WO1996037106A1 - Casein-based sprayable formulation for pest control - Google Patents

Abstract

The present invention relates to a casein-based sprayable formulation for pest control. A process of delivering a pest control agent in a sprayable pest control formulation is provided. The present invention also provides a method of protecting a pest control agent from environmental conditions, such as a washoff and ultraviolet degradation.

Description

CASEIN-BASED SPRAYABLE FORMULATION FOR PEST CONTROL

DESCRIPTION

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a casein-based sprayable formulation, a process of delivering a pest control agent in a sprayable pest control formulation, and a method of protecting a pest control agent from environmental conditions, including washoff and ultraviolet degradation.

BACKGROUND OF THE INVENTION

Myriad approaches have been pursued to control pests. Many of these methods and compositions are directed to control of pests that attack plants, most notably commercially valuable plants. Although much current agricultural research has pest control as its objective, pest destruction of plants and plant products is still a major problem.

Control of pests of plants, livestock, and households has been accomplished with the aid of chemical and biological control agents. Unfortunately, approaches using these agents may fail due to inadequate formulation of the pesticides. In particular, many formulations are adversely affected by major environmental hindrances. By way of example, rainfall can wash away control agent deposits and sunlight can inactivate the active agent.

Starch, flour and gluten have been studied extensively as materials to encapsulate pesticides. Encapsulation of the pesticides helps to maintain activity by protecting the active ingredient from harmful environmental conditions. Most of this work has been done with granular matrices in efforts to reduce the amount of chemical pesticide needed to control pests or to protect environmentally sensitive pesticides (usually biological control agents) and thus extend their activity. While efforts with these granular formulations have been successful, by far, the majority of pesticides are applied as sprayable formulations.

Formulations of this type are essential for the widespread use of biological control agents and for enabling the reduction of potentially environmentally hazardous chemical pesticides. Formulations that are effective with lower active ingredient rates are possible through the judicious use of protectants, attractants, or other additives that synergize ingredient activity. Current technologies for sprayable systems require solids rates of not less than 2% for a flour/sucrose formulation and 1% for a gluten formulation. This can be a detriment to the use of these adjuvants because certain spray applications may require high volumes of water. When high volumes of water are used to spray certain crops, for example, fruits and vegetables (³800 L/acre), the amount of solids required for significant improvement of persistence may exceed 4 kg per acre. This large amount leads to commercialization restrictions because of costs associated with shipping and handling of large amounts of bulky materials. In situations where low volumes are used, for example cotton (<40 L/acre), the solids rate is not as much of an issue. Therefore, for a formulation or adjuvant to be useful for the widest variety of applications, it is essential that low solids rates will provide the necessary protection of the active ingredient in the pesticide spray.

The use of skim milk as an additive for application of pest control agents was disclosed in Kolodny-Hirsch et al. 1993 and Nail et al. 1991. Nail et al. reported no benefit in terms of field persistence with up to 1% v/v skim milk in applications oϊCydia pomonella (L.), granulosis virus. Kolodny-Hirsch et al. indicated improved field persistence of Spodoptera exigua multinucleocapsid nuclear polyhedrosis virus with 6% skim milk added as an ultraviolet screen. Importantly, Kolodny-Hirsch and Nail used milk and not specifically casein in their formulations. Milk is a complex system comprising water, emulsified particles of fat and fatty acids, sugar (lactose), casein, serum proteins and other nutrients and vitamins. (Hawlev's Condensed Chemical Dictionary. Ν. Irving

Sax and Richard Lewis Sr., eds., Van Νostrand Reinhold, New York, 1987). Casein is the major protein component of milk consisting of various identifiable proteins. BRIEF SUMMARY OF THE INVENTION

The present invention provides, in one aspect, casein-based formulation for pest control comprising an effective amount of a pest control agent, casein and an alkalinizing agent. An aqueous solvent or water is added to prepare a sprayable pest control formulation.

The pH of the casein-based formulation casein-based is from about 7.5 to about 12.0. Preferably, the pH is from about 8.0 to about 10.0. This invention also provides a casein-based formulation for pest control comprising a pesticidally effective amount of a pest control agent, casein salt, and a cross- linking agent. A preferred cross-linking agent is ammonium zirconium carbonate. The preferred amount of casein or casein salt in the casein-based formulation for pest control is from about 0.1 percent to about 1.5 percent (weight/volume, w/v). Even more preferably, the amount of casein or casein salt in the casein-based formulation for pest control is from about 0.2 percent to about 1.0 percent (w/v). As used herein with regard to casein, the term "percent" refers to weight/volume percent. For example, a casein-based pest control formulation that contains 1% casein comprises 0.01 gram casein per mL of formulation.

