POTENTIATOR OF BACILLUS PESTICIDAL ACTIVITY

Abstract: The present invention relates to a factor that potentiates the pesticidal activity of a Bacillus related pesticide as well as methods for isolating and identifying said factor. The invention further relates to pesticidal compositions comprising the factor as well as methods of using the pesticidal compositions to control a pest and decrease the resistance of a pest to a pesticide and potentiate a Bacillus related pesticide. The factor of the present invention may be obtainable from a Bacillus fermentation. In a most specific embodiment said factor has the structure (Ia).

Description: POTENTIATOR OF BACILLUS PESTICIDAL ACTIVITY 1. FIELD OF THE INVENTION The invention is related to a factor which potentiates the pesticidal activity of a Bacillus related pesticide, a chemical pesticide and/or a virus with pesticidal properties, as well as methods for obtaining the factor. Furthermore, the invention relates to pesticidal compositions comprising the factor and a pesticidal carrier, or the factor and a Bacillus related pesticide, a chemical pesticide and/or a virus with pesticidal properties as well as methods of using such compositions. The invention further relates to a mutant or variant Bacillus strain in which such a factor is obtained in larger amounts or has a greater potentiating activity compared to the parental strain and methods for producing such mutant or variant strains. 2. BACKGROUND OF THE INVENTION Every year, significant portions of the world's commercially important agricultural crops, including foods, textiles, and various domestic plants are lost to pest infestation, resulting in losses in the millions of dollars. Various strategies have been used in attempting to control such pests. One strategy is the use of broad spectrum pesticides, chemical pesticides with a broad range of activity. However, there are a number of disadvantages to using such chemical pesticides. Because of their broad spectrum of activity, these pesticides may destroy non- target organisms such as beneficial insects and parasites of destructive pests. Additionally, these chemical pesticides are frequently toxic to animals and humans, and targeted pests frequently develop resistance when repeatedly exposed to such substances. Another strategy has involved the use of biopesticides, which make use of naturally occurring pathogens to control insect, fungal and weed infestations of crops. Biopesticides comprise a bacterium which produces a toxin, a substance toxic to the pest. Biopesticides are generally less harmful to non-target organisms and the environment as a whole than chemical pesticides. The most widely used biopesticide is Bacillus thuringiensis (B. t. ) . B. t. is a widely distributed, rod shaped, aerobic and spore forming microorganism. During its sporulation cycle, B. t . produces a protein(s) in a crystal form known as a crystal delta-endotoxin(s) , which kill insect larvae. B. t. , therefore, is very useful as an agricultural pesticide. Some strains, e.g. Bacillus thuringiensis subsp. Jcurstaki, and Bacillus thuringiensis subsp. aizawai, have been found to be specific for Lepidoptera. Bacillus thuringiensis subsp. israelensis has been found to be specific for Diptera (Goldberg, U.S. Patent No. 4,166,112). Other strains, e.g. Bacillus thuringiensis subsp. tenebrionis (Krieg et al., 1988, U.S. Patent No. 4,766,203), have been found to be specific for Coleoptera. The isolation of another coleopteran toxic Bacillus thuringiensis strain was reported in 1986 (Hernnstadt et al. Bio/Technology vol. 4, 305-308, 1986, US patent 4,764,372; 1988). This strain, designated "Bacillus thuringiensis subsp. san diego" , M-7, has been deposited at the Northern Regional Research Laboratory, USA under accession number ΝRRL B-15939. However, the assignee of the '372 patent, Mycogen, Corp. has publically acknowledged that Bacillus thuringiensis subsp. san diego is Bacillus thuringiensis subsp. tenebrionis. Furthermore, the '372 patent has been assigned to Νovo Νordisk A/S. Additionally, there has been disclosed a B. t . strain which is toxic against Lepidoptera and Coleoptera (PCT Application No. wo 90/13651) . The toxin disclosed in PCT Application No. wo 90/13651 has a molecular weight of 81 kd. During its sporulation cycle, B.t. produces a protein(s) in crystal form known as a crystal delta- endotoxin(s) having a molecular weight ranging from 27-140 kd, which upon ingestion kills insect larvae. Toxic activity may reside in one or more of such crystal proteins in a given B.t. " strain. Most delta-endotoxins are protoxins that are proteolytically converted into smaller toxic (truncated) polypeptides in the target insect midgut (Hόfte and Whiteley, 1989, Microbiol. Rev. 53:242-255). The delta-endotoxins are encoded by cry (crystal protein) genes. The cry genes have been divided into six classes and several subclasses based on structural similarities and pesticidal specificity. The major classes are Lepidoptera-specific (cryJ); Lepidoptera- and Diptera-specific ( cryll) ; Coleoptera-specific (cryJII) ; Diptera-specific ( crylV) (Hόfte and Whiteley, 1989, Microbiol. Rev. 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV by Tailor et al., 1992, Mol. Microbiol. 6:1211-1217); and Nematode- specific (referred to as cryV and cr Vl by Feitelson et al., 1992, Bio/Technology 10:271-275) . Delta-endotoxins have been produced by recombinant DNA methods. The delta-endotoxins produced by recombinant DNA methods may or may not be in crystal form. B. t . delta-endotoxin is water insoluble except at alkaline pH, and is almost always plasmid encoded. Some strains of Bacillus thuringiensis have been shown to produce a heat-stable pesticidal adenine-nucleotide analog, known as β-exotoxin or thuringiensin which is pesticidal alone (Sebesta et al., in H.D. Burges (ed.), Microbial Control of Pests and Plant Diseases, Academic Press, New York p. 249-281, 1981). β-exotoxin has been found in the supernatant of some Bacillus thuringiensis cultures. It has a molecular weight of 789 and is comprised of adenosine, glucose, and allaric acid (Lύthy 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 elanogaster, and Tetranychus cinnabarinus. The toxicity of β-exotoxin is thought to be due to inhibition of DNA- directed RNA poly erase by competition with ATP. It has been shown that β-exotoxin is encoded by a Cry plasmid in five Bacillus thuringiensis {B. t . ) strains and that β- exotoxin may be classified as type I or type II β-exotoxin (Levinson et al., 1990, J. Bacteriol. 172:3172-3179). β- exotoxin type I was found to be produced by B. t . subsp. thuringiensis serotype l, B.t. subsp. tolworthi serotype 9, and B.t. subsp. darmstadiensis serotype 10. β-exotoxin type II was found to be produced by B.t. subsp. morrisoni serotype 8ab and is active against Leptinotarsa texana. Other water soluble factors that have been isolated from B.t. include alpha-exotoxin (Luthv, 1980, FEMS Microbiol. Lett. 8:1-7); qamma-exotoxins, which are various proteolytic enzymes including lecithinases, chitinaseε, and proteases (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, Subcomittees on Pesticides and Related Compounds and Biological Phenomena); sigma exotoxin (Argauer et al., 1991, J. Entomol. Sci. 26:206-213); and anhydrothuringiensin (Coll. Czechoslovak Chem. Comm. 40, 1775, 1975). The art has strived to achieve increased mortality of B.t. formulations. Means have included searching for new strains with increased mortality, attempting to engineer present strains, and attempting to design more effective formulations by combining B.t. spores and/or crystals with new pesticidal carriers chemical pesticides, or enhancers (see, for example, U.S. Patent No. 5,250,515 y a trypsin inhibitor). It is therefore an object of the present invention to potentiate the pesticidal activity of pesticides. 