RU2264103C2 - Method for preparing insecticide preparation and preparation based on microorganisms bacillus thuringiensis - Google Patents

Abstract

FIELD: microbiological industry, microbiology, insecticides.

SUBSTANCE: invention relates to methods for producing agents for plants protection and against harmful insects. Invention proposes a method for preparing insecticide preparation and preparation based on microorganisms Bacillus thuringiensis. Method involves culturing microorganisms Bacillus thuringiensis, isolation of delta-endotoxin from biomass by addition of a solvent, removal of precipitate and mixing solution with a filling agent and sticker followed drying the end product. Insecticide preparation obtained by proposed method comprises the following components, wt.-%: active component - dissolved delta-endotoxin from Bacillus thuringiensis, 10-20%; alkali (NaOH or KOH), 3-8%; reducing agent - sodium sulfite or sodium sulfide, 1-8%; sticker - a stabilizing agent of working suspension molasses or dry buttermilk, or pectin, or whey, or hydrolyzed chitin, or ground fish glue, or pectin from beet or apple pulp, 3-20%; filling agent, for example, NaCl - up to 100%. Method provides preparing highly effective and rapidly acting insecticide preparation.

EFFECT: improved preparing method, valuable properties of preparation.

2 cl, 5 tbl, 6 ex

Description

The invention relates to the microbiological industry, to the production of plant protection products from harmful insects.

Many pesticidal preparations based on bacteria of the genus Bacillus contain spores and bacteria as an active principle, the presence of which, when treated against pests, provides long-term action and protection (SU 1713145, SU 1792281, RU 2128915). However, the environmental safety and feasibility of using insecticides containing bacteria and spores of microorganisms is doubtful. According to modern environmental concepts, excessive contamination of spores with environmental bacteria is undesirable, since it can upset the natural balance of soil, land and water flora and fauna. In the case of Bacillus thuringiensis, it has been established and generally accepted that spores practically do not contribute to the occurrence of pathology in insects. In this regard, the presence of spores in the preparations can be considered not only redundant, but also undesirable (Kvyatkovskaya I.Ya., Slinko V.N., Kalinina L.N. Dep.VINITI, M., 1989, 15 pp.).

Known methods for producing entomopathogenic preparations containing as an active principle a toxin in its pure form, i.e. without bacteria and spores (SU 204831, SU 1792613). However, these methods do not allow to completely isolate the target product from biomass to obtain a drug of sufficient activity against pests. In the copyright certificate SU 204831, the entire spectrum of toxins is isolated without isolating the necessary fraction, that is, the resulting preparation does not have the necessary selectivity. In SU 1792613, a preparation is obtained which is purified from concomitant macromolecular substances based on non-protein β-exotoxin. However, numerous published data on its toxicity are known not only for insects, but also for vertebrates, including humans. It not only exhibits direct toxicity, but can also cause teratogenic, mutagenic, possibly carcinogenic effects.

The proposed method involves the removal of β-exotoxin and other soluble toxins, thereby ensuring high environmental purity of the drug and its safety for vertebrates. The method allows to isolate protein-specific δ-endotoxin (delta-endotoxin) from the biomass of Bacillus bacteria that is highly specific and safe for vertebrates and beneficial insects. As a result, high environmental safety is achieved with a very high biological effectiveness (the drug is 10-30 times more effective than spore-containing).

Closest to the proposed method and preparation are a method for producing an insecticidal (entomopathogenic) preparation and the preparation described in patent RU 2062577. The method involves cultivating Bacillus thuringiensis bacteria, isolating delta-endotoxin from biomass, dissolving crystals in alkali, removing sediment and mixing with filler. The method allows by adding to the active fraction of alkali biomass to dissolve the active principle of the target product, namely, delta-endotoxin crystals. The separation of the precipitate allows you to get almost pure solution of delta-endotoxin, which increases the activity of the target product and, as a result, reduces its consumption. The use of alcohol bard as an adhesive, which is a biologically and chemically inert substance, simultaneously as a filler and adhesive simplifies the preparation technology and increases the efficiency of production of an insecticidal preparation.

However, this method does not allow to completely dissolve the delta-endotoxin, which is only protoxin. The presence of a crystalline protein partially dissolved as a result of alkaline hydrolysis reduces the activity of the drug, increases the duration of its action, and limits the range of insects - objects of control.

The proposed improved method includes cultivating the bacteria Bacillus thuringiensis, isolating the delta-endotoxin from the biomass by introducing a solvent, removing the precipitate and mixing the solution with a filler and adhesive with further drying of the target product. The method differs from the known one in that before isolation of the endotoxin delta from biomass, the biomass is subjected to centrifugation or separation to remove soluble components of the medium and microbial metabolites (β, γ, α-exotoxins) that do not have selective toxicity. Then, an aqueous solution of a mixture of alkali (0.16% sodium or potassium hydroxide) and 0.04-0.17% sodium sulfite or sodium sulfide is added to the obtained precipitate containing crystals, spores and vegetative cells, and the selected hydrolyzed one is used as an adhesive chitin, or molasses, or dry buttermilk, or whey, or ground fish glue, or pectin from beet or apple pulp.

