This invention relates to novel biologically - based pesticides and their use in reducing insect infesta¬ tions in plants. More specifically, this invention relates to the use of adjuvants mixed with the biopesticides, wherein the adjuvants comprise compounds which -are capable of binding tannins contained within or on the surface of th plant, thereby preventing the interference of the tannins with the biopesticide activity. Applications of this invention include, but are not limited to, pesticides suitable for use in reducing or preventing an infestation o lepidopteran insects on tannin containing plants such as cotton.

2. BACKGROUND OF INVENTION

2.1. Commercial Pesticides: General Considerations

Each year, significant portions of the world- ¹ s commercially important agricultural crops are lost to insec and other pest infestation. The damage wrought by these pests extends to all areas of commercially important plants including foods, textiles, and various domestic plants, and the economic damage runs well into the millions of dollars. Thus, protection of crops from such infestations is of paramount concern.

Broad spectrum pesticides are most commonly used for crop protection, but indiscriminate use of these agents can lead to disruption of the plant's natural defensive agents. .Furthermore, because of their broad spectrum of activity, the chemical pesticides may destroy non-target organisms such as beneficial insects and parasites of

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destructive pests. They are also frequently toxic to animals and humans and, thus, pose environmental hazards when applied.

Additionally, insects and other organisms have frequently- developed resistance to these pesticides when repeatedly exposed to them. In addition to reducing the utility of the pesticide, resistant strains of minor pests may become major infestation problems due to the reduction tσ of beneficial parasitic organisms. This is a major problem encountered in using broad spectrum pesticides.

What is needed is a biodegradable pesticide that combines a narrower spectrum of activity with an ability of

T5 maintaining its activity over an extended period of time, i.e., to which resistance develops much more slowly, or not at all. Biopesticides appear to be useful in this regard.

2.2. Biological Pesticides

20

Biopesticides, also called biorationals, make use of naturally occurring pathogens (diseases) to control insect, fungal, and weed infestations of agricultural crops. Such substances may comprise a bacterium which produces a substance, toxic to the infesting agent (a toxin) , with or

2E without a bacterial growth medium. Such bacteria can be applied, directly to the plants by standard methods of pesticide- application and will persist on the crops for an extended period of time, decreasing the need for repeat applications.

30

The use of biological methods of pest control was first suggested in 1895 when a fungal disease was discovered in silkworms. It was not until 1940, however, when spores of the milky disease bacterium Bacillus popilliae were used

35

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to control the Japanese beetle, that successful biological pest control was first achieved. In the late 1960's, the discovery of a new strain of a previously-known bacterium that produced a toxin fatal to caterpillars set the stage for commercial biopesticides. This bacterium, named Bacillus thuringiensis, also known as BT, is currently the most widely used biopesticide.

Bacillus thuringiensis fits well into current agricultural theories which support the use of naturally occurring organisms to suppress harmful insects. It is a widely distributed, rod-shaped, spore forming, aerobic, gra positive micro-organism and is characterized by producing, during the sporulation cycle, one or more proteinaceous parasporal crystals which are pathogenic to many insect species.

BT is a common inhabitant of the environment. It has no known adverse effect on life forms such as man, pets birds, fish, earthworms, most beneficial insects or plants. Its pathogenicity to sensitive insects is essentially due t the presence of a parasporal crystal, toxic to these insects, which may represent 30 to 40% of the dry weight of the BT cell at the time of sporulation. It is active only when ingested. Some hours after ingestion sensitive insect cease to feed and, therefore, damage to the plant is stopped. Most sensitive insects die after approximately 24 to 72 hours from toxemia due to the crystal proteins. This is sometimes accompanied by septicaemia as a result of the presence of germinating spores.

Thus, the principal pesticidial effect of BT is due to the toxin crystal which acts only after its ingestion. In Lepidopteran pests, the activation of the crystal is caused by a combination of alkaline pH and

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proteolytic enzymes in the gut. The reaction is dependent on the high gut pH of Lepidopteran larvae (pH greater than 7) which allows the release of the toxic components of the crystal. These proteins poison the mid-gut cells causing feeding to stop. The growth of bacteria released into the gut cavity can result in septicaemia which also may play a part in the death of the insect.