In another aspect, the casein-based formulation for pest control, including the pest control agent, adheres to a plant surface and, more preferably to a plant foliar surface. In another preferred embodiment, the formulation adheres to an external surface of an animal and, preferably skin, fur or hair.

In another aspect, the present invention provides a method of protecting a pest control agent from environmental conditions comprising admixing the pest control agent with a protective amount of casein, an alkalinizing agent and an aqueous solvent, preferably water. Alternatively, the pest control agent is admixed with casein salt, a cross-linking agent and an aqueous solvent, preferably water. The casein-based formulation for pest control is then exposed to environmental conditions, including ultraviolet radiation and water. The pest control agent is protected against environmental conditions, including radiation inactivation and washoff by rainfall or watering of the pest control agent.

A further aspect of this invention provides a process of delivering a pest control agent by spraying the casein-based formulation for pest control onto an external surface of an organism. The sprayable pest control formulation comprises a pesticidally effective amount of a pest control agent, water and solubilized casein. As described herein, and in particular the examples, the sprayable casein formulation is prepared by exposing casein to an alkalinizing agent, or alternatively, by exposing the salt of casein to a cross-linking agent.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a casein-based formulation for pest control. The formulation comprises a pest control agent, casein, and an alkalinizing agent. In another embodiment, the formulation comprises a pest control agent, casein salt, and a cross- linking agent. An aqueous solvent, preferably water, is added to the casein-based formulation to prepare a sprayable formulation.

When the formulation is prepared using casein and an alkalinizing agent, the pH of the formulation is between about 7.5 to about 12.0. Preferably, the pH is from about 8.0 to about 12.0. The alkalinizing agent partially solubilizes casein. Upon application of the formulation to a target surface, e.g. foliar surface, as the water begins to evaporate, the casein molecules cross-link with each other and form a film on the target surface. After the film has formed, because of the cross-linking, the film is no longer soluble in water and resists washoff. Furthermore, the film also provides protection against radiation inactivation, in particular, protection against ultraviolet degradation.

When casein salt is utilized, a cross-linking agent must be provided. Casein salt is readily soluble in aqueous solvents. If a cross-linking agent is not provided when casein salt is used, the resulting formulation does not provide resistance against wash off because casein has not been cross-linked. The addition of the cross-linking agent to the casein salt based formulation cross-links casein molecules to each other after the pest-control formulation has been applied to a target surface. The film formed upon evaporation of the aqueous solvent is resistant to wash off. Furthermore, the film also provides protection against radiation inactivation, in particular, protection against ultraviolet degradation. This invention provides a method of protecting a pest control agent from environmental conditions. The pest control agent is protected by the casein-based formulation against radiation inactivation and washoff by rainfall or watering. The method comprises admixing a pest control agent with a protective amount of casein, an aqueous solvent, and an alkalinizing agent to form a casein-based formulation for pest control. The casein-based formulation for pest control including the pest control agent is exposed to environmental conditions including exposure to UV radiation and exposure to water. Alternatively, the method comprises admixing a pest control agent with a protective amount of casein salt, an aqueous solvent, and a cross-linking agent to form a casein-based formulation for pest control and exposing the casein-based formulation to the environment. The casein-based formulation forms a film that resists washoff by rain or watering and also provides protection against radiation inactivation, including UN inactivation. The casein-based formulations for pest control preferably contain from about 0.1 percent to about 1.5 percent casein (w/v), and even more preferably between about 0.2 percent to about 1.0 percent casein (w/v).

Furthermore, this invention also provides a process of delivering a pest control agent by spraying a casein-based pest control formulation. The casein-based formulation for pest control is delivered by spraying the formulation onto the external surface of a target.

This invention provides sprayable formulations of chemical and microbial pesticides that are effective in low amounts and resist radiation inactivation and wash-off from rainfall or watering. Casein is used to disperse the pesticide and entrap it on leaf or other surfaces. The pesticide then remains viable under harsh environmental conditions. Casein-based formulations for pest control are disclosed for the delivery of biological or chemical pest control agents.