3. SUMMARY OF THE INVENTION The invention is related to a novel factor which unlike factors known in the art potentiates the pesticidal activity of a Bacillus related pesticide. Specifically, the factor of the present invention is a potentiator. As defined herein, a *potentiator" is a substance which has no significant pesticidal activity, e.g. having an LC 50 (LC 50 is the concentration of the substance required to kill 50% of the pests) of more than about 3000 μg/g as assayed by bioassay (see Section 6) but acts to increase the pesticidal activity of a Bacillus related pesticide at least about 50% and does not cause larval stunting. As noted in Section 2, other substances capable of enhancing pesticidal activity known in the art such as delta-endotoxins, trypsin inhibitors and exotoxins have pesticidal activity. In a specific embodiment, the factor is water soluble. As defined herein, a substance or compound is "water soluble" if at least about 1 mg of a substance can be dissolved in 1 ml of water. The factor may also potentiate the pesticidal activity of a chemical pesticide and/or a virus with pesticidal properties. As defined herein, "a Bacillus related pesticide" is a Bacillus (e.g. Bacillus thuringiensis or Bacillus subtilis) strain, spore, or substance, e.g. protein or fragment thereof having activity against or which kill pests; a substance that provides plant protection, e.g. antifeeding substance; or a microorganism capable of expressing a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kill pests (e.g. Bacillus thuringiensis delta- endotoxin) and an acceptable carrier (see Section 5.2., infra, for examples of such carriers) . The pest may be, for example, an insect, a nematode, a mite, or a snail. A microorganism capable of expressing a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kill pests inhabits the phylloplane (the surface of the plant leaves) , and/or the rhizosphere (the soil surrounding plant roots) , and/or aquatic environments, and is capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms and provide for the stable maintenance and expression of a Bacillus gene encoding a Bacillus protein or fragment thereof having activity against or which kill pests. Examples of such microorganisms include but are not limited to bacteria, e.g. genera Bacillus, Pseudomonas, Erwinia, Serratia, Klebsiella, Xantho onas , Strepto yceε , Rhizobium, Rhodopseudomonas , Methylophiliuε, Agrohacterium, Acetόbacter, Lactobacillus , Arthrobacter, Azotobacter, Leuconostoc, Alcaligenes, and Clostridium; algae, e.g. families Cyanophyceae, Prochlorophyceae , Rhodophyceae, D±nophyceae, Chrysophyceae, Prymnesiophyceae, Xanthophyceae , Raphidophyceae, Bacillariophyceae, Eustig atophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae; and fungi, particularly yeast, e.g. genera Saccharomyceε , Cryptococc s , Kluyvero yces , Sporόbolo yces , Rhodotorula, and Aureobasidium. As defined herein, "pesticidal activity" measures the amount of activity against a pest through killing or stunting of the growth of the pest or protecting the plant from pest infestation. The invention further relates to a mutant or variant Bacillus strain in which such a factor is obtained in large amounts compared to the parental strain as well as methods for obtaining such a mutant or variant. The invention further relates to pesticidal compositions comprising the factor and a pesticidal carrier as well as the factor and a Bacillus related pesticide, chemical pesticide and/or a virus with pesticidal properties. The invention further relates to methods of using the pesticidal compositions of the present invention. In one embodiment, the invention relates to a method for controlling a pest comprising exposing the pest to a pest-controlling amount of the composition. In another embodiment, the invention relates to a method for decreasing the resistance of a pest to a Bacillus related pesticide comprising exposing the pest to a composition comprising the factor and a pesticidal carrier. The invention further relates to a method for potentiating the pesticidal activity of a Bacillus related pesticide comprising administering to a pest exposed to a Bacillus related pesticide a composition comprising the factor and carrier in amount effective to potentiate the pesticidal activity of a Bacillus related pesticide. The invention is further directed to a method for obtaining "substantially pure" factor of the present invention comprising the steps of (a) fermentation of a Bacillus strain; (b) recovering the supernatant of (a) ; and (c) isolating the factor from the supernatant. As defined herein a "substantially pure" factor means a factor which contains less than 10% of contaminants, for example, delta-endotoxin protein. The invention is further directed to a method for identifying the factor of the present invention comprising (a) fermentation of a strain of Bacillus; (b) recovering the supernatant of the fermentation of (a) ; and (c) assaying the supernatant of (b) for potentia ion of a Bacillus related pesticide. 4. BRIEF DESCRIPTION OF THE FIGURES Figure l schematically shows the general procedure used for purifying Ia. Figure 2 shows the "C NMR spectrum of Ia. Figure 3 shows the proton NMR spectrum of Ia. Figure 4 shows the deduced structure of Ia. Proton (iH) and 13 C shifts are shown in ppm recorded in D 2 0. Figure 5 shows a synthetic scheme for obtaining structure Ia. 5. DETAILED DESCRIPTION OF THE TNVENTTON The present invention relates to a factor that potentiates the pesticidal activity of a Bacillus related pesticide. The factor may have a molecular weight of from about 350 to about 1200 or in a specific embodiment from about 350 to about 700. The factor of the present invention potentiates the pesticidal activity of a Bacillus related pesticide at least about 1.5 fold to optionally about 1000 fold, preferably from about 100 fold to about 400 fold. In a specific embodiment, the factor of the present invention potentiates the pesticidal activity of a Bacillus thuringiensis delta-endotoxin including but not limited to a Cryl (including but not limited to CrylA, CrylB, and CrylC) , Cryll, Crylll, CrylV, CryV, or CryV protein in full-length form or a proteolytically processed, truncated form, from about 1.5 fold to about 1000 fold. In a most specific embodiment, the factor of the present invention potentiates a B.t. delta-endotoxin from about 100 fold to about 400 fold. The factor may also potentiate the pesticidal activity of a chemical pesticide and/or a virus with pesticidal properties. The factor may also be water soluble, stable in water up to about 100°C for at least about 5 minutes, stable when subjected to direct sunlight for at least about 10 hours, and/or stable at a pH of about 2 for about 10 days. In a specific embodiment, the factor has from about 13 to about 24 carbons. In a more specific embodiment, the factor has from about 13 to about 19 carbons. In a most specific embodiment, the factor has about 13 carbons. In a specific embodiment the factor has the structure (I) in which Ri is a amino, hydroxy, Cι-ι 0 -alkyl, benza ide, 3,5- dinitrobenzamide p-nitrobenzamide, halobenzamide, alkylbenzamide, thiophene amide, furan amide, pyrrole amide, imidazole amide, pyrazole amide, Cι-ι 0 -alkylamino, Ci-io-dialkylamino, i-io-alkyl or benzyl ester, halogen, or Ci-io-alkoxy; R2 is (CH 2 ) n wherein n is an integer from 0-10; R 3 and R* are independently (NH) P wherein p is 0 or l; CH2X 1 R5 is OΪ-CHX2-CH 2 -CHX3-CHX*-CHX5-CHX 6 -CX or (CHOHjgCHsOH wherein χι, X2 , X*, X* , xs , x« and X7 are independently NH 2 , OH, acetyl, halogen or Cι- 10 -alkαxy and q is an integer from 1- 10 ; R 6 is a hydroxyl, amino, an 0.