RU2514023C1 - Strain bacillus thuringiensis var darmstadiensis n25 as means of integrated effect on harmful coleopteran insects and phytopathogenic fungi - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain Bacillus thuringiensis var. darmstadiensis №25 (BtH₁₀ №25) has insecticidal activity against pests - coleopteran insects and a wide range of antifungal activity against pathogenic fungi. It is deposited in State Scientific Institution All-Russia Institute for Agricultural Microbiology under the registration number RCAM01490. It can be used in the manufacture of polyfunctional means of protection of plants against harmful coleopteran insects and phytopathogenic fungi.

EFFECT: invention also enables to improve the germinating capacity of plants.

7 tbl, 1 ex

Description

The invention relates to the microbiological industry and can be used to protect plants from pests and diseases.

The protection of agricultural plants of vegetables, grains and fruit crops from diseases and harmful insects is a serious economic problem. Annual agricultural losses reach several billion rubles (Kandybin N.V., Patyka T.I., Ermolova V.P., Patyka V.F. Microbiocontrol of the number of insects and its dominant Bacillus thuringiensis. St. Petersburg, Pushkin, 2009. , 245 p.).

Among the most dangerous pests are hard-winged insects, in particular the Colorado potato beetle, which infects potatoes, eggplant, and tomatoes. The population of bushes of nightshade crops in different years can reach 50-70%, and crop losses 25-35%. To control the Colorado potato beetle, chemical insecticides that do not have selectivity of action are often used. They can accumulate in the environment and thereby worsen the ecological situation. Based on the bacteria of the Thuringiensis group, a number of entomocidal biological products have been created: Lepidocide, Bitoxibacillin, Dendrobacillin, Colorado, but they do not cover a whole group of mass harmful species of hard-winged insects.

The invention is known "Bacterial strain of Bacillus thuringiensis spp. thuringiensis for the production of exotoxin-containing bioinsecticides ”according to patent No. 2061376, application 93034131 with priority dated 07.07.1993, IPC A01N 63/00, C12N 1/20, published on 10.06.1996.

The patent describes a bacterial strain Bacillus thuringiensis 16-T50, deposited in VKPM under No. B-6404, which was obtained from strain 98 by multi-stage selection for signs of exotoxin formation, thermal stability of spores and phage resistance. An example of obtaining a culture fluid based on the specified strain.

The invention is known "The strain of Bacillus thuringiensis - producer of endotoxin against beetles and their cultivation" according to patent No. 2108384, application 95111008 with priority of 06/27/1995, IPC C12N 1/20, A01N 63/00, published on 04/10/1998.

The patent describes a bacterial strain Bacillus thuringiensis 16931, which is isolated from a dead winter scoop and deposited in VKPM under No. B-6649; Also described is a method for its cultivation. It has been shown that this strain is effective against beetles, including against the Colorado potato beetle.

The invention is known "The bacterial strain Bacillus thuringiensis H8, designed to control hard-winged insects" according to patent No. 2204598, application 2001128487 with a priority of 10.23.2001, IPC C12N 1/20, A01N 63/00, published 05.20.2003.

The patent describes the bacterial strain Bacillus thuringiensis H8 deposited in VKPM No. B-8057, which is obtained from the Bacillus thuringiensis H8 strain VKPM No. B-6306 by multi-stage selection based on increased synthesis of toxin against harmful coleopteran insects, in particular Colorado potato beetle, and spore formation to prevent environmental pollution by spores when using a biopesticide based on this strain. An example of obtaining a culture fluid based on the strain VKPM B-8057.

To combat pathogens of plants of fungal or bacterial origin, chemical agents are widely used: Fundazol, Alto, Folikur, etc. Their effectiveness is not more than 50-60% and they can cause resistance in pathogens and can accumulate in the environment. Currently, the use of bacteria as antagonists of phytopathogens, i.e. biological preparations are used, the basis of which are metabolites of bacterial origin that exhibit antagonistic activity against phytopathogenic fungi: Flavobacterin, Mizorin, Agat-25, Bactofit, etc. (Smirnov O.V., Grishechkina S.D. Studying the effect of biological preparations based on Bacillus thuringiensis on phytopathogenic fungi. Plant Protection Bulletin, No. 1, 2010. P.27-35).

