RU2514211C1 - BACTERIAL STRAIN Bacillus thuringiensis varthuringiensis N800/15 AS AGENT FOR PREPARING ENTOMOCIDAL BIOPREPARATION - Google Patents

Abstract

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

Description

The invention relates to biotechnology, namely to microbiological means of protecting agricultural, medicinal and forest crops from harmful insects, i.e. possessing entomocidal action.

It is known that as producers of pathogens use producer strains of bitoxibacellin, namely Bacillus thuringiensis subsp. thuringiensis 202, 98 and 620, capable of forming two entomocidal toxins: protein crystals of endotoxin released from the cells during lysis of their membranes, and water-soluble nucleotide exotoxin produced during the vegetative growth of culture (N.V. Kandybin, N.M. Barbashova , A.A. Stus, L.N. Kuznetsova, Comparative Effectiveness of Three Bacillus Thuringiensis Subsp.thuringiensis Strains / Bul. VNIISKHM No. 53, L., 1990, pp. 44-46). As a result of comparing the biological and entomocidal activity of these three strains, it was revealed that strains 98 and 620 exceeded strain 202 in their entomocidal activity.

Strain Bacillus thuringiensis subsp. thuringiensis 98 (BtH ₁ 98) is isolated from a dead pup of a cabbage scoop, is a producer of entomocidal endotoxin, and is stored in the collection of the All-Russian Research Institute of Genetics under No. TsMPL V-2452.

Strain BtH ₁ 98 has a sufficiently high entomocidal activity against the Colorado potato beetle and other harmful insects (prototype).

Bitoxibacillin is obtained by culturing strain BtH ₁ 98 on a nutrient medium containing ammonium phosphate, magnesium sulfate, calcium chloride, corn starch and fodder yeast, green molasses, with aeration for 48 hours, spore titer in the culture fluid of 3.5-3.75 billion / ml, exotoxin content 0.5 mg / ml. 1% sodium chloride is added to the resulting culture liquid and dried. Get Bitoxibacillin, which contains a spore of 65-72 billion / g and 1% exotoxin.

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

Bitoxibacillin based on the BtH ₁ 98 strain is obtained in dry form, and therefore, its production is energy-intensive and costly due to the drying operation. In addition, high temperatures during drying have a detrimental effect on endotoxin crystals, destroying them, which leads to a decrease in the entomocidal activity of Bitoxibacillin.

The task of the invention is to identify a strain based on an environmentally friendly bacterium from the group of Bacillus thuringiensis, suitable for creating a highly effective entomocidal preparation in liquid form against mass insect phytophages, pests of agricultural and forest crops and retaining its entomocidal activity during long-term storage.

The problem is solved in that a strain of Bacillus thuringiensis var. Is used as a means to obtain an entomocidal preparation. thuringiensis No. 800/15.

Strain Bacillus thuringiensis var. thuringiensis No. 800/15 (BtH ₁ 800/15) was isolated from the corpses of Colorado potato beetle larvae in the Leningrad Region and deposited on March 20, 2006 in the Departmental Collection of Useful Microorganisms of Agricultural Purpose of the Russian Agricultural Academy (VKSM) GNU VNISHM under registration No. 611 in the group of spore microorganisms, as evidenced by a certificate of deposit dated 02.07.2010 (Certificate attached). The strain is characterized by the following properties.

Cultural and morphological properties.

Gram-positive motile sticks, peritrichi, vegetative cells, single or in the form of short chains of 2-4 cells in a chain. The size of the cells of a one-day culture on fish agar (RA) is (2.5-3.0) × (0.8-0.9) microns. On the 3-4th day of growth on RA, oval-shaped spores are formed in the cells, located subterminally, with a size of (1.1-1.3) × (0.8-0.9) microns and parasporal endoxin inclusions (crystals) of regular bean-shaped size 1.1-1.3 microns.