Where applied to a living organism, the casein-based pest control formulation, including the pest control agent, adheres to an external surface of that organism. As used herein, the term "adhere" or any of its grammatical equivalents means that the formulation sticks to a target surface on which the formulation is applied. Exemplary surfaces to which a formulation of the present invention adheres include an external surface of a living organism and artificial surfaces such as those made of glass, metal, plastic, wood, and the like. In a preferred embodiment, a formulation of the present invention adheres to an external surface of a living organism such as a plant or animal. Where the living organism is a plant, a preferred external surface is any surface susceptible to damage by insects, and disease. Preferred external surfaces include the surfaces of a leaf, stem, branch, trunk, root, flower, fruit, vegetable, and seed. Where the living organism is an animal, a preferred external surface is skin, fur or hair.

Casein is an aggregate of several proteins together with phosphorous and calcium derived from milk (Hawlev's Condensed Chemical Dictionary. N. Irving Sax and Richard Lewis Sr., eds., Van Nostrand Reinhold, New York, 1987). Casein is almost insoluble in water and is available commercially. A second form of casein, known as casein salt, is readily soluble in water and is also available commercially.

The casein-based formulation can be prepared by two preferred processes. The first process comprises exposing casein to an alkalinizing agent and the second process comprises exposing casein salt to a cross-linking agent. When the casein-based formulation is prepared by the process comprising exposing casein to an alkalinizing agent, due to the low solubility of casein in water at a neutral pH, an alkalinizing agent such as sodium hydroxide, ammonium hydroxide, trisodium phosphate or potassium hydroxide is added to obtain an alkaline pH. The alkalinizing agent can be either dissolved in water prior to the addition of casein and pest control agent or can be included with the casein-active ingredient in a powder or liquid form. The alkalinizing agent solubilizes casein and as the casein-based formulation dries after application, casein molecules cross-link to form a film on the target surface that is resistant to washoff and protects the pest control agent from radiation inactivation. If the alkalinizing agent is included with the casein and active ingredient, a user simply adds the water to prepare the sprayable pest-control formulation.

In the second process, casein salt is exposed to a cross-linking agent. Because of the ionization of the amino acid side chains, casein salt is readily soluble in water. However, because of the solubility of the casein salt, casein salt alone is incapable of producing a pest control formulation that is resistant to washoff by rain or watering.

When casein salt is used to prepare the casein-based pest control formulation, the cross- linking agent cross-links casein molecules. Casein molecules become cross-linked as the aqueous solvent or water evaporates from the target surface. The cross-linked casein forms a film that is resistant to washoff and provides protection against UN radiation. The casein-based formulation for pest control prepared by exposing casein salt to a cross- linking agent is useful in a sprayable pest control formulation in that the solubilized casein provides resistance to radiation damage and to washoff by rainfall or watering.

Any compound that cross-links proteins can be used as a cross-linking agent. Protein cross-linking agents are well known in the art and include

dinitrodiphenylsulfone; l,5-difluoro-2,4-dinitrobenzene; l-fluoro-2-nitro-4-Azidobenzene; phenol-2, a-naphthol-2,4-disulfonyl chloride; adipate bis-(p-nitro-phenyl ester; carbonyl bis(methionine p-nitrophenyl ester; tartaryl diazide; tartryl bis-(glycylazide); succinate bis- (hydroxy-succinimide ester; Ν-(Azidonitrophenyl)g-aminobutyrate hydroxy-succinimide ester; 1,3-dibromoacetone; p-azidophenacyl bromide; l,l-bis-(diazo acetyl)-2- phenylethane; l-diazoacetyl-l-bromo-2-phenylethane; bis diazo benzidine; glutaraldehyde; polymethylene (n=3-12) di-imidates; di-methylsuberimidate; ethyl (chloroacetimidate); hexamethylene diisocyanate; toluene 2-isocyanate; 4-isothiocyanate; bis(maleidomethyl)ether; N,N'phenylene-dimaleimides; and N,N-Bis(p-Azido-o- nitrophenyl)l,3 diamino-2-propanol; imidoesters; and iminothiolanes. Exemplary cross- linking agents are zirconium carbonate salts, including sodium zirconium carbonate and potassium zirconium carbonate. A preferred cross-linking agent is ammonium zirconium carbonate (AZCote₅₈oo ®. trademark of Hopton Technologies, Inc., Rome, Georgia). In a preferred embodiment, the amount of cross-linking agent in the sprayable pest control formulation is from about 0.01 percent to about 0.10 percent. As used herein, the term "percent" refers to weight/volume percent. For example, a casein-based pest control formulation that contains 0.05% cross-linking agent comprises 0.05 gram cross-linking agent per 100 mL of formulation. As used herein, the term "pest control agent" indicates a substance that serves to repel or kill a pest from a living organism, decrease or inhibit the growth, development or destructive activity of a pest. A pest can be a plant, an animal or a microorganism. Exemplary pests include insects, spiders, nematodes, fungi, weeds, bacteria and other microorganisms. Thus, a pest control agent can be insecticide, a pesticide, a fungicide, a herbicide, antibiotic, an anti-microbial, a recombinant pest control agent and the like. A pest control agent can also be a mixture of two or more agents.

