Bacterial strain bacillus thuringiensis varthuringiensis n800/15 as agent for preparing entomocidal biopreparation. RU patent 2514211.

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 refers to biotechnology, namely to microbiological means of protection of agricultural, medicinal and forest crops from insects, i.e. with insecticidal action.

It is known that as entomopathogens use strains-producers bitoxibacillin, namely Bacillus thuringiensis subsp. thuringiensis 202, 620 98 and that can form two insecticidal toxin: protein crystals of endotoxin, are released from the cells in the process of lysis of their shells, and water-soluble exotoxin nucleotide nature produced in the process vegetative growth of culture (Nvention, Nambowa, ATS, Linkosalo. Comparative efficacy of three strains of Bacillus thuringiensis subsp. thuringiensis /bul. ARRIAM №53, Leningrad, 1990, p.44-46). As a result of comparison of biological and insecticidal activity of these three strains found that strains 620 98 and Excel in insecticidal activity strain 202.

The strain of Bacillus thuringiensis subsp. thuringiensis 98 (1 BtH 98) allocated from the dead dolls cabbage moths, is a producer of insecticidal toxin is stored in the collection of "Vniigenetika" # TMPL IN-2452.

Strain 1 BtH 98 has a sufficiently high insecticidal activity against the Colorado beetle and other pests (prototype).

Bitoxibacillin get cultivation strain BtH 1 98 on a nutrient medium, containing ammonium phosphate, magnesium sulfate, calcium chloride, starch corn and fodder yeast, green syrup, when aeration within 48 hours, a title dispute in the cultural liquid of 3.5-3.75 bln/ml, the content of exotoxin 0.5 mg/ml received the cultural liquid contribute 1% of sodium chloride and dried. Get Bitoxibacillin, which contains the dispute 65-72 billion/year and 1% of exotoxin.

The disadvantage strain Bacillus thuringiensis subsp. thuringiensis 98 (1 BtH 98) is that it quickly loses the insecticidal activity during storage.

Bitoxibacillin from the strain of BtH 1 98 receive in the dry form, therefore, its production is energy-intensive and costly due to the operation of drying. In addition, high temperatures have a detrimental impact on crystals of endotoxin, destroying them, which reduces insecticidal activity of Bitoxibacillin.

The task of the claimed invention is to identify the strain on the basis of environmentally friendly bacteria of the Bacillus group thuringiensis, suitable for creation of highly effective insecticidal preparation in liquid form against mass of insects-phytophagans, pests of agricultural and forest cultures and keeping the insecticidal activity during prolonged storage.

The problem is solved by the fact that as a means to obtain insecticidal drug use strain of Bacillus thuringiensis var. thuringiensis №800/15.

The strain of Bacillus thuringiensis var. thuringiensis №800/15 (1 BtH 800/15) allocated from the corpses of Colorado beetle larvae in the Leningrad region and deposited 20.03.2006, in Departmental collection of useful microorganisms agricultural purpose RAAS (BKSM) wildebeest of VYSHM under registration №611 in the group of spore microorganisms, which is confirmed by the certificate of Deposit of 02.07.2010, (certificate attached). The strain is characterized by the following properties.

Cultural morphological properties.

Gram moving sticks, peritrichia, vegetative cells of solitary or in short chains 2-4 cells in the chain. The size of cells one-day culture in fish agar (RA) is(2,5-3,0) × (0,8-0,9) mkm. For 3-4 days growth on RA in cells spores are formed oval subterminals size(1,1-1,3)×(0,8-0,9) mkm and personalnye enable endoxana (crystals) the right bobowicko form a $ 1.1-1.3 mkm.

On RA after 48 h at 26-28°With culture forms a flat colony grayish-white rounded with rough edges, fine-grained surface, diameter colony 22-24 mm Strain grows at a temperature of 20 to 34 C (opt 26-32 C) and with the measure of acidity pH 6,4-8,0 (opt to 6.8 to 7.2). The fish soup (RB) after 24 h increases with the education films (fog).

Biochemical properties.

Optional gone anaerobic. Gelatine nazivaet on the 5th day, casein splits, peptonizing milk with alkalization through 24-28 hours, then acidify and discolor the litmus paper. On environment Clark forms acetylenedicarbonic (AMK).