On the RA, after 48 h at 26-28 ° С, the culture forms flat grayish-white colonies of rounded shape with an uneven edge, the surface is fine-grained, the colony diameter is 22-24 mm. The strain grows at a temperature of 20-34 ° C (opt 26-32 ° C) and at a pH of 6.4-8.0 (opt 6.8-7.2). On fish broth (RB) after 24 hours it grows with the formation of a film (veil).

Biochemical properties.

Optional anaerobic. It degrades gelatin on the 5th day, casein breaks down, milk peptones with alkalization after 24-28 hours, then acidifies and bleaches the litmus. Clark forms acetylmethylcarbonyl (AMA).

It hydrates starch, uses citrates, and does not form pigment and urease. Indole and hydrogen sulfide does not use, reduces nitrates to nitrites.

It breaks down glucose, mannose, levulose, sucrose, maltose, cellobiose, glycerin and salicin to form an acid. Does not use galactose, arabinose, xylose, rhamnose, lactose, raffinose, mannitol, sorbitol, dulcite, inositol, inulin.

Antigenic properties.

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

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

The strain is resistant to production phages: K, M, N, E-5, I-77, D ₂ -D ₅ .

The strain has low toxicity for warm-blooded animals.

The strain grows on nutrient yeast polysaccharide media; stored in a lyophilized state or on the jambs of RA in test tubes at 4-5 ° C with periodic reseeding once every 6 months.

The strain BtH ₁ 800/15 has pronounced entomocidal properties and is promising as a means for the production of biological products for the protection of crops from mass insect phytophages.

Based on this strain of BtH ₁ 800/15, a technology was developed for the production of a new entomocidal biological product called Bitoxin by the authors.

An example of obtaining Bitoxin.

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

Corn flour 1,5-2,4 Fodder yeast 3.0-3.5 (NH ₄ ) ₂ HPO ₄ 0.5-0.6 MgSO ₄ × 7H ₂ O 0.02-0.04 CaCl ₂ × 2H ₂ O 0.15-0.20 Tap water up to 100.

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

For the inoculation of the medium in the flasks is a spore culture of strain BtH ₁ 800/15 obtained by washing 5-10 ml of physical. solution to a test tube with culture on mowed agar. The resulting suspension with the strain culture was shaken until a homogeneous suspension was obtained and introduced into flasks with prepared nutrient medium at the rate of 0.3-0.5 ml of suspension per 30-50 ml of medium. Inoculated flasks were placed on a rocking chair at 200-220 rpm and 28-30 ° С for 56-72 hours until the spores and endoxin crystals completely precipitated (verification was performed by microscopy). The culture fluid grown in flasks of the strain BtH ₁ 800/15 was used to inoculate the medium in fermenters.

As a nutrient medium for fermenters, any yeast polysaccharide medium can be used, optimally the same composition that was used to obtain the culture fluid in the flasks, i.e. based on fodder yeast and corn flour (composition indicated above).

At the same time, all other ingredients were poured into tap water heated to 40 ° C with constant stirring, the resulting suspension was heated to 80 ° C and kept for 30 min, after which the pH was adjusted to 7.8-8.0 and the medium was sterilized for 1 h at 140 ° C.

The sterilized nutrient medium for the fermenter was cooled to 30-35 ° C and the culture fluid of the BtH strain ₁ 800/15, previously grown in flasks, was added to it at the rate of 0.7-1.0% of the culture fluid from the volume of the nutrient medium in the fermenter.

In the fermenter, the cultivation process was carried out under the following conditions:

- cultivation temperature 28-30 ° C;

- rotation speed of the mixer 200-220 rpm;

- the pressure in the apparatus of 0.4-0.5 kgf / cm ² ;

- air supply mode:

from 0 to 12 hours - 0.2 V _air / V _medium / min;

from 12 to 24 hours - 0.5 V _air / V _environment / min;

from 24 to 42 hours - 0.8-1.0 V _air / V _medium / min;

from 42 h - until the end of fermentation - 0.5 V _air / V _medium / min.