- 1 0 alkoxy or benzoxy, benzyl ester, 3,5-dinitrobenzyl ester, p-nitrobenzyl ester, halobenzyl ester, alkylbenzyl ester, thiophene ester, furan ester, pyrrole ester, imidazole ester; or pesticidally acceptable salt thereof including but not limited to phosphate, sulfate, acetate, carbonate, and nitrate. In a most specific embodiment said factor has the structure (la) 5.1. OBTAINING THE FACTOR The factor of the present invention may be obtainable from a Bacillus fermentation (e.g. Bacillus subtilis, Bacillus cereus, and Bacillus thuringiensis) . In a specific embodiment, the factor of the present invention is obtainable from the supernatant of a Bacillus thuringiensis fermentation including but not limited to Bacillus thuringiensis subsp. Jcurstaki, Bacillus thuringiensis subsp. aizawai, Bacillus thuringiensis subsp. galleriae, Bacillus thuringiensis subsp. entomocidus, Bacillus thuringiensis subsp. tenebrionis, Bacillus thuringiensis subsp. thuringiensis, Bacillus thuringiensis subsp. alesti, Bacillus thuringiensis subsp. canadiensis, Bacillus thuringiensis subsp. darmstadiensis , Bacillus thuringiensis subsp. dendrolimαs , Bacillus thuringiensis subsp. finiti us, Bacillus thuringiensis subsp. kenyae, Bacillus thuringiensis subsp. morrisoni, Bacillus thuringiensis subsp. subtoxiόus, Bacillus thuringiensis subsp. toumanoffi and Bacillus thuringiensis subsp. israelensiε. In a preferred embodiment, the factor is obtainable from the supernatant of Bacillus thuringiensis subsp. kurstaki, Bacillus thuringiensis subsp. aizawai, or Bacillus thuringiensis subsp. galleriae or mutants thereof having substantially the same potentiating activity. In a specific embodiment, the factor is recovered from a cry- spo- mutant of Bacillus thuringiensis subsp. kurstaki . In one embodiment, the factor is obtainable from a mutant Bacillus, particularly a Bacillus thuringiensis in which the factor is produced in larger amounts or a mutant Bacillus thuringiensis in which the potentiating activity of the factor obtainable from the mutant is greater, as compared to the parental strain. A "parental strain" as defined herein is the original Bacillus strain before mutagenesis. To obtain such mutants, the parental strain may, for example, be treated with a mutagen by chemical means such as N-methyl-N'-nitro- N-nitrosoguanidine or ethyl methanesulfonate, gamm - irradiation, x-ray or UV-irradiation. Specifically, in one method of mutating Bacillus strains and selecting such mutants the parental strain is: i) treated with a mutagen; ii) the thus treated mutants are grown in a medium suitable for the selection of a mutant strain; iii) selection of a mutant strain. ii) the thus treated mutants are grown in a medium suitable for the selection of a mutant strain; iii) selection of a mutant strain. According to a preferred embodiment of this method, the selected colonies are grown in a normal production medium, and a final selection for strains capable of increased factor production is performed. Bacillus may be cultured using media and fermentation techniques known in the art (see, for example, Rogoff et al., 1969, J. Invertebrate Path. 14:122- 129; Dul age et al., 1971, J. Invertebrate Path. 18:353- 358; Dulmage et al., in Microbial Control of Pests and Plant Diseases, H.D. Burges, ed. , Academic Press, N.Y., 1980) . Upon completion of the fermentation cycle, the supernatant can be recovered by separating B.t. spores and crystals from the fermentation broth by means well known in the art, e.g. centrifugation and/or ultrafiltration. The factor of the present invention is contained in the supernatant which may be recovered by means well known in the art, e.g. ultrafiltration, evaporation, and spray- drying. This procedure is more specifically described in the sections which follow. Alternatively, the factor of the present invention may be obtained by chemical synthesis using procedures known in the art. As noted above in a specific embodiment, the factor has the structure (Ia) A synthetic scheme is shown in Figure 5 . 2 , 3 - Diaminopropionic acid (DAP) is protected using Boc (t- butylcarba ate) . The fully N-protected DAP may be converted to the imidizoyl by treatment with diimidazoic carbonate to give compound A (see Figure 5) . Compound B (see Figure 5) may be prepared from 3-amino-3-deoxy- aldonic acid by selective substitution of the terminal primary alcohol by using a benzene sulfonyl leaving group. Condensation of A and B will be achieved under Julia conditions (NaH, THF) followed by desulfurization with Raney nickel. The keto group in compound C (see Figure 5) may be stereoselectively reduced by chelation control using sodium tris acetoxyborohydride. Deprotected C may be treated with isocyanate D derived from DAP to give the corresponding ureido derivative which will be deprotected by hydrogenolysis to give compound (Ia) . in one embodiment, the amino groups of (la) obtained synthetically or from a Bacillus supernatant may be acylated with either acid chloride or acid anhydride in the presence of aqueous sodium hydroxide to obtain triacetamides, tribenzamides and trioctamides of (Ia) . Alternatively, the amino and hydroxy groups may be per acylated by first forming methyl esters using excess diazomethane, followed by per acylation on the amino and hydroxyl groups by treatment with an acyl anhydride (e.g. acetic anhydride, benzoic acid anhydride, n-octanoic acid anhydride) in pyridine. In another embodiment, starting with an acylated derivative, a mixed amide ester may be prepared by selective ethylation of the carboxyl followed by treatment with an acyl anhydride in pyridine. In another embodiment, the carboxylic acid may be derivatized to form alkyl or aryl esters or amide. In yet another embodiment there may be selective oxidations of the hydroxy substituents to ketone and chain shortened carboxylic acid analogs. In another embodiment, the amino groups of (la) 09630 may be protected as t-butyl carbamates by treatment with Boc anhydride in the presence of aqueous sodium carbonate, followed by selective protection of the hydroxyl groups using benzyl bromide in sodium hydride to give (X) shown below This fully protected derivative may be hydrolyzed at the ureido linkage using aqueous sodium hydroxide. This may be followed by selective acylation with for example amino acids, hydroxy carboxylic acids or dicarboxylic acid including but not limited to aspartic acid, glutamic acid, 2-aminoadipic acid, malic acid, adipic acid in the presence of N,N-dicyclohexylcarbodiimide (DCC) . N-Boc and O-benzyl groups may be removed with mild acid cleavage and hydrogenolysis over palladium on charcoal to obtain structure (II) below In yet another embodiment, the fully protected compound (X) may be treated with various isocyanates derived from amino acids including but not limited to glycine, alanine, valine, phenylalanine, phenylglycine, methionine, nor ethionine to obtain 9-ureido derivatives. Protecting groups may be removed as described above to obtain structure (III) in which R is a naturally occurring amino acid. In yet another embodiment, 2,3-diaminopropionic acid is protected at the 2-position using Boc in the presence of aqueous sodium carbonate to give N-2-Boc-2,3- diaminopropionic acid. This derivative may be acylated with per acetyl aldonic acids including but not limited to penta-O-acetyl-D-gluconic acid, penta-O-acetyl-D-mannoic acid, penta-O-acetyl-D-galactonic acid, hepta-0-acetyl-D- erythro-L-manno actoanoic acid in the presence of DCC, followed by solvolysis of the acetyl groups and Boc groups in the presence of ethanolic ammonia (Chem. Pharm. Bull. 1985, 33:509-514). Polyhydroxy amide analogs having the structure (IV) may be obtained with mild acid treatment in which n is 1-10. The same sequence may be applied using peracetylated aldaric acids, for example, D-glucaro- 6,3-lactone, to yield polyhydroxy with primary terminal carboxamide groups. In yet another embodiment, a protected derivative of 2,3-diaminopropionic acid is coupled with an isocyanate derived from an amino sugar derivative with a phosgene equivalent. The amino sugar in one embodiment 09630 may be a 1- or 2-aminohexose (e.g. 1-amlno-l- deoxysorbitol, glucosamine, mannosamine) and has the structure (V) in which n is 1-10. Purification of the factor of the present invention can be carried out by various procedures known in the art, including but not limited to chromatography (e.g. ion exchange, affinity, and size exclusion column chromatography), electrophoretic procedures, differential solubility, extraction, or any other standard technique known in the art. The potentiating activity of the factor of the present invention of the pesticidal activity of Bacillus related pesticide, virus having pesticidal activity, or chemical pesticide against various pests may be assayed using procedures known in the art, such as an artificial insect diet incorporated, artificial diet overlay, leaf painting, leaf dip, and foliar spray. Specific examples of such assays are given in Section 6, infra. 