KR19990022529A - How to prepare Bacillus insecticidal activity enhancer - Google Patents

Abstract

The present invention relates to a method for obtaining a factor that enhances the pesticidal activity of a virus having bacillus related insecticides, chemical insecticides and / or insecticidal properties.

Description

How to prepare Bacillus insecticidal activity enhancer

1. Field of Invention

The present invention relates to methods of obtaining and identifying factors that enhance the pesticidal activity of viruses with bacillus related pesticides, chemical pesticides and / or pesticidal properties.

2. Background of the Invention

Every year, millions of dollars are spent on pests that are harmful to agriculture, forests, and public health. Various methods have been used to control such pests.

One method is to use chemical pesticides with broad or varying activity. However, the use of chemical pesticides has many disadvantages. Specifically, because of their wide activity, these pesticides may destroy unwanted organisms such as beneficial insects and parasites against harmful pests. In addition, chemical pesticides are often toxic to animals and humans. In addition, the desired pest is often resistant to repeated exposure to such substances.

Another method is to control insect, fungus and weed infestation using biopesticides. Biological pesticides are naturally occurring etiologies and / or substances produced by these etiologies. The advantage of using biopesticides is that they are generally less harmful to organisms they are not intended for as a whole compared to chemical pesticides.

2.1. Bacillus thuringiensis

The most widely used biopesticide is Bacillus thuringiensis. Bacillus thuringiensis is a Gram-positive bacterium, a rod-form of motility that is widely distributed in nature, particularly in soil and insect-rich environments. Upon sporulation, Bacillus thuringiensis produces, by ingestion, a spore crystal inclusion body that is insecticidal against infectious insect larvae of the Lepidoptera, Diptera and Coleoptera orders. do. Inclusion bodies can vary in form, number and composition. They consist of one or more proteins (commonly referred to as delta-endotoxins) ranging in size from 27 to 140 kDa. Insecticidal delta-endotoxins are generally converted to smaller (cut) toxin polypeptides by the protease in the larvae intestine, which destroys the intestines and ultimately kills insects. Hofte and Whiteley, 1989, Microbiological Reviews 53: 242-255].

Several Bacillus thuringiensis strains are widely used as biological pesticides in the forest, agriculture and public health sectors. Bacillus thuringiensis subspecies kurstaki and Bacillus thuringiensis subspecies aizawai produce delta-endotoxins specific for lepidoptera. Delta-endotoxins specific for choloteera are produced by Bacillus thuringiensis subspecies tenebrionis (Krieg et al., 1988, US Pat. No. 4,766,203). In addition, Bacillus thuringiensis subspecies israelensis produces delta-endotoxins specific for Diphthera (Goldberg, 1979, US Pat. No. 4,166,112).

Also described are other Bacillus thuringiensis strains specific for Diphthera. Bacillus thuringiensis isolates toxic to Diphthera and Lepidoptera are described in Hodgman et al., 1993, FEMS Microbiology Letters 114: 17-22. SDS polyacrylamide gel electrophoresis of crystalline delta-endotoxin purified from the isolate revealed three protein classes associated with CryIA (b), CryIB and CryIIA toxins. In addition, Bacillus thuringiensis isolates that produce diphtheran-active crystals consisting of proteins of molecular weight 140, 122, 76, 72 and 38 kDa are described in Payne, 1994, US Pat. No. 5,275,815. EPO 480,762 describes five Bacillus thuringiensis strains, each active against diphtheran pests, each with a unique crystal delta-endotoxin pattern.

Several Bacillus thuringiensis strains have been described that have pesticidal activity against pests other than Lepidoptera, cholopterra and diphthera. Five Bacillus thuringiensis strains that produce delta-endotoxins that are toxic to nematodes are described in Edwards, Payne, and Soares, 1988, Eur. Pat. Appl. No. 0 303 426 B1. In addition, Bacillus thuringiensis strain, PS81F, which can be used to treat humans and animals that are hosts of parasitic protozoa, are described in Thompson and Gaertner, 1991, Eur. Pat. Appl. No. 0 461 799 A1. Also described are several Bacillus thuringiensis isolates that have activity against tick pests. These isolates produce crystals consisting of proteins ranging in molecular weight from 35 kDa to 155 kDa. Payne, Cannon, and Bagley, 1992, PCT Application No. WO 92/19106. In addition, activity against the tree of Hymenoptera [Payne, Kennedy, Randall, Meier, and Uick, 1992, Eur. Pat. Appl. No. 0 516 306 A2); Activity against pests of the neck of Hemiptera (Payne and Cannon, 1993, US Pat. No. 5,262,159); Activity against insecticidal pests (Hickle, Sick, Schwab, Narva, and Payne, 1993, US Pat. No. 5,262,159); And Bacillus thuringiensis strains having activity against pests of the order Phthiraptera (see Payne and Hickle, 1993, US Pat. No. 5,262,399). In addition, the blood-sucking parasite Damalinia eggs, isolated from Australian wool cuts, another Bacillus thuringiensis subspecies Kurstaki strain, WB3S, which is toxic to the Phthiraptera pest -16, see Drummond, Miller, and Pinnock, 1992, J. Invert. Path. 60: 102-103.

Delta-endotoxins are generally encoded by the cry (crystal protein) gene present on the plasmid. The cry gene is divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The main class is lepidoptera-specific (cryI); Lepidoptera- and Diphthera-specific (cryII); Choloptera-specific (cryIII); Diphthera-specific (cryIV) (Hofte and Whiteley, 1989, Microbiological Reviews 53: 242-255); Choloptera- and lepidoptera-specific (referred to as cryV gene by Taylor et al., 1992, Molecular Microbiology 6: 1211-1217) and nematode-specific (referred to as cryV and cryVI by Paytelsson et al., 1992, Bio / Technology 10: 271-275).

Delta-endotoxins are produced by recombinant DNA methods. The delta-endotoxin produced by the recombinant DNA method may or may not be in crystalline form.

Some strains of Bacillus thuringiensis have been found to produce solely insecticidal heat-stable insecticidal adenine-nucleotide homologs, known as β-exotoxin type I or thuringiencins. Sebesta et al., In H.D. Burges (ed.), Microbial Control of Pests and Plant Diseases, Academic Press, New York, 1980, pp. 249-281]. β-exotoxin was found in the supernatants of some Bacillus thuringiensis cultures. Its molecular weight is 701 and consists of adenosine, glucose and alaric acid. Farkas et al., 1977, Coll. Czechosslovak Chem. Comm. 42: 909-929; Luthy et al., In Kurstak (ed), Microbial and Viral Pesticides, Marcel Dekker, New York, 1982, pp. 35-72]. Its host range includes, but is not limited to, Musca domestica, Mamestra configurata Walker, Tetranychus urticae, Drosophila melanogasster ( Drosophila melanogaster) and Tetranychus cinnabarinus. The toxicity of β-exotoxin type I is thought to be due to the inhibition of DNA-derived RNA polymerase by competition with ATP. β-Exotoxin Type I is encoded by cry plasmids in five Bacillus thuringiensis strains. Levinson et al., 1990, J. Bacteriol. 172: 3172-3179. β-exotoxin type I is found in Bacillus thuringiensis subspecies thuringiensis serotype 1, Bacillus thuringiensis subspecies tollworthi serotype 9 and Bacillus thuringiensis subspecies darmstadiensis serotype 10 It was found to be produced by.