Exemplary pest control agents are dimilin (N~{[(4-chlorophenyl) amino} carbonyl} -2,6-difluorobenzamide), malathion ((dimethoxyphosphinothioyl)thio]butanedioic acid diethyl ester), carbaryl (1-naphthalenol methylcarbamate) and diazinon® (0,0-diethyl 0-[6-methyl-2-(l-methylethyl)-4- pyrimidinyl] phosphorothioate); 2,4-D (2,4-dichlorophenoxyacetate sodium salt), a 2,4-D ester (2,4-dichlorophenoxyacetate isopropyl ester); metolachlor (2-Chloro-N-(2-ethyl-6- methylphenyl)-N-(2-methoxy- 1 -benzenedicarboxylate); glyphosate (N-(phosphonomethyl) glycine); paraquat (1, l'-dimethyl-4, 4'-bipyridinium salt); and trifluralin (1, 1, 1- trifluoro- 2, b-dinitro-N, N-dipropyl-p-toluidine). Pesticides, insecticides, herbicides, fungicides, antimicrobials and antibiotics are commercially available. An exemplary list of such substances can be found in United States Patent No. 4,911,952, the disclosure of which is incorporated herein by reference.

Alternatively, a preferred pest control agent is a recombinant pest control agent. As used herein, a recombinant pest control agent is a pest control agent produced by the use of well known recombinant DNA technology (Sambrook. J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, 1989). Briefly, when a pest control agent is prepared by recombinant DNA techniques, the recombinant pest control agent is obtained from recombinant host cells which express the recombinant pest control agent. To achieve this, a specific oligonucleotide based upon the sequence of the desired pest control agent is prepared. The oligonucleotide is then inserted into an expression vector, such as any one of the many expression vectors currently available commercially. A host cell is then transformed or transfected with the vector, where it will direct the expression of the recombinant version of the pest control agent, which may then be purified from the recombinant host cell. Alternatively, the recombinant pest control agent is a substantially intact recombinant host cell. The preparation of oligonucleotide, vector and transformation of the host cell are within the skill of the ordinary artisan. Exemplary recombinant pest control agents are described in United States Patents 5,169,629 and 5,352,661, herein incorporated by reference.

A pest control agent can be a biological or chemical material. As used herein, the phrase "biological material" means a living organism or a substance isolated, produced or otherwise derived from a living organism (e.g., a toxin or a hormone). Thus, a biological pest control agent can be an inanimate form of a once living organism. The use of such a biological pest control agent is exemplified hereinafter in Examples 1-6 and 8-11. United States Patents 5,169,629 ('629) and 5,352,661 ('661) describe the preparation of a recombinant Bacillus thuringiensis (B. thuringiensis) toxin. Recombinant B. thuringiensis toxin is a preferred pest control agent.

U.S. Patent '629 and '661 disclose that recombinant B. thuringiensis toxin can be produced in a host cell. After the host cell produces a desired concentration of B. thuringiensis toxin, the host cell can be treated under conditions that prolong the activity of the toxin produced in the cell. When the host cells are treated under such conditions, the host cells remain substantially intact. U.S. patents '629 and '661 also disclose that the recombinant host cell can be applied to the environment in a living state. The use of a recombinant pest control agent, either treated or in a living state, in a casein-based formulation for pest control provides for improved protection of the pest control agent from environmental conditions. In particular, a pest control agent is protected from being washed away by water and protected from radiation degradation or inactivation by the sprayable casein-based formulation. The minimization of ultraviolet damage to a commercially available recombinant B. thuringiensis product is further disclosed in Examples 10-11 below. Both Mycogen MVP® and "cream" (See Example 8) provide B. thuringiensis toxins encapsulated in substantially intact cells. (MVP® is a trademark of

Mycogen Corp., San Diego, Ca.)

Exemplary biological pest control agents include a bacteria such as the bacterium

B. thuringiensis, Baculoviridae, e.g., Autographa californica nuclear polyhedrosis virus, protozoa such as Nosema spp., fungi such as Beauveria spp., and nematodes.

As used herein, the phrase "chemical material" means a synthetically prepared compound or composition. Exemplary chemical pest control agents include thiocarbonates, dinitroanilines, organophosphates, and alachlor.

