RU2692655C2 - Bacillus thuringiensis var. darmstadiensis 56 strain as polyfunctional agent for plant growing - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention represents bacterial strain p. Bacillus thuringiensis var. darmstadiensis 56, deposited under number RCAM 04725, as an agent for protecting plants from insect pests and fungal diseases, as well as stimulating growth thereof. In addition, the novel strain BtH56 and biopreparations produced on its basis extend the range of such agents.EFFECT: disclosed strain is an effective polyfunctional agent for use in plant growing of agriculture.1 cl, 7 tbl

Description

The invention relates to biotechnology and agriculture and relates to a new strain of bacteria p. Bacillus as a means to protect plants from insect pests and fungal diseases, as well as to stimulate their growth.

The intensification of agriculture has necessitated the use of biological plant protection products against harmful insects, which destroy up to 30% of the crop [1].

The most dangerous insects include beetles, in particular, the Colorado potato beetle, which infects potatoes, eggplants, tomatoes, and pepper. Potatoes are a favorite food plant. Beetles adapt well to a variety of environmental conditions, as well as in relation to food. Once on the tomato (on an unfavorable food plant for them) the beetles very quickly get used to it. Such a fast getting used to the new food is explained by the possibility of adaptive rearrangements of the digestive system associated with the activity of hydrolytic enzymes. For the Colorado potato beetle in the European part of the range is characterized by a constantly high population density. With fecundity of females (usually around 500, but in some cases more than 4000), the presence of three and even two generations, the offspring of one female theoretically could reach 30 million individuals by the end of the season [2].

To combat the Colorado potato beetle, mainly chemical insecticides are used: Aktar, Fastpas, Regent, Konfidor, Tanret, etc. However, as these drugs are used, insects become resistant to them, as a result of which their effectiveness decreases. This causes either to increase the dose of the drug, or to carry out the rotation of the insecticide. In addition, chemical insecticides are environmentally unsafe, do not possess selectivity of action, can accumulate in soil, water, and are dangerous to humans and warm-blooded animals.

Biological insecticides are used as alternatives: Bowerin, Bitoxibacillin, Colorado, Novodor, but they do not cover a whole group of mass harmful species of coleoptera.

Among the economically significant plant diseases are diseases caused by microscopic fungi [3]. To combat the pathogens of plants of fungal or bacterial origin, chemical fungicides are also widely used: Alto, Raksil, Fundazol, Folikur, the effectiveness of which pesticidal action is no more than 50-60%. But these drugs are able to accumulate in the environment, causing a phytotoxic effect. In addition, after their application, a period of regulated waiting is necessary before people and equipment appear on the treated areas. As an alternative means of protecting plants against fungal diseases, biological preparations are used which are based on bacterial-derived metabolites exhibiting antagonistic activity against their various pathogens: Flavobacterin, Mizorin, Agat-25, Baktofit, etc. [4].

Known invention "Insecticidal drug" Colorado "against Coleoptera and the bacterial strain Bacillus thuringiensis, used to obtain insecticidal drug for patent No. 2081583, application 93018092/13 with priority from 04/07/1993, IPC A01N 63/00, C12N 1/20 published on 06/20/1997

The patent describes a strain of Bacillus thuringiensis var. tenebrionis, deposited in VKPM under No. V-6306, on the basis of which the Colorado liquid bioinsecticide was created, containing 5-endotoxin as an active principle, active against the Colorado potato beetle, oxal leaf beetle, and red-breast milker. The preparation contains glycerin, tridecane oil, technical carbon as a photoprotector, as a surfactant - Slan 60 and neonol, as well as a perfume. The titer of the strain VKPM No. 6306 when cultivated on a yeast polysaccharide medium at 30 ° C after 48 h is 3.4 billion spores / ml, however, spontaneous phage yield is sometimes observed. The drug is recommended to use against larvae 1-2 age. The disadvantage of the "Colorado" is a relatively low efficiency, leading to high consumption rates: 4-5 kg / ha.

Known invention "Strain of Bacillus thuringiensis - producing endotoxin against Coleoptera and method of its cultivation" in patent No. 2108384, application No. 95111008 with priority from 06.26.1995, IPC C12N 1/20, A01N 63/00, published 04/10/1998 g .