Gidrolizuut starch, uses citrate, 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 Bacillus thuringiensis var. thuringiensis H-1 in dilution 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 production phages: K, M, N, eh-5, And-77, D 2-D 5 .

A strain of low toxicity to mammals.

The strain is growing on nutrient programresearch environments; is stored in a dried state or on the doorposts of RA in test tubes at 4-5 degrees Celsius with periodic re-seeding once in 6 months.

Strain 1 BtH 800/15 has pronounced insecticidal properties and promising as a means for production of biopreparation for protection of agricultural crops from the mass of insects-phytophagans.

On the basis of this strain BtH 1 800/15 developed technology of production of new insecticidal biological product, called by the authors Biaxin.

An example of retrieving Biotoxin.

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

Corn flour

1.5 and 2.4

Fodder yeast

(NH 4 ) 2 4 HPO

MgSO 4 x 7H 2 O

0,02-0,04

CaCl 2 x 2H2O

0,15-0,20

Water water

Using alkali (for example, 10% NaOH) bringing the pH of 7.4-7.8 on. Then this medium is poured 30-50 ml Erlenmeyer flask with a capacity of 750 ml and sterilized for 40 min at 120-122°C. After sterilization pH amounted to 6.8 to 7.2. Prepared nutrient medium cooled and seeded culture strain BtH 1 800/15.

For cultivation environment in flasks is spore culture strain BtH 1 800/15 received by flushing 5-10 ml of a physiologic saline on a test tube culture on the sloping agar. Received a suspension with the culture of the strain which was to obtain a homogeneous suspension and contributed to flasks prepared a medium rate of 0,3-0,5 ml suspension of 30-50 ml environment. Sown bulb was placed on the rocking chair at 200-220 rpm and 28-30 degrees C in 56-72 hours to complete rash dispute and crystal endoxana (audit was carried out by mikroskopiya). Grown in flasks culture fluid strain BtH 1 800/15 used for cultivation environment in fermenters.

As a nutrient medium for fermenters can be used any groupinvariant environment, preferably of the same composition, which was used to obtain the culture fluid in the war, i.e. on the basis of fodder yeast and corn flour (specified above).

In this first in the tap water, heated to 40 C, covered with constant stirring all the other ingredients, received a suspension were heated to 80 C and kept for 30 minutes, after which the pH brought to 7.8 to 8.0 and environment sterilized for 1 h at 140 degrees C.

Prosterilizovat nutrient medium for fermenter cooled up to 30-35 C and had made a culture fluid strain BtH 1 800/15 in advance grown in flasks, at the rate of 0.7-1.0% cultural liquid of the volume of the nutrient medium in the fermenter.

In the fermenter the cultivation process was carried out at the following conditions:

- temperature cultivation 28-30 Degrees;

- the mixer rotation speed 200-220 rpm;

- pressure in the apparatus of 0,4-0,5 kg/cm2 ;

- the mode of air supply:

from 0 to 12 h is 0.2 V, d / V environment /min;

from 12 to 24 h 0.5 V, d / V environment /min;

from 24 to 42 h - 0,8-1,0 V, d / V environment /min;

with 42 hours - until the end of the fermentation of 0.5 V, d / V environment /min

That is, fermentation is carried out in conditions of high aeration and finished at achievement of the content in the resulting drug 80-85% free dispute and crystals of endotoxin at a ratio of 1:1. At the same titer strain BtH 1 800/15 obtained in the liquid was not less than 3,8-4,0 mlrdsr/ml in the absence of foreign flora and virulent phage.

In this way? was received Biopreparat from the strain of BtH 1 800/15 (Biaxin), which is a liquid, easily diluted with water to the necessary working concentration, easy to use serial spraying equipment. Production of the drug factories, biofactories in the regions of the consumer does not require expenses for transportation and delivery for large distances. As Biaxin is liquid, the technology of production of the drug does not require large energy consumption for the operation of drying.

Were the laboratory tests of the efficacy of the drug from the strain of BtH 1 800/15 - Batocina in relation to the mass of insects-phytophagans crops.

So, for example, laboratory determination of the insecticidal activity of the drug from the strain of BtH 1 800/15 (Batocina) was performed on the Colorado potato beetle larvae of the 2nd age. While food (leaf potatoes) were treated with Batocina in dilutions 1:20; 1:100; 1:500, which corresponds to the concentration 5,0; 1,0; 0,2%, providing the death of the test object from 30 to 95%. In each option experience planted on 25 Colorado beetle larvae of the 2nd age (in triplicate). For comparison, in experiments under the same conditions used the drug on the basis of the strain prototype BtH 1 98 (Bitoxibacillin liquid, without drying), at the same concentrations. In the control of food processed water. The account of the death of Colorado beetle larvae held on the 7th day.