Also known is the invention “Consortium of antagonist strains for combating bacterial and fungal plant diseases” according to patent No. 2149552, application 98111359 with priority dated June 15, 1998, IPC A01N 63/00, C12N 1/20, С12Р 39/00, published 05/27/2000

A consortium consisting of four strains of soil antagonist bacteria: Bacillus polymixa, Bacillus thuringiensis, Pantoea agglomerans, Pantoea chlororatis, is intended to produce a drug against bacterial and fungal plant diseases. A preparation based on this consortium of bacterial strains can be used to effectively suppress the development of gray rot and powdery mildew in tomato and cucumber, bacterial cancer in clover, vascular bacteriosis in cabbage, angular spotting in cucumber, fusarium wilt in tomato, helminthosporosis in barley, and root rot in cotton. The pesticidal activity of the drug is comparable to the activity of chemical agents.

The invention is known "Bacillus thuringiensis bacterial strain exhibiting fungicidal activity, insecticidal activity and suppressing the virulent properties of phytopathogenic bacteria Ervina carotovorola" according to patent No. 2347809, application 2007132825 with priority dated 08/31/2007, IPC C12N 1/20, A01N 63/00, published February 2, 2009

The bacterial strain Bacillus thuringiensis was obtained by multistage selection from material isolated from natural samples, and deposited under the number VKPM B-9790. This strain has multiple pesticidal activity, namely, insecticidal and fungicidal activity against coleopteran insect larvae (Colorado potato beetle, large mealworm, oxalid and viburnum leaf beetles), as well as the ability to suppress the manifestation of virulent properties of phytopathogenic bacteria Ervina carotovorolapp. carotovorola.

A known bacterial strain Bacillus thuringiensis H10 No. 109 (BtH ₁₀ No. 109) is a producer of bacicola that combines insecticidal and antifungal properties (Kandybin N.V., Smirnov O.V., Barbashova N.M. New entomocidal preparation with a specific effect on beetles. // Materials of the All-Russian Scientific-Industrial Meeting, Krasnodar, August 21-24, 1994. Part 2. Pushchino, 1994. S.179-181) - prototype.

The results of a study of the effect of the bacterial strain Bacillus thuringiensis H10 No. 109 (BtH ₁₀ No. 109), the producer of bacicol on phytopathogenic fungi, are published in the articles:

- Grishechkina S.D., Smirnov O.V., Kandybin N.V. Fungistatic activity of various subspecies of Bacillus thuringiensis // Mycology and Phytopathology. Volume 36, issue 1, 2002, Mushrooms - pathogens;

- Grishechkina S.D., Smirnov O.V. Vegetative and field assessment of the antifungal effect of Bacillus thuringiensis // Bulletin of Plant Protection. All-Russian Institute for Plant Protection of the Russian Academy of Agricultural Sciences. St. Petersburg, Pushkin, 2010 No. 3. C 44-49.

The objective of the invention is to obtain a strain based on an environmentally friendly bacterium from the group of Bacillus thuringiensis with multifunctional properties: high insecticidal and antifungal activity against a wide group of massive harmful species of winged insects and growth-promoting effect on plants suitable for creating a highly effective entomocidal preparation.

The problem is solved in that a strain of Bacillus thuringiensis var. Is used as a means to obtain an entomocidal preparation of multifunctional action with insecticidal and antifungal activity and growth-promoting properties. darmstadiensis No. 25.

Strain Bacillus thuringiensis var. darmstadiensis No. 25 (BtH ₁₀ No. 25) was isolated from the corpses of the Colorado potato beetle in the Leningrad Region and deposited on September 12, 2012 at the Departmental Collection of Useful Microorganisms of Agricultural Purpose of the Russian Agricultural Academy (VKSM) GNU VNIISHM under registration number RCAM01490, which is confirmed by the certificate of deposit dated October 30, 2012 . (Help attached).

Strain BtH ₁₀ No. 25 has pronounced entomocidal properties against coleopteran insects - phytophages. The antifungal and growth-promoting properties of the strain were also revealed.

Strain BtH ₁₀ No. 25 is characterized by the following properties.

Cultural and morphological properties.