The patent describes a bacterial strain Bacillus thuringiensis 16931, which is isolated from a winter moth and deposited in VKPM under No. B-6649. It is shown that this strain is effective against Coleoptera, including against the Colorado potato beetle. When exposed to a culture liquid diluted 10 times on the larvae of the Colorado potato beetle 1-2 years old, their mortality is 100%.

Known invention of the Strain Bacillus thuringiensis var. thuringiensis No. 800/15 as a means for producing an entomocidal biological product "according to patent No. 2514211, application 2012143320 with priority of 10.10.2012, IPC C12N 1/20, A01N 63/00, published on April 27, 2014

Strain of Bacillus thuringiensis var. thuringiensis No. 800/15 (BtH ₁ 800/15) was isolated from the bodies of the larvae of the Colorado potato beetle and deposited with the VKSM GNU VNISHM under per. No. 611. The patent describes an example of obtaining a preparation of a liquid form, created on the basis of the strain BtH ₁ 800/15 (Bitoxin), which has an entomocidal activity against phytophagous insects.

The invention is known: “Bacillus thuringiensis bacterial strain, showing fungicidal activity, insecticidal activity and inhibiting virulent properties of Erwinia carotovora phytopathogenic bacteria” according to patent No. 2347809, application 2007132825 with priority from 08.31.2007, IPC C12N 1/20, A01N 63/00, published 27.02.2009

The bacterial strain Bacillus thuringiensis obtained by analytical selection of material isolated from natural samples and deposited under the number VKPM B-9790. The strain has insecticidal and fungicidal activity against the larvae of the Colorado potato beetle, a large mealworm, oxalic and Kalinov leaf beetles, as well as the ability to suppress the virulent properties of the phytopathogenic bacteria Erwinia carotovora spp.carotovora.

Known invention of the Strain Bacillus thuringiensis var. darmstadiensis No. 25 as a means of complex impact on harmful beetles and phytopathogenic fungi ”according to patent No. 2514023, application 2012156501, with priority from November 25, 2012, IPC A01N 63/00, C12N 1/20, published on April 27, 2014. (prototype).

The strain Bacillus thuringiensis H ₁₀ No. 25 (BtH ₁₀ No. 25) was isolated from the bodies of the Colorado potato beetle and deposited in the GNU VNIISM under per. No. RCAM 01490 The patent gives an example of obtaining a preparation based on this strain (Batsikolin), which has entomocytic properties against harmful Coleoptera and antifungal activity against phytopathogenic fungi.

The objective of the invention is to identify a new strain based on environmentally safe bacteria from the Bacillus thuringiensis group, suitable for use in agriculture and possessing multifunctional properties: entomocidal activity against various types of massive harmful coleoptera, antifungal activity against phytopathogenic fungi and growth-promoting effect on plants.

This problem is solved due to the fact that as a polyfunctional means for agriculture, which has entomocidal, antifungal activity and growth-promoting properties, use a new strain of Bacillus thuringiensis var. darmstadiensis 56.

Strain of Bacillus thuringiensis var. darmstadiensis 56 was isolated from the corpses of the Colorado potato beetle in the Leningrad region, followed by a multi-stage selection as a result of analyzing the physiological and economically valuable properties of the strain in a number of generations in stages:

- sowing culture on solid nutrient media (MPA, RA);

- study of the morphology of the colonies;

- microscopic analysis of the culture, taking into account the formation of crystals of endotoxin and spores (optimal ratio of 1: 1);

- assessment of exotoxinogenicity for BtH ₁₀ (test - object Musca domestica);

- assessment of entomocidal activity (test - the object of the 2nd instar larva of the Colorado potato beetle).

Strain of Bacillus thuringiensis var. darmstadiensis 56 (BtH ₁₀ 56) was deposited _on 12/01/2017 in the Departmental Collection of Useful Microorganisms for Agricultural Purposes (RCAM) FANO FGBNU VNIISM under registration number RCAM 04725, which is confirmed by the deposit certificate of 04/12/2017 (reference attached).

Morphological features and physiological and biochemical properties were studied standardly. Preliminary identification of bacteria was carried out according to the determinant Bergi. Strain BtH ₁₀ 56 characterized by the following properties.

Cultural and morphological properties.