The object of the present invention is also an insecticidal preparation obtained by the proposed method.

The drug has the following composition (wt.%):

Active principle - dissolved delta endotoxin Bacillus thuringiensis 10-20% Alkali (NaOH or KOH) 3-8% Reducer sulfite or sodium sulfide 1-8% Adhesive - stabilizer working suspension molasses, or dry buttermilk, or whey, or hydrolyzed chitin, or ground fish glue, or beet pectin or apple pulp 3-20% Filler, e.g. NaCl up to 100%

It has been experimentally established that the proposed technological methods can significantly reduce the dissolution time. So, according to experimental data in the method, according to RU 2062577, the dissolution time is 6-8 hours, while in the new method in the presence of a reducing agent, the time is reduced to 2-3 hours. At the same time, longer periods are impractical, because the toxin basically goes into a soluble state in 2-3 hours, and prolonging the time worsens the technological characteristics of the process (its duration). When using the proposed method, the yield of the drug increased by 1.5-2 times. Table 1 shows comparative data on the amount of toxin obtained by known and proposed methods.

Table 1 Dissolution time, min The amount of toxin in the supernatant mg / g dry weight Alkali (prototype) Alkali + reducing agent (new method) thirty 60.3 ± 11.0 136.3 ± 9.6 60 86.4 ± 6.5 156.2 ± 10.5

The proposed method allows to reduce the dissolution time. For example, when dissolved in alkali within an hour, about 90 mg of toxin from 1 g of dry bacterial biomass passes into the solution, and in the case of alkali + reducing agent, about 160 mg / g. Thus, the process parameters (time) are improved, because dissolution took place within 0.5 hours.

In addition, the activity of the product increases, namely, the rate of onset of the final effect when applied, since the product does not require activation in the intestine of the insect. Table 2 shows comparative data on the rate of exposure of insecticides to unpaired silkworms of the 3rd age.

table 2 A drug* Efficiency,% by hours 3 6 12 24 famous 5 17 72 96 way the proposed method contains 3% sulfite 23 58 93 100 sodium the proposed method contains 3% sulfide 24 57 95 100 sodium * Preparations contain 10% of the active substance.

An important result of reducing the duration of action, in addition to increasing the% of dead insects, is the reduction of plant damage.

The technical result of the proposed group of inventions is that the proposed method compared to the prototype allows to reduce the dissolution time of delta-endotoxin 2 times or more and to obtain a drug with greater insecticidal activity at the same time of dissolution in the intestine of the insect. Moreover, the delta-endotoxin contained in the preparation corresponds in activity to the toxin, which has acquired toxicity in the intestine of the insect, i.e. it can immediately act on sensitive intestinal cells, destroying them. This shortens the onset of action and enhances the overall effect (insect mortality).

The invention also allows to increase the selectivity and effectiveness of preparations based on Bacillus thuringiensis, to increase the yield of the target product by more complete dissolution of the crystals of delta-endotoxin and to expand the arsenal of agents used to combat harmful insects.

An insecticidal preparation can be obtained by the proposed method from virtually any strain of the species Bacillus thuringiensis, since it is known that all subspecies of this type of microorganism produce delta-endotoxin.

The invention is implemented as follows.

Example 1. Deep cultivation of a strain of you.thurigiensis, a subspecies of dendrolimus 502 was carried out in working fermenters at 28-30 ° C. Then, the active fraction was isolated by biomass separation. The pasty active fraction obtained after separation contains elements of the culture medium, bacterial cells, spores, active principle delta-endotoxin crystals and other metabolic products. The specified active fraction (precipitate) was dissolved in an aqueous solution of sodium sulfite at a concentration of 0.05% in 0.16% sodium hydroxide solution, with a volume ratio of 1: 4 to the solvent. Then the precipitate was separated by separation or centrifugation and an alkaline reducing agent solution was obtained containing the active principle, delta-endotoxin, as the main component. Next, dry whey is added to the solution at the rate of 20% for the total weight of the finished product and NaCl. After thorough mixing, the solution was dried by freeze-drying or spray drying.

Received a preparation of the following composition: the content of the active principle is the dissolved delta-endotoxin of Bacillus thuringiensis - 10%, NaOH - 3%, sodium sulfite - 1%, dry whey 20%. NaCl - the rest. The activity of the drug EA / mg. 14,000.