It is clear that the use of BT as an insecticide provides an effective and environmentally acceptable method of dealing with various strains of infesting pests. Thus, biological pest control, and use of BT in particular, has a number of distinct advantages. Biopesticides are generally nontoxic to man and other mammals. They are highly selective and spare beneficial plant insects. Resistance to them is less common and develops slowly. Some limitations, however, have not yet been overcome: when applied to crops, biopesticides are subject to rapid breakdown by sunlight; they can be washed off foliage by rain or irrigation. Additionally, they are often attacked and rendered inactive by substances secreted by the plant or contained within the plant. Indeed, in 1976, the Chemical Control Research Center conducted field tests of an aerial sprayed BT formulation containing Bacillus Thuringiensis and a variety of adjuvants including polyvinylpyrrolidone (PVP) , which was used as a "tacky film-* for aerial application promoting the adhesion of the formulation to the spruce trees. (Report CC-X-144 AERIAL FIELD TRIALS WITH A NEW FORMULATION OF BACILLUS THURINGIENSIS AGAINST THE SPRUCE BUDWORM, CHORISTONEURA FUMIFERANA, Morris et al. , May, 1976). However, the formulation was found to be ineffective in reducing the infestation.

Recent advances in genetic engineering have lessened some of the problems. For example, researchers have used a combination of recombinant and classical geneti

fe-/ -i __J»>O i

techniques to produce and identify novel strains of Bacillu thuringiensis. To produce genetically altered strains of BT, naturally occurring BT strains were collected from the soil, from plants and from grain storage silos and were screened for their ability to produce insect toxin. BT strains that produced high amounts of toxin and novel toxin proteins were then identified and isolated; classical genetic manipulation was then employed to further increase insecticidal activity.

In a process known as "'curing,'-' growth of the bacteria at elevated temperatures was used to induce strain of BT to lose genes which did not code for the production o the desired insect toxin. A second method called "conjuga¬ tion- ¹ ' (mixed culturing) was used to promote the exchange of genetic material between BT strains. Through careful and painstaking examination of cured and mixed cultured strains, new strains of BT with increased insecticidal activity were isolated. As a result, novel Bacillus thuringiensis strain with insecticidal activity up to 150 times greater than tha found in commercial strains can be produced.

The attack and binding of the bacteria and toxins by substances produced by or contained within the plant, however, has proven to be a major drawback to biopesticide use. Unlike chemical pesticides, which can be quite inert, biopesticides are comprised of living organisms or compounds produced by them and can thus, be easily killed or inactivated by various substances. Many of the substances produced by plants as a defense against diseases and/or other infestations, will also be toxic to the biopesticide bacteria or tend to bind any produced toxin; such a binding of toxin can occur even within the insect gut. Thus, in many cases the utility of biopesticides is severely limited.

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Fσr example, it is a well known fact that cotton leaves contain condensed tannins; researchers have proposed that these tannins function to protect this plant from certain forms of insect attack (see, e.g., Klocke and Chan, Insect Physiology, Vol. 28 No. 11, p. 911, 1982). Such tannins also exert an effect on BT, inactivating the toxins it produces (Luthy, et al., FEMS Microbiology Letters, 28, p. 31 (1985) , making the use of a BT based biopesticide less effective. In fact, while BT has been found to be quite useful in the control of tobacco budworm (H. Virescens) on tobacco, it is much less effective in controlling the same insect on cotton plants (Microbial and Viral Pesticides, E. Kurstak (ed.), 1982, pp 209-210).

2.3. Tannin Chemistry

Tannin compounds are derivatives of tannic acid and are characterized by cyclic structures coupled with hydroxyphenols. The tannins are divided into two main groups, condensed tannins and hydrolyzable tannins. The condensed tannins have been identified in many plants. Although the role of these tannins is not clear, it has been postulated that they act to protect the plant from insect, fungal, etc., attack (see Klocke and Chan, supra) .

Condensed tannins are characterized by the presence of a large number of phenolic groups, and, thus, will tightly bind to amides, such as those present in proteins. Any such attachment to a protein will induce a conformational stress which can eventually denature or precipitate it, or block its functional groups. Thus, plan tannins can act to inhibit the actions of any protein-based toxin produced by a bacterium such as BT. Also, ingestion of tannin-containing plants by insects will cause a high concentration of tannin in the insect's gut; such tannins

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can bind any ingested toxins. This property has been exploited to isolate various tannins and proteins from plan preparations (see Loomis, Methods in Enzymology, 3_1 ( 14 ) , p.

528) . To date, however, no one has designed a biopesticide system which preferentially binds the tannins to non-active components, leaving the unbound (active) component still active.