Vegetative cells are gram-positive, rod-shaped in size of 1.2-1.5 × 2.5-4.5 microns, single with peritrichous type of flagellation. Along with the spore, a paraspore crystal of a tetragonal, bipyramidal or ovoid form in size of 0.2-0.6 × 0.3-0.8 μm (edge length) is formed. The spores are oval, subterminal with a size of 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, the colonies form flat, rough, grayish-white colored irregularly rounded, opaque. The diameter of the colonies is 8-10 mm. On potato slices, growth is a moderate grayish-green color. When cultivated in liquid nutrient media, the strain grows at a temperature of 20-36 ° C (optimum 28-32 ° C) and with a pH of 6.2-8.0 (optimum 6.8-7.2) with shaking on a 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. It does not absorb galactose, arabinose, mannose, lactose, rhamnose, xylose, raffinose, attracts, sorbitol, dulcite, inositol, inulin.

Hydrolyzes esculin and starch, salicin does not break down, sodium citrate does not use as the sole carbon source. It forms amylase, proteinase, catalase and nitrate reductase. It does not form lecithinase, urease, lysine decarboxylase, phenylalanine deaminase, indole and hydrogen sulfide.

It does not form pigment.

The strain produces a water-soluble thermostable exotoxin.

Antigenic properties.

Gives a positive reaction of a flagellar antigen with a typical serum of Bacillus thuringiensis var. darmstadiensis (identified by this reaction and by the classification of Bacillus thuringiensis (1962, 1967, 1990) H. de Barjack. By serological properties, it belongs to the 10th serotype 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 of Bacillus thuringiensis var. darmstadiensis No. 25 (BtH ₁₀ No. 25) is not pathogenic for warm-blooded animals, belongs to hazard class 4 (low hazard) and meets the requirements for industrial microorganisms.

Strain BtH ₁₀ No. 25 grows on nutrient yeast polysaccharide media based on soy flour, corn starch, fodder yeast.

The strain is stored on slanted fish agar in test tubes at 4-5 ° C with periodic reseeding once every 6 months, in a lyophilized state in sealed ampoules and by cryopreservation in the Departmental Collection of Useful Agricultural Microorganisms of the Russian Agricultural Academy (RCAM).

Strain BtH ₁₀ No. 25 exhibits insecticidal activity against a number of dangerous crop pests - Coleoptera, such as Colorado potato beetle, cruciferous and bread fleas, leech, strawberry raspberry weevil, buckwheat weevil, rapeseed beetroot, leaf beetroot, leaf beetle, , horseradish, cabbage and elm) and others.

Strain BtH ₁₀ No. 25 is characterized by a wide spectrum of antifungal activity and inhibits the development of a number of phytopathogenic fungi: Botrytis cinerea Pers., Pythium spp., Bipolaris sorokiniana (Sacc.) Shoemaker, Verticillum dahliae Kleb., Rhizoctonia solani Kuhn, Alternaria alternata alternata Keissl, Sclerotinia sclerotiorum (Lib) de Bary, Fusarium avenaceum (Fr.) Sacc., F.oxysporum (Schlecht.), Snyd. et Hans., F. solani App. et Wr.

Based on the strain BtH ₁₀ No. 25, a technology for the production of a new entomocidal biological product has been developed.

An example of obtaining a drug based on strain BtH ₁₀ No. 25.

First, in flasks (1 l), yeast polysaccharide nutrient medium of the following composition is prepared,%: 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 - 0.01; the rest (up to 100) is water. Before sterilization, the pH of the medium 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 30 minutes. After sterilization, the pH of the medium is 6.8-7.2. The sterile culture medium is cooled to 30-35 ° C and inoculated with the culture of the strain from the test tube, for which 5-8 ml of sterile water or physiological saline is added to the test tube with the culture on the beveled RA. Using the same pipette, carefully grind the culture, shake it to a homogeneous suspension and add 0.2-0.5 ml to the flasks. The culture in the flasks is grown on a shaker at 220-250 rpm and a temperature of 28-30 ° C for 56-72 hours until the spores completely precipitate and endotoxin crystals form (verification is carried out by microscopy). The culture fluid grown in flasks of strain BtH ₁₀ No. 25 is used for inoculating the medium in fermenters.

