RU2192744C2 - Method of potentiation of pesticide activity of pesticide obtained on basis of microorganisms of genus bacillus - Google Patents

Abstract

FIELD: biotechnology, microbiology, pesticides. SUBSTANCE: invention relates to microbiological method of preparing potentiating agent of pesticide activity of pesticide. Method involves preparing substance of the structure (I) or its salt given in description by culturing microorganisms of genus Bacillus (Bacillus thuringiensis) and used in combination with pesticide obtained on the basis of microorganisms of genus Bacillus. Using the above indicated potentiating agent method ensures to enhance activity of crystalline delta-endotoxins of protein nature isolated from Bacillus thuringiensis by many times. EFFECT: improved method of potentiation. 11 cl, 4 dwg, 7 tbl

Description

 This application is a partial continuation of US application 08/295283, filed 08/23/1994, which in turn is a partial continuation of US application 08/146852, filed 03/11/1993, which is a partial continuation of US application 08/095240, filed July 20, 1993, which is a partial continuation of the application US 07/990202, filed 12/14/92, which is a partial continuation of the application US 07/971786, filed 05. 11. 1992.

FIELD OF THE INVENTION
The invention relates to a method for producing and identifying a potentiator that enhances the activity of a pesticide related to bacteria of the genus Bacillus, a pesticide obtained by chemical means and / or a virus having pesticidal properties.

State of the art
Millions of dollars are spent every year due to pests that damage agriculture, forests and human health. Several approaches are used to control the spread of pests.

 One of them is the use of chemical pesticidal agents with a wide range or spectrum of action. However, the use of chemical pesticides has several disadvantages. In particular, due to the wide spectrum of action, such pesticides can adversely affect other inappropriate organisms, such as beneficial insects and parasites of insect pests. In addition, chemical pesticides are often toxic to animals and humans. Further, pests to be destroyed often develop resistance to pesticides when used repeatedly.

 Another way is to use biopesticides to control insects, fungi and weeds. Biopesticides are natural pathogens and / or substances produced by these pathogens. The advantage of using biopesticides is that they are generally less hazardous to other organisms and the environment than chemical pesticides.

Bacillus thuringiensis
The most widely used biopesticide is Bacillus thuringiensis. Bacillus thuringiensis are motile, rod-shaped, gram-positive bacteria that are widespread in nature, especially in soil and other objects infected with insects. During sporulation, Bacillus thuringiensis forms parasporal crystalline inclusions that have insecticidal activity when swallowed by the larvae of sensitive insects of the orders Lepidoptera, Diptera, and Coleoptera. Inclusions can vary in size, quantity and composition. They include one or more proteins called delta endotoxins, which may have a mole. mass from 27 to 140 kDa. Insecticidal delta endotoxins break down under the action of proteases in the intestinal tract of the larva with the formation of smaller truncated toxic polypeptides that cause intestinal damage and ultimately insect death (Hofte and Whiteley, 1989, Microbiological Reviews 53: 242-255).

 Various strains of Bacillus thuringiensis are known to be used as biopesticides in forestry, agriculture and landscaping. Bacillus thuringiensis subsp. kurstaki and bacillus thuringiensis subsp. aizawai produce delta endotoxins specific for Lepidoptera. Coleoptera specific delta-endotoxin is produced by Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Patent 4,766,203). Further, Bacillus thuringiensis subsp. israelensis produces delta endotoxins specific for Diptera (Goldberg, 1979, US Patent 4,166,112).

 Other strains of Bacillus thuringiensis specific to pests of the Diptera order are also described. The Bacillus thuringiensis isolate has been found to be toxic to Diptera and Lepidoptera (Hodgman et al., 1993, FEMS Microbiology Letters, 114: 17-22). Using SDS-polyacrylamide gel electrophoresis of purified crystalline delta-endotoxin from the indicated isolate, three types of proteins were found corresponding to cryIA (b) -, cryIB- and cryIIA-toxins. Also described is an isolate of Bacillus thuringiensis, producing crystalline proteins with mol. mass 140, 122, 76, 72 and 38 kDa, active against insects of the order Diptera (Payne, 1994, US, patent 5275815). EP 480762 describes five B.t. strains, each of which is active against pests belonging to the Diptera order, and contains a unique crystalline endoxin.

 Several strains of Bacillus thuringiensis with pesticidal activity against pests not belonging to the orders Lepidoptera Coleoptera and Diptera are described. Five strains of Bacillus thuringiensis producing delta endotoxins toxic to nematodes have been characterized (Edwards, Payne, and Soares, 1988, EP Patent 0303426 B1). Also described is a strain of Bacillus thuringiensis PS81F, which can be used to treat humans and animals infected with parasitic protozoa (Thompson and Gaertner, 1991, EP, application 0461799 A2). In addition, certain Bacillus thuringiensis isolates that have tick activity have been identified. These isolates produce crystalline proteins with a molecular weight in a wide range, namely from 35 kDa to 155 kDa (Payne, Cannon and Bagley, 1992, PCT application WO 92/19106). Also described are strains of Bacillus thuringiensis active against pests of the Hymenoptera order (Payne, Kennedy, Randall, Meier and Uick, 1992, EP, application 0516306 A2); Hemiptera pests (Payne and Cannon, 1993, US Patent 5,262,159); trematode (Hickle, Sick, Schwab, Narva and Payne, 1993, US Patent 5262399); and pests of the order Phthiraptera (Payne and Hickle, 1993, US Patent 5,273,746). Further, it was found that another strain of Bacillus thuringiensis subsp. kurstaki WB3S-16, isolated from scraps of Australian sheep's wool, is toxic to Damalinia ovis lice, a pest of the Phthiraptera order (Drummond. Miller and Pinnock, 1992, J. Invert. Path. 60: 102-103).

 Delta endotoxins are encoded by cry genes (from the English. Crystal protein - crystalline protein), which are mainly localized in plasmids. cry-genes are divided into six classes and several subclasses depending on the relative homology of amino acids and pesticidal specificity. The main classes are: Lepidoptera-specific (cry I); Lepidoptera- and Diptera-specific (cry II); Coleoptera-specific (cry III); Diptera-specific (cry IV) (Hofte and Whiteley, 1989, Microbiological Reviews 53: 242-255); Coleoptera- and Lepidoptera-specific (assigned to the cry V genes by Taylor et al., 1992, Molecular Microbiology 6: 1211-1217); and nematode-specific (assigned to the cry V and cry VI genes by Feitelson et al., 1992, Bio / Technology 10: 271-275).

 Delta endotoxins are produced by recombinant DNA methods. Delta endotoxins obtained by recombinant DNA methods can be in crystalline form or not.

