METHODS FOR PRODUCING A POTENTIATOR OF BACILLUS PESTICIDAL

ACTIVITY

This application is a continuation-in-part application of application serial no. 08/295,283, filed August 23, 1994, which is a continuation-in-part of application serial no. 08/146,852, filed November 3, 1993, which is a continuation-in- part of application serial no. 08/095,240, filed July 20, 1993, which is a continuation-in-part of applcation serial no. 07/990,202, filed December 14, 1992, which is a continuation-in- part of application serial no. 07/971,786, filed November 5, 1992.

1. FIELD OF THE INVENTION

The invention is related to a method of obtaining and identifying a factor which potentiates the pesticidal activity of a Bacillus related pesticide, a chemical pesticide and/or a virus with pesticidal properties.

2. BACKGROUND OF THE INVENTION

Every year, pests detrimental to agriculture, forestry, and public health cause losses in the millions of dollars. Various strategies have been used to control such pests.

One strategy is the use of chemical pesticides with a broad range or spectrum of activity. However, there are a number of disadvantages with using chemical pesticides. Specifically, because of their broad spectrum of activity, these pesticides may destroy non-target organisms such as beneficial insects and parasites of destructive pests. Additionally, chemical pesticides are frequently toxic to animals and humans. Furthermore, targeted pests frequently develop resistance when repeatedly exposed to such substances.

Another strategy involves the use of biopesticides to control insect, fungal and weed infestations. Biopesticides are

naturally occurring pathogens and/or the substances produced bythese pathogens. The advantage of using biopesticides is that they are generally less harmful to non-target organisms and the environment as a whole compared to chemical pesticides.

2.1. Bacillus thuringiensis

The most widely used biopesticide is Bacillus thuringiensiε. Bacillus thuringiensis is a motile, rod-shaped, gram-positive bacterium that is widely distributed in nature, especially in soil and insect-rich environments. During sporulation, Bacillus thuringiensis produces a parasporal crystal inclusio (s) which is insecticidal upon ingestion to susceptible insect larvae of the orders Lepidoptera, Diptera, and Coleoptera. The inclusions may vary in shape, number, and composition. They are comprised of one or more proteins called delta-endotoxins, which may range in size from 27-140 kDa. The insecticidal delta-endotoxins are generally converted by proteases in the larval gut into smaller (truncated) toxic polypeptides, causing midgut destruction, and ultimately, death of the insect (Hόfte and Whiteley, 1989, Microbiological Reviews 53:242-255) .

There are several Bacillus thuringiensis strains that are widely used as biopesticides in the forestry, agricultural, and public health areas. Bacillus thuringiensis subsp. kurstaki and Bacillus thuringiensis subsp. aizawai produce delta-endotoxins specific for Lepidoptera. A delta-endotoxin specific for Coleoptera is produced by Bacillus thuringiensis subsp. tenebrionis (Krieg et al . , 1988, U.S. Patent No. 4,766,203). Furthermore, Bacillus thuringiensis subsp. israelensis produces delta-endotoxins specific for Diptera

(Goldberg, 1979, U.S. Patent No. 4,166,112).

Other Bacillus thuringiensis strains specific for dipteran pests have also been described. A Bacillus thuringiensis isolate has been disclosed which is toxic to Diptera and Lepidoptera (Hodgman et al . , 1993, FEMS Microbiology Let ters 114:17-22). SDS polyacrylamide gel electrophoresis of the purified crystal delta-endotoxin from this isolate revealed

three protein species which are related to CryΙA(b) , CrylB, and CryHA toxins. There has also been disclosed a Bacillus thuringiensis isolate which produces a dipteran-active crystal comprised of proteins with molecular weights of 140, 122, 76, 72, and 38 kDa (Payne, 1994, U.S. Patent No. 5,275,815). EPO 480,762 discloses five B. t . strains which are each active against dipteran pests; each also have a unique crystal delta- endotoxin pattern.