To grow the culture in the fermenter, any yeast polysaccharide nutrient medium can be used, optimally the same composition that was used to obtain the culture fluid in Erlenmeyer flasks (the composition is indicated above). In a fermenter of any capacity filled with 50% water, salts were first dissolved, then the calculated amount of corn starch was brewed at 80-85 ° С, cooled to 40 ° С, the calculated amount of soy flour, fodder yeast was added and mixed with water for 20 minutes . The pH of the medium was adjusted to 8.0 with 40% sodium hydroxide (caustic soda). Then the nutrient medium was heated to 80-85 ° C, the temperature was maintained for 30 minutes and sterilized at 1 atm for 1 hour, after which it was cooled to a temperature of 32-35 ° C. Before introducing seed from the fermenter, a sample of the medium was taken to control sterility. When viewed under a microscope in the field of view should not be the presence of microorganisms.

After that, 1-2% of the culture fluid from Erlenmeyer flasks was introduced into the nutrient medium in the fermenter.

The process of growing the culture in the fermenter was carried out at 28-30 ° C, continuously mixing the medium with a stirrer, the pressure in the fermenter 0.2-0.4 atm and the corresponding aeration:

from 0-3 hours - only mixing;

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

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

from 42 hours to 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 free spores and endotoxin crystals reached 80-90% of the total number of inclusions at a ratio of 1: 1. The spore titer was determined by the conventional serial dilution method. Moreover, the spore titer of strain BtH ₁₀ No. 25 in the resulting preparation is not less than (3.8-4.0) · 10 ⁹ spores / ml in the absence of extraneous microflora and virulent phage.

Thus, a bioproduct was obtained on the basis of strain BtH ₁₀ No. 25, which is a liquid that is easily diluted with water to the required working concentration, convenient for using serial spraying equipment. The technology of its production does not require large energy costs necessary for drying upon receipt of dry form preparations.

Laboratory tests were conducted on the insecticidal effectiveness of the drug based on strain BtH ₁₀ No. 25 in relation to mass insects - phytophages of agricultural crops.

Laboratory determinations of the entomocidal activity of the drug based on strain BtH ₁₀ No. 25 were carried out on larvae of a Colorado potato beetle of the 2nd age. In this case, the feed (potato leaves) was treated with an aqueous suspension of the strain at concentrations of 5, 1 and 0.2%. In each experiment, 25 larvae of a Colorado potato beetle of the 2nd age (four times) were planted. For comparison, in the experiments under the same conditions, we used a preparation based on the prototype strain BtH ₁₀ No. 109 (Batsikol) and BtH ₁ 800 (Bitoxibacillin liquid, without drying), at the same concentrations. The death of the Colorado potato beetle larvae was recorded on day 7.

The exotoxin content in the preparation was determined on housefly larvae according to the following procedure.

A preparation based on strain BtH ₁₀ No. 25 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 feed. Each concentration was tested in four parallel determinations.

For testing, in a glass jar with a capacity of 200 ml were added: 2 ml of the drug based on BtH ₁₀ strain No. 25 of the appropriate dilution; 11 ml of a 2.5% aqueous suspension of milk powder; 7 g of sterilized wheat bran. In the control experiment, 2 ml of sterile water was added to the cans instead of the diluted preparation. The food with the appropriate dilutions of the preparation in each jar was thoroughly mixed and 25 housefly larvae were placed there, the jar openings were tied with calico and kept in a thermostat at a temperature of (28 ± 1) ° С and relative humidity of 70-85%. On the 5th day, puparia were selected and left under the same conditions until the flies left.

For comparison, in the experiments under the same conditions, we used a preparation based on the prototype strain BtH ₁₀ No. 109 (Batsikol) and BtH ₁ 800 (Bitoxibacillin liquid, without drying), at the same concentrations.

Exotoxin activity was evaluated by the number of flies killed on day 8, adjusted for death in the control, determined by the formula:

X = (K-B) / K × 100, where

K is the number of flies flying out in the control;

B is the number of flies flying out in the experiment.

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

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

Lg LC ₅₀ = log C _m -δ (ΣL ₁ -0.5),

where C _m is the maximum of the tested concentrations;

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 results of entomocidal activity and the formation of exotoxin (LC ₅₀ values) obtained in these experiments for preparations based on the BtH ₁₀ strain No. 25, the prototype strain BtH ₁₀ No. 109 (Batsikol) and the analogue of BtH ₁ 800 (Bitoxibacillin liquid, without drying), presented in table 1.