The phrase "pesticidally effective amount" indicates that amount of a pest control agent sufficient to bring about the desired response (e.g., repel or kill a pest). When the pest control agent kills the pest, a "pesticidally effective amount" is that amount which, when delivered to an external surface of a living organism, results in a significant mortality rate of a pest when compared to the mortality rate of that same pest exposed to a living organism not treated with that agent. A pest control agent can further comprise an additive or adjunct such as a dispersant, a phagostimulant (a feeding stimulant), an attractant, an ultraviolet light protectant, a preservative and an inert filler. Examples of such additives can be found in United States Patent No. 4,911,952, the disclosure of which is incorporated herein by reference. In a preferred embodiment, the additive is an attractant or a phagostimulant. An attractant is preferably an aqueous, non-soluble, or hydrophobic substance that attracts a pest to the spray deposit. A phagostimulant is a substance that stimulates ingestion of the spray deposit.

A preferred attractant is a pheromone or a volatile feeding attractant such as p- methoxycinnamaldehyde. An exemplary and preferred phagostimulant is cucurbitacin obtained from the powdered, dried root of the buffalo gourd, or Coax®, a feeding stimulant containing cotton seed flour, sugar, vegetable lipid oil and ethoxylated ester (CCT Corporation, Litchfield Park, Az). Exemplary sugars are mono-, oligo- and polysaccharides containing from about 1 to about 50 saccharide units. In a preferred embodiment, a sugar is a disaccharide such as sucrose, molasses or corn syrup solids. Exemplary formulations comprising one or more of the above ingredients are described in detail hereinafter in Examples 1 through 11. Typically, the concentration of casein in a formulation of the present invention is from about 0.1 percent by weight/volume (grams/mL) to about 1.0 percent by weight/volume. More preferably, the concentration of casein is from 0.125 percent by weight/volume to about 0.5 percent by weight/volume. The only limitation on the concentration of casein is the solubility of casein.

The casein-based pest control formulation is prepared by admixing pest control agent, casein, an alkalinizing agent and an aqueous solvent at a temperature of from about

5°C to about 100°C and, preferably, at a temperature of from about 10°C to about 50°C.

The pest control agent, solubihzed casein and water can be admixed in any order. Solubihzed casein is preferably prepared by the process described above. When casein salt is the source of casein the casein-based formulation for pest control is prepared by admixing pest control agent, casein salt, a cross-linking agent and an aqueous solvent at a temperature of from about 5°C to about 100°C and, preferably, at a temperature of from about 10°C to about 50°C. The pH of the sprayable pest control formulations is between about 7.5 to about

12.0 when an alkalinizing agent is used. Alternatively, when a cross-linking agent in combination with casein salt, the pH of the resulting formulations is about neutral. Preferred alkalinizing agents include metal salts such as NaOH or KOH. A preferred cross-linking agent is ammonium zirconium carbonate. A sprayable formulation of the present invention can also comprise a buffer to maintain the pH at a predetermined value. Any buffer can be used so long as that buffer does not adversely affect the formulation or the pest control agent contained therein. A buffer can also be used as the alkalinizing agent. Thus, in one embodiment, formulation pH can be set and maintained with a form of buffering agents such as phosphate, carbonate, and borate. Other exemplary buffers are well known and are within the skill of an artisan.

A formulation of the present invention can also be prepared to comprise water- dispersible granules. In accordance with such an embodiment, a formulation comprises, in addition to a pest control agent, 1) an agglomerating agent that promotes formation of granules that contain casein 2) a dispersing agent that promotes separation of casein granules upon contact with the aqueous solvent, or 3) both an agglomerating and a dispersing agent. In a preferred embodiment, a dispersing or agglomerating agent is premixed with casein prior to the addition of the aqueous solvent. A preferred dispensing agent or an agglomerating agent is a vegetable oil such as corn oil or soybean oil. A preferred agglomerating agent is molasses.

A method of protecting a pest control agent from environmental conditions is also provided by the present invention. In particular, the pest control agent is protected from radiation degradation or inactivation and washoff by rainfall or watering.

The pest control agent is incorporated in a sprayable formulation comprising solubihzed casein and water as described herein. Degradation or inactivation of pest control agents by radiation, in particular by ultraviolet radiation, is decreased or minimized by a sprayable casein-based formulation. Furthermore, the addition of agents which provide additional protection against radiation damage, is contemplated.