5 .2 . COMPOSITIONS COMPRISING THE FACTOR The factor of the present invention described supra can be formulated along with a Bacillus related pesticide and an acceptable carrier into a pesticidal composition(s) that is for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, an e ulsifiable concentrate, an aerosol or impregnated granule. Examples of such Bacillus related pesticides preferably include but are not limited to pesticides produced by Bacillus thuringiensis subsp. Jurstaλi {B.t.k.) (marketed as DIPEL™ from Abbott Laboratories, Inc., JAVELIN™ from Sandoz, BIOBIT™ from Novo Nordisk A/S, FORAY™ from Novo Nordisk A/S, MVP™ from Mycogen, BACTOSPEINE™ from Novo Nordisk A/S, -and THURICIDE™ from Sandoz) and Bacillus thuringiensis subsp. aizawai {B.t. a.) (marketed as FLORBAC™ from Novo Nordisk A/S, and XENTARI™ from Abbott Laboratories, Inc.). Further examples include but are not limited to Bacillus thuringienεiε subsp. israelensiε {B.t.i.) (marketed as BACTIMOS™ from Novo Nordisk A/S and VECTOBAC™ from Abbott Laboratories, Inc.); Bacillus εphaericus (B. sphr.) (marketed as SPHERIMOS™ from Novo Nordisk A/S) ; Bacillus thuringiensis kurstaki/tenebrionis (marketed as FOIL™ from Ecogen) ; Bacillus thuringiensiε aizawai/kurεtaki (B.t.k. /B.t.k.) (marketed as CONDOR™ from Ecogen and AGREE™ from Ciba-Geigy) ; and Bacilluε thuringienεiε kurεtaki/kurεtaki (B.t.k. /B.t.k.) (marketed as CUTLASS™ from Ecogen) . The virus having activity against pests may be a baculovirus, e.g. Autograp a californica nuclear polyhedrosis virus (NPV) , Syngrapha falcifera NPV, Cydia pomonella GV (granulosis virus) , Heliothis zea NPV, Lymantria dispar NPV, Orgyia pεeudotεugata NPV, Spodoptera exigua NPV, Neodiprion lecontei NPV, Neodiprion sertifer NPV, Harrisina brillians NPV, and Endopiza viteana Clemens NPV. In compositions comprising the factor and a Bacilluε related pesticide, the factor may be present in the amount of at least about 0.002 g/BIU. In one embodiment, the factor may have structure Ia and/or may be present in an amount of at least about 0.1 g/BIU or 0.05 g factor per g Bacillus delta- endotoxin and spore, optionally to about 300 g/BIU or 150 g factor per g Bacillus delta -endotoxin and spore, preferably 2 g/Biu or 1 g factor per g Bacillus delta- endotoxin and spore. As 9630 defined herein "BIU" is billion international units as determined by bioassay. The bioassay compares the sample to a standard Bacillus reference material using Trichoplusia ni or other pest as the standard test insect. The potency is determined by dividing the reference standard LC S0 then multiplying by the reference standard potency. In another embodiment, the composition may comprise the factor of the present invention in substantially pure form or a supernatant from Bacillus in dry, concentrated, or liquid form and a suitable pesticidal carrier, examples of which are disclosed infra. This composition may be applied separately to a plant, e.g. transgenic plants. Specifically, the cort osition may be applied to a plant previously containing a Bacillus, e.g. B. t. gene. In another embodiment, the composition may be applied to a plant previously exposed to a Bacillus, e.g. B. t . composition present in the amount of at least about 0.01% up to about 60%. The composition comprising the factor and a pesticidally acceptable carrier in addition to controlling a pest may also be used to decrease the resistance of a pest to a pesticide. Alternatively, the composition may be used to potentiate a Bacillus related pesticide. The composition in one embodiment may be applied at the same time as the pesticide in an amount of at least about 2 g factor/BIU up to optionally about 300 g factor/BIU. In another embodiment, the composition may be applied up to about 24 hours after the pesticide as an adjuvant to extend the efficacy of residual pesticide. Such compositions disclosed above may be obtained by the addition of a surface active agent, an inert carrier, a preservative, a humectant, a feeding stimulant, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a U.V. protectant, a buffer, a flow agent, or other component to facilitate product handling and application for particular target pests. Suitable surface-active agents include but are not limited to anionic compounds such as a carboxylate, for example, a metal carboxylate of a long chain fatty acid; an N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty alcohol ethoxylates or salts of such esters; fatty alcohol sulphates such as sodium dodecyl sulphate, sodium octadecyl sulphate or sodium cetyl sulphate; ethoxylated fatty alcohol sulphates; ethoxylated alkylphenol sulphates; lignin sulphonates; petroleum sulphonates; alkyl aryl sulphonates such as alkyl-benzene sulphonates or lower alkylnaphthalene sulphonates, e.g. butyl-naphthalene sulphonate; salts of sulphonated naphthalene-formaldehyde condensates; salts of sulphonated phenol-formaldehyde condensates; or more complex sulphonates such as the amide sulphonates, e.g. the sulphonated condensation product of oleic acid and N- methyl taurine or the dialkyl sulphosuccinates, e.g. the sodium sulphonate or dioctyl succinate. Non-ionic 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 ethers, e.g. sorbitan fatty acid esters, condensation products of such esters with ethylene oxide, e.g. polyoxyethylene sorbitar fatty acid esters, block copolymers of ethylene oxide and propylene oxide, acetylenic glycols such as 2,4,7,9-tetraethyl-5-decyn-4,7- diol, or ethoxylated acetylenic glycols. Examples of a cationic surface-active agent include, for instance, an aliphatic mono-, di-, or polyamine as an acetate, naphthenate or oleate; an oxygen-containing amine such as an amine oxide of polyoxyethylene alkylamine; an amide- linked amine prepared by the condensation of a carboxylic acid with a di- or polyamine; or a quaternary ammonium salt . Exaπples of inert materials include but are not limited to inorganic minerals such as kaolin, phyllosilicates, carbonates, sulfates, phosphates or botanical materials such as cork, powdered corncobs, peanut hulls, rice hulls, and walnut shells. The compositions of the present invention can be in a suitable form for direct application or as a concentrate or primary composition which requires dilution with a suitable quantity of water or other diluent before application. The pesticidal concentration will vary depending upon the nature of the particular formulation, specifically, whether it is a concentrate or to be used directly. 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 will be administered at the labeled rate for the commercial product, preferably about 0.01 lb-5.0 lb per acre when in dry form and at about 0.01 pts-10 pts per acre when in liquid form. In a further embodiment, the Bacillus, e.g. B.t. crystal delta-endotoxin and potentiating factor can be treated prior to formulation to prolong the pesticidal activity when applied to the environment of a target pest as long as the pretreatment is not deleterious to the crystal delta-endotoxin or factor. Such treatment can be by chemical and/or physical means as long as the treatment does not deleteriously 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-infectives, such as zephiran chloride; alcohols, such as isopropranol and ethanol; and histological fixatives, such as Bouin's fixative and Helly's fixative (see, for example, Humason, Animal Tissue Techniques, W.H. Freeman and Co., 1967). The compositions of the invention can be applied directly to the plant by, for example, spraying or dusting at the time when the pest has begun to appear on the plant or before the appearance of pests as a protective measure. Plants to be protected within the scope of the present invention include but are not limited to cereals (wheat, barley, rye, oats, rice, sorghum and related crops), beets (sugar beet and fodder beet), drupes, pomes and soft fruit (apples, pears, plums, peaches, almonds, cherries, strawberries, raspberries, and blackberries), legviminous plants (alfalfa, beans, lentils, peas, soybeans) , oil plants (rape, mustard, poppy, olives, sunflowers, coconuts, castor oil plants, cocoa beans, groundnuts) , cucumber plants (cucumber, marrows, melons) , fibre plants (cotton, flax, hemp, jute) , citrus fruit (oranges, lemons, grapefruit, mandarins), vegetables (spinach, lettuce, asparagus, cabbages and other brassicae, carrots, onions, tomatoes, potatoes, paprika) , lauraceae (avocados, cinnamon, camphor) , deciduous trees and conifers (e.g. linden-trees, yew-trees, oak-trees, alders, poplars, birch-trees, firs, larches, pines), or plants such as maize, turf plants, tobacco, nuts, coffee, sugar cane, tea, vines hops, bananas and natural rubber plants, as well as ornamentals. In both cases, the preferred mode of application is by foliar spraying. It is generally important to obtain good control of pests in the early stages of plant growth as this is the time when the plant can be most severely damaged. The spray or dust can