 Some strains of Bacillus thuringiensis have been shown to produce a heat-resistant, pesticidal, adenine-nucleotide analog known as type I exotoxin, or “turingienzine,” which itself is a pesticide (Sebesta et al., In N. D. Burges ( ed.), Microbial Control of Pests and Plant Diseases, Academic Press, New York, 1980, pp. 249-281). β-Exotoxin type I was found in the suspension of some cultures of Bacillus thuringiensis. It has a molecular weight of 701 and is composed of adenosine, glucose and allaric acid (Farkas et al., 1977, Coll. Czechosslovak Chem. Comm. 42: 909-929; Lutny et al., In Kurstar (ed.), Microbial and Viral Pesticides, Marcel Dekker, New York, 1982, pp. 35-72). The circle of its owners includes, but is not limited to: Musca domestica, Mamestra configurata Walker, Tetranychus urticae, Drosophila melanogaster and Tetranychus cinnabarinus. Type I β-exotoxin toxicity is believed to be due to inhibition of DNA-dependent RNA polymerase in competition with ATP. It has been shown that type I β-exotoxin is encoded by a cry plasmid in five strains of Vasillus thuringiensis (Levinson et al., 1990, J. Bacteriol. 172: 3172-3179). It was found that type I β-exotoxin is produced by Bacillus thuringiensis subsp. thuringiensis serotype 1, Bacillus thuringiensis subsp. tolworthi serotype 9, and Bacillus thuringiensis subsp. darmstadinsis serotype 10.

 Another β-exotoxin classified as type II β-exotoxin is described (Levinson et al., 1990, J. Bacteriol. 172: 3172-3179). It was found that type II β-exotoxin is produced by Bacillus thuringiensis subsp. morrisoni is serotype 8 ab and is active against Leptinotarsa decemlineata. The structure of β-exotoxin type II is not completely known, but it differs significantly from the structure of β-exotoxin type I, in which the pseudouridine group occupies the position of adenine at the point of attachment to the ribose ring, which would otherwise be occupied by hydrogen. (Levinson, in Hickle and Finch (eds.), Analytical Chemistry of Bacillus thuringiensis, ACS Sumposium Series, Washington, D. C. 1990, pp. 114-136). Further, in the proton NMR spectrum, only one signal was obtained corresponding to the nucleotide base (at 7.95 ppm), and no signal of the anomeric protein of the ribose type (5.78 ppm) was received.

 Other water-soluble substances that have been isolated from Bacillus thuringiensis include alpha exotoxin, which is toxic to Musca domestica larvae (Luthy, 1980, FEMS Microbiol. Lett. 8: 1-7); gamma-exotoxins, which are various enzymes, including lecithinases, chitinases and proteases, the toxic effects of which are manifested only in combination with beta-exotoxin (Forsberg et al., 1976, Bacillus thuringiensis: Its Effects on Environmental Quality, National Research Council of Canada, NRC Associate Committee on Scientific Criteria for Environmental Quality, Subcommittees on Pesticides and Related Compounds and Biological Phenomena); sigma-exotoxin, which has a structure similar to that of beta-exotoxin, and is also active against Leptinotarsa decemlineata (Arganeer et al., 1991, J. Entomol. Sci 26: 206-213); and anhydroturinngienzine (Prystas et al., 1975, Coll. Czechosslovak Chem. Comm. 40: 1775).

Сущность изобретения
Усилия специалистов направлены на повышение активности пестицидов на основе В.t. Для этого ведется поиск новых штаммов с повышенным уничтожающим воздействием, предпринимаются попытки искусственного создания таких штаммов и разработки более эффективных препаратов путем комбинирования спор и кристаллов В. t. с новыми пестицидными носителями, химическими пестицидами или потенциаторами (усилителями) их активности (см., например, US, патент 5250515, касающийся ингибитора трипсина). Таким образом, предметом настоящего изобретения является усиление пестицидной активности соответствующих средств.Zwittermicin
A substance was isolated from Bacillus cereus that inhibits the growth of the plant pest Phytophthora medicaginis and reduces alfalfa infection (see, for example, US Pat. Nos. 4,877,738 and 4,878,936). No other activity was detected. The following formula characterizes the structure of zwittermicin A (He et al., Tet. Lett. 35: 2499-2502):

SUMMARY OF THE INVENTION
The efforts of specialists are aimed at increasing the activity of pesticides based on B.t. To do this, a search is underway for new strains with an increased destructive effect, attempts are made to artificially create such strains and develop more effective drugs by combining spores and B. t crystals. with new pesticidal carriers, chemical pesticides or potentiators (enhancers) of their activity (see, for example, US Pat. No. 5,250,515 regarding trypsin inhibitor). Thus, the subject of the present invention is to enhance the pesticidal activity of the respective agents.

The invention relates to a potentiation method that enhances the activity of a pesticide obtained on the basis of bacteria of the genus Bacillus, comprising the following stages:
a) culturing a strain of Bacillus under suitable conditions;
b) the selection of the specified potentiator from the supernatant of the culture obtained in stage a).

 The Bacillus strain, in particular, is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis and Bacillus thuringiensis. Preferably, the potentiator is prepared in substantially purified form. As used herein, the term “substantially purified potentiator” means that the potentiator contains less than 10% impurities, for example, protein delta endotoxin. Such a substantially purified potentiator can be obtained by isolation, for example column chromatography.

As used herein, the term "potentiator" means a substance (factor) that, without itself having significant pesticidal activity, for example, having an LC _{50 of} more than about 3000 μg / g, as determined by biological analysis (Section "Information confirming the possibility of carrying out the invention ") (LC ₅₀ is the concentration of the substance necessary to kill 50% of the pests), increases the pesticidal activity of the pesticide obtained on the basis of bacteria of the genus Bacillus by at least 50% and does not delay the development of larvae to. Other substances are known that can enhance the activity of pesticides, such as trypsin inhibitors and exotoxins with pesticidal activity.

 In a preferred embodiment, the target potentiator is water soluble. As defined herein, a substance or compound is "water soluble" if at least 1 mg of the substance can be dissolved in 1 mg of water. A potentiator is capable of enhancing the pesticidal activity of chemical pesticides and / or a virus with pesticidal properties.

 As used herein, the term “pesticide derived from bacteria of the genus Bacillus” means a strain of Bacillus (eg, Bacillus thuringiensis or Bacillus subtilis), spores or a substance, for example a protein or fragment thereof, which has anti-pest activity or is capable of killing pests, or a microorganism capable of Express a Bacillus gene encoding a Bacillus protein or fragment thereof that has anti-pest activity or is capable of killing pests (e.g., Bacillus thuringiensis delta-endotoxin) in combination with a suitable carrier. The pest can be an insect, nematode, tick or snail. Microorganisms capable of expressing the Bacillus gene encoding the Bacillus protein or its fragment, possessing activity against pests or capable of killing pests, live on the surface of plant leaves and / or in the rhizosphere (soil at the roots of plants) and / or in the aquatic environment and are able to successfully compete in a certain environment (crops or other insect habitats) with wild-type microorganisms and stably express the Bacillus gene encoding the Bacillus protein or its fragment, which have the activity of into pests or capable of destroying them. Examples of such microorganisms include, but are not limited to, bacteria, for example, the genera of Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacter, Lactobacter, Lactobacter, Lactobacter, Lactum ; algae, for example, of the families Cyanophyceal, Prochlorophyceae, Rhodophyceae, Dinophyceae, Chrysophyceal, Prymnesiophyceae, Xanthophyceal, Raphidophyceal, Bacillariophycee, Eustigmatophycee, Gryptophyceaeoma, Sp. , Rhodotorula and Awreobasidium.

 As used herein, the term “pesticidal activity” corresponds to a pest activity that is sufficient to kill or stop the growth of pests or to protect plants from being infected by pests.