3. SUMMARY OF INVENTION

It is generally an objective of this invention to provide a biopesticide suitable for use on plants which contain or produce tannins. It is further an objective of this invention to provide a biopesticide mixture which contains a component capable of competitively binding with said tannin, thereby leaving the active pesticide component free and, furthermore, which does not harm the plant or inactivate the biopesticide. It is also an objective of this invention to provide methods for reducing or preventin insect infestation on plants with the biopesticide mixture.

This invention provides novel biopesticide mixtures or formulations which contain an adjuvant capable of binding tannins. This adjuvant permits the use of such biopesticide mixtures in plants which contain or produce tannins, since it will competitively bind the tannins, preventing the binding and inactivation of the toxins produced. Surprisingly, however, the adjuvant does not adversely affect the pesticide activity. Thus, these biopesticide formulations will be more potent and longer- acting than those without the tannin-binding component.

Such formulations can be used on a wide variety o commercially important agricultural crops, particularly the angiosperms or seed-bearing plants. Such plants include, but are not limited to, food crops such as the cereals

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(wheat, corn, oats, barley, millets) , green vegetables (beans, peas, lettuce) , root vegetables (potatoes, carrots) and frui -bearing trees. Important textile crops such as cotton and timber crops such as oaks and other trees can also be treated. Thus, the formulations can be used over a wide array of commercially important plants.

The formulations are also effective against a large number of insects which commonly infest such plants. 0 Potential applications include, but are not limited to, control of insects generally classified in the following ordersr

Orthαptera - which includes grasshoppers, crickets, and roaches. 5 Diptera - which includes flies.

Hermoptera - which includes cicadas and aphids.

Coleoptera - which includes beetles and weevils.

Lepidoptera - which includes moths and butterflies.

In one embodiment, the sporulated culture of BT 0 bacteria (containing spores and crystals) is mixed with polyvinypyrrolidone (PVP) which acts as a tannin binder due to its amide linkages. Such a formulation can be mixed with suitable adjuvants as carriers, and applied directly to plants as" a liquid spray or a solid dust. Such formulations 5 have been shown to be effective against Lepidoptera infestations of cotton plants for nearly a week, while the BT alone rapidly lost its potency after 1-3 days.

It is postulated that this toxicity enhancement occurs due to competitive binding of the tannins by the PVP, 0 allowing more of the BT toxin to remain unbound- and, therefore, potent. Thus, use of these formulations can result in a longer-lived and more potent biopesticide on plants which produce high concentrations of tannins.

351.

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Other novel biopesticides provided by this invention comprise BT parasporal toxin crystals as the active pesticide component. Such compounds also exhibit broad range pesticidal activity and offer a wide degree of latitude in the preparation of the formulations.

Likewise, in addition to straight chain PVP polymers, copolymers and substituted polymers of PVP, as well as other compounds which can bind tannins, can be used as adjuvants. In particular, compounds possessing functional groups which can form H bonds with the tannin such as synthetic polya ides (e.g. Nylon) or proteins, especially those proteins rich in proline, which possesses an amine group are useful. Also useful are polyethylene glycols, polystyrene resins, borates, and urea. Thus, a wide array of biopesticides suitable for use on tannin containing plants is encompassed by this invention.

Additionally, this invention provides methods for reducing insect infestations in tannin - containing plants such as Angiosperms by utilizing the biopesticide compounds.

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4. DETAILED DESCRIPTION OF THE INVENTION

4.1. Chemistry of the Tannin Binding

The tannins contained in plants are, for the most part, condensed tannins, which are associated with substituted phenols. These phenolic groups give the tannin ^** distinctly acidic properties and, thus, these compounds wil_. be tightly bound by groups possessing basic functionalities, such as amide groups in proteins. Thus, these compounds ca bind and denature or precipitate proteins.

The tannins are contained within the plant cells and can also be found on plant surfaces. When released fro the cell, they act to bind and/or precipitate any proteins. Since any pest insect will feed on the plant, ingestion will cause disruption of these vacuoles and, consequently, release of the tannins. Ultimately, loss of the biopesti¬ cide ffectiveness is observed due to binding of either the parasporal crystal or its metabolites, particularly the toxin. To prevent this, the tannin must be, in some way, bound to a tannin-binding compound; such a compound must possess basic functionalities yet be inert toward both the plant and bacterial cells and toxin (otherwise, the pesticide would be useless) .