conveniently contain another pesticide if this is thought necessary. In a preferred embodiment, the composition of the invention is applied directly to the plant. The compositions of the present invention may be effective against pests including but not limited to pests of the order Lepidoptera, e . g. Achroia griεella, Acleris gloverana, Acleriε variana, Adoxophyes orana, Agrotis ipεilon, Alabama argillacea, Alsophila pometciria, Amyelois transi tella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, A chips sp. , Argyrotaenia sp. , At etis mindara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneura sp. , Cochylls hospes, Colias euxrythe e, Corcyra cephalonica, Cydia latiferreanus, Cydia po onella, Datana integerrima, Dendrolimus sibericus, Des ia funeraliε, Diaphania hyalinata, Diaphania ni tidaliε, Diatraea grandioεella, Diatraea εaccharaliε, Ennomoε εubsignaria, Eoreuma loftini, Epheεtia elutella, Eranniε tilaria, Estig ene acrea, Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chryεorrhoea , Euxoa meεεoria, Galleria mellonella, Grapholita moles ta, Harris ina americana, Helicoverpa subflexa, Helicoverpa zea, Heliothis virescenε, Hemileuca oliviae, Homoeoεoma electellum, Hyphantria cunea, Keiferia lycoperεicella, Lambdina fiεcellaria fiεcellaria, Lambdina fiεcellaria lugubroεa, Leucoma εaliciε, Lobeεia botrana, Loxostege sticticalis, Lymantria diεpar, Macalla thyrεiεaliε, Malacoεoma εp. , Mameεtra braεεicae, Mameεtra configurata, Manduca guinguemaculata, Manduca εexta, Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp. , Ostrinia nubilalis, Paleacrita vernata, Papilio cresphonteε, Pectinophora goεεypiella, Phryganidia califomica, Phyllonorycter blancardella, Pieriε napi, Pieriε rapae, Plathypena scabra, Platynota flouendana, Platynota stultana, Platyptilia car dui dactyl a, Plodia interpunctella , Plutella xyloεtella, Pontia protodice, Pεeudaletia unipuncta, Pεeudoplaεia includenε, Sabulodes aegrotata, Schizura concinna, Sitotroga cerealella, Spilonota ocellana, Spodoptera sp. , Thaumεtopoea pityocampa, Tineola biεεelliella, Trichopluεia ni, Odea rubigalis, Xylo ygeε curialiε, Yponomeuta padella; Diptera, e.g. Aedeε εp. , Andes vittatus, Anastrepha ludenε, Anastrepha suspensa, Anopheles barber i, Anopheles guadrimaculatus , Armigeres subalbatus, Calliphora stygia, Calliphora vicina, Ceratitiε capitata, Chironomus ten tans, Chrysoπya rufi fades, Cochliomyia macellaria, Culex sp. , Culiseta inornata, Dacus oleae, Delia antigua, Delia platura, Delia radicum, Drosophila elanogaster , Eupeodes corollae, Glossina auεteni, Glossina brevipalpis, Glosεina fuεcipes, Glosεina morsitans centraliε, Gloεsina moristans morsitans, Glossina moristans submorεi tanε, Gloεεina pallidipeε, Gloεsina palpalis ga bienεis, Glosεina palpalis palpalis, Glossina tachinoides, Haemagogus eguinus, Hae atobiuε irritanε, Hypoderma bovis, Hydoderma lineatum, Leucopis ninae, Lucilia cuprina, Lucilia sericata, Lutzo yia longlpaipiε, Lutzomyia εhannoni, Lycoriella mali, Mayetiola destructor, Musca autumnalis, Musca domestica, Neobellieria εp. , Nephrotoma εuturaliε, Ophyra aenescens , Phaenicia sericata, Phlebotomus εp. , Phor ia regina, Sabetheε cyaneus, Sarcophaga bullata, Scatophaga stercoraria, Stomaxyε calcitranε, Toxorhynchi teε a boinenεiε, Tripteroideε bambuεa; Coleoptera, e.g. Leptinotarεa sp. , Acanthoεcelideε obtectuε, Calloεobruchuε chinenεiε, Epilachna varivestis, Pyrrhalta luteola, Cylas formicarius elegantuluε, l/is ronotus oregonenεiε, Sitophiluε sp. , Cyclocephala borealiε, Cyclocephala im aculata, Macrodactylus subεpinoεuε, Popillla japonica, Rhizotroguε majaliε, Alphitobluε diaperinuε, Paloruε ratzeburgi, Tenebrio molitor, Tenebrio obεcuruε, Tribolium caεtaneum, Tribolium confuεum, Triboliuε deε true tor; Acari, e.g. Oligonychuε pratenεiε, Panonychus ulmi, Tetranychuε urticae; Hymenoptera, e.g. Iridomyrmex humiliε, Solenopεiε invicta; Isoptera, e.g. Reticulitermeε hesperus, Reticulitermes flavipes, Coptotermes formosanus, Zootermopsis anguεticolliε, Neoter es connexuε, Inciεitermeε minor, Incisitermes immlgrans; Siphonaptera, e.g. Ceratophyllus gallinae, Ceratophyllus niger, Nosopsyllus fasciatus, Leptopsylla segnis, Ctenocephalldes canis, Ctenocephalldes felis, Echicnophaga gallinacea, Pulex irri tans, Xenopsylla cheopis, Xenopsylla vexabilis, Tunga penetrans; Tylenchida, e.g. Melodidogyne incognita, Pratylenchus penetrans . The following examples are presented by way of illustration, not by way of limitation. 6. EXAMPLE: CHARACTERIZATION OF la As detailed herein, Ia is recovered and purified. The characterization of Ia is detailed infra. 6.1. RECOVERY AND PURIFICATION OF T* B. thuringiensis subsp. . urstaJei strain EMCC0086 (deposited with the NRRL as B-21147) is fermented for 72 hours at 30°C in a medium comprised of a carbon source such as starch, hydrolyzed starch, or glucose and a nitrogen source such as protein, hydrolyzed protein, or corn steep liquor. The production of Ia is detected at 13 hours into the fermentation. Peak activity is found to be at approximately 30 hours. Supernatant from a B. thuringiensiε subsp. kurεtaki fermentation is recovered by centrifugation and then is clarified by ultrafiltration through a 30 kDa MW- CO membrane using a Rhone Poulenc UF system. The 30 kDa filtration removed any remaining cell debris, crystal delta-endotoxin, spores, and soluble protein greater than 30 kDa molecular mass. The permeate is concentrated 10 fold by evaporation. The permeate is centrifuged and then 0.2μ filtered to further remove insolubles from the broth, leaving a clear broth containing la. The purification of Ia to homogeneity is achieved using a multi-step purification procedure shown schematically in Figure 1. In conjunction with the recovery protocol outlined above, the purification proceeded with a 5 kDa ultrafiltration step. The permeate from the 5 kDa ultrafiltration is adsorbed to a Sulfopropyl (SP) cation exchange resin and eluted with an ammonium acetate solution. The compound is then concentrated approximately 3OX by lyophilization, and the salt and other contaminants are removed with a BioRad P2 size exclusion column. The pool from the P2 column is run over a high resolution SP HPLC cation exchange column which yielded a homogeneous compound. The contaminating salt is removed by repeated lyophilization. Activity is monitored by a Spodoptera exigua micro-bioassay, and purity is determined by capillary electrophoresis. Sample consisting of 50 μl of Ia and 50 μl of CryΙA(c) protein (15 μg/ml) purified from BIOBIT™ FC (100 μl) , is applied to individual wells of a jelly tray containing 500 μl of solidified artificial insect diet. The trays containing the various samples are air dried. Two to four 2nd or early 3rd instar Spodoptera exigua are added to the wells containing the dried sample. The wells are sealed with mylar poked with holes and are incubated for 2-3 days at 30°C. Degree of stunting and percent mortality are then recorded. Typically, 5 replicate wells are run for each sample. 6.2. STRUCTURE ELUCIDATION The active compound is found to be water soluble but is not soluble in organic solvents. It is positively charged and reacted with ninhydrin as evidenced by silica thin layer chromatography. "C and proton NMR of the compound are shown in Figures 2 and 3, respectively. 13 C NMR experiments revealed the presence of 13 carbons. A DEPT experiment determined that there are three quaternary carbons (C) , seven methines (CH) , three methylenes (CH 2 ) and no methyl groups (CH 3 ) . Using proton coupling experiments such as 1-D decoupling and COSY, one large spin system containing eight carbons is identified. In addition, a smaller spin system consisting of two carbons is present. A two-dimensional carbon proton correlation experiment (HETCOR) enabled assignment of each proton resonance in the molecule to its attached carbon (see Figure 4) . Treatment of the active compound (13 mg) with acetic anhydride in pyridine resulted in the formation of an acetylated derivative which is much less polar. This derivative is purified by HPLC to give 3 mg of pure acetylated derivative. Mass spectroscopy analysis revealed that the derivative has 7 acetates and a molecular weight of 690, which gives a molecular weight of 396 and indicates that an even number of nitrogens are present. Also, fragments of 6 acetates and 5 acetates are detected. High resolution data for 5 and 6 acetate daughter ions are 645.2594 (6 acetates) and 607.2519 (5 acetates) which indicate the following molecular formula for la, Cι 3 H 2θ N 6 O e . Treatment of the active compound (13 mg) with 6 N HC1 gave a derivative which is ninhydrin positive. These results indicate the presence of amide bonds. The derivative had the same Rf value as determined by thin layer chromatography as 2,3-diaminopropionic acid. These results along with NMR data, suggest the presence of 2,3 diaminopropionic acid. The following structure has been elucidated for la: include 2 carboxyls (1 amidated) , a urea, three aminos, and four hydroxyls. It contains seven chiral centers. 