 The potentiator obtained according to the claimed invention can be included in a composition containing a potentiator and a pesticidal carrier, as well as a potentiator and pesticide derived from bacteria of the genus Bacillus, a chemical pesticide and / or virus with pesticidal properties. Such compositions can be used to control pests and reduce the resistance of pests to a Bacillus-based pesticide, including contacting the pests with said composition containing a potentiator and a carrier, or enhancing the activity of a Bacillus-based pesticide.

The invention also relates to a method for identifying said potentiator, comprising the following steps:
a) culturing a strain of Bacillus;
b) the selection of the supernatant of the culture obtained in stage a); and
c) a study of the supernatant on the ability to enhance the activity of a pesticide obtained on the basis of Bacillus.

List of drawings
1 schematically shows the basic methodology used for purification of Ia.

 Figure 2 shows the 13C NMR spectrum of Ia.

 Figure 3 shows the proton NMR spectrum of Ia.

 Figure 4 shows the results of nOe experiments with acetylated derivative Ia.

Information confirming the possibility of carrying out the invention
The claimed potentiator enhances the activity of a pesticide based on bacteria of the genus Bacillus and may have a molecular weight of from about 350 to about 1200, or, preferably, from about 350 to about 700.

 The factor enhances the activity of the pesticide based on bacteria of the genus Bacillus at least about 1.5-1000 times, preferably 100-400 times. In a specific embodiment, the potentiator enhances the pesticidal activity of Bacillus thuringiensis delta endotoxin, including but not limited to cry I (cry IA, cry IB and cry IC), cry II, cry III, cry IV, cry V or cry VI protein full length or subjected to proteolysis, or having a truncated shape, about 1.5-1000 times. In particular, the potentiator enhances the effect of delta-endotoxin B. approximately 100-400 times. In addition, the potentiator can enhance the pesticidal activity of a chemical pesticide and / or virus with pesticidal properties.

The factor may be water soluble, stable in water when heated to 1000 ^{° C.} for at least 5 minutes, stable when exposed to direct sunlight for at least about 10 hours, and / or stable at a pH of about 2 for 10 days. The factor has 13 carbon atoms. In addition, the potentiator can have ¹ H NMR oscillations at (1.5, 3.22, 3.29, 3.35, 3.43, 3.58, 3.73, 3.98, 4.07, 4, 15, 4.25, 4.35.

 In a most preferred embodiment, the potentiator has a structure Ia or is the corresponding salt.

Соль должна обладать способностью усиливать действие пестицида на основе бактерий рода Bacillus.

The salt must be able to enhance the action of a pesticide based on bacteria of the genus Bacillus.

Getting a potentiator
The potentiator can be obtained using strains of microorganisms of the genus Bacillus (for example, Bacillus subtilis, Bacillus lichemiformis and Bacillus thuringiensis) when grown in rocking flasks or a fermenter. In one embodiment of the invention, the potentiator can be obtained from the supernatant of a Bacillus thuringiensis culture, including (but not limited to these microorganisms), Bacillus thuringiensis subspecies kurstaki, Bacillus thuringiensis subspecies aizawai, Bacillus thuringiensis subspecies galleriae, Bacillus thuringiisisensis bisillusis thuringiensisis bisillusis thuringiensisis bisillusis thuringiensisensis subspecies thuringiensis, Bacillus thuringiensis subspecies alesti, Bacillus thuringiensis subspecies canadiensis, Bacillus thuringiensis subspecies darmstadiensis, Bacillus thuringiensis subspecies dendrolimus, Bacillus thuringiensisiensis thisiensensiensis thiensensiensis thiensensiensis thiensis thiensis thiensensi thiensensi thiens teniensensi s subspecies, toumanoffi and Bacillus thuringiensis subspecies israelensis. Preferably, the potentiator is obtained from the supernatant of Bacillus thuringiensis subsp. kurstaki, Bacillus thuringiensis subsp. aizawai or Bacillus thuringiensis subsp. gallerial or their mutants having basically the same enhancing activity. In a specific embodiment, the potentiator is isolated from the cry-Spo mutant of Bacillus thuringiensis subsp. kurstaki.

 Bacillus can be cultured using media known to those skilled in the art and fermentation technology (see, for example, Rogoff et al., 1969, J. Invertebrate Path. 14: 122-129; Dulmage et al., In Microbial Control of Pests and Plant Diseases, HD Burges, ed., Academic Press, NY 1980). At the end of the cycle, the supernatant is isolated, separating B.t. and crystals from the culture fermentation medium in a known manner, for example by centrifugation and / or ultrafiltration. The potentiator contained in the supernatant can be isolated using known methods, for example by ultrafiltration, evaporation and spray drying. The cleaning procedure is described in more detail in the following sections.

 The potentiator is cleaned by various methods known to those skilled in the art, including (but not limited to) column chromatography (e.g., ion exchange, affinity and gel chromatography), electrophoretic methods, re-dissolution, extraction, and any other standard well-known methods.

 The property of the potentiator to enhance the activity of pesticides based on bacteria of the genus Bacillus, the pesticidal activity of viruses, the activity of chemical pesticides against various pests can be determined and studied using known methods, such as the use of artificial diet for insects, leaf treatment (surface and immersion in solution), the use of sprays for application to leaves, etc. Special examples of such studies are given below in the Section "Example: Characteristic Ia".

Potentiator Compositions
The resulting potentiator can be used either alone or in combination with pesticides based on bacteria of the genus Bacillus, which, as indicated above, can be a Bacillus strain, spores, a protein or its fragment, or another substance with activity against pests or contributing to their destruction or protecting plants from pests; with a chemical pesticide and / or entomopathogenic virus and a suitable carrier in the pesticidal composition (s), which, for example, can be a solution, emulsion, powder, dispersible granules, wettable powder, emulsifiable concentrate, aerosol or impregnated granules. Examples of such strains of Bacillus are (but are not limited to): Bacillus thuringiensis subsp. kurstaki (referred to as DIPEL ^TM from Abbott Laboratories, Inc., JAVELIN ^TM from Sandoz, BIOBIT ^TM from Novo Nordisk A / S, FORAY ^TM from Novo Nordisk A / S, BIOCOT ^TM from Novo Nordisk A / S, MVP ^TM from Mycogen, BACTOSPEINE ^TM from Novo Nordisk A / S, and THURICIDE ^TM from Sandoz); Bacillus thuringiensis subsp. aizawai (denoted as FLORBAC ^TM from Novo Nordisk A / S, and XENTARI ^TM from Abbott laboratories, Inc.); Bacillus thuringiensis subsp. enebrionis (referred to as NOVODOR ^TM from Novo Nordisk A / S, TRIDENT ^TM from Sandoz and M-TRAK ^TM and M-ONE ^TM from Mycogen); Bacillus thuringiensis subsp. israelensis (designated either BACTIMOS ^™ or SKEETAL ^™ from Novo Nordisk A / S, TEKNAR ^™ from Sandoz, and VECTOBAC ^™ from Abbotts Laboratories, Inc.); Bacillus thuringiensis subsp. kurstaki / tenebrionis (referred to as FOIL ^TM from Eoogen); Bacillus thuringiensis subsp. kurstaki / aizawai (denoted as CONDOR ^TM from Ecogen and AGREE ^TM from Ciba-Geigy); and Bacillus thuringiensis subsp. kurstaki / kurstaki (referred to as CUTLASS ^TM from Ecogen). The Bacillus protein can be selected from the group including (without limitation indicated) cry I, cry II, cry III, cry IV, cry V, cry VI. A chemical pesticide may, for example, be an insect growth regulator such as difluorobenzuron, carbomate such as thiodicarb and methomyl, organophosphate such as chlorpyrifos, pyrethroid such as cypermethrin and esfenvalerate, inorganic fluoride such as cryolite, and pyrrole. The entomopathogenic virus may be a baculovirus, for example, nuclear polyhedrosis virus (NPV), Autographa californica, Syngrapha falcifera NPV, Cydia pomonella GV (granulosis virus), Heliothis zea, NPV, Lymantria dispar NPV, Orgyia pseudotsugata NPV, Spiopodoptera lepodopteopoda Neodiprion sertifer NPV, Harrisina brillians NPV and Endopiza viteana Clemens NPV.