That is, the fermentation process was carried out under high aeration conditions and was completed when the content of 80-85% free spores and endotoxin crystals in the resulting preparation was reached at a ratio of 1: 1. Moreover, the titer of the BtH strain ₁ 800/15 in the obtained liquid preparation was not less than 3.8-4.0 billion spores / ml in the absence of extraneous microflora and virulent phage.

In this way? a biopreparation was obtained on the basis of the BtH ₁ 800/15 strain (Bitoxin), which is a liquid that is easily diluted with water to the required working concentration, convenient for using serial spraying equipment. Production of the drug in factories, biofactories in the regions of the consumer will not require the cost of its transportation and delivery over long distances. Since Bitoxin is a liquid, the production technology of the drug also does not require large energy costs for the drying operation.

Laboratory tests of the effectiveness of the drug based on the BtH strain ₁ 800/15 - Bitoxin in relation to mass insect phytophages of agricultural crops were conducted.

For example, laboratory determinations of the entomocidal activity of a preparation based on the BtH strain ₁ 800/15 (Bitoxin) were carried out on larvae of a Colorado potato beetle of the 2nd age. In this case, the feed (potato leaves) was treated with Bitoxin at a dilution of 1:20; 1: 100; 1: 500, which corresponds to concentrations of 5.0; 1.0; 0.2%, ensuring the death of the test object from 30 to 95%. In each experiment, 25 larvae of the Colorado potato beetle of the 2nd age (in triplicate) were planted. For comparison, in the experiments under the same conditions, a preparation was used based on the prototype strain BtH ₁ 98 (Bitoxibacillin liquid, without drying), at the same concentrations. In the control, the feed was treated with water. The death of the Colorado potato beetle larvae was recorded on day 7.

Laboratory tests of the entomocidal activity of Bitoxin (a preparation based on the BtH strain ₁ 800/15) on caterpillars of the 2nd age of unpaired silkworm were also conducted. The experiments were carried out in glass jars with a volume of 0.8 l. In this case, the feed (branches of apple trees with leaves) was treated with Bitoxin in five concentrations: 2.0; 1.0; 0.5; 0.25; 0.125%. In each experiment, 25 caterpillars of unpaired silkworms of the 2nd age were planted (in triplicate). For comparison, in the experiments under the same conditions, a preparation was used based on the prototype strain BtH ₁ 98 (Bitoxibacillin liquid, without drying) in the same concentrations. In the control, the feed was treated with water. Accounting for the death of unpaired silkworm caterpillars was carried out on day 7.

The experiments to determine the exotoxin content in the Bitoxin preparation were evaluated on housefly larvae according to the following procedure.

A preparation based on the BtH strain ₁ 800/15 (Bitoxin) in an amount of 20 ml was centrifuged for 15 min at 8000 rpm. The resulting centrate was autoclaved at a pressure of 0.1 MPa and a temperature of 105 ° C for 20 minutes. Then prepared dilutions of the drug: 1: 2; 1: 4; 1: 8; 1:16; 1:32, which corresponds to concentrations 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 Bitoxin appropriate dilution;

- 11 ml of a 2.5% aqueous suspension of milk powder;

- 7 g of sterilized wheat bran.

In the control, instead of Bitoxin, a preparation was used based on the strain prototype BtH ₁ 98 (Bitoxibacillin liquid, without drying) in the same concentrations. Food with appropriate dilutions of the preparation in each jar was thoroughly mixed and 25 larvae of housefly were placed there. Exotoxin activity was evaluated by the number of dead housefly larvae on day 8 of the experiment.

According to the results of the experiments, for each of them the LK ₅₀ indicator of the drug dose was determined (in%, in μl / g of feed, ...), which causes 50% of insect death.