6.3 . PROPERTIES OF la The isolated Ia is found to potentiate the activity of Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensiε subsp. aizawai crystal delta - endotoxin pesticidal proteins toward Spodoptera exigua regardless of the form of the pesticidal proteins . The pesticidal activity of formulated B. t.k. , isolated crystals, full-length (130 kDa molecular mass) or truncated CrylA proteins (-65 kDa molecular mass) are all potentiated. The activity of Cryll and CrylC inclusions are also potentiated. It is also found to potentiate the activity of the individual truncated CryΙA(a) , (b) , and (c) proteins . Incubation time of Ia with the Cry protein is not found to be critical for bioactivity. However, la is inactive alone. The level of potent iation is found to be 100-200 fold for the truncated CrylA proteins, Cryll and CrylC inclusions and approximately 320 fold with full- length CrylA (c) (see Tables I and II respectively) . Specifically, for full-length protein, 0.75 μg/ml CrylA(c) produced the same insect mortality/stunt score when Ia is included as 240 μg/ml of CrylA (c) alone. In the case of the truncated CrylA ( c) , an OD 28o of 0.0006 gave the same stunt score in combination with Ia as the same sample of CryiA(c) tested alone with an OD 2 so of 0.075. Cryll inclusions, at a concentration of 0.6 μg/ml gave the same stunt score and similar mortality in combination with Ia as Cryll protein alone at 75 μg/ml, a 125 fold potentiation. CrylC inclusions, at 0.3 μg/ml with the addition of Ia gave similar mortality and stunt score as 75 μg/ml of the CrylC protein alone, which reflects a 250 fold level of potentiation. The concentration of CrylA protein that produced stunting yielded mortality on addition of Ia. la is found to be stable upon boiling for 5 minutes, but loses all activity upon autoclaving (>190C) . Further, it is stable when subjected to direct sunlight for at least 10 hours. Ia is stable at pH 2 for 3 days, but unstable at pH 12. It is found to lose all activity when exposed to periodic acid or concentrated HC1. TABLE I POTENTIATION EFFECTS OF la WITH PURIFIED TRUNCATED Bt PROTEIN ■Bt Protein Spodoptera Exiaua Type la Mortality* Stunt Scoret Cryla (a) 0.055 0/5 2 .2 0.040 0/5 2.2 0. 020 0/5 2. 0 0.020 + 2/5 0. 0 0. 010 + 0/5 0 . 2 0.005 0/5 0. 0 0.0025 0/5 0.4 0.0012 0/5 1. 8 0. 0006 0/5 1. 6 CrylA (c) 0.075 0/5 3.4 0. 040 0/5 2.6 0.020 0/5 2, 0.020 + 1/5 0, 0. 010 + 0/5 0, 0.005 + 1/5 0. 0.0025 + 2/5 2. 0. 0012 + 0/5 1. 0. 0006 + 1/5 1. None ΝA + 0/5 4. None ΝA 0/5 4. * Mortality = # insects dead/# total insects after 2 days t Stunt score is defined by the average size of the live insect larvae at the end of the bioassay: .0 = untreated control, 3.0 = 75% size of untreated control, 2.0 = 50% size of untreated control, 1.0 = 25% size of untreated control, 0.0 = no growth or size unchanged from start of experiment. TABLE II POTENTIATION EFFECTS OF Ia WITH Bt PROTEIN * Mortality = # insects dead/# total insects after 2 days t Stunt score is defined by the average size of the live insect larvae at the end of the bioassay: 4.0 = -untreated control, 3.0 = 75% size of untreated control, 2.0 = 50% size of untreated control, 1.0 = 25% size of untreated control, 0.0 = no growth or size unchanged from start of experiment. • 30- 6.4. EVALUATION OF OTHER SUBSPECIES OF Bacillus t-.hurlnσiensis AND OTHER SPECIES OF Bacilli Several Bacillus species are evaluated for production of Ia. The strains are fermented for 72 hours at 30°C in a medium comprised of a carbon source such as starch, hydrolyzed starch, or glucose and a nitrogen source such protein, hydrolyzed protein, or corn steep liquor. The supernatants- are tested for Ia production using the Spodoptera exigua micro-bioassay described supra . B. thuringiensis subsp. aizawai strain EMCC0087 (deposited with the NRRL as NRRL B-21148) and B. thuringiensis subsp. galleriae (deposited with the NRRL) are found to produce Ia in about the same concentration as B. thuringiensis subsp. kurstaki . Ia is also produced in B. εubtilis, B. cereuε, B. t . subsp. aleεti , B. t. subsp. canadiensis, B. t. subsp. darmstadiensis, B. t . subsp. dendrolimus, B. t . subsp. entomociduε, B. t . subsp. fini timuε, B. t . subsp. iεraelenεis, B. t . subsp. kenyae, B. t . subsp. morriεoni , B. t . subsp. subtoxicus, B.t. subsp. tenebrioniε, B. t . subsp. thuringiensis, and B. t . subsp. toumanoffi, B. cereuε, B . εubtiliε, and B. thuringienεiε subsp. kurεtaki cry- spo- mutant as determined by capillary electrophoresis. Specifically, a Bec man P/ACE Capillary Electrophoresis System equipped with a 50 μm x 57 cm uncoated capillary, 0.2 M phosphate pH 6.8 buffer, voltage at 15KV, and detection at 200 nm is used for quantifying the level of Ia. Sample volumes are 20 nl with a run time of 25 minutes. A standard curve is generated using purified Ia as the standard at levels of 1.25 mg/ml, 0.625 g/ml, 0.3125 mg/ml, 0.156 mg/ml, and 0.078 mg/ml. A linear calibration curve is generated. The resultant y = mx + b 9630 equation is used to generate the concentration of Ia in each sample. Before each run, the capillary is flushed with running buffer (0.2 M phosphate, pH 6.8) for three minutes. After each 25 minute run, the capillary is flushed with 1 N NaOH for 1 minute, filtered HPLC water for l minute, 0.5 M phosphoric acid for 3 minutes, and filter HPLC water for 1 minute. The area under each peak is integrated and the peak area is determined and a final concentration is calculated from the standard curve. 6.5. EVALUATION OF B. t . PRODUCTS The amount of la present in various commercially available B.t. products is determined by capillary electrophoresis described in Section 6.4, supra. 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 obtained from Ciba- Geigy; MVP™ is obtained from Mycogen and CUTLASS™ is obtained from Ecogen. The results are shown in Table III, infra and indicate that la is present in varying quantities ranging from less than 0.001 g Ia/BIU to 0.71 g ia/BIU. TABLE III la IN Bacillus thuringiensis PRODUCTS 6.6. DIET INCORPORATION BIOASSAYS B. t.k. activity is determined by an artificial diet incorporation bioassay using third instar Spodoptera exigua larvae, second instar Helicoverpa zea larvae, third instar Spodoptera frugiperda larvae, second instar Helioth±ε virescens larvae, third instar Trichoplusia ni larvae, third instar Pseudoplusia includens larvae, third instar Plutella xylostella larvae, third instar Spodoptera li ttora riε, and third instar Mamestra brassicae larvae. To determine the level of potentiation by adding za to B. t . products, and establish the range of insects that are affected, diet incorporation bioassays are performed. In the experiments with high concentrations of Ia against Spodoptera exigua (7.4-23.7 g Ia/BIU) , purified la (70% active ingredient, 30% acetate counter ion) is used to potentiate BIOBIT™ FC (FC represents flowable concentrate) . The remaining data presented in Table IV shows the potentiation of BIOBIT™ HPWP (high potency wettable powder) with Ia (0.658% active ingredient) . S. li ttoral iε and M. braεεicae are tested using FLORBAC™ HPWP and Ia. The various B.t. products are weighed and Ia is added to give 0.1 to 237 g ia/BIU. The volume is adjusted with 0.1% Tween™. The samples are sonicated for 1 minute and then diluted to final volume. Neat samples (without Ia) and reference substances are prepared as well. Reference substances include B.t..Jo HD-l-S-1980 (obtained from the NRRL) which is assigned a potency of 16,000 international units (IU) per milligram and JAVELIN™ WG which has been assigned a potency of 53,000 Spodoptera Units/mg (SU) . Standard artificial diet composed of water, agar, sugar, casein, wheat germ, methyl paraben, sorbic acid, linseed