In a composition containing an activity enhancing potentiator and a pesticide derived from Bacillus bacteria, the potentiator may be present in an amount of at least 0.1 g / BIU or 0.05 g of potentiator per 1 g of Bacillus delta-endotoxin and spores, about 300 g / BIU or 150 g of potentiator per 1 g of Bacillus delta endotoxin and spores, preferably 2 g / BIU or 1 g of potentiator per 1 g of Bacillus delta endotoxin and spores. “BIU” means here a billion international units defined by bioassay. A bioassay involves comparing a sample with standard Bacillus material using Trichoplusia ni or another insect as a standard insect for testing. The enhancing activity is determined by dividing the reference standard LC ₅₀ and multiplying by the value of the enhancing activity of the reference standard.

 In another embodiment, the composition may include a purified potentiator or supernatant obtained by growing Bacillus in a dry, concentrated, or liquid form and a pesticide-acceptable carrier, examples of which are described herein. This composition can be applied to a single plant, for example a transgenic plant. In particular, the composition can be used on plants already containing and expressing the Bacillus thuringiensis gene. Alternatively, the composition may be used on plants previously exposed to a composition based on Bacillus thuringiensis. In another embodiment, the composition can be used in other habitats of pests belonging to the order Diptera, for example in water or soil. The target substance (activity potentiator) is included in the composition in the concentration range from about 0.001% to 60% (by weight).

 A composition consisting of a potentiator and a carrier acceptable for the pesticide, in addition to controlling the pests, can also be used to reduce the resistance of the pests to the pesticide. Alternatively, the composition can be used to enhance the action of a pesticide derived from bacteria of the genus Bacillus. The composition in one case can be applied simultaneously with the pesticide in an amount of at least about 2 g of the substance potentiator / BIU to about 300 g of the substance potentiator / BIU. In another case, the composition can be used as an adjuvant in the next 24 hours after applying the pesticide to prolong the action of the remaining pesticide.

 Such compositions described above can be prepared by adding a surfactant, an inert carrier, a preservative, a humectant, a food stimulant, an attractant, an encapsulating agent, a binding agent, an emulsifier, a dye, a UV tread, a buffer, a flow agent, or another component to facilitate manipulation of the product and its use in relation to certain pests.

 Suitable surfactants include anionic compounds, such as carboxylates, for example long chain fatty acid metal carboxylates; N-acyl sarcosinate; phosphoric acid mono-or diesters with fatty alcohol ethoxylates or salts of these esters; fatty alcohol sulfates such as sodium docetyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated alkyl phenol sulfates; lingin sulfates; petroleum sulfonates; alkylaryl sulfonates, such as alkylbenzenesulfonates or lower alkylnaphthalene sulfonates, for example, butylnaphthalene sulfonate; salts of sulfonated naphthalene formaldehyde condensates; salts of sulfonated phenol-formaldehyde condensates or more complex sulfonates, such as amide sulfonates, for example, the sulfonated condensation product of oleic acid and N-methyltaurine, or dialkyl sulfosuccinates, for example sodium sulfonate or dioctyl succinate. Nonionic reagents include condensation products; fatty acid esters, fatty alcohols, fatty acid amides or alkyl or alkynyl substituted phenols with ethylene oxide, polyhydric alcohol fatty acid esters, for example sorbitan fatty acid esters; condensation products of these esters with ethylene oxide, for example polyoxyethylene sorbite fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylene glycols such as 2,4,7,9-tetraethyl-5-decin-4,7-diol, or ethoxylated glycols acetylene series. Examples of cationic surfactants include, for example, aliphatic mono-, di- or polyamine, say acetate, naphthenate or oleate; an oxygen-containing amine, such as polyoxyethylene alkylamide amine; an amide-containing amine obtained by condensation of a carboxylic acid with a di- or polyamine; or quaternary ammonium salt.

 Examples of inert carriers are inorganic, mineral materials such as kaolin, mica, gypsum, fertilizer, phyllosilicates, carbonates, sulfates or phosphates; organic substances - sugar, starch, cyclodextrins; or botanical materials - wood processing products, cork, powdered corn cobs, rice husks, peanut and walnut shells.

 Compositions that are the subject of the present invention, can be obtained both in a form suitable for direct use, and in the form of a concentrate or primary composition, which requires dissolution in an appropriate amount of water or another solvent before use. The concentration of the pesticide may vary depending on the nature of the composition and, in particular, on whether it is a concentrate or a form that is already ready for use. The composition contains from 1 to 98% of a solid or liquid inert carrier and 0-50%, preferably 0.1-50% of a surfactant. The use of these compositions will be carried out in accordance with the standards established for a commercial product, preferably from 1.84 g to 0.92 kg per ha (from 4.54 g to 2.27 kg per acre) in dry form and from 0.0019 liters to 4.76 liters per ha (from 0.0047 liters to 11.75 liters per acre) in liquid form.

 Further, pretreatment of the crystalline delta endotoxin of Bacillus thuringiensis and / or the potentiator can be carried out before preparing the composition for prolonging the action of the pesticide when applied in a pest infected environment, since the pretreatment does not adversely affect the crystalline delta endotoxin or potentiator. Such processing can be carried out both by chemical and / or physical methods that do not have undesirable effects on the properties of the composition (s). Examples of chemical reagents include, but are not limited to: halogenating agents; aldehydes, for example formaldehyde and glutaraldehyde; anti-infectious agents, for example, marshmallow; alcohols, for example isopropanol and ethanol; and histological fixators, such as Bowen's fixative and Helly's fixative (see, for example, Humason, Animal Tissue Techniques, W.H. Freeman and Co., 1967).