As used herein, the term "radiation" refers to energy in the form of electromagnetic waves that a pest control agent is exposed to during use and after application. For the purposes of the present invention, radiation that a pest control agent is exposed to during use is ultraviolet, visible and infrared radiation.

EXAMPLES

Formulations

Typically, unmodified casein (1 g) was mixed in a solution of 200 mL of KOH (0.05% w/v) in a Waring blender for a final concentration of 0.5% (w/v) and kept at ambient temperature for 1 hour before use. After mixing, the pH was 8.6, and 40 mg of B. thuringiensis technical powder was then added. Casein-salt, which is water soluble at neutral pH, was used, typically, at a concentration of 0.5% (w/v) with a cross-linking reagent solution, 0.25% (v/v)of ammonium zirconium carbonate 30% (w/v). The percent casein used in specific experiments are described in further detail in the examples below. Flour/sucrose and commercial formulations were compared to casein formulations. The flour/sucrose formulation (2% w/v) consisted of grinding pregelantinized flour 961 (Illinois Cereal Mills, Paris, PL) with sucrose in a 1 to 1 mixture and dissolving it in water at the desired concentration (w/v), and adding the technical powder form of B. thuringiensis. Commercial formulations were assayed for comparison with new formulations including Dipel® 2X (Trademark of Abbott Laboratories), XenTari® (B. thuringiensis, subsp. aizawai, Trademark of Abbott Laboratories), MVP® formulated product (Lot number 211202 IX). For each of the following examples, the formulations were mixed in 100 mL quantities. Unless otherwise stated, technical powder of Bacillus thuringiensis subsp. kurstaki (69,000 International Units/mg) was supplied by Abbott Laboratories (Chemical and Agricultural Products Division, North Chicago, IL ) and was added to each formulation to provide a final concentration of 13,800 International Units/mL.

Preliminary assays showed this concentration to be a discriminating dose of B. thuringiensis for comparing simulated rain with no rain treatments. Casein formulations were made with several additional preparations of B. thuringiensis. Dipel 2X was mixed at 20 mg/50mL (12,800 IU/mL) and XenTari was mixed at 100 and 500 mg/50mL in order to apply equivalent rates of active agent at application volume of 47 and 235 1/ha. MVP and MVP Stabilized Cream-K-175 (technical material for the MVP formulation) were mixed to provide final concentrations of 1.6 mg/mL for the formulated MVP and 0.8 mg/mL for the MVP cream.

The final spray solutions were applied directly to the plants. Formulations were mixed by stirring to prevent settling of B. thuringiensis immediately prior to application. Components of various formulations were tested individually as formulations to assay for insecticidal activity or resistance to environmental factors.

Laboratory Studies Rainfastness and solar stability of various casein formulations were measured in the laboratory based on a leaf bioassay. Cotton plants, Gossypium hirsutum L. T)ES 119', were grown in a glass house in 15 cm diameter plastic pots. Two plants were grown in each pot in a peat moss-vermiculite soil mixture. When the plants had five or more fully expanded true leaves, pots were selected and paired for treatments so that each treatment had 10 large true leaves. Cotyledon leaves and immature true leaves were trimmed from the plants. After trimming, the remaining leaves were washed with tap water to remove soil and plant exudates that may interfere with larval survival during the assay. Plants were allowed to dry before the application of formulations.

Rainfastness: Formulations were made as previously described with solids rates up to 0.5 % w/v of casein. Formulations were applied to plants in a spray chamber

(Research Track Sprayer, DeVries Manufacturing, Hollandale, MN). This chamber held up to eight pots of plants on a stationary platform and applied the formulations through a spray apparatus that passed over the plants. Formulations were applied through a flat fan 8002ss nozzle (Spray Systems, Wheaton, EL) at 4.9 kg/cm² and a track speed setting of

3.0 km/h (1.9 mph) to apply 36 mL total volume while travelling the 2.0 m linear spray track. This rate was calculated to be equivalent to 235 L/ha. As a variation to this standard application procedure, a lower volume of application was also used. The low volume application equivalent to 47 1/ha was achieved by using a 8001 nozzle, at 2.5 kg/cm² pressure and 6.4 k/h track speed to apply 7 mL in a single pass. For experiments with the low volume application, the normal volume application was achieved by spraying five passes for a total application of 35 mL. The concentration of B. thuringiensis was adjusted in the formulation mix to provide the equivalent amount of active agent as in the normal application although the concentration of casein was maintained as a percentage of the total volume applied for individual treatments. Spray residue was allowed to dry before the application of simulated rain. An untreated control was included with each experiment to assess larval mortality due to handling.