oil, cellulose, salts, and vitamins are prepared in a 20 L heated kettle. This provides enough diet to test 10 to 12 saπqples with seven different concentrations of each test substance. The B.t. solutions are serially diluted to give 16 ml aliquots. Each aliquot is added to 184 g of molten diet. The mixture is subsequently homogenized and then poured into a plastic tray bearing 40 individual cells. Three control trays are prepared for each batch of diet. Once the diet has cooled and solidified, one insect of a known age (2-3 instar) is added to each cell, and the trays are covered with a perforated sheet of clear mylar. The trays are placed on racks and incubated for four days at 28°C and 65% relative humidity. After four days, insect mortality is rated. Each tray is given a sharp blow against a table top, and larvae that did not move are counted as dead. Percent mortality is calculated and the data is analyzed via parallel probit analysis. LC 50 s, LC 90 s, the slope of the regression lines, coefficient of variation, and potencies are estimated. Samples are run a minimum of 3 times or until three potencies are within 20% of a calculated mean for each sample. To calculate the increase in activity associated with each concentration of Ia, the LC 50 of the B.t./Ia sample is corrected to reflect the amount of B.t. in the sample. The LC 50 s of the paired neat samples are divided by the corrected LC 50 values to give the fold reduction in LC 50 associated with Ia. The following procedure is used to assay for Lobeεia bothrana . Vine grapes attacked by Lobeεia bothrana are collected in an unsprayed field and larva is removed. A dilution series of Ia (250 μg/ml, 500 μg/ml, and 1000 μg/ml) is made in water. One larva is put in the middle of the petri dish. If the larva is observed to drink, it is moved into a petri dish with freshly cut grape berries. The larvae are stored at 22 β C for 3-4 days. As shown in Table IV, significant reductions in LC 50 s are observed for all species. TABLE IV Diet Incorporation Bioassayβ Increase in activity reduction in T-ft^ Helicoverpa zea 3.2 12.6 (BIOBIT™ HPWP) Heliothis virescens 3.2 4.2 (BIOBIT™ HPWP) Lobesia bothrana 2.0 3.0 (BIOBIT™ HPWP) Spodoptera littoralis 2.0 8.6 (FLORBAC™ HPWP) Mamestra brassicae 2.0 4.9 (FLORBAC™ HPWP) The potentiation of various products on Spodoptera exigua by la is determined using diet incorporation bioassays described supra. Amounts of Ia added/Blu product are shown in Table V, infra. Ia/B.t. product mixture is incorporated into an agar-based wheat germ casein diet. The insects are placed on the diet for four days and held at 28°C. Mortality is recorded and analyzed using probit analysis. LC 50 , LC9 0 an ό potency are calculated from matched product lacking Ia. The results shown in Table V indicate that Ia potentiate various B. t .k. and B.t.a. products obtained from various sources. The B.t. strains contained in these products are described in Section 5.2., supra. TABLE V Potentiation of B. t. Products on Spodoptera exigua g la per BIU Increase in activity Product Fold reduction in T-T^ BACTOSPEINE™ WP CONDOR™ AGREE™ CUTLASS™ MVP™ FLORBAC™ HPWP DI EL™ 2X JAVELIN™ WG XENTARI TV 6.7 . FOLIAR BIOASSAYS Foliar bioassays are performed with second instar Spodoptera exigua larvae on broccoli plants using BIOBIT™ FC and la . The ratio of la to BIOBIT™ FC is the same 2 g Ia/BIU BIOBIT™ FC. The treatments are applied to broccoli plants via a track sprayer in a carrier volume of 20 gallons per acre. Leaves are excised from the plants after the spray deposit had dried, and infested with second instar Spodoptera exigua larvae . The results Spodoptera exigua larvae. The results are shown in Table V , infra. 100% mortality is observed at a rate of 8.7 BlU/hectare BIOBIT™ FC + Ia, while BIOBIT™ FC alone killed 92% of the larvae at 58.8 BlU/hectare and 8% at 17.6 BlU/hectare. Treated plants are also placed in direct sunlight for eight hours, after which leaves are excised and infested. After eight hours in sunlight, BIOBIT™ FC alone at 58.8 BIU/hectare s gave 27% mortality, while BIOBIT™ FC + la gave 100% mortality at 8.7 BlU/hectare. A foliar assay done with early fourth instar larvae had BIOBIT™ FC alone with 75% mortality at 52 BlU/hectare, and BIOBIT™ FC (FC is flowable concentrate) + la gave 100% mortality at 13 Biu/hectare. 2 2 2 2 4 4 6.6.2. FIELD TRIALS Field trials on garbonzo beans (Spodoptera exigua) demonstrated that BIOBIT™ FC alone at 70 BlU/hectare gave 51% control while 2 g Ia/BIU BIOBIT™ FC at 40 BlU/hectare provided 89% control (relative to no control . Field trials on sweet corn (Spodoptera frugiperda) demonstrated that at 39.5 BlU/hectare, 2 g Ia/BIU BIOBIT™ FC provided 84% control. 6.9. RESISTANCE RATIOS Colonies of susceptible and resistant Plutella xylostella are bioassayed. Resistant moths are field collected samples from Florida that have developed B.t. resistance following intensive exposure to JAVELIN™ WG. BIOBIT™ HPWP with la is analyzed using a leaf-dip bioassay. Resistance to JAVELIN™ and XENTARI™ is assayed without Ia. Six cm diameter cabbage leaf disks are dipped for 10 seconds into one of eight different concentrations of B.t. products or B.t./la formulations. Concentrations range from 1 to 1000 ppm. The leaf disks are allowed to air dry for two hours and placed in plastic petri dishes with second instar (0.2 to 0.4 mg) larvae. Twenty five insects/dose/day are replicated twice to give 50 insects/dose. After 72 hours at 27°C, mortality is recorded. Dose mortality regression is analyzed with probit analysis. Resistance ratios are calculated by dividing the LC 50 and LC 90 values of the susceptible moths. The results are shown in Table VII and indicate that the BIOBIT™ HPWP potentiates with 2 g Ia/BIU and 4 g Ia/BIU. Specifically, with 4 g Ia/BIU there is a 2 fold decrease in the LC 50 resistance ratio and a 10 fold decrease in the LC 90 resistance ratio. TABLE VTI Plutella xylostella (B. t.k. Resistant) Resistance Ratios The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. 7 . DEPOSIT OF MICROORGANISMS The following strains of Bacillus thuringiensis have been deposited according to the Budapest Treaty in the Agricultural Research Service Patent Culture Collection (NRRL) , Northern Regional Research Center, 1815 University Street, Peoria, Illinois, 61604, USA. strain Accession Number Deposit pat EMCC0086 NRRL B-21147 October 6, 1993 EMCC0087 NRRL B-21148 October 6, 1993 The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of each deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Claims: WHAT IS CLAIMED IS! 1. A factor which potentiates the pesticidal activity of a Bacillus related pesticide. 2. The factor according to claim 1 in which said factor further potentiates the pesticidal activity of a virus having activity against a pest. 3. The factor according to claim 1 in which the factor is stable up to about 100°C in water for at least about 5 minutes. 4. The factor according to claim 1 in which the factor is stable when subjected to direct sunlight for at least about 10 hours. 5. The factor according to claim 1 in which the factor is stable at a pH of about 2 for about 10 days. 6. The factor according to claim 1 in which the factor has a molecular weight from about 350 to about 700. 7. The factor according to claim 1 in which the factor is soluble in water. 8. The factor according to claim 1 in which the factor potentiates the pesticidal activity of the Bacillus related pesticide at least about 1.5 fold. 9. The factor according to claim 1 in which the Bacillus related pesticide comprises a Bacilluε strain and an acceptable carrier. 630 10. The factor according to claim 9 in which the Bacillus strain is Bacillus thuringiensis. 11. The factor according to claim 1 in which the Bacillus related pesticide comprises a spore of a Bacillus strain and a pesticidally acceptable carrier. 12. The factor according to claim 11 in which the Bacillus strain is Bacillus thuringiensis. 13. The factor according to claim 1 in which the Bacillus related pesticide comprises a protein from a Bacillus strain or fragment thereof having activity against a pest and a pesticidally acceptable carrier. 14. The factor according to claim 13 in which the protein from a Bacilluε strain is a Bacilluε thuringiensis delta-endotoxin. 