different purposes, for both agricultural and medical purposes. The culturing medium contains molasses, raw sugar, potassium nitrate (KNO3), potassium phosphate (KH2PO4), magnesium sulphate (MgSO4 x 7H2O) and tap water in a given ratio.

EFFECT: invention increases the range of fusicoccins.

4 ex

Antibiotic peptides // 2472805

FIELD: chemistry.

SUBSTANCE: peptides and peptide derivatives have general formula Sub1-X1NX2X3PVYIPX4X5RPPHP-Sub2, where Sub1, X1, X2, X3, X5, Sub2 are given in the claim. The disclosed peptides or peptide derivatives have at least one of the following advantages compared to natural apidecine peptides: (i) longer half-life in mammal serum owing to higher protease stability, (ii) high antimicrobial activity with respect to one or more bacterial strains, particularly human pathogens or fungi, or other microbial infections, (iii) demonstrate a wide range of antimicrobial activity, (iv) cause slower development of resistance in microorganisms and (v) are not toxic for human cells, including erythrocytes.

EFFECT: improved properties of peptides.

21 cl, 8 dwg, 7 tbl, 6 ex

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