Another β-exotoxin, classified as β-exotoxin type II, is described in Levinson et al., 1990, J. Bacteriol. 172: 3172-3179. β-Exotoxin Type II was found to be produced by Bacillus thuringiensis subspecies morrisoni serotype 8ab and is active against Leptinotarsa decemlineata. The structure of β-exotoxin type II is not completely known, but β in that the pseudouridine residue is at the position of the adenine (where attachment to the ribose ring is at a position that can be occupied by the protons). -Significantly different from the structure of exotoxin type I [Levinson, in Hickle and Finch (eds.), Analytical Chemistry of Bacillus thuringiensis, ACS Symposium Series, Washington, DC, 1990, pp. 114-136. In addition, there is only one signal in the proton NMR spectrum corresponding to the nucleoside base (at 7.95 ppm) and no ribose-type anomer protein signal (5.78 ppm).

Other water-soluble substances isolated from Bacillus thuringiensis are α-exotoxins that are toxic to the larvae of Musca domestica. Luthy, 1980, FEMS Microbiol. Lett. 8: 1-7; Forsberg et al., 1976, Bacillus thuringiensis: Its, a variety of enzymes, including resitiase, chitinase and protease, which are expressed only in combination with β-exotoxin or delta-endotoxin. Effects on Environmental Quality, National Research Council of Canada, NRC Associate Committee on Scientific Criteria For Environmental Quality, Subcomittees on Pesticides and Related Compounds and Biological Phenomena; Sigma exotoxin, which has a structure similar to β-exotoxin and is also active against Leptinotarsa decemlineata (Prystas et al., 1975, Coll. Czechosslovak Chem. Comm. 40: 1775].

2.2. Zwittermicin

The substance was isolated from Bacillus cereus, which inhibits the growth of the plant disease Phytophthora medicaginis and reduces the infection of alpha waves (US Pat. Nos. 4,877,738 and 4,878,936). . No other activity is described. The following formula describes Zwittermicin A. He et al., Tet. Lett. 35: 2499-2502.

3. Purpose of invention

The technique is to increase the mortality of Bacillus thuringiensis formulations. Its method is to find new strains with increased mortality, engineer the strains, and design more effective formulations by combining Bacillus thuringiensis spores and crystals with new pesticidal carriers, chemical insecticides or enhancers. US Pat. No. 5,250,515, trypsin inhibitor. Accordingly, it is an object of the present invention to enhance the pesticidal activity of insecticides.

4. Summary of the Invention

The present invention comprises the steps of (a) culturing the Bacillus strain under suitable conditions;

(b) recovering the factor from the culture supernatant of step (a).

In certain embodiments, the Bacillus strain is selected from the group consisting of Bacillus subtilus, Bacillus licheniformis and Bacillus thuringiensis. In a preferred embodiment, the factors are in substantially pure form. As described herein, “substantially pure” factor means a factor containing less than 10% contaminants, eg, delta-endotoxin protein. Such substantially pure factors can be obtained by separating the factors, for example by column chromatography.

The factor obtained is a reinforcing agent. As defined herein, an "enhancer" has a significant pesticidal activity, for example LC ₅₀ (LC ₅₀ is the concentration of the substance required to kill 50% pests) as assayed by biological quantitation, about 300 μg / A substance that does not exhibit more than g, but acts to increase the insecticidal activity of the Bacillus related insecticide by at least about 50% and does not inhibit the development of the larvae. As mentioned in part 2, other substances capable of increasing pesticidal activity known in the art, such as trypsin inhibitors and exotoxins, have pesticidal activity.

In certain embodiments, the factor is water soluble. As defined herein, a substance or compound is “water soluble” as long as at least about 1 mg of substance can be dissolved in 1 ml of water. Factors may also enhance the pesticidal activity of chemical pesticides and / or viruses with bactericidal properties.

As defined herein, a "bacillus related pesticide" is a bacillus (eg, Bacillus thuringiensis or Bacillus subtilis) strain, spore, or substance, eg, a protein thereof that is active against or kills pests. Or microorganisms and acceptable carriers capable of expressing fragments, or Bacillus genes encoding Bacillus proteins or fragments thereof (eg, Bacillus thuringiensis delta-endotoxin) that are active against or kill pests [see: 5.2 below, examples of such a carrier]. Pests can be, for example, insects, nematodes, mites or snails. Microorganisms that are capable of expressing Bacillus genes that are active against pests or kill Bacillus proteins or fragments thereof that are capable of killing pests may be phylloplane (surface of plant leaves) and / or rhizospheres (soil around plant roots) and / or Proper maintenance of the Bacillus genes encoding Bacillus proteins or fragments thereof that live in the aquatic environment, can successfully compete with wild-type microorganisms in certain environments (crops and other insect habitats) and are active against or kill pests. Provide expression. Examples of such microorganisms include, but are not limited to, bacteria such as, for example, the genus Bacillus, Pseudomonas, Erwinia, Serratia, and Klebsiella. Genus Klebsiella, genus Xanthomonas, genus Streptomyces, genus Rhizobium, genus Alcaligenes and genus Clostridium; Birds, for example Cyanophceae, Primnesiophyceae, Xanthophyceae, Raphidophyceae, and Bacillariophyceae Eustigmatophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae; And fungi, in particular yeasts such as Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula Genus and Aureobasidium genus.

As defined herein, "sterilization activity" measures the amount of activity against a pest that kills or inhibits the growth of a pest or protects a plant from pest infection.

The factors obtained can be formulated into compositions comprising the factors and pesticidal carriers and bacillus associated pesticides, chemical pesticides with pesticidal activity and / or viruses. These compositions can be used to control pests and to expose pests to compositions comprising factors and pesticidal acceptable carriers to reduce the insect's resistance to bacillus-associated pesticides or to enhance the pesticidal activity of bacillus-related pesticides. .

The invention also comprises the steps of (a) culturing a Bacillus strain;

(b) recovering the culture supernatant of step (a); and

(c) assaying the supernatant of step (b) for enrichment of the Bacillus related pesticide.

5. Brief Description of Drawings

1 schematically depicts the general process used for the purification of Ia.

2 shows the ¹³ C NMR spectrum of Ia.

3 shows the proton NMR spectrum of Ia.

4 shows the results of nOe experiments on acetylated derivatives of Ia.

6. Detailed description of the invention

Factors of the present invention enhance the pesticidal activity of a Bacillus related insecticide and may have a molecular weight of about 350 to about 1200 or in certain embodiments about 350 to about 700.