15. The factor according to claim 14 in which the factor potentiates the pesticidal activity of the Bacillus thuringiensis delta-endotoxin from about 100 to about 400 fold. 16. A factor which has the structure (I) in which Ri is a amino, hydroxy, Cj*.-ιo-alkyl , benzamide, 3,5-dinitrobenzamide, p-nitrobenzamide, halobenzamide, alkylbenzamide, thiophene amide, furan amide, pyrrole amide, imidazole amide, pyrazole amide, Cι-ι 0 _ alkylamino, Ci-io-dialkylamino, Cι-ι 0 -alkyl or benzyl ester, halogen, or Ci-ιo-alkoxy; R 2 is (CH 2 ) n wherein n is an integer from 0-10; R3 and R 4 * are independently (NH) P wherein p is 0 or 1; CH2X 1 0 R5 is CH-CHX2-CH 2 -CHX3-CHX4-CHX5-CHX6-CX7 or (CHOH) q CH 2 OH wherein χι, X 2 , χ3, χ4, χs f χ6 and χ7 are independently ΝH 2 , OH, acetyl, halogen or Cι- 10 -alkoxy and q is an integer from 1-10; R 6 is a hydroxyl, amino, an Cι- i0 alkoxy or benzoxy, benzyl ester, 3,5-dinitrobenzyl ester, p-nitrobenzyl ester, halobenzyl ester, alkylbenzyl ester, thiophene ester, furan ester, pyrrole ester, imidazole ester; or pesticidally acceptable salt thereof. 17. The factor according to claim 16 in which the factor has the structure (Ia) 18. A factor which potentiates the pesticidal activity of a Bacillus related pesticide in which said factor or derivative thereof is obtained by (a) fermentation of a Bacillus strain; (b) recovering the supernatant of the fermentation of (a) ; and (c) isolating the factor or derivative thereof from the supernatant of (b). 19. The factor according to claim 18 in which the factor is obtained from a fermentation supernatant of Bacillus εubtilis. 20. The factor according to claim 18 in which the factor is obtained from a fermentation supernatant of Bacillus cereus. 21. The factor according to claim 18 in which the factor is obtained from a fermentation supernatant of Bacillus thuringiensiε. 22. The factor according to claim 21 in which the factor is obtained from a fermentation supernatant of a subspecies of a Bacilluε thuringiensis selected from the group consisting of Bacillus thuringienεiε subsp. kurεtaki, Bacillus thuringiensiε subsp. aizawai, Bacillus thuringiensiε subsp. galleriae, or mutants thereof having substantially the same pesticidal activity. 23. The factor according to claim 21 in which the factor is obtained from a fermentation supernatant of Bacillus thuringiensis subsp. kurstaki or mutant thereof having pesticidal activity. 24. The factor according to claim 21 in which the factor is obtained from a fermentation supernatant of a cry-spo- mutant of Bacilluε thuringiensis subsp. kurs taki . 25. A pesticidal composition comprising (a) a factor which potentiates the pesticidal activity of a Bacillus related pesticide and (b) a Bacillus related pesticide, in which the factor is present in said composition in the amount of at least about 0.1 g per BIU. 26. A pesticidal composition comprising (a) a factor which potentiates the pesticidal activity of a Bacillus related pesticide and (b) a Bacillus related pesticide, in which the factor is present in said composition in the amount of at least about 0.05 g per g Bacillus delta-endotoxin and spore. 27. The composition according to claim 25 or 26 in which said composition further comprises a virus having activity against a pest and said factor further potentiates the pesticidal activity of a virus having activity against a pest. 28. A pesticidal composition comprising (a) a factor which potentiates the pesticidal activity of a Bacillus related pesticide and (b) a Bacillus related pesticide in which the Bacillus is selected from the group consisting of Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai, in which the factor is present in said composition in the amount of at least about 0.1 g per BIU. 29. A pesticidal composition comprising (a) a factor which potentiates the pesticidal activity of a Bacillus related pesticide and (b) a Bacillus related pesticide in which the Bacillus is selected from the group consisting of Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai, in which the factor is present in the amount of at least about 0.05 g per g Bacillus delta-endotoxin and spore. 30. A pesticidal composition comprising (a) a factor which potentiates the pesticidal activity of a virus having activity against a pest and (b) a virus having activity against a pest. 31. A pesticidal composition comprising (a) a factor which potentiates the pesticidal activity of a Bacillus related pesticide and (b) a pesticidal carrier, in which the factor is present in said coπqposition in the amount of at least about 0.01% by weight. 32. A method for controlling a pest comprising exposing the pest to a pest-controlling effective amount of the pesticidal composition of claim 25, 26, 28, 29, 30, or 31. 33. The method according to claim 32 in which the pest is present on a plant. 34. The method according to claim 33 in which the plant is a transgenic plant. 35. The method according to claim 34 in which the transgenic plant contains a Bacillus thuringiensis gene. 36. The method according to claim 34 in which the plant has previously been exposed to a Bacillus related pesticide. 37. A method for decreasing the resistance of a pest to a Bacillus related pesticide comprising exposing the pest to an amount of the composition of claim 31 effective in decreasing the resistance of the pest. 9630 38. A method for potentiating the pesticidal activity of a Bacillus related pesticide comprising administering to a pest exposed to a Bacillus related pesticide the composition of claim 31 in an amount effective to potentiate the pesticidal activity of said Bacillus related pesticide. 39. A method for obtaining" a substantially pure factor which potentiates the pesticidal activity of a Bacillus related pesticide comprising (a) fermentation of a strain of Bacilluε; (b) recovering the supernatant of the fermentation of (a) ; and (c) isolating the factor from the supernatant of (b) to obtain the substantially pure factor. 40. The method according to claim 39 in which the factor is isolated from the supernatant by column chromatography. 41. A method for identifying a factor which potentiates the pesticidal activity of a Bacillus related pesticide comprising (a) fermentation of a strain of Bacillus; (b) recovering the supernatant of the fermentation of (a) ; and (c) assaying the supernatant of (b) for potentiation of a Bacillus related pesticide. 42. A mutant or variant of a Bacillus strain which produces a factor which potentiates the pesticidal activity of a Bacillus related pesticide, in which the potentiating activity of said factor produced by the mutant or variant is greater than the activity of a factor 630 mutant or variant is greater than the activity of a factor produced by a corresponding parental strain. 43. A method for obtaining a mutant or variant of a Bacillus strain which produces a factor which potentiates the pesticidal activity of a Bacillus related pesticide, in which the potentiating activity of said factor produced by the mutant or variant is greater than the activity of a corresponding parental strain comprising (a) treating the parental strain with a mutagen, (b) growing the mutated parental strain on a medium suitable for the selection of a mutant strain; and (c) selection of the mutant strain. 44. A mutant or variant of a Bacillus strain which produces a factor which potentiates the pesticidal activity of a Bacillus related pesticide, in which the amount of said factor produced by the mutant or variant is greater than the amount of factor produced by a corresponding parental strain. 45. A method for obtaining a mutant or variant of a Bacillus strain which produces a factor which potentiates the pesticidal activity of a Bacillus related pesticide, in which the amount of said factor produced by the mutant or variant is greater than the amount of factor produced by a corresponding parental strain comprising (a) treating the parental strain with a mutagen, (b) growing the mutated parental strain in a medium suitable for the selection of a mutant strain; and (c) selection of the mutant strain.

Inventors: LIDSTER WILLIAM DAVID, MACINTOSH SUSAN CARYL, MANKER DENISE CAROL, STARNES ROBERT LEE, LIDSTER, WILLIAM, DAVID, MACINTOSH, SUSAN, CARYL, MANKER, DENISE, CAROL, STARNES, ROBERT, LEE
Applicants: NOVO NORDISK ENTOTECH INC, NOVO NORDISK ENTOTECH, INC.
Application year: 1993
Main classification: A01N63
IPC codes: A01N47, A01N61, A01N63, C07C237, C07C275, C07D207, C07D307, C07G99, C12N1, C12P13, C12R1