Example 2. The method is implemented according to example 1. As a solvent, an aqueous solution of sodium sulfite in a concentration of 0.17% in a 0.16% solution of caustic potassium was used. Molasses was used as an adhesive and a filler.

Received a preparation of the following composition: the content of the active principle is the dissolved delta-endotoxin of Bacillus thuringiensis - 20%, KOH - 6%, sodium sulfite - 8%, molasses - 5%. NaCl - the rest. The activity of the drug EA / mg. 20,000.

Example 3. The method is implemented according to example 1. As a solvent, an aqueous solution of sodium sulfide at a concentration of 0.06% in 0.16% sodium hydroxide solution was used. As an adhesive and a filler, ground fish glue was used.

Received a preparation of the following composition: the content of the active principle is the dissolved delta-endotoxin of Bacillus thuringiensis -15%, NaOH - 5%, sodium sulfide - 3%, ground fish glue - 10%. NaCl rest. The activity of the drug EA / mg. 19000.

Example 4. The method is implemented according to example 1. As a solvent, an aqueous solution of sodium sulfide at a concentration of 0.04% in 0.16% potassium hydroxide solution was used. Hydrolyzed chitin was used as an adhesive and a filler.

Received a preparation of the following composition: the content of the active principle is the dissolved delta-endotoxin of Bacillus thuringiensis -15%, KOH - 6%, sodium sulfide - 5%, hydrolyzed chitin - 3%. NaCl - the rest. The activity of the drug EA / mg. 19000.

Example 5. The method is implemented according to example 1. An aqueous solution of sodium sulfide at a concentration of 0.08% in a 0.16% solution of caustic potassium was used as a solvent. Dry buttermilk was used as an adhesive and filler.

Received a preparation of the following composition: the content of the active principle is the dissolved delta-endotoxin of Bacillus thuringiensis - 10%, KOH - 6%, sodium sulfide - 3%, dry buttermilk - 20%. NaCl - the rest. The activity of the drug EA / mg. 14,000.

Example 6. The method is implemented according to example 1. As a solvent, an aqueous solution of sodium sulfite in a concentration of 0.17% in a 0.16% solution of caustic potassium was used. As an adhesive and a filler, pectin from beet or apple pulp was used.

Received a preparation of the following composition: the content of the active principle is the dissolved delta-endotoxin of Bacillus thuringiensis - 20%, KOH - 8%, sodium sulfite - 6%, pectin from beet or apple pulp - 3%. NaCl - the rest. The activity of the drug EA / mg. 20000

In laboratory conditions, the physical properties of the known (RU2062577) and insecticidal preparations obtained according to Examples 1-6 were compared. The experiments were carried out in five replicates. The wettability and stability of the working suspension of a new bacterial preparation and a known preparation were compared. Evaluation of the wettability and stability of the drug was carried out according to a well-known method (see Sternshs M.V. On methods for assessing the physical parameters of bacterial insecticides // agricultural biology, 1985, No. 9, pp. 120-123). The reliability of the results was evaluated by analysis of variance. The results are shown in table.3.

The data presented in table 3 show that the adhesive ability and stability of the working suspension of the bacterial preparation obtained by the proposed method is not inferior to those of the drug obtained in a known manner. This is due to the fact that biologically and chemically inert components that increase the adhesive ability of the drug are also used as filler and adhesive. In addition, the high physical properties of the drug are also due to the fact that the particles of the obtained target product are smaller than the particles of the known product, since the method allows to achieve a more complete dissolution of the crystals. Therefore, the proposed method allows to expand the possibility of using as stabilizers and adhesives in the production of insecticidal preparations of waste food and microbiological industries.

In laboratory conditions, the insecticidal activity of the bacterial insecticidal preparation obtained by the claimed method was determined, compared with the preparation obtained in a known manner, on cabbage white caterpillars. The experiments were carried out in five replicates. Caterpillars were infected orally and planted in cages for feed, 15 animals each. The reliability of the results was evaluated by analysis of variance. The results are presented in table 4. Obtained by the proposed method, the drug has a significantly greater insecticidal activity than the drug obtained in a known manner. The concentration of the proposed drug in the working suspension, providing 100% death of the tracks, is 2 times less than the concentration of the known drug. This allows you to significantly reduce the consumption rate of the drug and increase economic efficiency when using insecticides.

The analysis of the data in Table 4 also shows that it is not advisable to introduce more than 20% of the active principle into the preparation, since 100% caterpillar death is achieved at this concentration. An increase in concentration will also lead to a rise in the cost of the drug. When the concentration of the drug in the working suspension is less than 10%, its effectiveness is significantly reduced.

The insecticidal activity of the preparation obtained by the claimed method was compared with the insecticidal activity of the preparation obtained in a known manner, determining the concentration causing death on the third day of 50% of insects (caterpillars of the cabbage whitework participating in the experiment (LC50). The results are shown in Table 5.