Table 1 The biological activity of strains based on BtH Strain-based drug Entomocidal activity: LC ₅₀ , for L ₂ Colorado potato beetle,% Exotoxin content: LC ₅₀ for L ₂ housefly, µl / g feed BtH ₁₀ No. 25 0.27 2.98 BtH ₁₀ No. 109 0.38 4.79 BtH ₁ No. 800 0.29 3.05

From the data given in table 1, it follows that the preparation based on the BtH ₁₀ 25 strain in entomocidal activity for Colorado potato beetle larvae is 1.4 times more effective than the preparation based on the BtH ₁₀ 109 prototype strain, and it exceeds it by 1 in terms of exotoxin content , 6 times. Comparison of the BtH ₁₀ 25 strain with the BtH ₁ 800 strain shows that they are close analogues in terms of entomocidal activity and exotoxin content.

Laboratory experiments were conducted to determine the insecticidal activity of strain BTN ₁₀ No. 25 against leaf beetles (Chrysomelidae): rapeseed, cabbage, horseradish, oriental mustard, elm.

First, by the method described above in the Example, a preparation was obtained on the basis of strain BTN ₁₀ No. 25, which was then diluted with distilled water to concentrations: 1.0; 0.2 and 0.04%. The leaves of rapeseed, horseradish, mustard, cabbage, elm were dipped into the preparation in Petri dishes in appropriate dilutions. After drying, leaf cuttings were wrapped with a wet swab, placed in penicillin vials of water and transferred to crystallizers. 25 leaves of larvae of the corresponding leaf beetles were planted on the leaves of each variant of the plant; The experiments were performed at room temperature, each version of the experiment in triplicate. For comparison, in the experiments under the same conditions, we used a preparation based on the prototype strain BtH ₁₀ No. 109 (Batsikol) at the same concentrations. The insecticity of the strains was evaluated by the amount (dose) of the drug, causing 50% death of the larvae (LC ₅₀ ) on the 5th day of the experiment. The results of the experiments are shown in table 2.

table 2 Insecticidal activity of BtH ₁₀ strains against leaf beetles Strain-based drug The dose of the drug, causing 50% death of the larvae, (LC ₅₀ ),% Oriental mustard leaf beetle Rapeseed leaf beetle Cabbage leaf beetle Horseradish leaf beetle Ilm leaf beetle BtH ₁₀ No. 25 0,062 0.056 0.05 0.06 0,07 BtH ₁₀ No. 109 0.086 0,077 0,064 0.08 0.09

The results presented in table 2 indicate a high entomocidal activity of strain BtH ₁₀ No. 25 against leafworm larvae in different cultures. The activity of the strain BtH ₁₀ No. 25 is 1.3-1.4 times higher than the activity of the strain of the prototype BtH ₁₀ No. 109.

The effectiveness of the drug based on strain BtH ₁₀ No. 25 was also tested in the field on potatoes against the Colorado potato beetle in the farms of the Leningrad Region: ZAO Predportovy, OPH Suida, as well as the Stavropol Territory (STAZR) on an area of 1 ha; 0.5 ha and 3 ha, respectively.

Potato crops populated by the Colorado potato beetle with the ratio of larvae by age: L ₁ = 58.6%; L ₂ = 36.5%; L ₃ = 4.9%, was treated with a liquid preparation obtained as described above in the Example by the method based on strain VTN ₁₀ No. 25. For comparison, in the experiments under the same conditions we used preparations based on the prototype strain BtH ₁₀ No. 109 (Batsikol) and the analogue strain BtH ₁ 800 (Bitoxibacillin liquid, without drying), at the same concentrations. The average value of the larvae on 25 bushes was taken into account before treatment and on the 5th and 10th days after treatment. The treatment was carried out with a tractor sprayer, the dose of the drug was 15-20 l / ha, the flow rate of the working fluid was 400 l / ha. The results of field experiments in the farms of the Leningrad region and the Stavropol Territory are shown in table 3.