Several Bacillus thuringiensis strains have been described which have pesticidal activity against pests other then Lepidoptera, Coleoptera, and Diptera. Five Bacillus thuringiensis strains have been disclosed which produce delta- endotoxins that are toxic against nematodes (Edwards, Payne, and Soares, 1988, Eur. Pat. Appl. No. 0 303 426 Bl) . There has also been disclosed a Bacillus thuringiensis strain, PS81F, which can be used to treat humans and animals hosting parasitic protozoans (Thompson and Gaertner, 1991, Eur. Pat. Appl. No. 0 461 799 A2) . Several Bacillus thuringiensis isolates have also been disclosed with activity against acaride pests. These isolates produce crystals comprised of proteins with molecular weights in the (wide) range of 35 kDa to 155 kDa (Payne, Cannon, and Bagley, 1992, PCT Application No. WO 92/19106). There have also been disclosed Bacillus thuringiensis strains with activity against pests of the order Hymenoptera (Payne, Kennedy, Randall, Meier, and Uick, 1992, Eur. Pat. Appl. No. 0 516 306 A2); with activity against pests of the order Hemiptera (Payne and Cannon, 1993, U.S. Patent No. 5,262,159); with activity against fluke pests (Hickle, Sick, Schwab, Narva, and Payne, 1993., U.S. Patent No. U.S. 5,262,399; and with activity against pests of the order Phthiraptera (Payne and Hickle, 1993, U.S. Patent No.

5,273,746). Furthermore, another strain of Bacillus thuringiensis subsp. kurstaki , WB3S-16, isolated from Australian sheep wool clippings, has been disclosed that is toxic to the biting louse Damalinia ovis , a Phthiraptera pest (Drummond, Miller, and Pinnock, 1992, J. Invert . Path . 60:102-103).

The delta-endotoxins are encoded by cry (crystal protein) genes which are generally located on plasmids. The cry

genes have been divided into six classes and several subclasses based on relative amino acid homology and pesticidal specificity. The major classes are Lepidoptera-specific i cryi) • Lepidoptera-and Diptera-specific ( cryll) ; Coleoptera-specific (cryTXl); Diptera-specific ( crylV) (Hόfte and Whiteley, 1989, Microbiological Reviews 53:242-255); Coleoptera- and Lepidoptera-specific (referred to as cryV genes by Tailor et al . , 1992, Molecular Microbiology 6:1211-1217); and Nematode- specific (referred to as cryV and cryVI genes 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.

Some strains of Bacillus thuringiensis have been shown to produce a heat-stable pesticidal adenine-nucleotide analog, known as β-exotoxin type I 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, 1980, pp. 249-281) . β-exotoxin type I has been found in the supernatant of some Bacillus thuringiensis cultures. It has a molecular weight of 701 and is comprised of adenosine, glucose, and allaric acid (Farkas et al . , 1977, Coll . Czechosslovak Chem. Comm . 42:909-929; Lϋthy 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, Tetraπychus urticae, Drosophila melanogaster, and Tetranychus cinnabarinus . The toxicity of β- exotoxin type I is thought to be due to inhibition of DNA- directed RNA polymerase by competition with ATP. It has been shown that β-exotoxin type I is encoded by a cry plasmid in five

Bacillus thuringiensis strains (Levinson et al . , 1990, J. Bacteriol . 172:3172-3179) . β-exotoxin type I was found to be produced by Bacillus thuringiensis subsp. thuringiensis serotype 1, Bacillus thuringiensis subsp. tolworthi serotype 9, and Bacillus thuringiensis subsp. darmstadiensis serotype 10. Another β-exotoxin classified as β-exotoxin type II has been described (Levinson et al . , 1990, J. Bacteriol .

172:3172-3179). ^β -exotoxin type II was found to be produced by Bacillus thuringiensis subsp. morrisoni serotype 8ab and is active against Leptinotarsa decemlineata . The structure of β- exotcxin type II is not completely known, but is significantly different from that of β-exotoxin type I in that a pseudouridine moiety is in the place of adenine in which attachment to the ribose ring is at a position that would otherwise be occupied by a proton (Levinson, in Hickle and Finch (eds.), Analytical Chemistry of Bacillus thuringiensis, ACS Symposium Series, Washington, D.C., 1990, pp. 114-136). Furthermore, there is only one signal in the proton NMR spectrum corresponding to the nucleoside base (at 7.95 ppm) , and does not have a ribose-type anomeric protein signal (5.78 ppm).

Other water soluble substances that have been isolated from Bacillus thuringiensis include alpha-exotoxin which is toxic against the larvae of Musca domestica (Luthy, 1980, FEMS Microbiol . Lett . 8:1-7); gamma-exotoxins, which are various enzymes including lecithinases, chitinases, and proteases, the toxic effects of which are expressed only in combination with beta-exotoxin or delta-endotoxin (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 which has a structure similar to beta-exotoxin, and is also active against Leptinotarsa decemlineata (Argauer et al . , 1991, J-. Entomol . Sci . 26:206-213); and anhydrothuringiensin (Prystas et al., 1975, Coll . Czechosslovak Chem . Comm. 40:1775) .