Simulated rain consisted of the application of 5 cm of tap water at 50 psi (3.5 kg/cm²) with the same spray chamber described previously fitted with a full jet FL- 5VC (Spray Systems Co., Wheaton, IL) spray nozzle. To simulate rain, the traveling spray apparatus continuously traversed the chamber back and forth until the desired amount of rain was applied as measured with a rain gauge placed inside the spray chamber. Plants were allowed to dry overnight before assaying. Solar Stability: 100 mL of formulation was spread onto a 33 cm² leaf area which had been marked onto a cotton leaf while still on the plant. For each formulation, 20 areas were treated. Ten areas received solar treatment and the other 10 remained in the laboratory under ambient light conditions. The solar treatment consisted of placing the cotton plants under a Suntest CPS light source so that 10 marked areas were between 30 and 36 cm from the light. Clear plastic (Tefcel T², American Durafilm, Holliston, MA) was placed between the light and the plants to avoid excessive drying of the leaf tissue. Light intensity readings taken with a LiCor solar spectroradiometer demonstrated that energy in the 300-800nm range was not lost due to the plastic cover. Exposure time was 8 hours at a dial setting of 7.5. After exposure, treated areas of the leaves were excised and assayed for insecticidal activity.

Insects: Unless otherwise stated, neonate European corn borer (Ostrinia nubilalis (Hϋbner)) were used in experiments and formulations were compared based on larval mortality. European corn borer eggs were obtained from our laboratory colony reared according to standard methods. This colony was routinely supplemented with weekly shipment of eggs from the USDA-ARS Corn Insects Research Unit, Ames, IA. Neonate beet armyworm and cabbage looper were from colonies maintained in the laboratory and were also used for the assays. The beet armyworm (Spodoptera exigua (Hϋbner)) colony was established from eggs supplied by Auburn University, Auburn, AL and maintained on artificial diet. The cabbage looper (Trichoplusia ni (Hubner)) colony was initiated with eggs supplied by the USDA Biocontrol Laboratory, Columbia, MO and was also maintained on artificial diet.

Bioassav: Ten circular disks (33 cm²) were cut from the cotton leaves for each treatment. Leaf disks were placed individually on filter paper in a 100 x 15 mm petri dish with the treated side-down. Ten neonate European corn borer were transferred to each leaf disk, and then the dish was sealed with two wraps of parafilm. Dishes were placed in a dark incubator at 27-30° C for three days. After incubation, the number of live and dead larvae were counted on each leaf disk and mortality was calculated. The same procedure was used to assay against beet armyworms and cabbage looper. When larval mortality in the control was high (>15%) then the mortalities for the treatments were corrected using Abbott's formula (Abbott 1925). However, mortality of larvae feeding on untreated leaves was <5%, and thus actual mortalities were reported without correction for control mortality.

Field Studies

The relative impact of actual sunlight on persistence of insecticidal activity of B. thuringiensis was determined by establishing a small plot replicated field test on cabbage. Casein formulation (0.5 % w/v) was prepared as previously described. Treatments were applied with a C0₂ charged sprayer at the rate of 33 gallons of water per acre (308 L/ha (band application directed at the row at 19.3 mL/m of row)), 16 billion International Units (BIU) of B. thuringiensis per acre (39.52 per hectare), a pressure of 30 psi (2.1 Kg/cm₂) and a ground speed of approximately 4 mph (6.4 km/h). After treatments were applied and allowed to dry, plastic shields were erected over the tops of the plants to either screen out all light (black plastic) or screen out only rain (clear plastic). In addition, some plants were left uncovered. At 1, 2, 4, and 7 days after application, leaf tissue was removed from each formulation by cover treatment and brought into the laboratory. Leaf disks were cut out, placed in plastic petri dishes lined with filter paper, and 10 neonate cabbage looper were added. After 3 days, percentage mortality was assessed.

The percentage original activity remaining (% OAR) was based on a ratio of paired treatments. The mortality of a formulation exposed to artificial rain or light treatment is divided by the mortality of the corresponding formulations not exposed to this treatment. This measure is more reflective of actual protection then % mortality alone because mortality of larvae before exposure may vary with formulation or trial due to insect fitness and the feeding stimulant properties of the formulations. Data were analyzed for many of the experiments by statistical analysis of variance using a general linear model, PC-SAS version 6.08. Treatment means were separated using least significant difference (P<0.05).