The factors of the present invention enhance the pesticidal activity of Bacillus related pesticides from about 1.5 times to optionally about 1000 times, preferably from about 100 times to about 400 times. In certain embodiments, a factor of the invention is, but is not limited to, CryI (including but not limited to CryIA, CryIB, and CryIC), CryII, CryIII, CryIV, CryV, or full length forms or proteolysis Enhances the insecticidal activity of Bacillus thuringiensis delta-endotoxin, including the cleaved form of CryVI, treated by about 1.5- to 1000-fold. In the most particular embodiment, the factor of the present invention enhances Bacillus thuringiensis delta-endotoxin from about 100 to about 400 times. In addition, the factors of the present invention may enhance the pesticidal activity of chemical pesticides and / or viruses having pesticidal properties.

In addition, the factors of the present invention are water soluble, ie, stable to water at temperatures of about 100 ° C. or less for at least about 5 minutes, stable when exposed to direct sunlight for at least about 10 hours, and / or about 10 days. Stable at a pH of 2. The factor of the present invention includes 13 carbons. In addition, the factors of the present invention may have ¹ H NMR shifts at δ 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 the most particular embodiment, said factor is formula la or a salt thereof.

Salts of the present invention may enhance Bacillus related insecticides.

6.1. Obtaining Factors

Factors of the present invention may be obtained in shake flasks or fermenters from Bacillus strains (e.g., Bacillus subtilis, Bacillus licheniformis and Bacillus thuringiensis). In the present invention, the factors of the present invention include, but are not limited to, Bacillus thuringiensis subspecies kurstaki, Bacillus thuringiensis subspecies aizawai, Bacillus thuringiensis subspecies galleriae, and Bacillus turine Giensis subspecies etomocidus, Bacillus thuringiensis subspecies tenebrionis, Bacillus thuringiensis subspecies thuringiensis, Bacillus thuringiensis subspecies alesti, Bacillus thuringiensis subspecies Canadiensis, Bacillus thuringiensis subspecies Darmstadien Darmstadiensis, Bacillus thuringiensis subspecies dendrolimus, Bacillus thuringiensis subspecies finitimus, Bacillus thuringiensis subspecies kenyae, Bacillus thuringiensis subspecies morrisoni, Bacillus thuringiensis subspecies subtoxicus, Bacillus thuringiensis subspecies toumanoffi and Bacillus thuringiensis subspecies can be obtained from supernatants of Bacillus thuringiensis cultures including israelensis. In a preferred embodiment, the factor can be obtained from the supernatant of Bacillus thuringiensis subspecies Kurstaki, Bacillus thuringiensis subspecies Aizawa, or Bacillus thuringiensis subspecies Galleria or variants thereof substantially having the same enhancing activity. In certain embodiments, the factors of the present invention are Recovered from the cry ^- spo ^- variant of the Silus thuringiensis subspecies Kurstaki.

Bacillus can be cultured using culture and fermentation techniques known in the art. Rogoff et al., 1969, J. Invertebrate Path. 14: 122-129; Dulmage et al., 1971, J. Invertebrate Path. 18: 353-358; Dulmage et al., In Microbial Contral of Pests and Plant Diseases, H. D. Burges, ed., Academic Press, N. Y., 1980]. Once the cycle is complete, the supernatant can be recovered by separating Bacillus thuringiensis spores and crystals from the culture (fermentation) broth by techniques known in the art, such as centrifugation and / or ultrafiltration. The factors of the present invention are contained in the supernatants that can be recovered by methods known in the art, such as ultrafiltration, evaporation and spray-drying. This process is described in more detail below.

Purification of the factor includes, but is not limited to, chromatography (eg, ion exchange, affinity and size fractional column chromatography), electrophoretic processes, fractional dissolution, extraction, or other standard techniques known in the art. It can be performed by various processes.

Reinforcing activity of Bacillus-related pesticides, viruses with pesticidal activity, or factors with pesticidal activity of chemical pesticides against various pests is known in the art, including, for example, artificial insect diet, artificial dietary application, leaf painting, Assay can be used using leaf dipping and leaf spray. Specific examples of such assays are provided in the following seven sections.

6.2. Compositions Containing Factors

Factors obtained alone; With Bacillus strains, spores, proteins or other substances thereof that are active against pests or kill pests or protect plants from pests as described above; It can be formulated with pesticide compositions such as suspensions, solutions, emulsions, powders, dispersible granules, wet powders, emulsifiable thickeners, aerosols or soaked granules together with chemical pesticides and / or insecticide viruses and acceptable carriers. . The Examples of such Bacillus strains, without limitation, Bacillus Turin group N-Sys subspecies Kur Starkey (kurstaki) (Abbott Labo gelato Leeds, Inc. Corporation under the trade name DIPEL ^TM, Novo Nordisk A / S, Inc. under the trade name BIOBIT ^TM, Novo Nordisk Commercially available under the trade names FORAY ^™ , Novo Nordisk A / S's BIOCOT ^™ , Mycogen's tradename MVP ^™ , Novo Nordisk A / S's trade name BACTOSPEINE ^™ , and Sandos' trade name THURICIDE ^™ ); Bacillus thuringiensis subspecies aizawai (commercially available under the tradename FLORBAC ^™ from Novo Nordisk A / S and the tradename XENTARI ^{™ from} Abbott Laboratories, Inc.); Bacillus thuringiensis subspecies tenebrionis (available under the tradename BVODOR ^™ from Novo Nordisk A / S, the tradename TRIDENT ^{™ from} Sandos, and the trade names M-TRAK ^™ and M-ONE ^™ from Mycogen); Bacillus (sold by Novo Nordisk A / S, Inc. under the trade name BACTIMOS SKEETAL ^TM or ^TM, Sandoz, Inc. under the trade name ^TM TEKNAR and Abbott Labo gelato Leeds, Inc. Corporation under the trade name ^TM VECTOBAC) Turin group N-Sys subspecies Israel alkylene sheath (israelensis); Bacillus thuringiensis Kurstaki / Tennebrionis (available under the tradename FOIL ^™ from ECOGEN); Bacillus thuringiensis Kurstarki / Aisawa (available under the trade name CONDOR ^{™ from} ECOGEN Co., Ltd. and AGREE ^TM under the trade name of Shiba Gai Corporation) and Bacillus thuringiensis Kurstaki / Kurstaki (available under the trade name CUTLASS ^TM from ECOGEN Co.) do. Bacillus related proteins can be selected from the group consisting of, but not limited to, CryI, CryII, CryIII, CryIV, CryV and CryVI. Chemical pesticides include, for example, insect growth regulators such as diflubenzuron, carbamates such as thiodicarb and metomil, organophosphates such as chlorpyriphos, pyrethroids such as cipermethrin and espen valerate, creole Inorganic fluorine and pyrrole such as light. Insect pathogenic viruses include baculoviruses such as Autographa californica nuclear polyhedrosis virus (NPV), Syngrapha falcifera NPV, Lymantra dispar NPV, Orgyia pseudotsugata NPV, Spodoptera exigua NPV, Neodiprion lecontei NPV, Neodiprion sertifer NPV, Harishina Brillians brillians) NPV and Endopiza viteana Clemens NPV.