2.2. ZWITTEFMICIN

A substance has been isolated from Bacillus cereus which inhibits the growth of the plant pathogen Phytophthora medicaginis and reduces the infection of alfalfa (see, for example, U.S. Patent Nos. 4,877,738 and 4,878,936). No other activity was disclosed. The following structure has been elucidated for zwittermicin A (He et al . , Tet. Lett. 35:2499-

2502 ) :

3. OBJECTS OF THE INVENTION

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 crystals with new pesticidal carriers chemical pesticides, or enhancers (see, for example, U.S. Patent No. 5,250,515, a trypsin inhibitor) . It is therefore an object of the present invention to potentiate the pesticidal activity of pesticides.

4. SUMMARY OF THE INVENTION

The invention relates to a method for obtaining which a factor which potentiates the pesticidal activity of a Bacillus related pesticide comprising (a) culturing a Bacillus strain under suitable conditions;

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

In a specific embodiment, the Bacillus strain is selected from the group consisting of Bacillus subtilus, Bacillus licheniformis, and Bacillus thuringiensis . In a preferred embodiment, the factor is in substantially pure form. As defined herein a "substantially pure" factor means a factor

which contains less than 10% of contaminants, for example, delta-endotoxin protein. Such a substantially pure factor may be obtained by isolating said factor, e.g., by column chromatography. The factor obtained 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 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 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, Xanthomonas, Streptomyces , Rhizobiu , Rhodopseudomonas , Methylophilius, Agrobacterium, Acetobacter, Lactobacillus , Arthrobacter, Azotobacter , Leuconostoc, Alcaligenes, and Clostridium; algae, e.g. families Cyanophyceae , Prochlorophyceae , Rhodophyceae , Dinophyceae, Chrysophyceae , Prymnesiophyceae, Xanthophyceae, Raphidophyceae, Bacillariophyceae, Eustigmatophyceae, Cryptophyceae, Euglenophyceae, Prasinophyceae, and Chlorophyceae; and fungi, particularly yeast, e.g. genera Saccharomyces , Cryptococcus , Kluyveromyces , Sporobolomyces , 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 factor obtained may be formulated into a composition 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. These compositions may be used for controlling a pest, decreasing the resistance of a pest to a Bacillus related pesticide comprising exposing the pest to a composition comprising the factor and a pesticidally acceptable carrier, or potentiating the pesticidal activity of a Bacillus related pesticide.

The invention is also directed to a method for identifying said factor comprising (a) culture of a strain of Bacillus;

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

(c) assaying the supernatant of (b) for potentiation of a Bacillus related pesticide.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 schematically shows the general procedure

used for purifying la.

Figure 2 shows the ¹³ C NMR spectrum of la. Figure 3 shows the proton NMR spectrum of la. Figure 4 shows the results of nOe experiments on the acetylated derivative of la.

6. DETAILED DESCRIPTION OF THE INVENTION

The factor potentiates the pesticidal activity of a Bacillus related pesticide and 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 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 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) , CryII, Crylll, CryIV, CryV, or CryVI 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 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/o stable at a pH of about 2 for about 10 days. The factor may have 13 carbons. Additionally, the factor may have iH NMR shifts at δl.5, 3.22, 3.29, 3.35, 3.43, 3.58, 3.73, 3.98, 4.07,

4.15, 4.25, 4.35.

In a most specific embodiment, said factor has the structure la or salt thereof.

The salt would be capable of potentiating a Bacillus related pesticide.

6.1. OBTAINING THE FACTOR

The factor may be obtainable from a Bacillus strain (e.g. Bacillus εubtilis, Bacillus licheniformis, and Bacillus thuringiensis) in shake flasks or a fermentor. In a specific embodiment, the factor is obtainable from the supernatant of a Bacillus thuringiensis culture including but not limited to

Bacillus thuringiensis subsp. kurstaki , 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. den roliiπus, Bacillus thuringiensis subsp. finitimus, Bacillus thuringiensis subsp. kenyae, Bacillus thuringiensis subsp. morrisoni, Bacillus thuringiensis subsp. subtoxicus, Bacillus thuringiensis subsp. toumanoffi and Bacillus thuringiensis subsp. israelensis. 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 .