There were also conducted laboratory tests insecticidal activity Bioxin (preparation on the basis of the strain BtH 1 800/15) on the caterpillars of the 2nd age moth. Experiments were performed in glass banks by the volume of 0.8 l While food (branches of Apple trees with leaves) were treated with Batocina five concentrations: 2,0; 1,0; 0,5; 0,25; 0,125%. In each option experience planted 25 moth caterpillars of the 2nd age (in triplicate). For comparison, in experiments under the same conditions used the drug on the basis of the strain prototype BtH 1 98 (Bitoxibacillin liquid, without drying) in the same concentrations. In the control of food processed water. The account of the death of caterpillars of the moth held on the 7th day.

Experiments on determination of the content of the exotoxin in drug Biaxin assessed on the larvae of house flies by the following method.

The drug is based on the strain BtH 1 800/15 (Biaxin) in 20 ml centrifuged 15 min at 8000 rpm Received centrate autoclaved at a pressure of 0.1 MPa and a temperature of 105 OC for 20 minutes Then prepared for dilution: 1:2; 1:4; 1:8; 1:16; 1:32, that corresponds to a concentration of 50; 25; 12,5; 6,25; 3,125 MKL/g feed (each three - time).

For a test in a glass jar with a capacity of 200 ml were made:

- 2 ml of the drug Biaxin appropriate breeding;

- 11 ml of 2.5%water suspension of dried milk;

- 7 g sterilized wheat bran.

In control instead of Batocina used the drug on the basis of the strain prototype BtH 1 98 (Bitoxibacillin liquid, without drying) in the same concentrations. Food with appropriate dilutions of the drug in each Bank are thoroughly mixed and placed there by 25 larvae house flies. The activity of exotoxin was assessed by the number of dead larvae house flies on the 8th day experience.

According to the results of the experiments for each of them was defined indicator LC 50-dose (in %, in the MKL/g feed, ...)that causes 50% of the death of the insect.

The value of LC 50 was calculated by the formula Cerberus (see, for example, a monograph "ECOLOGY of BACILLUS THURINGIENSIS" authors Vratica, Teatega, Kiev, ed. PGAA, 2007, page 217):

lg 50 =lgC m-s(ΣL 1 -0,5),

where m is the maximum tested concentration;

Sigma logarithm relationship of each previous concentration to follow (the logarithm of the ratio of dilution);

ΣL 1 - the sum of the relations in the number of insects that have died from this concentration, the total number of insects that were exposed this concentration.

Obtained in these experiments, the values of LC 50 for drug from the strain of BtH 1 800/15 (Batocina) and preparation on the basis of the strain prototype BtH 1 98 (Bitoxibacillin), characterizing the insecticidal activity, is presented in table 1.

Table 1.

Biological activity of drugs on the basis of 1 BtH

LK of 50 %, for 2 L

LK of 50 %, for 2 L

The content of the exotoxin:

Colorado beetle

LC 50 , MKL/g feed for 2 L

house flies

The drug is based on

strain 1 BtH 98

(Bitoxibacillin -

prototype)

New drug

on the basis of

strain 1 BtH 800/15

(Biaxin)

From the data in table 1 it follows that the drug from the strain of BtH 1 800/15 (Biaxin) insecticidal activity for larvae Colorado potato beetles and caterpillars of the moth in 2,1 times more preparation on the basis of the strain prototype BtH 1 98 (Bitoxibacillin), but by the content of exotoxin he surpasses it 2.24 times.

In addition, there were conducted laboratory experiments on conservation insecticidal activity strains BtH 1 800/15 and BtH 1 98 after their long-term storage. The results of the experiments are given in table 2.

Table 2.