Table 3 The effectiveness of drugs based on BtH strains on potatoes against the Colorado potato beetle in field experiments Strain-based drug Area, ha Dose of the drug, l / ha The death of larvae,%: after 5 days after 10 days ZAO Predportovy, Leningrad Region BtH ₁₀ No. 25 1,0 20,0 80.2 98.9 BtH ₁₀ No. 109 1,0 20,0 60,4 88.4 BtH ₁ No. 800 1,0 20,0 63.5 89.2 OPH "Suida" of the Leningrad Region BtH ₁₀ No. 25 0.5 15.0 75.9 99.1 BtH ₁₀ No. 109 0.5 15.0 68.5 86.5 BtH ₁ No. 800 0.5 15.0 64.7 87.3 Stavropol Territory (STAZR) BtH ₁₀ No. 25 3.0 15.0 83.5 97.9 BtH ₁₀ No. 109 3.0 15.0 70.1 86.3 BtH ₁ No. 800 3.0 15.0 72.3 88.0 Control without processing 1.52-fold increase in numbers

The data in table 3 indicate the high efficiency of the drug based on the strain BtH ₁₀ No. 25 against the Colorado potato beetle, exceeding the effectiveness of drugs based on analog strains.

Were conducted as the vegetative-field test drugs based on strain ₁₀ BtH №25, obtained by the above method, the prototype strain ₁₀ BtH №109 and analog strain BtH ₁ №800 on strawberries before flowering against strawberry Malin weevil on the plot area of 100 m ² . Used a manual sprayer, the flow rate of the drug is 10 l / ha, the flow rate of the working fluid is 400 l / ha. Damage to strawberry plants by weevil was taken into account before treatment and on the 10th, 20th day after treatment with the drug.

The results of the experiment are shown in table 4.

Table 4 The effectiveness of drugs based on BtH strains against strawberry raspberry weevil Strain-based drug The defeat of strawberries,% Before processing 10th day 20th day BtH ₁₀ No. 25 6.1 6.8 9,4 BtH ₁₀ No. 109 5.9 7.9 14,4 BtH ₁ No. 800 5.8 14.7 23.5 Control (no processing) 5.3 18.9 28,2

The results presented in table 4 indicate a high activity of the drug based on strain BtH ₁₀ No. 25 against strawberry raspberry weevil. The defeat of strawberries by a weevil during its treatment with a preparation based on a strain of BtH ₁₀ No. 25 is 1.5 times lower than when treated with a preparation based on a prototype strain BtH ₁₀ No. 109, 2.5 times lower than when processing a preparation based on a BtH strain ₁ No. 800, and 3 times lower in comparison with the control.

To evaluate the insecticidal activity of the preparation obtained on the basis of the BtH strain ₁₀ No. 25 as described above in the Example, field experiments were carried out against various species of beetles in crops. Tests against cruciferous fleas, cabbage leaf beetle and cabbage scoops were performed in 20 m ² plots with rutabaga, rapeseed, cabbage, horseradish and horseradish against horseradish leaf beetle. For processing, a manual sprayer was used, the consumption of the preparation was 8-12 l / ha, the flow rate of the working fluid was 400 l / ha. The effectiveness of the strain was evaluated by% insect death on the 5th and 10th days after treatment of plants with the drug. The results of the experiment are shown in table 5.

Table 5 The effectiveness of the drug based on strain BtH ₁₀ No. 25 against various types of insects on crops Insects Culture Dose of drug consumption, l / ha The death of insects,% on the 5th day on the 10th day Cruciferous fleas (genus Phyllotreata) Swede 12 73,4 95.7 rape 12 71.6 94.5 Cabbage Scoop (Mamestra brassicae L.) Cabbage 10 89.3 one hundred Cabbage leaf beetle (Phaedon cochleariae F.) Cabbage 8 82.5 98.9 horseradish 10 79.9 99.6

The data in table 5 indicate a high insecticidal activity of the drug based on strain BtH ₁₀ No. 25 against various species of insects in various crops.

The antifungal activity of strain BtH ₁₀ No. 25 against various phytopathogenic fungi was determined by the method of agar blocks in Petri dishes (Methods of experimental mycology / edited by V.I. Bilai. Kiev, 1982.). The preparation based on the BtH strain ₁₀ No. 25, obtained by the method indicated above in the Example, was introduced into the Chapek medium melted and cooled to 40 ° C. Blocks cut from a 10-day culture of test mushrooms were placed on the surface of the frozen agar. The control was medium without the addition of the drug. For comparison, in the experiments under the same conditions we used preparations based on the prototype strain BtH ₁₀ No. 109 (Batsikol) and BtH ₁ 800 (Bitoxibacillin liquid, without drying) at the same concentrations.