In a composition comprising the substance and a Bacillus related insecticide, the substance comprises about 0.1 g / BIU or a factor of 0.05 g / g of Bacillus delta-endotoxin and spores to about 300 g / BIU or g of Bacillus delta-endotoxin and spores. It may be present in an amount of 150 g of sugar substance, preferably 2 g / BIU or 1 g of substance per g of Bacillus delta-endotoxin and spores. As defined herein, "BIU" is a billion international units measured by biological quantification. Biological quantification compares samples with standard Bacillus-related substances using Trichoplusia ni or other pests as the standard test insect. Efficacy is measured by dividing the reference standard LC ₅₀ and then multiplying by the relevant standard efficacy.

In another embodiment, the compositions of the present invention may comprise a Bacillus supernatant in a substantially pure form or anhydrous concentrated or liquid form and a pesticidal acceptable carrier, examples of which are described below. The composition of the present invention can be applied separately to plants, for example transgenic plants. Specifically, the composition may be applied to plants that contain and express the Bacillus thuringiensis gene in advance. In another embodiment, the composition can be applied to plants that have been previously exposed to the Bacillus thuringiensis composition. In another embodiment, the composition can be applied to other environments of the twin insect (s), such as water or soil. The material of the present invention is present in the composition at a concentration of about 0.001% to about 60% (w / w).

Apart from pest control, compositions comprising the material and pesticidal acceptable carriers can be used to reduce the resistance of the pest to pesticides. On the other hand, the composition can be used to enhance Bacillus-related pesticides. In one embodiment, the composition may be applied in an amount of from 2 g / BIU to about 300 g / BIU of material, simultaneously with the pesticide. In another embodiment, the composition can be applied as an adjuvant within about 24 hours after spraying the pesticide to extend the efficacy of the remaining pesticide.

Such compositions described above include surfactants, inert carriers, preservatives, wetting agents, dietary stimulants, attractants, encapsulating agents, binders, emulsifiers, dyes, U.V. Prophylactic protectants, buffers, flow agents, or other ingredients are added to facilitate product manipulation and, in particular, application to the desired pest.

Suitable surfactants include, for example, carboxylates such as metal carboxylates of long chain fatty acids; N-acylcarcosinates; Mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; Fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium cetyl sulfate; Ethoxylated alkylphenol sulfates; Lignin sulfonate; Petroleum sulfonate; Alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower alkyl naphthalene sulfonates such as butyl-naphthalene sulfonate; Salt or sulfonated naphthalene-formaldehyde condensates; Salts of sulfonated phenol-formaldehyde condensates; Or one or more complex sulfonates such as amide sulfonates, for example sulfonated condensation products of oleic acid and N-methyl taurine or dialkyl sulfosuccinates, such as sodium sulfonate or dioctyl succinate. Nonionic agents include condensation products of fatty acid esters, fatty alcohols, fatty acid amides or fatty-alkyl- or alkenyl-substituted phenols with ethylene oxide, fatty esters of polyhydric alcohol esters, for example sorbitan fatty acid esters, and such esters. Condensation products with ethylene oxide such as polyoxyethylene sorbitan fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylene alcohols such as 2,4,7,9-tetraethyl-5-decine-4 , 7-diol, or ethoxylated acetylene glycols. Examples of cationic surface-active agents include, for example, fatty mono-, di-, or polyamines such as acetates, naphthenates or oleates; Oxygen-containing amines such as amine oxides of polyoxyethylene alkylamines; Amide-linked amines prepared by condensation of carboxylic acids with di- or polyamines; Or quaternary ammonium salts.

Examples of inert materials include minerals such as kaolin, mica, gibsome, puttylizers, phyllosilicates, carbonates, sulfates, or phosphates; Organics such as sugars, starches or cyclodextrins; Or vegetable substances such as wood, cork, powdered corn stalks, rice bran, peanut shells, and walnut bark.

The compositions of the present invention may be present in a form suitable for the designated field or as a concentrate or base composition which must be diluted with a suitable amount of water or other diluent prior to application. Insecticidal concentrations may vary depending on the nature of the particular formulation, specifically concentrate or directly used. The composition contains 1 to 98% of a solid or liquid inert carrier and 0 to 50%, preferably 0.1 to 50% of a surfactant. These compositions may be administered in the indicated ratios for commercial products, preferably about 0.01 to 5.0 pounds per acre for anhydrous form and 0.01 to 25 pints per acre for liquid form.

In a further embodiment, the Bacillus thuringiensis crystalline delta-endoxin and / or factor is preformulated to sustain pesticidal activity when applied to the environment of the target pesticide, unless the pretreatment is harmful to the crystalline delta-endotoxin or surface. Can be processed. Such treatment may be carried out by chemical and / or physical means so long as the treatment does not adversely affect the properties of the composition (s). Examples of chemical reagents include, but are not limited to, halogenating agents; Aldehydes such as formaldehyde and glutaraldehyde; Anti-infective agents such as zephyran chloride; Alcohols such as isopropranol and ethanol; And histological fixatives such as Bowin fixatives and Heli fixatives. Humason, Animal Tissue Techniques, W.H. Freeman and Co., 1967].

The compositions of the present invention can be applied directly to plants, for example by spraying or spraying, if the pests start appearing on the plants before the emergence of the pests as a protective means. Plants protected within the scope of the present invention include, but are not limited to, cereals (wheat, barley, rye, oats, rice, sugar cane and related crops), sugar beets (sugar beet and fodder beets), nuclear fruits, fruit and soft fruit. (Apples, pears, plums, almonds, cherries, strawberries, raspberries and blackberries), legumes (alpha, green beans, lentils, peas, soybeans), oil plants (flat, mustard, poppy, olives, sunflowers, Coconuts, castor oil plants, cocoa beans, peanuts, gourds (cucumbers, pumpkins, melons), fiber plants (cotton, flax, hemp, jute), citrus fruits (orange, lemon, grapes, tangerines), vegetables (spinach, Lettuce, asparagus, cabbage and other brassica, carrots, onions, tomatoes, potatoes), lauraseade (avocado fruit, cinnamon, camphor), deciduous and coniferous trees (linden, yew, oak, alder, poplarse, Plants such as birch, fir, larch, pine), or corn, The digwa plants, tobacco, walnuts, coffee, sugar cane, tea, vines, hops, bananas and natural rubber plants, and scenic stream is included. The composition may be applied to leaves, furrows, spray granules, shelters, or the like, or to soil absorption. In general, it is important to control pests well in the early stages of plant growth because the period at which plants are most damaging is the early stages of plant growth. Sprays or fines can typically contain another pesticide if deemed necessary. In a preferred embodiment, the composition of the present invention is applied directly to the plant.