Biological characteristics of strains BtH 1 after their long-term storage

Indicators

The original values

After 2 years

BtH 1 98

BtH 1 800/15

BtH 1 98

BtH 1 800/15

BtH 1 98

BtH 1 800/15

Title dispute, bln/ml

The content of the exotoxin: LC 50 , MKL/g feed for L 2 bedroom flies

The insecticidal activity: LC 50 , %, for the L 2 Colorado beetle

From the data presented in table 2, shows that in 2 years the storage of the insecticidal activity of the strain prototype BtH 1 98 against Colorado potato beetle decreased by 15%, and in strain BtH 1 800/15 - only 7%, which confirms the possibility of its long-term storage. However in 2 years storage strain BtH 1 800/15 92% more strain prototype 1 BtH 98, but by the content of exotoxin and insecticidal activity against Colorado potato beetle 2.1 times exceeds it.

The effectiveness of the drug from the strain of BtH 1 800/15 (Batocina) was also tested in field conditions in the farms of the Leningrad region: SE OPH "Kaleici"OPH "Suyda" and OPH "Predportovaya", on potatoes against the Colorado beetle. The results of field experiments in the farms of the Leningrad region are given in table 3.

Table 3.

The effectiveness of the drug from the strain of BtH 1 800/15 on potatoes against the Colorado beetle in farms lenobl

Treatment option

The treatment area, ha

The consumption rate of the drug, l/ha

The rate of consumption of the working fluid

The average number of the pest (ind. per 1 plant)

Processing efficiency, % accounting for a day

Before treatment

Day accounting

SE OPH "Kaleici"

Control (without treatment)

The increase in num. pest 30%

OPH "Suyda"

Control (without processing

The increase in num. pest 31%

OPH "Predportovaya"

Control (without treatment)

The increase in num. pest 37%

Note: * - some larvae left in the soil.

In table 3 the data confirm the high insecticidal activity Biotoxin - preparation on the basis of the strain BtH 1 800/15.

The drug is based on the strain BtH 1 800/15 (Biaxin) safe for warm-blooded animals.

The virulence and the dissemination of strain BtH 1 800/15 studied in a one-time daily agar culture in the saline solution into the stomach white mice and white rats at doses of 10 6 , 10 7 , 8 and 10 10 9 and intraperitoneally on 10 6 , 10 7 , 8 and 10 10 9 microbial cells at the animal. The experience is used by 12 animals per dose (6 males and 6 females). In the period of monitoring of clinical symptoms of the disease in animals have not been observed, the death was absent. Within 30 days after the administration of culture of microorganisms, animals were killed by inhalation of CO 2 and method prints did the planting of blood and internal organs (lungs, liver, kidneys and spleen) on Petri dishes with agar medium. The growth of culture in the sowing of animal organs in both ways of introduction has not been detected.

Toxicity strain BtH 1 800/15 evaluated by intraperitoneal administration to mice suspension agar cultures of microorganisms, prepared to sterile physiological solution and inactivated by heating at 60 C for 30 minutes at concentrations of 10 7 , 8 and 10 10 9 microbial cells at the animal (6 individuals per dose).

Toxigenicity strain BtH 1 800/15 studied in mice by intraperitoneal and intragastric administration of sterile filtrate cultural Noi fluid filtration through the filter Millipor with pore size 0,22 microns) 3 and 7 subsistence crops at doses of 0.3 ml 0.6 ml, and 1.0 ml (6 individuals per dose). Animals of the control group was injected sterile liquid nutrient medium in the same amounts. The death of mice was not. LD 50 has not been established. In both the ways she exceed 1.0 ml per animal for 3 and 7 per diem cultural liquids.

That is, in terms of virulence, dissemination, toxicity and talk of sidenote strain of Bacillus thuringiensis var. thuringiensis №800/15 (1 BtH 800/15) not pathogenic for animals or, in accordance with the Methodological guidelines of the Ministry of health №2620-82 and # 4263-87, strain BtH 1 800/15 belongs to the 4th class of danger (low-hazardous) and meets the requirements of industrial microorganisms.

The results for the security of this biological preparation are consistent with the international practice of wide use of bioinsecticides based on strains of a group of bacteria Bacillus thuringiensis.

Thus, based on the above it can be argued that the drug liquid forms from the strain of BtH 1 800/15 (Biaxin) has high insecticidal activity against dangerous mass pest insects-phytophagans, characterized safety for humans and warm-blooded animals, and that the strain BtH 1 800/15 retains its the insecticidal activity during prolonged storage.

And this, in turn, characterized by a strain of Bacillus thuringiensis var. thuringiensis №800/15 (1 BtH 800/15) as promising as a tool for the production of insecticidal biological product.

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