One compound meeting these criteria is polyvinyl- pyrrolidone (PVP) , a polymer which is comprised of the following subunits:

CH — CH ₂ -

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Such a compound possesses an amide nitrogen as part of the pyrrolidone ring and will, thus, competitively bind tannins

When mixed with a biopesticide and contained in a suitable carrier, the tannin-binding compound will competitively bind the tannin, preventing its attachment to the proteins of the biopesticides, thereby enhancing its toxic action. In this way, the biopesticide will exhibit enhanced activity and persistence on such plants.

In addition to straight chain PVP, copolymers and substituted polymers of PVP can also be effective tannin binding agents. Such compounds are marketed under a variet of trade names and include, but are not limited to, the following:

vinyl pyrollidone/vinyl acetate copolymers PVP VAE 735, PVP VAI 735 (mole ratio 70/30) PVP VAE 635 (mole ratio 60/40) PVP VAE 535, PVP VAI 535 (mole ratio 50/50) PVP VAE 335, PVP VAI 335 (mole ratio 30/70) (note: E indicates that the polymer is dissolved in ethanol, I indicates isopropanol)

alkylated vinyl pyrollidone polymers GANEX V 216 (avg. mol. wt. 7300) GANEX V 220 (avg. mol. wt. 8000) GANEX V 516 (avg. mol. wt. 9500) GANEX P 904 (avg. mol. wt. 16,000)

vinyl pyrollidone/styrene copolymers POLECTRON 430

vinyl pyrollidone/dimethylaminoethylmethacrylate copolymers

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copolymer 845 copolymer 937 copolymer 958

vinyl pyrrollidone/quarternized dimethyl- aminoethylmethacrylate copolymers

Gafquat 734

Gafquat 755

Gafquat 755 N

polyvinylpyrrollidone

PVP K-15 (avg. mol. wt. 10,000)

PVP K-30 (avg. mol. wt. 40,000)

PVP K-60 (avg. mol. wt. 220,000)

PVP K-90 (avg. mol. wt. 700,000)

POLYCLAR AT

POLYCLAR 10

In addition to PVP and its copolymers and substituted polymers, any substance having a plurality of amine, amide, or imine or any other functionalities capable of forming H-bonds with the tannins can be a suitable tannin-binding substance in the practice of this invention. This includes proteins such as whey, generic cotton protein and soy protein, but especially proteins rich in proline such as gelatin and other collagens. Also useful are synthetic polyamides such as nylon; polyethylene glycols; polystyrene resins, such as Amberlite XAD-4; borates; and urea. The only criterion for selection of such a substance is that it have a greater affinity for tannin than the BT toxin since the binding is competitive.

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4.2. Composition of the Formulations

The biopesticide mixtures consist essentially of the actual biological pesticide the tannin-binding agent an a suitable carrier. The biological pesticide can be sporulated BT, a mixture of the BT spores and toxin, or the parasporal toxin crystals of BT individually or in a mixtur with one another; other pesticide components can be added t obtain a broader spectrum of effectiveness. The formulations may be administered as a dust or as a suspension in oil (vegetable or mineral) or water, a wettable powder or in any other material suitable for agricultural application, using the appropriate carrier adjuvants. Suitable carriers can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g., natural or regenerated minera substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

The mixtures containing a solid or liquid adjuvant, are prepared in known manner, e.g., by homogeneo¬ usly mixing and/or grinding the active ingredients with extenders, e.g., solvents, solid carriers, and in some case surface active compounds (surfactants) . Spray drying can also be used to obtain a homogenous mixture.

Suitable liquid carriers are vegetable oils, such as coconut oil or soybean oil, mineral oils or water. The solid carriers used, e.g., for dusts and dispersible powders, are normally natural mineral fibers such as calcite, talcum, kaolin, or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, seplolite o

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bentonite; and suitable nonsorbent carriers are materials such as silicate or sand. In addition, a great number of pregranulated materials or inorganic or organic mixtures can be used, e.g., especially dolomite or pulverized plant residues.

Depending on the nature of the active ingredients to be formulated, suitable surface-active compounds are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants.

Suitable anionic surfactants can be both water- soluble soaps and water-soluble synthetic surface active compounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted ammonium salts of higher fatty acids (C -C ) , e.g., the sodium or potassium salts of oleic or stearic acid, or natural fatty acid mixtures which can be obtained, e.g. , from coconut oil or tallow oil. Further stable surfactants are also the fatty acid methyltaurin salts as well as modified and unmodified phospholipids.

More frequently, however, so-called synthetic surfactants are used , especially fatty sulfonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylaryl- sulfonates.