 The compositions obtained according to the present invention can be applied directly to plants, for example, by spraying or spraying at a time when the pest has already begun to appear on the plant, or before the appearance of pests for protective protection. Plants that may be protected in accordance with this invention include (but are not limited to): cereals (wheat, barley, rye, oats, rice, sorghum and the corresponding crops); beetroot (sugar beets and fodder beets); stone fruits, fruit crops and crops with soft fruits (apple trees, pears, plums, peaches, almonds, cherries, strawberries, raspberries, blackberries); legumes (alfalfa, beans, lentils, peas, soy); oilseeds (rapeseed, mustard, poppy seeds, olives, sunflowers, coconut palms, plants producing castor oil, cocoa trees, peanuts); cucumber (cucumber, zucchini, melon); fibrous plants (cotton, flax, hemp, jute); citrus fruits (oranges, grapefruits, tangerines); vegetable crops (spinach, salad, asparagus, cabbage, etc. cruciferous, carrots, onions, tomatoes, potatoes); laurel trees (avocado, cinnamon tree, camphor tree); deciduous and coniferous trees (linden, yew, oak, alder, poplar, birch, spruce, larch, pine) or plants such as maize, peat plants, tobacco, walnut, coffee tree, sugarcane, tea bush, vine, hops , banana tree, rubber trees, and also decorative plants. The composition can be applied to leaves, arable furrow, dispersed in the form of granules, applied to the soil or soaked with soil. It is extremely important to ensure proper pest control in the early stages of plant growth, as it is at this time that the plant can be especially seriously damaged. The spray liquid or spray powder may include another pesticide, if deemed necessary. Preferably, the composition is applied directly to the plant.

 The compositions obtained according to this invention can also be directly used for treating reservoirs, lakes, rivers, streams, still water and other objects that are infected with pests of the Diptera order, especially when it relates to human health. The composition can be applied by spraying, spraying, by watering, etc.

 The compositions obtained according to the present invention can be effective against pests of the Lepidoptera order, for example, Achroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta kiaotaella Antheraea pernyi Anticarsia gemmatalis Archips sp. Argyrotaenia sp. Athetis mindara sibericus, Desmia funeralis, Diaphania hyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Ephestia elutella, Erannis tiliaria, Estigmene acrea, Eulia salubricola, Eupoecilia amb ctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisina virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantria cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria lisugis lyria lyri Malacosoma sp., Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp., Ostrinia nubilalis, Paleacriti lla phi phi phi rpri calphora phila phi rphi calphi philippes Pieris rapae, Plathypena scabra, Platynota flouendana, Platynota sultana, Platyptilia carduidactyla, Ptodia interpunctella, Plutella xylostella, Pontia protodice, Pseudaletia unipuncta, Pseudoplusia includens, Sabulooptopletaotarataopleotaegraotaotraoda tetraopleta ineola bisselliella, Trichoplusia ni, Udea rubigalis, Xylomyges curialis, Yponomeuta padella; order Diptera, e. g. , Aedes sp., Andes vittatus, Anastrepha ludens, Anastrepha suspensa, Anopheles barberi, Anopheles quadrimaculatus, Armigeres subalbatus, Calliphora stygian, Calliphora vicina, Ceratitis capitata, Chironomus tentans, Chrysomyauliaeciauliaifiaeciauliaeciauliaeciauliaeciaecia leoma tomiauliaifiaecia leoma tomiaifiaecia roma , Delia antiqua, Dilia ptatura, Delia radicum, Drosophila melanogaster, Eupeodes corollae, Glossina austeni, Glossina morsitans morsitans, Glossina morsitans submorsitans, Glossina pallidipes, Glossina palma palpalis gambiensis, Glossemalogus palompalis gambiensis, Glossina palma palpalis gambiensis, Glossina palma palpalis gambiensis, Glossina palma palpalis gambiensis Hypoderma lineatum, Leucopis ninae, Lucilia cuprina, Lucilia sericata, Lutzomyia longlpaipis, Lutzomyia shannoni, Lycoriella mali, Mayetiola destructor, Musca autumnalis, Musca domestica, Neobellieria sp., Phyoma phisra, Phi phra serum Sabethes cyaneus, Sarcophaga bullata, Sarcophaga stercoraria, Stomo xys calcitrans, Toxorhynchites amboinensis, Tripteroides bambusa. However, the compositions of the invention can also be effective against insect pests of the Coleoptera order, for example, Leptinotarsa sp., Acanthoscelides obtectus, Callosobruchus chinensis, Epilachna varivestis, Pyrrhalta luteola, Cylas formicarius elegantulus, Listronotud oregonensis, Sitophalphis spulocisphalus spulocisphalus sprucephalus sprucephalus oregonensis. subspinosus, Popillia japonica, Rhizotrogus majalis, Alphitobious diaperinus, Palorus ratzeburgi, Tenebrio molitor, Tenebrio obscurus, Tribolium castaneum, Tribolium confusum, Tribolius destructor; Acari, e.g., Oligonychus pratensis, Panonychus ulmi, Tetranychus urticae; Hymenoptera, e.g. Iridomyrmex humilis, Solenopsis invicta; Isoptera, e.g. Reticulitermes hesperus, Reticulitermes flavipes, Coptotermes formosanus, Zootermopsis angusticollis, Neotermes connexus, Incisitermes minor, Incisiterimes immigrans; Siphonaptera, e.g. Ceratophyllus gallinae, Ceratophyllus niger, Nosopsyllus fasciatus, Leptopsylla segnis, Ctenocephlides canis, Ctenocephlides felis, Echicnophaga gallinacea, Pulex irritans, Xenopsylla cheopis, Xenoplla, Xenoplla and Tylenchida, e.g. Melodidogyne incognita, Pratylenchus penetrans.

 The following examples illustrate but do not limit the invention.

Example: characteristic Ia
As indicated herein, product Ia is isolated and purified. Its characteristic is given below.

Isolation and purification Ia
Strain B. thuringiensis subsp. kurstaki EMCC0086 (deposited in NRRL under the number B-21147) was cultured at 30 ^{° C.} for 72 hours in a medium containing starch or glucose as a carbon source and a protein, hydrolyzed protein or liquid corn extract as a nitrogen source. Ia formation begins 13 hours after the start of fermentation. Peak activity is detected after approximately 30 hours.

 After fermentation, the culture supernatant of B. thuringiensis subsp. Is separated by centrifugation. kurstaki, then purified by ultrafiltration through a 30 kDa-MW-CO membrane using a Rhone Poulenc UV system. 30 kDa filtration removes any remaining cellular debris, delta-endotoxin crystals, spores and soluble protein with a molecular weight of more than 30 kDa. The permeate is concentrated 10 times by evaporation, centrifuged and then filtered (pore size of the membrane 0.2 μm) to further remove insoluble residues from the solution to obtain a purified solution containing Ia.

Purification of Ia to a homogeneous state is carried out using a multi-stage procedure, schematically depicted in figure 1. In accordance with the above procedure, purification is continued from the 5 kDa ultrafiltration step. Received after 5 kDa ultrafiltration permeate
adsorb on Sulfopropyl (SP) -cation exchange resin and elute with a solution of ammonium acetate. The product is then concentrated approximately 30 times by lyophilization, and saline and other impurities are removed using a BioRad P2 column (size exclusion). The volume obtained from the P2 column is passed through a high resolution cation exchange column (SP HPLC), resulting in a homogeneous preparation. Contaminating salt impurities are removed by re-mofilization.

Activity is checked by microbiological examination using Spodoptera exigua, and the degree of purification is determined by capillary electrophoresis. A sample containing 50 μl of Ia - and 50 μl of cry IA (c) - protein (15 μg / ml) isolated from BIOBIT ^™ FC (100 μl) is added to individual wells of a plate containing 500 μl of a solid artificial insect nutrient base. Tablets containing various samples are air dried. From 2 to 4 Spodoptera exigua larvae at the 2nd and early 3rd stages of development are added to the wells with the dried samples. The holes are closed with a polyester film with holes and the plates are incubated for 2-3 days at 30 ^o C. Then the degree of developmental delay and the percentage of larvae death are determined (Mylar). Typically, 5 wells are used to test each sample.