The value of LC _{50 was} calculated according to the Kerber formula (see, for example, the monograph “ECOLOGY OF BACILLUS THURINGIENSIS” by V.F. Patyk, T.I. Patyk, Kiev, publ. PHAA, 2007, 217 pp.):

lgLK ₅₀ = lgC _m -σ (ΣL ₁ -0.5),

where C _m is the maximum tested concentration;

σ is the logarithm of the ratio of each previous concentration to the next (logarithm of the ratio of dilutions);

ΣL ₁ is the sum of the ratios of the number of insects that died from a given concentration to the total number of insects that were exposed to this concentration.

The LC ₅₀ values obtained in these experiments for a preparation based on a strain of BtH ₁ 800/15 (Bitoxin) and a preparation based on a strain of the prototype BtH ₁ 98 (Bitoxibacillin) characterizing their entomocidal activity are presented in Table 1.

Table 1. The biological activity of drugs based on BtH ₁ LC ₅₀ ,%, for L ₂ LC ₅₀ ,%, for L ₂ Exotoxin Content: A drug Colorado potato beetle gypsy moth LK ₅₀ , μl / g feed for L ₂ house fly Drug based strain BtH ₁ 98 0.51 0.42 6.82 (Bitoxibacillin - prototype) New drug based 0.24 0.2 3.05 strain BtH ₁ 800/15 (Bitoxin)

From the data given in table 1, it follows that the preparation based on the BtH ₁ 800/15 strain (Bitoxin) by entomocidal activity for larvae of the Colorado potato beetle and unpaired silkworm caterpillars is 2.1 times more active than the preparation based on the strain prototype BtH ₁ 98 (Bitoxibacillin) and in terms of exotoxin content it exceeds it by 2.24 times.

In addition, laboratory experiments were carried out to preserve the entomocidal activity of strains BtH ₁ 800/15 and BtH ₁ 98 after long-term storage. The results of the experiments are shown in table 2.

Table 2. Biological characteristics of strains of BtH ₁ after their long-term storage Indicators Initial values After 1 year After 2 years Strain BtH ₁ 98 BtH ₁ 800/15 BtH ₁ 98 BtH ₁ 800/15 BtH ₁ 98 BtH ₁ 800/15 Spore titer, billion / ml 2.11 4.0 2.05 3.92 1.98 3.8 Exotoxin content: LC ₅₀ , μl / g of feed for L ₂ house fly 5.04 2.9 5,4 3.0 6.4 3.05 Entomocidal activity: LC ₅₀ %, for L ₂ Colorado potato beetle 0.54 0.28 0.58 0.29 0.62 0.3

From the data shown in table 2, it can be seen that after 2 years of storage, the entomocidal activity of the strain prototype BtH ₁ 98 in relation to the Colorado potato beetle decreased by 15%, and that of strain BtH ₁ 800/15 only 7%, which confirms the possibility of it long-term storage. At the same time, after 2 years of storage, the strain BtH ₁ 800/15 is 92% more technologically advanced _{than the} strain prototype BtH ₁ 98, and in terms of exotoxin content and entomocidal activity against the Colorado potato beetle it is 2.1 times higher.

The effectiveness of the drug based on the BtH ₁ 800/15 strain (Bitoxin) was also tested in the field at the farms of the Leningrad Region: GP Kalozhytsya farm, Suida farm and Predportovy farm, on potatoes against the 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 based on the strain BtH ₁ 800/15 on potatoes against the Colorado potato beetle on the farms of Len.obl. Processing option Area of treatment, ha The consumption rate of the drug, l / ha Working fluid flow rate The average number of pests (copies per 1 plant) Processing efficiency,% on the day of accounting l / ha Before processing Accounting day 7 fourteen 7 fourteen GP OPH "Kalozhitsy" Bitoxin 3 fifteen 300 26.7 1,0 1,6 96.4 95.9 Control (no processing) 0.25 - - 19.3 30,4 25.1 * Increase in number 30% pest OPH "Suida" Bitoxin 2 12 400 28,4 1,5 0.9 94.7 96.8 Control (no processing 0.25 - - 26,4 36.7 34.6 * Increase in number pest by 31% OPH "Preportovy" Bitoxin 3 fifteen 400 30,2 3.0 0.8 90.0 97.4 Control (no processing) 0.25 - - 23,4 28.1 30.9 Increase in number pest by 37% Note: * - part of the larvae went into the soil.