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

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

X - the degree of inhibition of the growth of fungal colonies, cm

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

The results of the experiment are presented in table 6.

Table 6 Antifungal activity of drugs based on BtH strains Type of phytopathogenic fungus Inhibition of growth of fungal colonies after 3 days.,% BtH ₁₀ No. 25 BtH ₁₀ No. 109 BtH ₁ No. 800 Botrytis cinerea Pers one hundred 83 42 Pythium spp. 86 70 23 Bipolaris sorokiniana 75 61 eleven Verticillum dahliae Kleb. 52 49 0 Rhizoctonia solani kuhn 43 28 9 Alternaria alternata (Fries), Keissl 36 31 0 Fusarium avenaceum (Fr.) (Sacc) 51 44 0 Fusarium oxysporum (Schlecht.) Snyd. et hans. 53 49 0 F.solani App. et Wr. 26 19 0

From the results presented in table 6, it follows that the antifungal effect of the drug based on the strain BtH ₁₀ No. 25 is 14-17% higher than the drug based on the prototype strain BtH ₁₀ No. 109, and several times exceeds the effect of the drug based on strain of the 1st serotype BtH ₁ No. 800 for all types of phytopathogens presented. At the same time, a 100% effect of inhibiting the growth of the fungus occurs when the drug based on the BtH strain ₁₀ No. 25 on the fungus Botrytis cinerea - the causative agent of gray rot.

In the growing experiments on tomatoes (cultivar "Gribovsky") against the infectious background, the antifungal effect of strain BtH ₁₀ No. 25 against the fungus Fusarium oxysporum was 70%, while in the control plant damage reached 100%.

Assessment of phytoregulatory activity of strain BtH ₁₀ No. 25, i.e. its ability to exert a growth-promoting effect on plants was carried out in laboratory experiments. The seeds of agricultural plants: tomato, cabbage, lettuce, cucumber, zucchini were soaked before sowing for 3 hours in a preparation based on the BtH ₁₀ strain No. 25 obtained by the method described above in the Example, for comparison, in a preparation based on the BtH ₁₀ strain prototype strain No. 109 , 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, after 3 weeks the length of sprouts and roots was controlled. The results of the experiment are presented in table 7.

Table 7 Phytoregulatory activity of preparations based on BtH ₁₀ strains during presowing soaking of seeds of agricultural crops The cult
ra % to control Seed germination Height of seedlings Root length BtH ₁₀ No. 25 BtH ₁₀ No. 109 BtH ₁₀ No. 25 BtH ₁₀ No. 109 BtH ₁₀ No. 25 BtH ₁₀ No. 109 Tomato 104 94 156 143 112 110 Cabbage 98 96 149 140 158 150 Salad 110 107 123 113 136 128 Zucchini 135 121 159 140 162 144 Beet 108 98 103 98 114 102 Cucumbers 115 110 104 101 121 106

The test results of the drug based on the strain BtH ₁₀ No. 25, presented in table 7, showed that this strain does not have phytotoxicity. Moreover, it stimulates the germination of seeds, the growth of seedlings and plant roots, i.e. has a growth-promoting property. Moreover, the phytoregulatory (growth-promoting) activity of the inventive strain BtH ₁₀ No. 25 is 3-19% more than that of the prototype strain BtH ₁₀ No. 109.

Thus, the inventive strain of bacteria Bacillus thuringiensis var. darmstadiensis No. 25 (BtH ₁₀ No. 25) has multifunctional properties: high insecticidal and antifungal activity against a wide group of mass harmful species of hard-winged insects and growth-promoting effect on plants and is suitable for the production of highly effective entomocidal biological product for protecting crops from harmful mass insects and phytophage insects phytopathogenic fungi.

Claims (1)

The bacterial strain Bacillus thuringiensis var. Darmstadiensis No. 25, deposited in the collection of the GNU VNIISCHM of the Russian Agricultural Academy (Departmental collection of useful microorganisms for agricultural purposes) under registration number RCAM01490, as a multifunctional plant protection product from harmful coleopteran insects and phytopathogenic fungi.