EXPLANATIONS ON THE STRUCTURE
The active compound has been found to be soluble in water and insoluble in organic solvents. It is positively charged and reacts with ninhydrin, as evidenced by thin-layer chromatography on silica. ¹³ C and proton NRM spectra of the compounds are shown in FIGS. 2 and 3, respectively. ¹³ C NMR experiments revealed the presence of 13 carbon atoms (relative to 3- [trimethylsilylpropionic acid]) in the compound. The DEPT experiment showed that the compound contains three Quaternary carbon atoms (C), seven methine (CH) groups, three methylene (CH ₂ ) groups, and there are no methyl groups (CH ₃ ). Using proton-binding experiments, such as 1-D rupture and COZY, one large spin system was identified containing eight carbon atoms. In addition, a smaller spin system consisting of two carbon atoms was discovered. An experiment on correlation of a proton of a carbon atom (NMVS) made it possible to correlate the resonance of each proton in a molecule with the corresponding carbon atom.

Treatment of the active compound (13 mg) with acetic anhydride in pyridine leads to the formation of an acetylated derivative, which is much less polarized. This derivative is purified using HPLC to obtain 3 mg of pure acetylated derivative. Mass spectroscopic analysis showed that the derivative has 7 acetagroups and a molecular weight of 690. This indicates that the molecular weight of the starting active compound is 396 and it contains several nitrogen atoms. In addition, fragments containing 6 and 5 acetagroups were revealed. The data of high-resolution experiments for 5 and 6 acetate daughter ions are 645.2594 (6 acetagroups) and 607.2519 (5 acetagroups). This allows us to establish the molecular formula of compound Ia: C ₁₃ H ₂₈ N ₆ O ₈ .

Treatment of active compound (13 mg) 6 N HCl leads to the formation of a derivative that is positive for ninhydrin. This indicates the presence of amide bonds. The derivative has the same R _f value determined by thin-layer chromatography as 2,3-diaminopropionic acid. These results, along with NMR data suggest the presence of 2,3-diaminopropionic acid.

 Another method used for Ia analysis is called “NОе” (Nuclear Overhauser Effect), it allows you to determine the proximity of the distance between protons in space. The nOe method is applied to the acetylated derivative Ia. In the two-dimensional "nOe" experiment (NOESY) effects (NOEs) are observed between the N-H proton at 8.06 ppm and 5.17 ppm (figure 4).

Данное соединение может быть классифицировано как уреидоамид. Заместители - 2 амидные группы, мочевина, две аминогруппы и пять гидроксильных групп. Оно содержит семь хиральных центров.The following structural formula was established for Ia

This compound can be classified as ureidoamide. Substituents are 2 amide groups, urea, two amino groups and five hydroxyl groups. It contains seven chiral centers.

Ia Properties
It was found that Ia enhances the activity of crystalline delta-endotoxins of protein nature isolated from Bacillus thuringiensis subsp. kurstaki and bacillus thuringiensis subsp. aizawai with pesticidal activity against Spodoptera exigua, regardless of the form of pesticidal proteins. The factor enhances the pesticidal activity of B. tk, isolated crystals, full-length proteins (molecular weight 130 kDa) and truncated cry IA proteins (molecular weight about 65 kDa). The activity of cry II and cry IC inclusions is also enhanced. An increase in activity was also found for individual cry IA proteins (a), (b), (c). It was also shown that the interaction time of Ia and cry protein is not critical for the manifestation of bioactivity. However, Ia itself is not active. It was found that the level of amplification for truncated cry IA proteins and cry IC and cry II inclusions is 100-200 and approximately 320 for full length cry IA (c) (see Tables 1 and 2, respectively). In particular, for full-length proteins, it was shown that the addition of Ia to 0.75 μg / ml cry IA (s) provides the same mortality (insect retardation) as cry IA (c) proper at a dose of 240 μg / ml. In the case of the truncated protein cry IA (c) at OD ₂₈₀ = 0,0006 and in combination with Ia, the developmental delay effect was the same as if cry IA (c) was used alone in a dose corresponding to OD ₂₈₀ = 0,075. Inclusions of cry II at a concentration of 0.6 μg / ml and in combination with la provided the same degree of insect retardation and mortality as cry II protein alone at a dose of 75 μg / ml, i.e. a 125-fold increase in activity was observed. The use of cry IC inclusions at a dose of 0.3 μg / ml with the addition of Ia provides the same mortality / delay in insect development as the use of one cry IC protein at a dose of 75 μg / ml, which corresponds to a 250-fold increase in activity. The concentration of cry IA protein, which leads to developmental delay, ensured insect death when Ia was added. Using the bioassays described in the Section "Isolation and Purification of Ia", it was found that Ia is stable when boiled for 5 minutes, but loses activity during autoclaving (> 190 ^o С). Further, it is stable when exposed to direct sunlight for at least 10 hours. Ia remains stable at pH 2 for 3 days and is not stable at pH 12. It was found that Ia completely loses activity in iodic acid or in concentrated HCl.

Assessment of other subspecies of Bacillus thuringiensis and other species of the genus Bacillus
Assess the possibility of obtaining Ia based on several species of Bacillus. The strains were cultured at 30 ^{° C.} for 72 hours in a medium containing starch, hydrolyzed starch or glucose as a carbon source, and a protein, hydrolyzed protein or liquid corn extract as a nitrogen source. In the supernatants, the presence of Ia is determined by microbiological examination using the Spodoptera exigua described above. It was found that B. thuringiensis subsp. aizawai strain EMCC 0087 (deposited in NRRL under N B-21148) and B. thuringiensis subsp. gallerriae (deposited in NRRL) produce Ia in the same amount as B. thuringiensis subsp. kurstaki.

 Using capillary electrophoresis, it was found that Ia produce B. subtilis, B. cereus, B.t. subsp. alesti, B.t. subsp. canadiensis, B.t. subsp. darmstadiensis, B. t. subsp. dendrolimus, B.t. subsp. entomocidus, B. t. subsp. finitimus, B. t. subsp. israelensis, B.t. subsp. kenyae, B.t. subsp. morrisoni, B. t. subsp. subtoxicus, B.t. subsp. tenebrionis, B.t. subsp. thuringiensis, B. t. subsp. toumanoffi, B. cereus, B. subtilis and B. thuringiensis subsp. kurstaki cry-spo-mutant.

 In particular, to determine the amount of Ia, a Beckman P / ACE capillary electrophoresis system was used, equipped with a capillary (50 μm • 57 cm), a capacity of 0.2 M phosphate buffer, pH 6.8, a device with a voltage of 15 kV, and a device for determining absorption at 200 nm. Use samples with a volume of 20 nl, the study time (run) is 25 minutes.

 The calibration curve is obtained using the following amounts of Ia as standard: 1.25 mg / ml, 0.625 mg / ml, 0.3125 mg / ml, 0.156 mg / ml, 0.078 mg / ml. Then build a line graph. To determine the concentration of Ia in each sample, the obtained dependence y = mx + b is used.