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; Dulmage 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 cycle, the supernatant can be recovered by separating B.t. spores and crystals from the culture (fermentation) broth by means well known in the art, e.g. centrifugation and/or ultrafiltration. The factor 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.

Purification of the factor 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 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 7, infra.

6.2. COMPOSITIONS COMPRISING THE FACTOR

The factor obtained can be formulated alone; with a Bacillus related pesticide, which as defined, supra, is a

Bacillus strain, spore, protein or fragment, or other substance, thereof, with activity against or which kills pests or protects plants against a pest; with a chemical pesticide and/or an entomopathogenic virus 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 emulsifiable concentrate, an

aerosol or impregnated granule. Examples of such Bacillus strains include, but are not limited to, Bacillus thuringiensis subsp. kurstaki (marketed as DIPEL™ from Abbott Laboratories, Inc., JAVELIN™ from Sandoz, BIOBIT™ from Novo Nordisk A/S, FORAY™ from Novo Nordisk A/S, BIOCOT™ from Novo Nordisk A/S, MVP™ from Mycogen, BACTOSPEINE™ from Novo Nordisk A/S, and THURICIDE™ from Sandoz); Bacillus thuringiensis subsp. aizawai (marketed as FLORBAC™ from Novo Nordisk A/S, and XENTARI™ from Abbott Laboratories, Inc.); Bacillus thuringiensis subsp. tenebrionis (marketed as NOVODOR™ from Novo Nordisk A/S,

TRIDENT™ from Sandoz, and M-TRAK™ and M-ONE™ from Mycogen); Bacillus thuringiensis subsp. israelensis (marketed as either BACTIMOS™ or SKEETAL™ from Novo Nordisk A/S, TEKNAR™ from Sandoz, and VECTOBAC™ from Abbott Laboratories, Inc.); Bacillus thuringiensis kurstaki /tenebrionis (marketed as FOIL™ from

Ecogen) ; Bacillus thuringiensis kurstaki/aizawai (marketed as CONDOR™ from Ecogen and AGREE™ from Ciba-Geigy) ; and Bacillus thuringiensis kurstaki /kurstaki (marketed as CUTLASS™ from Ecogen) . The Bacillus related protein may be selected from the group including, but not limited to, Cryl, Cryll, Crylll, CrylV,

CryV, and CryVl. The chemical pesticide may be, for example, an insect growth regulator such as diflubenzuron, a carbamate such as thiodicarb and methomyl, an organophosphate such as chlorpyrifos, a pyrethroid such as cypermethrin and esfenvalerate, inorganic fluorine such as cryolite, and a pyrrole. The entomopathogenic virus may be a baculovirus, e.g., Autograpήa californica nuclear polyhedrosis virus (NPV) , Syngrapha falcifera NPV, Cydia po onella GV (granulosis virus) , Heliothis zea NPV, ymantria dispar NPV, Orgyia pseudotsugata NPV, Spodoptera exigua NPV, Neodiprion lecontei

NPV, Neodiprion sertifer NPV, Harrisina brillians NPV, and Endopiza viteana Clemens NPV.

In compositions comprising the substance and a Bacillus related pesticide, the substance may be present in the 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 substance per g Bacillus delta-endotoxin and

spore, preferably 2 g/BIU or 1 g substance per g Bacillus delta- endotoxin and spore. As 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 LC50 then multiplying by the reference standard potency.

In another embodiment, the composition may comprise the factor in substantially pure form or a supernatant from Bacillus in dry, concentrated, or liquid form and a pesticidally acceptable carrier, examples of which are disclosed, infra. This composition may be applied separately to a plant, e.g., transgenic plants. Specifically, the composition may be applied to a plant previously containing and expressing a Bacillus thuringiensis gene. In another embodiment, the composition may be applied to a plant previously exposed to a Bacillus thuringiensis composition. In another embodiment, the composition may be applied to other environments of a dipteran pest(s), e.g., water or soil. The substance is present in the composition at a concentration of about 0.001% to about 60% (w/w) .

The composition comprising the substance 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 substance/BIU up to optionally about 300 g substance/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 anionic compounds such as a carboxylate, for example, a metal carboxylate of a long chain fatty acid; a 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 or 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, fatt 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.