As follows from the data given in table 5, the drugs obtained by the proposed method are more effective compared to the drug obtained in a known manner.

In the summer of 2001, the drug was tested to protect the forest from Lepidoptera leaf-eating pests (silkworm nun) against caterpillars of 1-2 age on a pine mixed with hardwood. The test area was 15 ha. Used the preparations obtained in examples 1-3. For treatments, the GARD-M-20 aerosol generator was used. The consumption rate of the preparations according to the examples was 0.01 kg / ha with a flow rate of 0.2 l / ha. The effect of the drug was compared with the action of bacterial lepidocide (consumption rate 1 kg / ha) and chemical Decis (0.05-0.06 kg / ha). The preparation obtained in Example 2 showed, when counted on the 5th day, the effectiveness was 92%, lepidocide-80% and Decis-92%. Tests confirmed the high insecticidal activity of the drug, comparable to the action of chemical insecticides. During the tests, data were also obtained on the safety of the drug for beneficial entomofauna (hymenopteran insects pollinators and entomophages). As a result of the treatment, none of the identified species was affected, while as a result of the action of the chemical preparation Decis, 10 species disappeared from 17. The economic assessment showed that the use of the proposed preparation by aerosol method was 4-7 times cheaper than lepidocide in the same way and 1400 times cheaper than by air.

It is known that insufficiently effective doses of drugs, as well as their duration of action, cause some affected insects to survive, which leads to the accumulation of mutations in insect populations. At the same time, the effectiveness from subsequent applications of such drugs decreases and contributes to an increase in adaptive abilities in insects. The present invention allows to obtain a highly effective fast-acting insecticidal preparation. And also to increase the selectivity and effectiveness of preparations based on Bacillus thuringiensis, increase the yield of the target product by completely dissolving the crystals of delta-endotoxin and expand the arsenal of products used to control harmful insects.

Table 3
Physical properties of drugs A drug Physical properties wettability, s adhesive ability,% working suspension stability,% prototype drug 60 66 89 the preparation of example 1 53 64 90 the preparation of example 2 60 70 92 the preparation of example 3 56 65 93 the preparation of example 4 fifty 67 89 the preparation of example 5 fifty 60 89 the preparation of example 6 53 65 90

Table 4
Insecticidal activity of a bacterial preparation obtained by the claimed method on caterpillars of cabbage white. A drug The required concentration of the drug in the working suspension,% The death of caterpillars per day depending on the concentration of the working suspension,% I-day III day drug by 0.02 48 85 prototype 0.05 76 100 drug by 0.01 fifty 84 example 1 0,025 78 100 drug by 0.01 74 90 example 2 0,025 97 100 drug by 0.01 70 88 example 3 0,025 89 100 drug by 0.01 69 87 example 4 0,025 87 100 drug by 0.01 68 93 example 5 0,025 95 100 drug by 0.01 88 100 example 6 0,025 100 100

Table 5
Data on determining the concentration that causes death on the third day of 50% of insects (larvae of cabbage white (LK50)). A drug LC50,% on day III X10 ^-3 prototype drug 0.4 the preparation of example 1 0.08 the preparation of example 2 0.02 the preparation of example 3 0.15 the preparation of example 4 0.14 the preparation of example 5 0.14 the preparation of example 6 0.14 the preparation of example 7 0.06 the preparation of example 8 0.14

Claims (2)

1. A method of obtaining an insecticidal preparation, comprising culturing the bacteria Bacillus thuringiensis, isolating delta endotoxin from biomass by introducing a solvent, removing sediment and mixing the solution with filler and adhesive, and then drying the target product, characterized in that before isolation of delta endotoxin from biomass an active fraction is isolated - a precipitate, which is dissolved in an aqueous solution of a reducing agent in alkali, while sodium sulfite or sodium sulfide in a concentration of 0.04-0.17%, and hydrolyzed chitin, or molasses, or dry buttermilk, or whey, or ground fish glue, or pectin from beet or apple pulp, are used as an adhesive. 2. An insecticidal preparation based on bacteria Bacillus thuringiensis, containing, as an active principle, dissolved delta-endotoxin, and in the composition of target additives a solvent, an adhesive stabilizer, a filler, characterized in that it is obtained according to the method according to claim 1 and has the following composition, wt .%: Active principle - dissolved delta endotoxin Bacillus thuringiensis 10-20% Alkali-NaOH or KOH 3-8% The reducing agent is sulfite or sodium sulfide 1-8% Adhesive stabilizer working suspensions - molasses, or dry buttermilk, or whey or hydrolyzed chitin, or ground fish glue, or pectin from beet or apple pulp 3-20% Filler Up to 100%