 Before each test, the capillary is washed with run buffer (0.2 M phosphate, pH 6.8) for three minutes. After each 25-minute run, the capillary is washed with 1 N. NaOH for 1 minute, filtered with HPLC using water for 1 minute, 0.5 M phosphoric acid - 3 minutes, filtered with HPLC water - 1 minute.

 The area under each peak is integrated and the total area of the peaks is determined, and the final concentration is calculated from the calibration curve.

 Evaluation of products derived from B.t.

The amount of Ia present in various commercially available products is determined by capillary electrophoresis. BACTOSPEINE ^TM , JAVELIN ^TM , NOVODOR ^TM , SPHERIMOS ^TM , BACTIMOS ^TM , FORAY ^TM , FLORBAC ^TM and BIOBIT ^TM are obtained from Novo Nordisk A / S. XENTARI ^TM and DIPEL ^TM are obtained from Abbott Laboratories. AGREE ^{™ is} obtained from Ciba-Geigy; MVP ^{™ is} obtained from Mycogen and CUTLASS ^{™ is} obtained from Ecogen.

 The research results are carried out in table. 3; they show that Ia is present in various amounts, ranging from less than 0.001 g of Ia / BIU to 0.071 g of Ia / BIU.

Nutrient bioassays
The activity of S. tk is determined by the biomethod using synthetic insect growth media using Spodoptera exigua larvae, at the third stage of development, Helicoverpa zea second stage larvae, Spodoptera frugiperda third stage larvae, Heliothis virescens second stage larvae, Trichoplusia ni third stage larvae stages of Pseudoplusia includens, third stage larvae of Plutella xylostella, third stage larvae of Spodoptera littoralis and third stage larvae of Mamestra brassicae.

In order to determine the degree of increase in activity caused by the addition of Ia to B. t.-products, and to establish a circle of insects that are susceptible to this effect, biological tests are carried out using special nutrient media for insects. In experiments using high concentrations of Ia against Spodoptera exigua (7.4-23.7 g Ia / BIU), purified Ia (70% of the active ingredient, 30% of the acetate ion) is used to enhance the activity of BIOBIT ^™ FC (FC is a fluid concentrate ) The data obtained are presented in table. 4 show an increase in the activity of BIOBIT ^™ HPWP (highly active wettable powder) in combination with Ia (0.658% of the active ingredient). S. littoralis and M. brassicae are tested using FLORBAC ^™ HPWP in combination with Ia.

Various B. t.-products are weighed, and Ia is added in an amount of from 0.1 to 237 g of Ia / BIU. The volume was adjusted with 0.1% Tween ^™ . Samples are sonicated for 1 minute and then diluted to the final volume. Clean samples (without Ia) and control samples are also prepared. Control samples include B. tk HD-1-S-1980 (obtained from the NRRL collection), whose activity corresponds to 16,000 international units (IU) per milligram, and JAVELIN ^™ WG, whose activity is 53,000 Spodoptera Units / mg (SU).

Standard synthetic culture media, including water, agar, sugar, casein, wheat germ, methyl paraben, sorbic acid, flax seed oil, cellulose, salts and vitamins, are prepared in a 20-liter boiler when heated. This provides a sufficient volume of culture medium for testing 10-12 samples with seven different concentrations of each test substance. B.t. solutions are sequentially diluted to give 16 ml aliquots. Each aliquot is added to 184 g of molten medium. The mixture is sufficiently homogenized and then poured into a plastic tablet having 40 individual cells. Three control plates are prepared for each batch of medium. As soon as the medium cools and becomes solid, one insect of a known age (larvae of the 2-3rd generation) is placed in each cell, the plates are covered with a transparent perforated polyester film (mylar). The tablets are mounted on shelves and incubated for four days at 28 ^{° C.} and 65% relative humidity.

Four days later, determine how many insects died. Each tablet is shaken sharply on the surface of the table and fixed larvae are counted as dead. The percentage of dead larvae is calculated and the data analyzed in parallel independent experiments. Calculate LC ₅₀ s, LC ₉₀ s, slope of the regression lines, coefficient of variability and activity level. Samples are examined at least three times or until the three activity values obtained fall within the 20% range of the calculated activity value for each sample. In order to calculate the increase in activity provided by each concentration of Ia, the LC _{50 of the} B.t. / Ia sample is adjusted for the amount of B. t. in the sample. The LC ₅₀ value of the paired “pure” samples is divided by the adjusted LC ₅₀ values to determine the order of decrease in the LC ₅₀ associated with Ia. For the study of Lobesia bothrana using the following methodology. Grapes affected by Lobesia bothrana are harvested in a field not treated with pesticides and the larvae are removed. Serial dilutions of Ia in water are prepared (250 μg / ml, 500 μg / ml and 1000 μg / ml). One larva is placed in the center of the Petri dish. If it is observed that the larva is drinking, it is transferred to a Petri dish with freshly picked grapes. The larvae are kept at 22 ^o C for 3-4 days.

As shown in the table. 4, there is a significant decrease in LC ₅₀ values for all samples.

The enhancement of the effects of various products on Spodoptera exigua due to Ia is determined biologically using culture media as described above. The amount of added Ia / BIU product are given in table. 5. A mixture of Ia / B. The t.-product is introduced into an agarized nutrient medium containing wheat germ and casein. Insects are placed on this medium for a 4-day period and kept at 28 ^o C. The death of insects is recorded and investigated using probabilistic studies. LC ₅₀ , LC ₉₀ and the increase in activity is determined taking into account the corresponding product not containing Ia. The results presented in table. 5 show that Ia enhances the activity of various B. tk and Bt. products derived from various sources. B.t. strains containing these products are described in the preceding tables.

Biological studies using leaves
Foliar biological tests are carried out on second-stage Spodoptera exigua larvae using broccoli plants using BIOBIT ^™ FC and Ia. The ratio of Ia and BIOBIT ^TM FC is the same - 2 g of Ia / BIU BIOBIT ^TM FC. The treatment of broccoli plants is carried out by directed spraying of the drug in a carrier volume of 30.64 liters per ha (75.7 liters per acre). After the remainder of the sprayed liquid dries up, leaves with second-stage Spodoptera exigua larvae are cut from the plants. The research results are presented in table. 6. 100% insect mortality is observed when treated with a dose of 8.7 BIU / hectare BIOBIT ^TM FC + Ia, while BIOBIT ^TM FC by itself destroys 92% of the larvae at a dose of 58.8 BIU / hectare and 8% of the larvae at a dose 17.6 BIU / hectare. Treated plants are also exposed to direct sunlight for 8 hours, after which infected leaves are torn off. After such an 8-hour exposure to light, BIOBIT ^TM FC alone provides 27% insect mortality at a dose of 58.8 BIU / hectare, while BIOBIT ^TM FC + Ia provides 100% mortality at a dose of 8.7 BIU / hectare. Foliar studies using early fourth-stage larvae show that BIOBIT ^™ FC alone provides 75% mortality of larvae at a dose of 52 BIU / hectare and 100% mortality at a dose of 13 BIU / hectare when combined with BIOBIT ^TM FC (FC stands for "fluid concentrate") + Ia.

Field test
Field experiments on beans (Spodoptera exigua insect) show that BIOBIT ^TM FC alone at a dose of 70 BIU / hectare provides 51% control, while 2 g of Ia / BIU BIOBIT ^TM FC at a dose of 40 BIU / hectare provide 89 % control (compared to the untreated field). JAVELIN ^TM WG at a dose of 45 BIU / hectare provides 51% control.