The fatty sulfonates or sulfates are usually in the forms of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and generally contain a -C alkyl, e.g., the sodium or calcium salt of

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dodecylsulfate, or of a mixture of fatty alcohol sulfates, obtained from fatty acids. These compounds also comprise the salts of sulfonic acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic aci groups and one fatty acid radical containing about 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylben- zenesulfonic acid, dibutylnaphthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g., salts of the phosphoric acid ester of an adduct of p-nonylphenol wit 4 to 14 moles of ethylene oxide.

Nonionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohol or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Other suitable non-ionic surfactants are the wate soluble adducts of polyethylene oxide with alkypropylene glycol, ethylenediaminopolypropylene glycol and alkylpoly- propylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glyco ether groups and 10 to 100 propylene glycol ether groups.

Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil, glycol ethers, polypropylene/polyethylene oxide adducts, tributyl- phenoxypolyethoxyethanol,ethylene glycol octylphenoxypoly- ethoxynethanol and the Triton series of surfactants. Fatty

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acid esters of polyoxyethylene sorbitan, such as pσlyoxyethylene sorbitan trioleate, are also suitable non- ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which contain, as substituents on the nitrogen, at least one ^c -C__ alkyl radical and, ^' as further substituents, lower unsubstituted or halogenated alkyl benzyl, or hydroxylated lower alkyl radicals. The salts are preferably in the form of halides, methyl sulfates or ethylsulfates, e.g., stearyltrimethylammonium chloride.

In a preferred embodiment, PVP is mixed 1:100 in a water carrier. The concentration of the BT in the water can be varied to achieve the desired application rate, but the concentration of PVP remains constant. Other carriers can be used as conditions warrant.

4.3. Methods for Reducing Insect Infestation Using the BT/Tannin-Binding Compound Formulations

The above mixtures can be applied directly to the plants infested with the insects by any convenient means including aerial spray, dusting, ground spraying, and fogging. Once applied, the BT toxic activity will persist for an extended period of time, thus providing protection against reinfestation and eliminating the need for frequent reapplications to continue protection.

The amount of tannin binding compounds used in such formulations must be sufficient to effectively and competitively bind the plant tannins. For longer term protection, additional tannin binder can be added, but this eventually decreases the cost effectiveness of the use of such formulations. Additionally, a practical limit exists

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since, eventually, the supply of toxin becomes exhausted. For PVP, its concentration in the overall formulation can range from 0.1 to 10% (weight/volume) and preferably ranges from 0.2 to 5%. A wide variety of PVP compounds can be use including PVP K-15, PVP K-30, PVP K-60 and PVP K-90 as defined by the Fikentscher's K scale. Copolymers and substituted polymers of PVP are also useful as are other tannin binding substances described supra. The precise concentration ranges will vary as other tannin binding substances are substituted for the PVP.

5. EXAMPLES

5.1. Effect of PVP on BT Activity

The effect of PVP on the biopesticide activity on BT against cabbage looper (T^ ni) was examined by feeding these larvae cotton leaves coated with BT or BT/PVP mixtures; cabbage leaves (which contain low concentrations of tannins) were used as a control. The BT, when applied, was used at its approximate LC-50. After 3 days, the results were as follows:

_SU B _S TITUTE SHEET

No. Insects Dead

Treatment BT (out of 15 Total) cabbage yes 14 none 8 cotton yes 8 none 8 cotton + PVP K-15 yes 15 none 11 0 cotton + PVP-insol, yes 14 none 10

PVP-insol. - a cross-linked form of PVP also called

T5 polyvinylpolypyrrolidone and marketed as Polyclar by GAF

The results indicate that the presence of PVP, either in straight chain or .in crosslinked form, positively enhances the activity of BT.

20

Subsequently, the LC-50 of BT (alone) and BT -i- PVP K-15 (10%) toward tobacco budworm (H. virescens) was determined in a similar manner; cotton leaves coated with varying quantities of BT or BT/PVP K-15 were fed to the organisms for 3 days; based on these results the LC-50

25 values were calculated:

BT alone 8.0 (95% confidence interval range

4.4 - 14.8)

BT/PVP K-15 0.43 (95% confidence interval range

30

0.05-0.82)

Thus, the addition of PVP K-15 produces a dramatic effect on BT toxicity; nearly 18.6 - fold enhancement is observed over the control.