Examples of inert materials include inorganic mineral such as kaolin, mica, gypsum, fertilizer, phyllosilicates, carbonates, sulfates, or phosphates; organic materials such as sugar, starches, or cyclodextrins; or botanical materials such

as wood products, cork, powdered corncobs, rice hulls, peanut 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 pound to 5.0 pounds per acre when in dry form and at about 0.01 pint to 25 pints per acre when in liquid form.

In a further embodiment, the Bacillus thuringiensis crystal delta-endotoxin and/or 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 substance.

Such treatment can be by chemical and/or physical means as long as the treatment does not deleteriously affect the properties o 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' 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) , leguminous 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), lauraceae (avocados, cinnamon, camphor), deciduous trees and conifers (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. The composition can be applied by foliar, furrow, broadcast granule, "lay-by", or soil drench application. 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 can also be applied directly to ponds, lakes, streams, rivers, still water, and other areas subject to infestation by dipteran pests, especially pests of concern to public health. The composition can be applied by spraying, dusting, springling, or the like.

The compositions of the present invention may be effective against insect pests of the order Lepidoptera, e.g., Achroia grisella, Acleris gloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophila pometaria,

Amyelois transitella, Anagasta kuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis, Archips εp . , Argyrotaenia sp . , Athetis mindara, Bombyx mori , Bucculatrix thurberiella, Cadra cautella, Choristoneura sp. , Cochylis hospes, Colias eurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella, Datana integerrima, Dendrolimus sibericus, Desmia funeralis, Diaphania hyalinata, Diaphania

nitidalis, Diatraea grandiosella, Diatraea saccharaliε, Ennomoε subεignaria, Eoreuma loftini , Ephestia elutella, Erannis tiliaria, Estigmene acrea, Eulia εalubricola, Eupoecilia ambiguella , Euproctiε chrysorrhoea, Euxoa meεεoria, Galleria mellonella , Grapholi ta moleεta , Harriεina americana, Helicoverpa εubflexa, Helicoverpa zea, Heliothis vireεcenε, Hemileuca oliviae, Homoeoεoma electellum, Hyphantria cunea, Keiferia lycoperεicella, Lambdina fiεcellaria fiεcellaria, Lambdina fiεcellaria lugubroεa, Leucoma saliciε, Lobesia botrana, Loxostege εticticaliε, Lymantria dispar, Macalla thyrεisalis, Malacoεoma εp. , Mameεtra braεεicae, Mameεtra configurata, Manduca quinquemaculata, Manduca εexta, Maruca teεtulaliε, Melanchra picta, Operophtera brumata, Orgyia εp . , Oεtrinia nubilaliε, Paleacrita vernata, Papilio creεphonteε, Pectinophora goεsypiella, Phryσanidia californica, Phyllonorycter blancardella, Pieriε napi , Pieriε rapae, Plathypena εcabra, Platynota flouendana, Platynota sultana, Platyptilia carduidactyla, Plodia interpunctella, Plutella xylostella, Pontia protodice, Pεeudaletia unipuncta, Pseudoplusia includenε , Sabulodeε aegrotata, Schizura concinna, Sitotroga cerealella, Spilonota ocellana, Spodoptera sp. , Thaurnεtopoea pityocampa, Tineola biεεelliella, Trichoplusia ni , Udea rubigalis, Xylomyge curialis, Yponomeuta padella; order Diptera, e.g., Aedes εp. , Andes vittatus, Anaεtrepha ludenε, Anaεtrepha suspenεa, Anopheles barberi, Anopheles qruadrimaculatus, Armigereε εubalbatuε, Calliphora stygian, Calliphora vicina, Cerati tiε capi tata, Chironomuε tentanε, Chryεomya rufifacieε, Cochliomyia macellaria, Culex εp . , Culiseta inornata, Dacuε oleae, Delia antiqua, Delia platura, Delia radicum, Droεophila melanogaster, Eupeodeε corollae, Gloεεina auεteni , Glosεina brevipalpiε,