The composition of the present invention may be used in the order of Lepidoptera, for example, Acroia grisella, Acleris gloverana, Acleris variana, Adoxophys aura Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria, Amyelois transitella, Anagasta quenni Anagasta kuehniella, Anasia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips ), Argyrotaenia species, Athetis mindara, Bombyx mori, Buculcultrix thurberiella, Cadra cautella, Corey Choristoneura species, Cochylis hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Sidi Po Cydia pomonella, Dataana integerrima, Dendrolimus sibericus, Desmia funeralis, Diaphania hyalinata, Diaphania nitidali Diaphania nitidalis, Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria, Eoreuma loftini, Ephestia elutella, Erannis tiliaria, Estigmene acrea, Eulia salubricola, Eupoecilia ambiuela lia ambiguella, Euprotis chrysorrhoea, Euxos messoria, Galleria mellonella, Grappolita molesta, Harrisina americana Helicobpa subflexa, Helicobpa zea, Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantria cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellaria lugubrosa Leucoma salicis, Lobesia botrana, Loxostege sticticalis, Lymantria dispar, Macalla thyrsisalis, Malacosoma species, Mamestra brassicae, Mamestra configurata, Manduka quinquemaculata, dumplings Manuca sexta, Maruca testulalis, Melanchra picta, Operaperhtera brumata, Organia species, Austrian nubilalis Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes, Pectinophora gossypiella, Phryganidia californica, Phyllonolic Phyllonorycter blancardella, Pieris napi, Pieris rapae, Platypena scabra, Platinota flouendana, Platinta snow Platynota sultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Schdalele Pseudaletia unipuncta, Pseudoplusia includens, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella , Spilonoota ocellana, Spodoptera species, Thaurnstopoea pityocampa, Tineola bisselliella, Trichoflucia ni ), Udea rubigalis, Xylomyges curialis, Yponomeuta padella; Diptera, for example Aedes species, Andes vittatus, Anastrepha ludens, Anastrepha suspensa, Anopeles barberry ( Anopheles barberi, Anopheles quadrimaculatus, Armigeres subalbatus, Califora stygian, Califora vicina, Seratitis capita (Ceratitis capitata), Chironomus tentans, Chrysomya rufifacies, Cochliomyia macellaria, Culex species, Coolista innornata (Culiseta inornata), Dacus oleae, Delia antiqua, Delia platura, Delia radicum, Drosophila melanogaster, Euphedes Eupeodes corollae, Glossina austeni, Glossina brevipalpis, Glossina fuscipes, Glossina morsitans centralis, Glossina Glossina morsitans morsitans, Glossina moristans submorsitans, Glossina pallidipes, Glossina papalis gambiensis pal, Glossina palpalis palpalis, Glossina tachinoides, Haemagogus equinus, Haematobia irritans, Hypoderma bovis, Hypoderma bovis Hypoderma lineatum, Leucopis ninae, Lucilia cuprina, Lucilia seric ata), Lutzomyia longlpaipis, Lutzomyia shannoni, Lycoriella mali, Mayetiola destructor, Muska Otumnalis Musca autumnalis, Musca domestica, Neoobellieria spp., Nephrotoma suturalis, Ophyra aenescens, Paenicia sericata sericata, Phlebotomus species, Phormia regina, Sabethes cyaneus, Sarcophaga bullata, Scatophaga stercoraria, It may be effective against insect pests of Stomoxys calcitrans, Toxorhynchites amboinensis, and Tripteroides bambusa. However, the compositions of the present invention can also be used in the Coleoptera tree, for example, Leptinotarsa species, Acanthoscelides obtectus, Callosobruchus chinensis. , Epilachna varivestis, Pyrrhalta luteola, Silas formicarius elegantulus, Listronotus oregonensis, Cytophile Sitophilus species, Cyclocephala borealis, Cyclocephala immaculata, Macrodactylus subspinosus, Popillaia japonica, resortogus Rhizotrogus majalis, Alphitobius diaperinus, Palorus ratzebrugi, Tenebrio Molitor brio molitor, Tenebrio obscurus, Tribolium castaneum, Tribolium confusum, Tribolius destructor; Acari, for example Oligonychus pratensis, Panonicus ulmi, Tetranychus urticae; Hymenoptera, for example Iridomyrmex humilis, Solenopsis invicta; Isoptera, for example Reticulitermes hesperus, Reticulitermes flavipes, Coptotermes formosanus, Jutermopsis eye Zotermopsis angusticollis, Neotermes connexus, Incisitermes minor, Incisitermes immigrans; Siphonaptera, such as Ceratophyllus gallinae, Ceratophyllus niger, Nosopsyllus fasciatus, Leptopsylla segnis, Ctenocephalides canis, Ctenocephalides felis, Echicnophaga gallinacea, Pulex irritans, Xenopsylla cheopis Xenopsylla vexabilis, Tunga penetrans; And insect pests of Tylenchida, for example Melodydogyne incognita, Pratylenkus penetrans.

The following examples are presented for illustrative purposes and are not intended to be limiting.

7. Example: Characteristics of Ia

As described in detail herein, Ia is recovered and purified. Characterization of Ia is described in detail below.

7.1. Recovery and Purification of Ia

Bacillus thuringiensis subspecies kurstaki strain EMCC0086 (deposited as B-21147 in NRRL) was soaked in a carbon source such as starch, hydrolyzed starch or glucose, protein, hydrolyzed protein or corn soaked at 30 ° C. for 72 hours. It is fermented in a medium composed of a nitrogen source such as liquid. Production of Ia is detected 13 hours after fermentation. Peak activity is found at about 30 hours.

Supernatants from Bacillus thuringiensis subspecies Kurstaki fermentation are recovered by centrifugation and then fractionated by ultrafiltration through a 30 kDa MW-CO membrane using the Rhone Poulenc UF system. 30 kDa filtration removes all residual cell fragments, crystal delta-endotoxins, spores, and soluble proteins greater than 30 kDa molecular weight. The permeate is concentrated 10 times by evaporation. The permeate is centrifuged, followed by 0.2 micron filtration to further remove insoluble material from the broth to obtain a clear broth containing Ia.

Purification of Ia until homogeneous is carried out using the multistage purification process shown schematically in FIG. 1. In conjunction with the recovery process shown above, purification is carried out using a 5 kDa ultrafiltration step. Permeate from 5 kDa ultrafiltration is adsorbed to sulfopropyl (SP) cation exchange resin and eluted with ammonium acetate solution. The compound is then concentrated about 30-fold by lyophilization and salts and other contaminants are removed with a BioRad P2 size fractionation column. The pool from the P2 column is run on a high resolution SP HPLC cation exchange column to obtain a homogeneous compound. Contaminated salts are removed repeatedly by lyophilization.

Activity is monitored by Spodoptera exigua microbiopsy and purity is measured by capillary electrophoresis. Samples consisting of 50 μl of Ia and 50 μl of CryIA (c) protein (15 μg / ml) purified from BIOBIT ^™ FC (100 μl) are applied to each well of a freezing tray containing solidified artificial insect food. The trays containing the various samples are air dried. Two to four second or early tertiary sporofterae excigua are added to the wells containing the dried sample. The wells are sealed with perforated mylar and incubated at 30 ° C. for 2-3 days. Then, the rate of development inhibition and lethality is recorded. Typically, five wells are run for each sample.