Vegetative cells are gram-positive, rod-shaped, (1.2–1.5) × (2.5–4.5) μm in size, single with a peritrichous flagellation. Along with the spore, a parasporal crystal is formed of a tetragonal, bipyramidal or ovoid shape with a size of (0.2-0.6) × (0.3-0.8) μm along the length of the edge. Spores are oval, subterminal size (0.6-0.8) × (1.4-1.6) microns. One spore and one endotoxin crystal are formed in the cell. In fish broth (RB) growth is good, forms a film. When the strain is grown on fish agar (RA), after 48 hours at 28–30 ° C, flat, rough, grayish-white, irregularly rounded, non-transparent colonies form colonies. The diameter of the colonies of 8-10 mm. On slices of potato growth is moderate grayish-green. When cultivated in liquid nutrient media, the strain grows at a temperature of 20-36 ° C (optimum 28-32 ° C) and with the pH of the medium pH 6.2-9.0 (optimum 6.8-7.2) at a rotation speed of rocking chair 220 rpm The formation of spores and endotoxin crystals ends after 60-72 hours of growth.

Biochemical properties.

Optional anaerobic.

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

From glucose, maltose, cellobiose forms acid, but not gas. Does not assimilate galactose, arabinose, mannose, lactose, rhamnose, xylose, raffinose, mannitol, sorbitol, dulcite, inositol, inulin.

Hydrolyzes esculin and starch, salicin does not cleave, does not use sodium citrate as the sole carbon source. Forms amylase, proteinase, catalase and nitrate reductase. Does not form lecithinase, urease, lysine decarboxylase, phenylalanine disaminase, indole and hydrogen sulfide.

Pigment does not form.

The strain produces a water-soluble thermostable exotoxin.

Antigenic properties.

Gives a positive reaction to flagellate antigen with typical Bacillus thuringiensis var serum. darmstadiensis (identified by this reaction and according to the classification of Bacillus thuringiensis (1990) H.de Barjack. By serological properties refers to 10 serotypes of Bacillus thuringiensis.

The strain is resistant to the main production phages: M, K, E-5, D ₂ -D ₅ .

In terms of virulence, toxicity and toxigenicity, the strain Bacillus thuringiensis var. darmstadiensis 56 (BtH ₁₀ 56) is not pathogenic for warm-blooded animals, belongs to the 4th hazard class (low hazard) and meets the requirements for industrial microorganisms.

The strain is stored on agar nutrient media: meat-peptone (MPA), fish agar (RA) in test tubes under liquid paraffin, in sodium chloride crystals, by cryopreservation at a temperature of minus 80 ° C.

In the process of research it was found that for the cultivation of the BtH ₁₀ 56 strain various nitrogen sources can be used: soybean, barley, pea, oatmeal. This ensures productivity in the range of (2.7-4.4) ⋅10 ⁹ spores / ml of culture liquid, the optimal indicators were obtained using pea and soy flour (4.1-4.35) 10 ⁹ spores / ml.

The bacterial strain Bacillus thuringiensis var. darmstadiensis 56 (BtH ₁₀ 56) has pronounced entomocidal properties against Coleoptera phytophages, antifungal properties against phytopathogenic fungi and has a growth-promoting effect on plants.

At the same time, the BtH ₁₀ 56 strain has an entomocidal action against a wide range of phytophagous pests (Colorado potato beetle, potato cow, cruciferous flea beetles, leaf beetles, weevils) inhabiting various crops (vegetables, grains and berries). It shows efficacy against phytopathogens on a number of crops in various zones and with different growing technologies (including against gray rot on strawberries, fusarium wilting of tomatoes, flax, and rhizoctoniosis on potatoes. The strain stimulates the germination of seeds (for example, cabbage, tomato, cucumbers, squash, beet).

Below is an example of obtaining a biological product with polyfunctional properties based on the strain BtH ₁₀ 56, named by the authors Entobact.

First, in flasks (1 l) prepare yeast polysaccharide nutrient medium of the following composition, wt. %: Soy flour - 2.5; corn starch - 2.5; feed yeast - 0.5; K ₂ HPO ₄ - 0.3; MgSO ₄ × 7H ₂ O — 0.02; CaCl ₂ × 2H ₂ O — 0.1; the rest (up to 100) is water. The pH of the medium before sterilization is 7.4-7.8.