Gloεεina fuεcipeε, Gloεεina morεitanε centraliε, Glossina morεitanε morεitanε, Glossina moriεtanε εubmorεitanε, Gloεεina pallidipes, Glosεina palpaliε gambienεiε, Gloεεina palpaliε palpaliε, Gloεsina tachinoides, Haemagoguε equinuε, Haematobia irri tanε, Hypoderma boviε, Hypoderma lineatum, Leucopiε ninae, Lucilia cuprina, Lucilia εericata, Lutzomyia longlpaipiε, Lutzomyia εhannoni , Lycoriella mali , Mayetiola deεtructor, Muεc

autumnalis, Musca domestica, Neobellieria εp. , Nephrotoma εuturaliε, Ophyra aeneεcenε, Phaenicia sericata, Phlebotomus εp. , Phormia regina, Sabetheε cyaneuε, Sarcophaga bullata, Scatophaga εtercoraria, Sto oxyε calcitranε, Toxorhynchiteε amboinenεiε, Tripteroides bambuεa . However, the compositions of the invention may also be effective against insect pests of the order Coleoptera, e.g., Leptinotarεa εp. , Acanthoεcelideε obtectus, Callosobruchuε chinenεiε, Epilachna variveεtiε, Pyrrhalta luteola, Cylaε formicariuε elegantuluε, Liεtronotus oregonenεiε, Sitophiluε sp. , Cyclocephala borealiε, Cyclocephala immaculata, Macrodactyluε subspinosuε, Popillia japonica, Rhizotroguε majaliε, Alphitobiuε diaperinuε, Paloruε ratzeburgi, Tenebrio molitor, Tenebrio obεcuruε, Tribolium caεtaneum, Tribolium confuεum, Triboliuε deεtructor; Acari, e.g., Oligonychuε pratenεiε, Panonychuε ulmi, Tetranychuε urticae; Hymenoptera, e.g., Iridomyrmex humiliε, Solenopsiε invicta; Isoptera, e.g., Reticulitermeε heεperuε, Reticulitermeε flavipes, Coptotermeε formoεanuε, Zootermopεiε anguεticollis , Neotermeε connexuε, Inciεitermeε minor, Incisitermes immigrans; Siphonaptera, e.g., Ceratophylluε gallinae, Ceratophylluε niger, Noεopεylluε faεciatuε, Leptopεylla εegniε, Ctenocephalideε caniε, Ctenocephalideε feliε, Echicnophaga gallinacea, Pulex irritanε, Xenopεylla cheopiε, Xenopsylla vexabilis, Tunga penetranε; and Tylenchida, e.g., Melodidogyne incognita, Pratylenchus penetranε.

The following examples are presented by way of illustration, not by way of limitation.

7. EXAMPLE: CHARACTERIZATION OF la As detailed herein, la is recovered and purified. The characterization of la is detailed infra.

7.1. RECOVERY AND PURIFICATION OF la

B. thuringienεiε subsp. kurεtaki 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 la is detected at 13 hours into the fermentation. Peak activity is found to be at approximately 30 hours.

Supernatant from a B . thuringienεiε subsp. kurstaki 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 la 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 la 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.

7.2. 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. ¹³ 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 (referenced to 3-[trimethylsilyl propionic acid) . 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 carbon proton correlation experiment (HMBC) enabled assignment of each proton resonance in the molecule to its attached carbon.

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 for the active compound and indicates that an even number of nitrogens are present. Also, fragments containing 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 ₂ 8N6θ ₈ . Treatment of the active compound (13 mg) with 6 N HCl gave a derivative which is ninhydrin positive. These results indicate the presence of amide bonds. The derivative had the same R _f 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.

Another technique used to analyzed la is nOe (Nuclear

Overhauser Effect) which can detect proximity of protons to one another through space. nOe is carried out on an acetylated derivative of la. In a two dimensional nOe experiment (NOESY) , NOEs are observed between an N-H proton at 8.06 ppm and the 5.17 proton (Figure 4) .

The following structure has been elucidated for la

It can be classified as a ureido amide. Constituents include 2 amides, a urea, two aminos, and five hydroxyls. It contains seven chiral centers.

7.3. PROPERTIES OF la

The isolated la is found to potentiate the activity of Bacillus thuringiensis subsp. kurstaki and Bacillus thuringienεiε 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 CrylA(a) , (b) , and (c) proteins. Incubation time of la with the Cry protein is not found to be critical for bioactivity. However, la is inactive alone. The level of potentiation is found to be 100-200 fold for the truncated CrylA proteins, Cryll and CrylC inclusions and approximately 320 fold with full-length CryΙA(c) (see Tables I

and II respectively). Specifically, ^■ for full-length protein, 0.75 μg/ml CryΙA(c) produced the same insect mortality/stunt score when la is included as 240 μg/ml of CrylA(c) alone. In the case of the truncated CrylA(c) , an OD _2ao of 0.0006 gave the same stunt score in combination with la as the same sample of CrylA(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 la as Cryll protein alone at 75 μg/ml, a 125 fold potentiation. CrylC inclusions, at 0.3 μg/ml with the addition of la 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 la. la is found to be stable by bioassay as described in

Section 7.1. 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. la 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 HCl.