The data in table 3 confirm the high entomocidal activity of Bitoxin, a drug based on the BtH strain ₁ 800/15.

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

Virulence, dissemination, toxicity and toxigenicity of the strain BtH ₁ 800/15 were studied in accordance with the Methodological guidelines of the USSR Ministry of Health No. 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.

The virulence and dissemination of strain BtH ₁ 800/15 was studied with a single administration of daily agar culture in physiological saline in the stomach of white mice and white rats at doses of 10 ⁶ , 10 ⁷ , 10 ⁸ and 10 ⁹ and intraperitoneally at 10 ⁶ , 10 ⁷ , 10 ⁸ and 10 ⁹ microbial cells per animal. In the experiment, 12 animals were used per dose (6 males and 6 females). During the period of observation of the clinical symptoms of animal disease, no deaths were observed. 30 days after the introduction of the culture of microorganisms, the animals were sacrificed by inhalation of CO ₂ and the method of imprints was inoculated from blood and internal organs (lungs, liver, kidneys and spleen) onto Petri dishes with agar medium. The growth of culture in seeding from animal organs with both methods of administration was not detected.

The toxicity of strain BtH ₁ 800/15 was evaluated by intraperitoneal administration to mice of a suspension of an agar culture of microorganisms prepared in sterile saline and inactivated by heating at 60 ° C for 30 minutes at concentrations of 10 ⁷ , 10 ⁸ and 10 ⁹ microbial cells per animal (6 individuals per dose).

The toxicity _{of the} BtH strain ₁ 800/15 was studied in mice by intraperitoneal and intragastric administration of a sterile filtrate of culture liquid (filtration through a Millipor filter with a pore size of 0.22 μm) of 3 and 7 day old cultures in doses of 0.3 ml, 0, 6 ml and 1.0 ml (6 animals per dose). The animals of the control groups were injected with sterile liquid nutrient medium in the same volumes. The death of mice was not. LD _{50 has} not been installed. With both methods of administration, it exceeds 1.0 ml per animal for 3 and 7 day old culture fluids.

That is, according to indicators of virulence, dissemination, toxicity and current-sigenosti strain Bacillus thuringiensis var. thuringiensis No. 800/15 (BtH ₁ 800/15) is not pathogenic for warm-blooded animals and, in accordance with the Methodological instructions of the Ministry of Health No. 2620-82 and No. 423-87, the strain BtH ₁ 800/15 belongs to hazard class 4 (low hazard) and meets the requirements for industrial microorganisms.

The safety results of this biological product are consistent with world practice on the widespread use of bioinsecticides based on bacterial strains of the Bacillus thuringiensis group.

Thus, based on the foregoing, it can be argued that the liquid preparation based on the BtH strain ₁ 800/15 (Bitoxin) has high entomocidal activity against dangerous mass pests - phytophage insects, is safe for humans and warm-blooded animals, and that the BtH strain ₁ 800/15 retains its entomocidal activity during prolonged storage.

And this, in turn, characterizes the strain of Bacillus thuringiensis var. thuringiensis No. 800/15 (BtH ₁ 800/15) as promising as a means for the production of an entomocidal biological product.

Claims (1)

The bacterial strain Bacillus thuringiensis var. thuringiensis No. 800/15, deposited at the GNU VNIISKhM (Departmental Collection of Useful Microorganisms of Agricultural Purpose - VKSM) under registration number 611 in the group of spore microorganisms, as a means for obtaining an entomocidal biological product.