The prepared medium is poured into Erlenmeyer flasks of 40-50 ml and sterilized in an autoclave at a temperature of + (120-125) ° C for 40 minutes. The pH of the medium after sterilization is 6.8-7.2. Sterile nutrient medium is cooled to 30-35 ° C and seeded with strain culture from a test tube. For this, 5-8 ml of sterile water or saline are added to a culture test tube on oblique fish agar. With the same pipette, gently triturate the culture, shake until a homogeneous suspension and make 0.2-0.5 ml in flasks. The culture in flasks is grown on a rocking chair at a rotation speed of 220-250 rpm and a temperature of 28-30 ° C for 56-72 hours until the spore is completely rained and the formation of endotoxin crystals (as checked by microscopy). Grown in flasks, the culture fluid of the strain BtH ₁₀ 56 is used for seeding the environment in fermenters.

For growing the culture in the fermenter, any nutrient medium can be used, optimally - the same composition that was used to obtain the seed culture liquid in Erlenmeyer flasks (the composition is indicated above). In the fermenter of any capacity filled with 25% water, salts are first dissolved, then the calculated amount of corn starch is brewed at 80-85 ° C, cooled to 40 ° C, the calculated amount of soy flour is added, then water is added to 50% of the fermenter’s volume and stirred with water for 20 minutes. The pH of the medium is adjusted to 8.0 using 40% caustic soda (caustic soda) and sterilized at 1 atm for 1 hour, after which it is cooled to 32-35 ° C. Before introducing the inoculum from the fermenter, a sample of the sterility is taken. When viewed under a microscope in the field of view should not be the presence of microorganisms.

After that, 1-2% of culture liquid from Erlenmeyer flasks is added to the nutrient medium in the fermenter.

The process of growing the culture in the fermenter is carried out at a temperature of 28-30 ° C, continuous mixing of the medium using a stirrer, at a pressure in the fermenter of 0.2-0.4 atm and appropriate aeration:

up to 3 hours - only mixing;

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

from 12-42 hours - 1.0V air / V medium / min;

from 42 hours before the end of fermentation - 0.5V air / V medium / min.

That is, the fermentation process is carried out under conditions of high aeration and ends when the ratio of 1: 1 is reached, the number of free spores and endotoxin crystals with a total amount of 70-80% of the total number of inclusions. The titer of the dispute is determined by the conventional method of serial dilution. At the same time, the spore titer of the BtH ₁₀ 56 strain in the resulting preparation is not less than (2.8-4.2) ⋅10 ⁹ spores / ml in the absence of extraneous microflora and virulent phage.

Thus, the biological product Entobact was obtained on the basis of the strain of bacteria BtH ₁₀ 56, which is a liquid that is easily diluted with water to the necessary working concentration, convenient for applying serial spraying equipment. The technology of its production does not require large energy consumption required for drying upon receipt of preparations of the dry form.

Entomocidal properties of the strain Bacillus thuringiensis var. The following experiments confirm darmstadiensis 56 (BtH ₁₀ 56) against Coleoptera phytophagous insects.

Laboratory tests were carried out on the efficiency of the preparation based on the strain BtH ₁₀ 56 (Entobact) against mass insect-phytophagous agricultural crops: the larvae of the Colorado potato beetle and the 2nd-age potato cow (L ₂ ). The feed (potato leaves) was treated with an aqueous suspension of the BtH ₁₀ 56 strain (Entobact) in concentrations of 5.0, 1.0, and 0.2%. In each variant of the experiment, 25 larvae of the Colorado potato beetle or potato cow of the 2nd age were planted (fourfold). For comparison, in experiments with the same conditions at the same concentrations, a preparation based on the prototype BtH ₁₀ strain No. 25 (Batsikolin) and the preparation of the strain analog BtH ₁ No. 800/15 (Bitoxin) were used. Accounting for the death of the larvae of the Colorado potato beetle and potato cows was carried out on day 7.

The content of exotoxin in the preparation was determined on the larvae of the room fly by the following method.

The preparation based on the BtH ₁₀ 56 strain (Entobact) 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 the 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 feed. Each concentration was tested in four parallel determinations.

For testing, a 200 ml glass jar was used to add: 2 ml of the preparation based on strain BtH ₁₀ 56 (Entobact) of the appropriate dilution; 11 ml of 2.5% - aqueous suspension of powdered milk; 7 g of sterilized wheat bran. In the control experiment, 2 ml of sterile water was added to the banks instead of the diluted drug. Food with the appropriate dilutions of the preparation in each jar was thoroughly mixed and 25 larvae of the housefly were placed there, the openings of the cans were tied with a calico and kept in a thermostat at a temperature of (28 ± 1) ° C and a relative humidity of 70-85%). On the 5th day, puparia were chosen and left under the same conditions until the flies were flown.