TABLE I POTENTIATION EFFECTS OF la WITH PURIFIED TRUNCATED Bt PROTEIN Bt Protein SoodoOt ra Exiσua

Type OD280 Ja Mortality* Stunt smrst

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 2.8 0.020 + 1/5 0.0

0.010 + 0/5 0.2

0.005 + 1/5 0.0

0.0025 + 2/5 2.0

0.0012 + 0/5 1.0 0.0006 + 1/5 1.0

None NA + 0/5 4.0

None NA - 0/5 4.0

* 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 .

TABLE II POTENTIATION EFFECTS OF la WITH Bt PROTEIN Bt Protein Spodoptera Exi a

Type μσ/ml la Mortality* Stunt Score CryΙA(c) 240 - 1/5 0.5

120 - 0/5 2.2

60 - 0/5 2.2

30 - 0/5 4.0

60 + 5/5 30 + 5/5

15 + 4/5 0.0

3 + 4/5 1.0

0.8 + 2/5 1.6 Cryll 300 - 1/5 0.8

150 - 2/5 0.7

75 - 1/5 0.2

38 - 0/5 0.8

19 - 0/5 1.6 9 0/5 1.8

5 - 1/5 4.0

38 + 3/5 1.0

19 + 2/5 0.5

9 + 3/5 0.0 5 + 1/5 0.5

2.4 + 1/5 0.0

1.2 + 3/5 0.5

0.6 + 2/5 0.3 Cryll 300 - 2/5 0.3

150 - 2/5 0.0

75 - 1/5 0.8

38 - 0/5 3.2

38 + 5/5 19 + 5/5

9 + 5/5

5 + 4/5 0.0

2.4 + 1/5 0.0

1.2 + 5/5 0.6 + 3/5 1.5

0.3 + 2/5 1.3

None NA - 0/5 4.0

None NA + 0/5 4.0

* 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.

7.4. EVALUATION OF OTHER SUBSPECIES OF Bacilluε thuringi ensis AND OTHER SPECIES OF Baci l li

Several Bacillus species are evaluated for production of la. 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 la production using the Spodoptera exigua micro- bioassay described supra. B. thuringiensiε subsp. aizawai strain EMCC0087 (deposited with the NRRL as NRRL B-21148) and B. thuringienεiε subsp. galleriae (deposited with the NRRL) are found to produce la in about the same concentration as B. thuringienεiε subsp. kurεtaki . la is also produced in B. εubtiliε, B. cereuε , B. t . subsp. alesti , B . t . subsp. canadienεiε, B. t . subsp. darmεtadiensiε , B. t . subsp. dendroli uε , B. t . subsp. entomociduε , B . t . subsp. finitimus, B . t . subsp. israelenεiε, B. t . subsp. kenyae, B . t . subsp. morriεoni , B. t . subsp. subtoxicus, B.t. subsp. tenebrioniε, B. t . subsp. thuringienεiε, and B.t. subsp. toumanoffi , B . cereuε, B . εubtiliε, and B. thuringienεiε subsp. kurεtaki cry- spo- mutant as determined by capillary electrophoresis.

Specifically, a Beckman 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 la. Sample volumes are 20 nl with a run time of 25 minutes.

A standard curve is generated using purified la as the standard at levels of 1.25 mg/ml, 0.625 mg/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 equation is used to generate the concentration of la 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 1 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.

7.5. RVAT.UATION 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 0.071 g Ia/BIU.

TABLE III la IN Bacillus thuringienεiε PRODUCTS

7.6. 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 Heliothis virescens larvae, third instar Trichopluεia ni larvae, third instar Pεeudopluεia includenε larvae, third instar Plutella xyloεtella larvae, third instar Spodoptera littoraliε, and third instar Mameεtra braεεicae larvae.

To determine the level of potentiation by adding la 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 la 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 la (0.658% active ingredient). S. li ttoraliε and M. braεεicae are tested using FLORBAC™ HPWP and la.