35

STITUTE Sn__£_r

5.2. Persistence of BT Activity

The persistence of BT activity was examined on Stoneville 213 cotton, using formulation EG 2035 with and without 1% PVP K-15, compared with a commercial formulation of DIPEL against H.virescens. The pesticides were applied by tractor as an aqueous solution using a CO sprayer; periodically, leaves were excised from plants and were each infested with one larva, the results were obtained 96 hours after this infestation. The results were as follows:

Application % Mortality .1"

Treatment Rate 1 3

(Kg/Ha) (days after application)

EG 2035 + 1% PVP 1 100 100 96.7 96.7

EG 2035 + 1% PVP 2 100 100 100 93.3

EG 2035 1 100 100 83.3 76.7

EG 2035 2 100 100 100 90

DIPEL ² + PVP 1 100 83.3 83.3 80

DIPEL ² 1 100 96.7 76.7 70

H-O (control) 23.3 3.3 3.3 3.3 1% PVP 19.7 3.3 3.3 3.3

_ All percentages were obtained using 30 replicates

DIPEL is a BT insecticide produced and marketed by Abbott Laboratories.

The results reveal that the PVP exerts a significant effect on the activity of both EG 2035 and DIPE (both of which contain BT toxin) , and the activity is still present after 7 days.

Further experiments were then conducted under field conditions; each pesticide was applied at 0.1 lb in 9.2 gallons H O/acre at a 40 psi spray pressure. Several

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different BT strains were used and the effectiveness against both H.virescens and H. zea were examined. The results were as follows :

% Mortality (N = 30) Treatment 0 days 6 - days

HV HZ HV HZ no PVP 1% PVP no PVP 1% PVP no PVP 1% PVP no PVP 1% PVP

DIPEL WP 100 97 93 83 73 77 7 17

EG 2038 100 100 100 100 50 87 17 47

EG 2012 100 100 70 100 40 90 13 37

EG 2095 100 100 100 100 27 73 0 20

EG 2101 100 100 100 100 67 87 7 57

EG 2035 100 100 90 87 50 87 17 47

Again, significant enhancement of biopesticide activity after 6 days can be observed when 1% PVP is added.

5.3. PVP - Molasses Tests

In tests similar to the field tests of 5.2, the following solutions were applied at a rate of 0.1 lb. in 9.2 gal. H-O/acre at a 30 psi spray pressure. The effectiveness against H^ zea larvae were examined 3 and 7 days after treatmen .

SUBSTITUTE SHEET

% Mortality (N = 30)

Treatment 3 days 6 day

EG 2038 10 3

EG 2038 + 1% PVP 27 7

EG 2038 + 1% molasses 40 27

EG 2038 + 1% PVP + 1% molasses 57 33

EG 2095 40 0

EG 2095 + 1% PVP 77 33

EG 2095 + 1% molasses 67 17

EG 2095 + 1% PVP + 1% molasses 87 57 control (no treatment) 0 0

It appears that both additives clearly enhance performance separately; but this effect is greatly increased when both are combined.

5.4. Effect of PVP on BT Toxicity

To assess the effect of PVP on BT toxicity toward H. virescens, a series of experiments were conducted using leaves from various cotton cultivars having varying tannin contents. Briefly, leaf discs were isolated from each cultivar and dipped in aqueous formulations of EG-2095, ranging from 1.25 - 20 micrograms/ml in concentration. The formulations also contained a surfactant (Triton X-100) , and duplicate formulations (with and without 10% by weight, PVP K-15) were examined.

The discs were then incubated with H. virescens and, after 3 days, the number of dead larvae were tallied. From this data, the LC-50 of the formulation was determined for each cultivar. The results were as follows:

^' _S UBSTITUTE SHEEl

LC 50 LC 50

Cultivar Tannin Content (no PVP) (+ 10% PVP)

(E 1/1)

TX 401 7 - 39.9

ST 825 10 - 11.5

LANK 57 10 - 40.9

TX 1.055 20 - 14.5

TX 1124 24 — 5.3

The formulations containing no PVP had no toxic effect on the H. virescens, indicating that the concentra¬ tion was too low to observe an effect. Therefore, a second experimental series was conducted using a broader BT concentration range (5-80 micrograms/ml for the + PVP formulations and 80-1280 micrograms/ml for the formulations without PVP) . Seedlings and cabbage containing very low amounts of tannin were used as controls. The results were as follows:

No PVP Formulations TABLE 2 Content LC-50 Range

Cultivar Tannin (E 1/1) LC-50 Lower Limit Upper Limi

TX 401 7 47 0.08 119

ST 825 10 71 11.5 129 LANK 57 10 117 128 889

TX 791 15 137 1 312

TX 1055 20 260 136 444

TX 1124 24 237 114 1239

Cabbage Very Low 6.3 2.9 9.2

ST 213

Seedling Very Low 26.3 18.3 39.7

SUBSTITUTE ) i .*

+10% PVP Formulations

Cultivar Tannin E 1/1 LC-50 Lower Limit Upper Limi

TX 401 7 3.75 0.3 7.4

ST 825 10 6.9 3.2 10.3

LANK 57 10 2 0 6.5

TX 791 15 3.9 0.23 8.1

TX 1055 20 12.9 8.9 109

TX 1124 24 8.5 4.3 12.8

Cabbage Very Low 6.9 2.7 11

ST 213

Seedling Very Low 5.7 2.3 8.6

A comparison of the data reveals that the PVP significantly lowers the LC-50 of the formulations as the tannin content increases. Briefly, the LC 50 was decreased by the following amounts:

Cultivar Tannin E 1/1 LC-50 Ratio (-PVP/+PVP)

TX 401 7 12 . 53

ST 825 10 10 . 29

LANK 57 10 58 . 50

TX 291 15 35 . 13

TX 1055 20 20 . 16

TX 1124 24 27 . 88

Cabbage Very 'Low 0.91

ST 213 Seedling Very Low 4.61

Two additional experiments were conducted utilizing Ecogen BT formulations EG 2038 (at a 10 micrograms/ml treatment rate) and EG 2348 (at a 2

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microgram/ml treatment rate) using mature Stoneville 213 cotton plants, and the same experimental procedure. However, instead of using cultivars with varying tannin contents, in this experimental series the quantity of PVP (PVP K-30) was varied. Out of 30 replicates, the number of H. virescens killed was recorded. The results were as follows:

Number Dead/Total

% PVP

Eco. Strain No. O 0.5 1 2 5 7 10

EG 2038 5/30 23/30 19/30 27/30 30/30 30/30 21/30

EG 2348 0/30 8/30 8/30 18/30 15/30 12/30 8/30

controls 10% PVP (no BT) 1/15

0.05% Triton (no BT/PVP) 1/15

Both formulations show a steady enhancement of activity until the 2-5% PVP level is attained. Above this value, th potency of the formulation levels-off, and begins to drop, at 10% PVP, both formulations show significantly decreased activity.

5.5. Field Trials on the Effectiveness of BT-Based Treatments on L. Dispar

Field trials were conducted by the aerial exposur of white oak saplings to various BT formulations. Each formulation was applied to five saplings. At 0, 2, 4, and days after such exposure, five leaves were aseptically collected from each tree and implanted into agar blocks in compartmentalized composite jelly trays. One gypsy moth

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larva was immediately placed in each compartment which was then sealed and incubated at ambient temperature for 48 hours. Larvae mortalities were recorded. The results were as follows :

Sarple Time

_- ^* _atrι_nt

BT Strain -±e F 0 __y 2 Day 4 Day 7 Cey

(ι_g ml)

2000 - ι.ooo ^a 1.000 1.000 .976

EG 2061 200 - .936 .728 .848 .968

2000 + 1.000 1.000 1.000 .992

200 + .984 .928 .960 .992 2000 1.000 .002 .984 1.000

EG 2035 200 - .880 .600 .888 .872

2000 + 1.000 .944 1.000 .984

^■ 200 + 1.000 .774 .984 .984 4000 1.000 .944 .992 .984

2000 - .968 .968 .928 .968

Cάpal 200 - .576 .430 .592 .592

4000 + 1.000 .960 .980 1.000

2000 + 1.000 .936 .952 1.000

200 + .896 .766 .824 .912 Cσ-±rol .017 .200 .088 .200

1.00 = 100% rrtxtality ^b + = ccir-taired 0.8% (w/v) FVP K-30

As shown, it can be seen that the mixtures of BT and PVP consistently exhibit high mortality rates, even after seven days. Further, even at the low application rates (200 ug/ml.) , the BT/PVP composites exhibited high mortality

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rates. Thus, the inclusion of a tannin binding compound in the BT formulations enhances activity and persistence. While formulations without PVP did exhibit similar activity and persistence (e.g. the HD 263-1 at 2000 !g/ml application rate) , there was a much wider variation among the effects of the non-PVP-containing formulations than the PVP-containing ones. The variations observed can be attributed to the application procedure used (wherein some trees may have received more formulation than others) and also to varying weather conditions. In general, however, the PVP-containing formulations did exhibit enhanced activity and persistence as compared with the non-PVP-containing ones.