For comparison, in experiments with the same conditions and at the same concentrations, a preparation based on the BtH ₁₀ prototype strain No. 25 (Batsikolin) and a preparation based on the analogue strain BtH ₁ No. 800/15 (Bitoxin) were used.

The activity of exotoxin was estimated by the number of flies killed on day 8, corrected for death in the control was determined by the formula:

X = (KV) / K × 100, where

K - the number of flies taken off in the control; In - the number of flies, took off in the experience.

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

The value of LC _{50 was} calculated using the Kerber formula (see, for example, [5]):

Lg LC ₅₀ = lg C _m -δ (ΣL ₁ -0,5),

where: C _m - the maximum of the tested concentrations;

σ is the logarithm of the ratio of each previous concentration to the next (logarithm of the multiplicity 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 values of LC ₅₀ for preparations based on: BtH ₁₀ 56 (Entobact) strain, as well as prototype BtH ₁₀ strain No. 25 (Batsikolin) and BtH ₁ analog strain No. 800/15 (Bitoxin), characterizing their biological (entomocytic) activity, presented in table 1.

From the results presented in table 1, it follows that the preparation based on the strain BtH ₁₀ 56 (Entobact) on entomocidal activity (LC ₅₀ ) with respect to the larvae of the potato cow is 1.5 and 1.6 times more active, respectively, of the preparations based on the strain of the prototype BtH ₁₀ No. 25 (Batsikolin) and strain analog BtH ₁ No. 800/15 (Bitoxin), and it exceeds them by 1.3 times in the content of exotoxin. At the same time, the values of entomocidal activity against the larvae of the Colorado potato beetle of all the tested preparations based on Bt strains are comparable.

Field small-scale experiments were also conducted on potatoes against the potato ladybug Henosepilachna vigintictomaculate Motch in Primorsky Krai, Ussuri region, in the podzolic soils zone. Potato crops inhabited by a potato cow with a ratio of larvae by age: L ₁ = 35.7%; L ₂ = 62.4%; L ₅ = l, 9%, was treated with a liquid preparation Entobact on the basis of strain BtH ₁₀ 56, obtained as described above in the Example method. For comparison, the Batsikolin preparations based on the prototype BtH ₁₀ strain No. 25 and Bitoxin based on the BtH ₁ analog strain No. 800/15 were used in the experiments under the same conditions. The average value of potato bug larvae on 25 bushes was taken into account before processing, as well as on the 5th and 10th days after processing. The treatment was performed with a hand sprayer, the dose of the drug was 15 l / ha, the flow rate of the working fluid was 400 l / ha. The results of field experiments are shown in table 2.

The results data presented in Table 2 indicate the high efficacy of the preparation based on the strain BtH ₁₀ 56 (Entobact) against potato cow in field experiments. Its effectiveness is 15 and 16% higher than the effectiveness of preparations based on the BtH ₁₀ prototype strain No. 25 (Batsikolin) and the preparation based on the BtH ₁ analog strain No. 800/15 (Bitoxin), respectively. At the same time, the yield increase during the treatment with Entobact based on the BtH ₁₀ 56 strain was higher by 21.7 and 26.7% than with the treatment with Batsikolin and Bitoxin, respectively.

Field experiments were conducted to investigate the entomocidal efficacy of Entobact preparation, obtained on the basis of the BtH ₁₀ 56 strain in the above-mentioned Example, in various cultures against various species of insects, phytophages.

The cultivation of plots located in different regions of the country (Leningrad region, Stavropol and Primorsky Krai) was carried out by spraying using a manual sprayer. The dose of the drug was in the range of 12-20 l / ha. The flow rate of the working fluid was 300-400 l / ha. At the same time, depending on the type of crop and the main type of insect phytophagans on it, the consumption of the drug was: on cabbage against white flowers, on raspberries against raspberry mites, on cereals against cereal grass and on flower crops in greenhouses (rose, carnation, chrysanthemum ) against thrips: 12, 15, 20 and 20 l / ha, respectively. Pest culture of the crops was taken into account before treatment, as well as on the 10th day after treatment, calculating the% of their deaths and thus determining the biological activity of the drug Entobact obtained on the basis of the BtH ₁₀ 56 strain. The results of the experiments are presented in Table 3.