The various B.t. products are weighed and la 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 la) and reference substances are prepared as well. Reference substances include B . t . k. 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 samples 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, LC90S, 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 la, the LC ₅₀ of the B.t./la 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 la.

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 la (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'C for 3-4 days. As shown in Table IV, significant reductions in LC _so s are observed for all species.

TABLE IV Diet Incorporation Bioassays

Increase in activity

Insect σ la per BIU Fold reduction in LC^n

Spodoptera exigua 0.1 1. 5 (BIOBIT™ HPWP) 0.2 1. 7

2.0 4. 3

4.0 7. 5

Spodoptera exigua 7.4 13 (BIOBIT™ FC) 15 26

30 34

118 59

237 79

Spodoptera frugiperda 0.2 2. .2 (BIOBIT™ HPWP) 0.8 3. .9

2.0 7. ,2

4.0 11. .6

Trichoplusia ni 0.1 1. .1 (BIOBIT™ HPWP) 0.2 1. .2

2.0 2, .0

4.0 3, .1

Pseudoplusia includens 0.1 0 (BIOBIT™ HPWP) 0.2 1 .2

0.8 2 .1

2.0 2 .4

4.0 3 .4

Plutella xylostella 0. 1 .6 (BIOBIT™ HPWP) 0. 1 .3 2.0 1 .4 4.0 1 .9

Helicoverpa zea 3.2 12.6 (BIOBIT™ HPWP) Heliothis virescens 3.2 4.2 (BIOBIT™ HPWP)

Lobes ia bothrana 2.0 3.0 (BIOBIT™ HPWP)

Spodoptera littoralis 2.0 8.6 (FLORBAC™ HPWP)

Wajnestra 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 la added/BIU 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. LC50, LCg ₀ and potency are calculated from matched product lacking la. The results shown in Table V indicate that la 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

σ la oer BIU Increase in activity

Product Fold reduction in Ccn

BACTOSPEINE™ WP 0.4 1.04

1.7 2.3

CONDOR™ 0.4 2.4 1.7 5.1

AGREE™ 0.4 1.1 1.7 1.6

CUTLASS™ 0.4 1.1 1.7 2.5

MVP™ 0.4 6.0 1.7 7.7 2.0 12.1

FLORBAC™ HPWP 0.2 1.1 0.8 2.0

DIPEL™ 2X 0.2 1.2 0.8 2.3 2.0 3.9

JAVELIN™ WG 0.2 0 0.8 1.08 2.0 2.9

XENTARI™ 0.2 1.2 0.8 1.6 2.0 2.4

7.7. FOLIAR BTOASSAYS

Foliar bioassays are performed with second.instar Spodoptera exigua larvae on broccoli plants using BIOBIT™ F _C and la. The ratio of la to BIOBIT™ FC is the same 2 g Ia/BI _U 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 are shown in Table VI, infra . 100% mortality is observed at a rate of 8.7 BlU/hectare BIOBIT™ FC + la, 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 BlU/hectare 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 BlU/hectare.

TABLE VI

Foliar Bioassavs

Treatment BTU/hectare % mortalitv larval instar

BIOBIT™ FC 58.8 92% 2

BIOBIT™ FC 17.6 8% . 2

BIOBIT™ FC + la 8.7 100% 2

BIOBIT™ FC +

8hr sunlight 58.8 27% 2

BIOBIT™ FC + la

+ 8hr sunlight 8.7 100% 2

BIOBIT™ FC 52 75% 4

BIOBIT™ FC + la 13 100% 4

7 . 8 . 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 treatment) . JAVELIN™ WG at 45 BlU/hectare gave 51% control.

Field trials on sweet corn (Spodoptera frugiperda) demonstrated that at 39.5 BlU/hectare, 2 g Ia/BIU BIOBIT™ FC provided 84% control.

7.9. 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 la. 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 VII Plutella xyloεtella (B . t . k. Resistant) Resistance Ratios

LC50 RR LC90 RR

PRODUCT TESTED

JAVELIN™ WG 302 . 6 3829 .7

BIOBIT™ HPWP 20 . 5 98 . 5

23 . 2 88 .0

2 . 0 g Ia/BIU BIOBIT™ HPWP

4 . 0 g Ia/BIU 10 . 4 11 .5 BIOBIT™ HPWP

XENTARI™ 9 .7 8 .2

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.

8. DEPOSIT OF MICROORGANISMS

The following strains of Bacilluε thuringiensiε 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

EMCC0086 NRRL B-21147 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.