7.2. Structure description

The active compounds have been found to be water soluble but insoluble in organic solvents. It turns positive and reacts with ninhydrin as demonstrated by silica thin layer chromatography. ¹³ C and proton NMR of this compound are shown in FIGS. 2 and 3, respectively. ¹³ C NMR experiments revealed 13 carbons present (see 3-trimethylsilyl propionic acid). DEPT experiments determined that there are three quaternary carbons (C), seven methines (CH), three methylenes (CH ₂ ) and no methyl groups (CH ₃ ). Proton coupling experiments such as 1-D decoupling and COSY are used to identify one large spin system containing eight carbons. There is also a smaller spin system of two carbons. Carbon correlation experiments (HMBC) allow the assignment of each proton resonance in a molecule to its attached carbon.

Treatment of the active compound (13 mg) with acetic anhydride in pyridine results in less polarized acetylated derivatives. This derivative is purified by HPLC to give 3 mg of pure acetylated derivative. Mass spectral analysis showed that the derivative had seven acetates and a molecular weight of 690 (the molecular weight of the active compound is 396), indicating that a large number of nitrogen was present. In addition, fragments containing 6 acetate and 5 acetate were detected. The 5 and 6 acetate daughter ions are 645.2594 (6 acetate) and 607.2519 (5 acetate) representing the molecular formula C ₁₃ H ₂₈ N ₆ O ₈ for Ia.

Treatment of the active compound (13 mg) with 6N HCl provides ninhydrin positive derivatives. These results indicate the presence of amide bonds. Art derivative is the R _f value as determined by thin layer chromatography, R _f is the same as the value of 2, 3-diaminopropionic acid. These results suggest the presence of 2,3 diaminopropionic acid with NMR data.

Another technique used in the analysis of Ia is nOe (nuclear overhauler effect), which can detect proton proximity to each other over space. nOe is carried out on the acetylated derivatives of Ia. In a two-dimensional nOe experiment (NOESY), NOE is observed between N-H protons and 5.17 protons at 8.06 ppm (FIG. 4).

The following structural formula describes formula (Ia).

It can be classified as ureido amide. The constituent comprises two amides, urea, two amino and five hydroxyls. It contains seven chiral centers.

7.3. Characteristics of Ia

Isolated Ia has been shown to enhance the activity of Bacillus thuringiensis subspecies Kurstarki and Bacillus thuringiensis subspecies aizawai crystalline delta-endotoxin pesticidal protein, regardless of the form of the pesticidal protein. The insecticidal activity of formulated Btk, isolated crystals, full length (molecular weight 130 kDa) or truncated CryIA protein (molecular weight about 65 kDa) was all enhanced. The activity of CryII and CryIC inclusion bodies was also enhanced. It has also been found to enhance the activity of the individual cleaved CryIA (a), (b) and (c) proteins. The incubation time of Ia with the Cry protein was found to be insignificant for bioactivity. However, Ia is inactive alone. Enrichment levels were found to be 100-200 fold for truncated CryIA protein, CryII and CryIC inclusions and about 320 fold for full length CryIA (c) (see Tables I and II, respectively). Specifically, when Ia alone contains 240 μg per ml of CryIA (c) for the full length protein, 0.75 μg per ml of CryIA (c) shows the same insect lethality / development value. In the case of a CryIA (c) cutting and provides the same to the sample compared to that of the OD ₂₈₀ is 0.075 when blended with the Ia, the same growth is OD ₂₈₀ of 0.0006 Low levels of the tested alone CryIA (c). CryII inclusions provide the same developmental inhibition values and similar mortality at concentrations of 0.6 μg / ml when combined with Ia, while at 75 μg / ml when used alone, and 125 times enhanced. At 0.3 μg / ml with Ia, the CryIC inclusion body alone gives CryIC protein a similar mortality and developmental inhibition value as 75 μg / ml, which means that it is 250-fold enhanced. The concentration of cleaved CryIC protein showed mortality following Ia addition.

Ia is boiled for 5 minutes 7.1. It was found to be stabilized by biopsy as described in the section, but lost all activity upon autoclaving (> 190 ° C). It also stabilizes when exposed to direct sunlight for at least 10 hours. Ia is stable for 3 days at pH 2 but unstable at pH 12. Exposure to periodic acid or concentrated HCl has been found to lose all activity.

7.4. Explanation of Other Subspecies of Bacillus thuringiensis and Other Bacilli Species

Several Bacillus species are evaluated for the production of Ia. The strain is fermented at 30 ° C. for 72 hours in a medium consisting of carbon source such as starch, hydrolyzed starch or glucose and nitrogen source such as protein, hydrolyzed protein or corn steep liquor. Supernatants are tested for Ia production using the Spodotera exigua microbiopsy described above. Bacillus thuringiensis subspecies Aizawai strain EMCC0087 (deposited as NRRL B-21148 in NRRL) and Bacillus thuringiensis subspecies galleriae (deposited in NRRL) have the same concentration as Bacillus thuringiensis subspecies Kurstaki Was found to produce Ia.

In addition, Ia is the ratio as measured by capillary electrophoresis. Subtilis, b. Cereus, Bacillus thuringiensis subspecies Alessi, Bacillus thuringiensis subspecies canadienssis, Bacillus thuringiensis subspecies Darmstadiensis, Bacillus thuringiensis subspecies dendrolimus, Bacillus thuringiensis subspecies entomosidus, Bacillus thuringin Gynesis subspecies Fininimus, Bacillus thuringiensis subspecies Israelensis, Bacillus thuringiensis subspecies Keniae, Bacillus thuringiensis subspecies Morrison, Bacillus thuringiensis subspecies subtoxins, Bacillus thuringiensis subspecies Teneblionis , Bacillus thuringiensis subspecies Thuringiensis and Bacillus thuringiensis subspecies Kurstaki cry- spo- variants.

Specifically, the level of Ia is quantified using a Beckman P / ACE capillary electrophoresis system with 50 μm × 57 cm uncoated capillary, 0.2 M phosphate pH 6.8 buffer, a voltage of 15 KV and detection at 200 nm. Sample volume is 20 nl at 25 min run time.

Standard curves are generated using Ia purified at standard levels of 1.25 mg / ml, 0.625 mg / ml, 0.3125 mg / ml, 0.156 mg / ml and 0.078 mg / ml. Linear measurement curves are formed. The equation y = mx + b is used to represent the Ia concentration in each sample.

Prior to each operation, the capillary is ejected with working buffer (0.2M phosphate, pH 6.8) for 3 minutes. After operating for 25 minutes, the capillary is ejected with 1N NaOH for 1 minute and with filter paper HPLC water for 1 minute, 0.5 M phosphoric acid for 3 minutes and filter paper HPLC water for 1 minute. The area under each peak is integrated, the peak area is measured and the final concentration is calculated from the standard curve.