5.6. Field Trials on the Effectiveness of BT-based Treatments for Control of Cabbage Looper

The effectiveness of BT/PVP formulations for the control of cabbage looper was examined in field trials on broccoli and cabbage naturally-infested with the insect. The tests were conducted over an extended period of time and the formulatins were applied from overhead by a backpack sprayer using a hand held boom. In each application, the sprayer was pressurized to 40 psi with CO to assure good dispersion. For the first application, the formulation was applied at a rate of 20 GPA, subsequent applications were at 47.4 GPA.

Four applications were made, each seven days apart. Two trials were conducted. In the first, which was conducted on broccoli, all formulations contained 0.6% (w/v) PVP K-30. Seven days after each application, but prior to the subsequent application, the number of medium and large- sized larvae were counted in randomly sampled plants. The results are summarized below:

_-

MEAN NO. OF CABBAGE LOOPER LARVAE PER PLANT ON BROCCOLI

(Seven Days Post-Treatment) 1,2

Formulation Application No, No. 1 2 3 4 Averag

EG 2038 0.5 3.4 6.0 1.3 2.8

EG 2012 0.5 4.0 2.0 1.5 2.0

EG 2035 0.1 0.8 5.7 0.8 1.9

Dipel 0.2 0 7.0 1.0 2.1

EG 2094 0 0.4 2.7 2.0 1.3

EG 2058 0.4 1.6 5.3 3.3 2.7

Control 0.4 8.8 40.0 23.5 18.2

Evaluations made 7 days post-application on Medium & Large-Sized Larvae;

2

Mean of 4 Replicates (1-10 plants counted per replicate) is reported.

As shown, all six formulations exhibited a simila effect on the number of larvae as compared with the control This is especially apparent in the last column which presents the four-week average, the range is from a low of 1.3 larvae to a high of 2.8, as compared with the 18.2 larv of the control.

In the second trial, the applications were made a more frequent intervals and the number of medium and large size larvae per 25 randomly selected cabbage heads was recorded just prior to the application of the subsequent treatment (e.g., the first count counting occurred 5 days after application 1) ; the last counting of larvae occurred

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7 days after application 4. All formulations contained 1% (w/v) PVP K-30 except for the DIPEL formulation, which contained no PVP. The results are summarized below:

CABBAGE LOOPER LARVAE ;/,*I-_aT-ENT ^"

Ecogen Application No. (days apart) Form No. 1 2(5 days) 3(3 days) 4(6 days)

EG 2038 6 6 13 16 10.2

EG 2012 12 17 5 7 10.2

EG 2035 7 8 7 21 10.7

Dipel 14 19 10 15 -14.5

EG 2094 3 8 6 7 6.0

EG 2058 11 10 8 10 9.8

Check 16 28 31 41 ^• 29.0

No. of Cabbage Looper Larvae or per 5 heads/plot x 5 replicates, e.g., 6 larvae per 25 heads.

As shown, all of the formulations except the DIPEL one exhibited a similar effect on the number of larvae, ranging from 6.0-10.7 for the four-treatment average. The DIPEL formulation, on the other hand, consistently exhibited less of an effect (as demonstrated by the large numbers of larvae present after each treatment) . Since all six formulations exhibited similar behavior in the first trial, the presence of PVP clearly enhanced the activity of DIPEL.

5.7. Tannin-Binding Capacity of Various Substance

A series of competitive binding experiments to assess the tannin-binding ability of several substances, including PVP ' s, as compared with remazol blue BSA (BBSA) .

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In each series, the BBSA and tannin were incubated with varying quantities of the test competitor and the ratio of amounts of BBSA in the assay to the amount (weight) of competitor required to achieve 50% inhibition of BBSA tanni binding was determined. The results were as follows:

Material BBSA/K50 (rel. binding constan

BSA 0.5

PVP K-15 10.7

PVP K-30 12.3

PVP K-60 15.2

PVP K-90 14.7

GANEX P904 9.2

GELATIN 1.9

UREA 0.0235

Briefly, the data reveals that PVP is an excellen binding agent, increasing in effectiveness as the molecular weight is increased up to PVP K-60. After this point, the efficiency shows a decrease. Gelatin, Ganex P904, and urea are also able to compete with BBSA, although they are somewhat less efficient.

It is apparent that many modifications and variations of this invention as hereinabove set forth may b made without departing from the spirit and scope thereof.

The specific embodiments described above are given by way o example only and the invention is limited only by the terms of the appended claims.

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