The data of table 3 indicate a high entomocidal activity of the preparation based on the BtH ₁₀ 56 strain against various insect phytophagous species on various agricultural and flower cultures.

The antifungal properties of the bacterial strain Bacillus thuringiensis var. darmstadiensis 56 (BtH ₁₀ 56) in relation to various phytopathogenic fungi that excite plant diseases, in particular: Bipolaris sorokiniana (gelmintosporiosis or dark brown spot) - on cereals (wheat, barley, rye); Rhizoctonia solani Kuhn (Rhizoctoniosis) - on potatoes; Botrytis cinerea Pers (gray rot) - on the berries (strawberries, raspberries, currants, gooseberries); Pythium spp. (pith root rot) - on cereals, peas, beets.

Antifungal activity of the BtH ₁₀ 56 strain against various pathogenic fungi was determined by the method of agar blocks in Petri dishes [6]. A preparation based on the BtH ₁₀ 56 strain, obtained as described above in the Example, was added to the Chapek medium melted and cooled to 40 ° C. Blocks cut from a 10-day culture of the tested fungi were placed on the surface of frozen agar. The control was the medium without the addition of the drug. For comparison, in the experiments under the same conditions and at the same concentrations, preparations based on the prototype BtH ₁₀ strain No. 25 (Batsikolin) and the analog strain BtH ₁ No. 800/15 (Bitoxin) were used.

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

X = (D _to -D _about ) / D _to × 100, where

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

D _to and D _about - the diameter of the colonies of the fungus in the control and experience, respectively.

The results of the experiments are presented in table 4.

From the results presented in table 4 it follows that the antifungal effect of the drug Entobact on the basis of the BtH ₁₀ 56 strain is 14–18% higher than that of the Batsikolin preparation on the basis of the prototype BtH ₁₀ 25 strain, and several times higher than the effect of the Bitoxin preparation on the basis of strain BtH ₁ No. 800/15 for all represented species of phytopathogens. At the same time, a 100% effect of inhibiting the growth of the fungus arises under the action of the preparations Entobact and Batsikolin on the fungus Botrytis cinerea Pers - the causative agent of gray rot on the berries.

The growth-promoting property of the bacterial strain Bacillus thuringiensis var. darmstadiensis 56 (BtH ₁₀ 56).

The ability of the strain BtH ₁₀ 56 to exert a plant-promoting effect on plants was investigated in laboratory experiments. The seeds of agricultural plants: lettuce, cucumber, zucchini, pumpkin, before sowing were soaked for 3 hours in the preparation of Entobact based on the BtH ₁₀ 56 strain obtained as described above in the Example, and also for comparison, soaked in the prototype BtH strain ₁₀ No. 25, and in control - in water. After soaking, the seeds were germinated in rolls of filter paper at a temperature of 26 ° C and a humidity of 86%. After 5 days, seed germination was taken into account, and after 3 weeks they controlled the length of sprouts and roots.

The results of the experiments are presented in table 5.

The test results presented in Table 5 showed that Entobact, based on the BtH ₁₀ 56 strain, stimulates seed germination, growth of seedlings and plant roots, which confirms the growth-promoting properties of the BtH ₁₀ 56 strain. At the same time, the growth-promoting activity of the new BtH ₁₀ 56 strain is 5- 22% more than the prototype strain BtH ₁₀ No. 25.

Field experiments were also conducted to investigate the ability of the BtH ₁₀ 56 strain to have a growth-promoting effect on plants (tops) and potato yield.

The experiments were carried out by pre-sowing soaking of potato tubers for planting in Entobact preparation on the basis of BtH ₁₀ 56 strain, obtained by the method described above in Example, and also for comparison were soaked in Batsikolin preparation on the basis of BtH ₁₀ prototype strain No. 25, and in control water. After soaking, sowing tubers were planted in the ground and grown in the usual manner. 1.5 months after planting, the potato tops were cut, the number and height of the stems, the weight of the plants were measured, and the resulting potato harvest was determined.

The results of field experiments are shown in table 6.

The results of the experiments are shown in table 6, indicate a growth-stimulating effect of the drug Entobact on the basis of strain BtH ₁₀ 56 on the tops and yield of potatoes while soaking tubers in it. At the same time, the weight of the plants, the height of the stems and the yield during the presowing treatment of tubers with Entobact are higher by 19.4; 17; 16% of the relevant indicators when soaking the Batsikolin preparation based on the prototype BtH ₁₀ strain No. 25. The control variant with pre-sowing soaking of tubers in water is inferior to Entobact based on the BtH ₁₀ 56 strain, respectively, by 27, 23 and 22%.