7.5. Evaluation of Bacillus thuringiensis product

The amount of Ia present in various commercially available Bacillus products is determined by capillary electrophoresis as described in section 6.4 above. BACTOSPEINE ^™ , JAVELIN ^™ , NOVODOR ^™ , SPHERIMOS ^™ , BACTIMOS ^™ , FORAY ^™ , FLORBAC ^™ and BIOBIT ^™ are obtained from Novo Nordisk A / S. XENTARI ^™ and DIPEL ^™ are obtained from Abbott Laboratories. AGREE ^™ is from Ciba-Geigy, MVP ^™ is from Mycogen and CUTLASS ^™ is from Ecogen.

The results are shown in Table III below, indicating that Ia is present in different amounts ranging from 0.001 g Ia / BIU to 0.071 g Ia / BIU.

7.6. Feeding Biopsy

B.T.K. Its activities were three-week-old Spodoptera exigua larvae, two-week-old Helicoberra Zea larvae, three-week-old Spodovtera pruperifera larvae, two-week-old Heliotis nonresense larvae, three-week-old Tricoccussia larvae, Sia influence larvae, three-week-old flutella xyllostella larvae, three-week-old Spodoptera litoralis and three-week-old Mamestra brassica are used by artificial food introduction biopsies.

Feed introductory biopsies are performed to determine the level of fortification with the addition of Ia to the Bacillus thuringiensis product and to establish the extent of the affected insects. In experiments with high concentrations of Ia (7.4-23.7 Ia / BIU) against Spodoptera excigua, BIOBIT ^™ (FC was used to determine the flow concentration using purified Ia (70% active gradient, 30% acetate count ions). It is enhanced. Residual data presented in Table IV show fortification of BIOBIT ^™ HPWP (high potency wet powder) using Ia (0.658% active gradient). s. Litoralis and M. Brassica is tested using FLORBAC ^™ HPWP and Ia.

Various B.T. The product is quantified and Ia is added to yield 0.1 to 237 g / BIU of Ia. Volume is adjusted with 0.1% Tween ^TM. Samples are sonicated for 1 minute and then diluted to final volume. Pure samples (no Ia) and reference materials are also prepared. Reference substances include Btk HD-1-S-1980 (obtained from NRRL) and JAVELIN ^™ WG, designated as potency of 53,000 sporofera units / mg (SU), designated as potency of 16,000 international units (IU) per milligram. .

Standard artificial foods consisting of water, agar, sugar, casein, wheat germ, methyl parabens, sorbic acid, washed oil, cellulose, salts, and vitamins are prepared in a 20 L heated kettle. This is a diet sufficient to test 10 to 12 samples using each test substance at seven different concentrations. B.t. Sterile dilution of the solution yields a 16 ml aliquot. Each aliquot is added to 184 g of dissolved food. The mixture is homogenized continuously and then poured into a plastic tray containing 40 individual cells. Three control trays are prepared for each food batch. Once the food has cooled and solidified, one insect of known age (2 to 3 weeks old) is added to each cell and the tray is covered with a perforated transparent mylar sheet. The tray is placed on a shelf and incubated at 28% relative humidity at 28 ° C. for 4 days.

After 4 days, insect mortality is assessed. Each tray provides a sudden wind at the top of the table, and the stationary larvae count as dead. Lethality is calculated and data analyzed by parallel probit analysis. LC ₅₀ , LC ₉₀ , slope, deviation and efficacy of the regression line are evaluated. The sample is run until 3 agonists are present within 3 minutes or 20% of the mean calculated for each sample. To calculate the increase in activity associated with each concentration of Ia, the LC ₅₀ of the Bt / Ia sample is correlated to reflect the amount of Bt in the sample. By dividing by two pure sample interaction of the LC ₅₀ of A LC _50, to obtain the reduction ratio of the LC ₅₀ associated with Ia.

The following procedure is used to assay Lobesia bothrana. Collect vines from vines attacked by Lovesia Botlana and remove larvae. Serial dilutions of Ia (250 μg / ml, 500 μg / ml and 1000 μg / ml) are prepared in water. Place any larvae in the middle of the Petri dish. When larvae are eaten, they are transferred to a Petri dish with freshly cut grapes. Larvae are stored at 22 ° C. for 3-4 days.

As shown in Table IV, a significant decrease in LC ₅₀ was observed in all species.

Enhancement of the various products for Spodoftera excigua by Ia is measured using the food introduction biopsy described above. The amount of Ia added / BIU product is shown in Table V below. The Ia / Bt product mixture is introduced into the agar-based wheat germ casein feed. Insects are placed in the prey for 4 days and maintained at 28 ° C. Record mortality and analyze using Probit Analysis. LC ₅₀ , LC ₉₀ and enhancement capacity are calculated from the binding product without Ia. The results presented in Table V indicate that Ia enhances various Btk and Bta products obtained from various sources. Bt strains contained in these products are described in section 5.2 above.

7.7. Leaf biopsy

Leaf biopsies are performed with 2 week old Spodoptera psigua larvae on broccoli flora using BIOBIT ^™ FC and Ia. The ratio of Ia to BIOBIT ^™ FC is the same 2g Ia / BIU BIOBIT ^™ FC. The treatment is applied to broccoli plants by track spraying at a carrier dose of 20 gallons per acre. After spraying, the larvae are removed from the plants, the deposits are dried and infected with the two-week-old Sofodotera exigua larvae. The results are shown in Table VI below. 100% mortality was observed at a rate of 8.7BIU / hr BIOBIT ^™ FC + Ia and BIOBIT ^™ FC alone killed 92% at 58.8BIU / hr and 8% at 17.6BIU / hr. In addition, the treated plants are exposed to direct sunlight for 8 hours, and then the larvae are separated and infected. After 8 hours in the sun, BIOBIT ^™ FC alone gives a 27% mortality at 58.8BIU / hr, while BIOBIT ^™ FC + Ia provides 100% mortality at 8.7BIU / hr.

Leaf assays are performed with early 4 week old larvae, with BIOBIT ^TM FC alone giving 75% mortality at 52BIU / hr and BIOBIT ^TM FC (FC being flow concentration) + Ia giving 100% mortality at 13BIU / hr. .

Leaf biopsy process BIU / hr % Mortality Larvae BIOBIT ^TM FC 58.8 92% 2 BIOBIT ^TM FC 17.6 8% 2 BIOBIT ^TM FC + Ia 8.7 100% 2 BIOBIT ^TM FC +8 hours sun ray 58.8 27% 2 BIOBIT ^TM FC + Ia + 8 Hour Sun Rays 8.7 100% 2 BIOBIT ^TM FC 52 75% 4 BIOBIT ^TM FC + Ia 13 100% 4

7.8. Packing test

While the BIOBIT ^TM FC alone trials (Spodoptera guar) for teaching Article kidney bean provides 51% relief from 70BIU / hr, 2g Ia / BIU BIOBIT TM FC is to provide a 89% relief from 40BIU / hr Proved (compared to no treatment). JAVELIN ^™ WG provides 51% relief at 45BIU / hr.