Field experiments on sweet corn (Spodoptera frugiperda insect) show that 2 g of Ia / BIU BIOBIT ^TM FC at a dose of 39.5 BIU / hectare provide 84% control.

Stability factors
Colonies of susceptible and resistant Plutella xylostella are examined. Sustainable moths are individuals collected in the fields of Florida that have already acquired resistance to B.t. due to heavy use of JAVELIN ^TM WC. BIOBIT ^™ HPWP in complex with Ia was investigated by the method of immersed leaves. Resistance to JAVELIN ^™ and XENTARI ^™ are studied without the addition of Ia. Leaf discs of cabbage with a diameter of 6 cm are immersed for 10 seconds in one of eight B.t. with different concentrations or in a mixture of these drugs with Ia. Concentration varies from 1 to 1000 ppm. Leaf discs are allowed to air dry for two hours and placed in plastic Petri dishes with second-stage larvae (0.2-0.4 mg). Twenty-five insects / dose / day are replicated twice to obtain 50 insects / dose. After incubation for 72 hours at 27 ^{° C.} , mortality is recorded. The reduction in lethal dose is investigated by probabilistic analysis. Stability factors are calculated by dividing the LC ₅₀ and LC ₉₀ values for susceptible individuals. The research results are given in table. 7. They show that BIOBIT ^™ HPWP enhances activity when used together with 2 g of Ia / BIU and 4 g of Ia / BIU. In particular, when using 4 g of Ia / BIU, there is a twofold decrease in the LC ₅₀ stability coefficient and a 10-fold decrease in the LC ₉₀ stability coefficient.

 The described and claimed invention should not be limited to the private options for its implementation described here, since the examples are provided only to illustrate some aspects of the present invention. It is contemplated that any equivalent embodiments of the invention are included within its scope. Indeed, various modifications of the invention, in addition to those disclosed in the present description, obviously follow from it and are clear to a person skilled in the art. Such modifications are also covered by the claimed claims.

 Cited in the present description, information sources are provided solely as references.

Deposition of microorganisms
In accordance with the Budapest Treaty, the following strains of Bacillus thuringiensis were deposited with the Agricultural Collection Service Patent Collection (NRRL), Northern Regional Research Center, 1815 University Street, Peoria, Illinois, 61604, USA. Strain EMCC0086; NRRL Number B-21147; date of deposit - October 6, 1993.

 The strains are deposited subject to the guaranteed availability of culture during the period preceding the grant of a patent to any person determined by the Commissioner of the Patent and Trademark Office in accordance with 37 C.F. R. 1.44 and 35 U.S.C. 122. A pure culture of the deposited strain is submitted for deposit. The deposit and its descendants are available in accordance with the requirements of patent laws of other countries to which the application is filed. However, it should be understood that the availability of deposited culture does not give the right to reproduce the subject of the invention in violation of the rights provided by a patent granted on behalf of the state.

Claims (11)

1. A method of potentiating the pesticidal activity of a pesticide obtained on the basis of bacteria belonging to the genus Bacillus, comprising adding to the pesticide a potentiator obtained by cultivating a strain of bacteria of the genus Bacillus, followed by isolation of the potentiator from the culture supernatant obtained in the previous step, characterized in that they use a potentiator which has structure I

and is characterized by ¹ H NMR shifts at approximately the following points: δ 1.5, 3.22, 3.29, 3.35, 3.43, 3.58, 3.73, 3.98, 4.07, 4 , 15, 4.25 and 4.35 and ¹³ With shifts at approximately the following points: δ 31.6, 37.2, 51.1, 53.3, 54.0, 54.4, 61.5, 61, 6, 64.1, 65.6, 158.3, 170.7 and 171.3, or a salt thereof.  2. The method according to p. 1, characterized in that the cultivation is carried out by a fermentation process.  3. The method according to p. 1, characterized in that the selection of the specified potentiator from the supernatant is carried out by column chromatography.  4. The method according to p. 1, characterized in that the strain belonging to the genus Bacillus is a strain of Bacillus thuringiensis.  5. The method according to p. 4, characterized in that the strain of Bacillus thuringiensis is selected from the group comprising strains of Bacillus thuringiensis subspecies aizawai, Bacillus thuringiensis subspecies alesti, Bacillus thuringiensis subspecies canadiensis, Bacillus thuringiensis subspecies colusriusensus bisillusis thuringiensis coreus thmeiensis thmeiensis coreus thmeiensis thmeriensis thmeriensis coreus thmeiensis subspecies dakota, Bacillus thuringiensis subsp darmstadiensis, Bacillus thuringiensis subsp dendrolimus, Bacillus thuringiensis subsp entomocidus, Bacillus thuringiensis subsp finitimus, Bacillus thuringiensis subsp galleriae, Bacillus thuringiensis subsp indiana, Bacillus thuringiensis subspecies israelensis, Bacillus thuringiensis subsp kenyae, Bacillus thuringiensis subspecies kumamotoensis, Bacillus thuringiensis subspecies kurstaki, Bacillus thuringiensis subspecies kyushuensis, Bacillus t huringiensis subspecies japonensis, Bacillus thuringiensis subsp mexcanensis, Bacillus thuringiensis subspecies morrisoni, Bacillus thuringiensis subsp neoleonensis, Bacillus thuringiensis subsp nigeriae, Bacillus thuringiensis subsp ostriniae, Bacillus thuringiensis subsp pakistani, Bacillus thuringiensis subsp pondicheriensis, Bacillus thuringiensis subsp shandongiensis, Bacillus thuringiensis subsp silo, Bacillus thuringiensis subspecies sotto, Bacillus thuringiensis subsp subtoxicus, Bacillus thuringiensis subsp tenebrionis, Bacillus thuringiensis subsp thompsoni, Bacillus thuringiensis subsp tochigiensis, Bacillus thuringiensis subsp tohokuensis, Bacillus thuringiensis subsp tolworthi, Bacillus thuringiensis subspecies toumanoffi, Bacillus thuringiensis subsp wuhanensis, Bacillus thuringiensis subsp yunnanensis.  6. The method according to p. 4, characterized in that the strain of Bacillus thuringiensis is a strain of Bacillus thuringiensis subspecies kurstaki.  7. The method according to p. 1, characterized in that the pesticide obtained on the basis of bacteria of the genus Bacillus is the delta-endotoxin of Bacillus thuringiensis or a fragment thereof having pesticidal activity.  8. The method according to p. 7, characterized in that the delta-endotoxin of Bacillus thuringiensis or a fragment thereof having pesticidal activity is selected from the group consisting of cry I, cry II, cry III, cry IV, cry V and cry VI.  9. The method according to p. 8, wherein the Bacillus thuringiensis delta-endotoxin or a fragment thereof having pesticidal activity is a cry IA delta-endotoxin or a fragment thereof having pesticidal activity.  10. The method according to p. 8, wherein the Bacillus thuringiensis delta-endotoxin or a fragment thereof having pesticidal activity is a cry IC delta-endotoxin or a fragment thereof having pesticidal activity.  11. The method according to p. 1, characterized in that the pesticide obtained on the basis of bacteria of the genus Bacillus, is a spore of Bacillus thuringiensis.

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