Thus, the strain of bacteria Bacillus thuringiensis var. darmstadiensis 56 (BtH ₁₀ 56), besides pronounced enomocidal activity against a wide group of mass harmful insect species, has a high antifungal activity against various pathogenic fungi that cause crop diseases, as well as a growth-promoting effect on plants, that is, it has polyfunctional properties.

In addition, laboratory experiments were carried out to determine the preservation of the titer of the spores of the BtH ₁₀ 56 strain and the content of exotoxin in Entobact after 6 months of storage at various positive temperatures: 5, 15, 25, 30 ° C without preservative additives in the control and with the addition of 0.5% sodium citrate (Na ₃ C ₆ H ₅ O ₇ ) or 10% sodium chloride (NaCl) as a preservative. Baseline values: the spore titer of the BtH ₁₀ 56–3.5 910 ⁹ / ml strain, and the exotoxin content, which was estimated using the LC ₅₀ index for L ₂ house flies, was 3.21 μl / g of feed.

The results of the experiment are presented in table 7.

From the data in Table 7 it follows that during storage of the preparation on the basis of the BtH ₁₀ 56 strain for 6 months at temperatures of + (5-15) ° C, with or without preservative, its spore titer was 97.1-92 , 2%) of the original value, i.e. decreased slightly; and only at high storage temperatures + (25-30) ° C, it decreased by 12-17.2% compared with the original.

At the same time, the level of exotoxin, determined through the indicator LC50, remained high in all variants of the experiment - within 3.35-3.71 μl / g of feed compared to the initial one - 3.21 μl / g of feed.

The results of the described experiments show that the drug Entobact on the basis of the BtH ₁₀ 56 strain can be stored at different positive temperatures from 5 ° C to 30 ° C for at least 6 months, largely preserving its biological activity.

In terms of virulence, dissemination, toxicity and toxigenicity, the strain Bacillus thuringiensis var. darmstadiensis No. 56 (BtH ₁₀ 56) is not pathogenic for warm-blooded animals and in accordance with the “Guidelines ...” No. 2620-82 and No. 4263-87, the BtH ₁₀ 56 strain belongs to hazard class 4 (low hazard) and satisfies the requirements imposed on industrial microorganisms. The safety results of this biological product are consistent with the global practice of widespread use of bio-insecticides based on bacteria strains of the Bacillus thuringiensis group.

Thus, experimentally it was found that the newly identified strain of the bacteria Bacillus thuringiensis var. darmstadiensis 56 (BtH ₁₀ 56) possesses simultaneously high entomocidal activity against a wide group of mass harmful insect species - phytophages, high antifungal activity against various pathogenic fungi - pathogens of agricultural crops, and growth-promoting effect on plants, i.e. It is an effective polyfunctional tool for use in crop farming.

In addition, the new strain BtH ₁₀ 56 and the biologics obtained on its basis expand the arsenal of such means.

LITERATURE

1. Kandybin N.V., Patyka T.I., Yermolova V.P., Patyka V.F. Microbiocontrol of insect numbers and its dominant Bacillus thuringiensis. St. Petersburg, Pushkin, 2009, 245 p.

2. Bogdanov-Patkov N.N. Colorado potato beetle Septinatarsa desemlineata say and its quarantine value. L. Type. “Pec. The Yard, 1947, 200 p.

3. Muller E., Leffler V. Mycology. Translation from it. M., Mir, 1995, 114 p.

4. Smirnov OV, Grishechkina S.D. Studying the effect of biopreparations based on Bacillus thuringiensis on phytopathogenic fungi. Plant Protection Bulletin, №1, 2010, p. 27-35.

5. Patyka V.F., Patyka T.I., Ecology of Bacillus thuringiensis, Kiev, ed. PGAA, 2007, 217 p.

6. Methods of experimental mycology, edited by Bilai VI, Kiev, ed. Naukova Dumka, 1982, 552 p.

Claims (1)

Strain of Bacillus thuringiensis var. darmstadiensis 56, deposited in FANO FGBBU VNIISHM under the number RCAM 04725, as a polyfunctional means for crop production, with entomocytic, antifungal activity and growth-promoting properties.