Field tests on sweet corn (Spodoftera pruperiferda) provided 84% relief from 2g Ia / BIU BIOBIT ^™ FC at 39.5BIU / hr.

7.9. Immunity ratio

Sensitive and resistant Colonies of flutella xylostella are bioassayed. Resistant moths are collected from the field and samples are collected from Florida that exhibited Bt resistance after strong exposure to JAVELIN ^™ WG. BIOBIT ^™ HPWP with Ia is analyzed using a leaf-immersion bioassay. Resistance to JAVELIN ^™ and XENTARI ^™ is assayed without using Ia. Slices of 6 cm diameter cabbage leaves are immersed in 8 Bt products or Bt / Ia formulations of different concentrations for 10 seconds. The concentration ranges from 1 to 1000 ppm. The leaf pieces are air dried for 2 hours and placed in a plastic Petri dish with 2 week old (0.2-0.4 mg) larvae. Repeat 25 insects / dose / day twice to obtain 20 insects / dose. After 72 hours at 27 ° C., lethality is recorded. Dose lethality degeneration is analyzed via probit analysis. The ratio of resistance is calculated by dividing the LC ₅₀ and LC ₉₀ values of the sensitive moth. The results are shown in Table VII, indicating that BIOBIT ^™ HPWP is enhanced by Ia 2g / BIU and Ia 4g / BIU. Specifically, the use of Ia 4g / BIU was shown to double the LC ₅₀ resistance rate and decrease the LC ₉₀ resistance rate by 10 times.

Flutella xylostella (B.t.k. resistant strain) resistance ratio Trial product LC ₅₀ RR LC ₉₀ RR JAVELIN ^TM WG 302.6 3829.7 BIOBIT ^TM HPWP 20.5 98.5 Ia 2.0g / BIUBIOBIT ^TM HPWP 23.2 88.0 Ia 4.0g / BIUBIOBIT ^TM HPWP 10.4 11.5 XENTARI ^TM 9.7 8.2

Because certain aspects of the disclosure are intended to illustrate some aspects of the invention, the invention described and claimed herein should not be limited in scope by the specific aspects described herein. All equivalent aspects are also considered to be within the scope of this invention. Indeed, various modifications of the invention in addition to those presented and described herein will be apparent to those of ordinary skill in the art from the foregoing detailed description. Such modifications are also considered to be within the scope of the appended claims.

Various references are cited herein, the contents of which are incorporated herein by reference in their entirety.

8. Microbial Deposit

The following Bacillus thuringiensis strains were deposited in the Agricultural Research Service Patent Culture Collection (NRRL), 61604 Peoria University Street, 1815 Northern Legendary Research Center, Illinois, USA, in accordance with the Budapest Treaty.

Strain Accession Number Deposit Date

EMCC0086 NRRL N-21147 1993. 10. 6.

The strain was applied to 37 C.F.R. §1.14 and 35 U.S.C. It has been deposited under § 122 under the conditions that make access to culture available so that rights are granted. The deposits are substantially pure cultures of each deposited strain. The deposit may be used as required by foreign patent law in the country in which the application is filed with the other country or its allies. However, it should be understood that the availability of the deposit does not constitute a license to carry out the invention in the derogation of patent rights granted by government authorities.

Claims (13)

(a) culturing a Bacillus strain and (b) recovering the factor from the culture supernatant of step (a), to obtain a factor that enhances the pesticidal activity of the Bacillus related pesticide. The method of claim 1, wherein the strain of step (a) is cultured in fermentation medium. The method of claim 1, further comprising separating the factor from the supernatant by column chromatography. The method of claim 1, wherein the ¹ H NMR shift of the factor is present at about δ1.5, 3.22, 3.29, 3.35, 3.43, 3.58, 3.73, 3.98, 4.07, 4.15, 4.25, and 4.35, and the ¹³ C shift is about δ31. 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. The method of claim 1 wherein the factor is Formula I or a salt thereof. The method of claim 1, wherein the Bacillus strain is a Bacillus thuringiensis strain. The Bacillus thuringiensis strain according to claim 6, wherein the strain of Bacillus thuringiensis subspecies is Bacillus thuringiensis subspecies aizawai, Bacillus thuringiensis subspecies alesti, Bacillus thuringiensis subspecies canadiensis, and Bacillus thuringiensis subspecies. Colmeri, Bacillus thuringiensis subspecies coreanensis, Bacillus thuringiensis subspecies dakota, Bacillus thuringiensis subspecies darmstadiensis, Bacillus thuringiensis subspecies dendrolimus ), Bacillus thuringiensis subspecies entomocidus, Bacillus thuringiensis subspecies finitimus, Bacillus thuringiensis subspecies galleriae, Bacillus thuringiensis subspecies indiana, Bacillus Thuringiensis subspecies israelensis, Bacillus thuringiensis subspecies Kenya (k enyae, Bacillus thuringiensis subspecies kumamotoensis, Bacillus thuringiensis subspecies kurstaki, Bacillus thuringiensis subspecies kyushuensis, Bacillus thuringiensis subspecies japonensis, Bacillus thuringin Gienssis subspecies mexcanensis, Bacillus thuringiensis subspecies morrisoni, Bacillus thuringiensis subspecies neoleonensis, Bacillus thuringiensis subspecies nigeriae, Bacillus thuringiensis Subspecies ostriniae, Bacillus thuringiensis subspecies pakistani, Bacillus thuringiensis subspecies pondicheriensis, Bacillus thuringiensis subspecies shandongiensis, Bacillus thuringiensis subspecies (silo), Bacillus thuringiensis Ajong soto, Bacillus thuringiensis Species subtoxicus, Bacillus thuringiensis subspecies tenebrionis, Bacillus thuringiensis subspecies thompsoni, Bacillus thuringiensis subspecies tochigiensis, Bacillus thuringiensis subspecies Tohocuensis (tohokuensis), Bacillus thuringiensis subspecies tolworthi, Bacillus thuringiensis subspecies toumanoffi, Bacillus thuringiensis subspecies wuhanensis and Bacillus thuringiensis subspecies yunnanensis The method is selected from the group consisting of strains. The method of claim 6, wherein the Bacillus thuringiensis strain is a Bacillus thuringiensis subspecies kurstaki strain. The method of claim 1, wherein the Bacillus related pesticide comprises Bacillus thuringiensis delta-endotoxin or a pesticidal active fragment thereof. The method of claim 10, wherein the Bacillus thuringiensis delta-endotoxin or pesticidal active fragment thereof is selected from the group consisting of CryI, CryII, CryIII, CryIV, CryV, and CryVI. The method of claim 11, wherein the Bacillus thuringiensis delta-endotoxin or pesticidal active fragment thereof is a CryIA delta-endotoxin or pesticidal fragment thereof. The method of claim 11, wherein the Bacillus thuringiensis delta-endotoxin or pesticidal active fragment thereof is a CryIC delta-endotoxin or pesticidal active fragment thereof. The method of claim 1, wherein the Bacillus related insecticide comprises Bacillus thuringiensis spores.