Example 1

A formulation with casein was analyzed to determine whether it had detrimental effects on the viability of B. thuringiensis spores added to the formulation as the active control agent for insects. The insecticidal activity also was determined for each of the formulations in the experiment. Colony forming units (CFUs) that represent the number of viable spores for each formulation were determined by diluting an aliquot from each formulation to 10^"* and plating the dilutions on nutrient agar. After a 24 hr incubation period, the number of individual colonies was counted and used to calculate the number of CFUs for each formulation. The insecticidal activity was expressed as the mortality of

European corn borer larvae fed cotton leaf disks from plants treated individually with the formulations.

The casein formulation of B. thuringiensis did not adversely affect spore viability (Table 1). CFU's were the same for technical powder and the casein formulation. Also, casein provided a high level of insecticidal activity compared with other formulations based on larval mortality.

Table 1. Colony forming units (CFUs/mL) of formulations and mortality of neonate European corn borer exposed to cotton leaf disks treated with the formulations containing Bacillus thuringiensis (Bt).

Trt# Formulation CFUs/mL % Mortality

1 Casein KOH/Tech Bt, pH = 10.4 23,000,000 91

2 Tech. Bt in KOH, pH = 11 .6 22,150,000 73

3 Tech. Bt in water, pH = 5.5 2,757,500 73

Example 2

The casein formulation was compared with other formulations of B. thuringiensis for resistance to wash-off. The casein formulation was prepared by mixing 2 g of casein in 220 mL of 0.05% KOH solution. After the casein dissolved, 44 mg of B. thuringiensis technical powder was added, and the final pH of the solution was 8.5. This formulation was compared with a commercial formulation (Dipel 2X), technical powder in water, and a Mirasperse®/sucrose formulation of B. thuringiensis (Mirasperse is a trademark of A. Staley Co. Decatur, IL).

After receiving greater than 2 inches (5 cm) of simulated rain, the casein formulation expressed 60 % mortality against European corn borer (Table 2), which was greater than all the other treatments exposed to the simulated rain. Miraspers/sucrose is a specific starch/sucrose formulation and showed little resistance to wash-off by simulated rain. Both Dipel and Technical powder lost most of the insecticidal activity when exposed to simulated rain.

Table 2. Mortality of European corn borer when exposed to cotton leaves treated with formulation of Bacillus thuringiensis.

Trt# Formulation (% Solids, w/v) Rain, In. %Mortality

1. Dipel 2X 0 100

2. Dipel 2X 2.3 (5.9 cm) 22

3. Technical powder 0 77

4. Technical powder 2.3 (5.9 cm) 12

5. Mirasperse/sucrose (1%) 2.3 (5.9 cm) 7

6. Mirasperse/sucrose (2%) 2.3 (5.9 cm) 0

7. Mirasperse/sucrose (4%) 2.3 (5.9 cm) 2

8. Casein (0.5%) 2.3 (5.9 cm) 60

9. Control 0 0

Example 3

Several formulations were compared that differed in concentration of casein and the formulations were exposed to simulated rain. The casein formulations were also compared to flour/sucrose formulations. The casein formulation was prepared by mixing 2 g of casein in 220 mL of 0.05% KOH solution. After the casein dissolved, 44 mg of B. thuringiensis technical powder was added. The final pH of the solution was 8.5.

The casein formulation showed good efficacy as expressed by high insecticidal activity (mortality > 90%) when not exposed to simulated rain (Table 2). There were no significant differences (LSD, P > 0.05) among no rain treatments of casein, flour/sucrose, and technical powder formulations. When exposed to simulated rain, casein formulations with as little as 0.25 % w/v resisted wash-off better then the flour/sucrose formulation and technical powder. Casein at 0.5% resisted wash-off better than 0.25% casein formulation.

In this experiment, there was no additional benefit from a formulation containing 1% casein. Thus, casein at 0.5% is likely to be the best formulation in this formulation because this was the lowest concentration that provided resistance to wash-off

Table 3. Larval mortality and percentage of original activity remaining (% OAR) for European corn borer exposed to cotton leaves sprayed with formulations of Bacillus thuringiensis with and without simulated rain.

Trt# Formulation % Solids Rain, In. % Mortality % OAR

1 Casein 0.25 0 94.0

2 Casein 0.25 2 (5 cm) 43.8 46.6

3 Casein 0.5 0 93.0 4 Casein 0.5 2 (5 cm) 70.3 75.6

5 Casein 1.0 0 96.0 6 Casein 1.0 2 (5 cm) 61.4 64.0

7 Flour/sucrose 2.0 0 100 8 Flour/sucrose 2.0 2 (5 cm) 11.9 11.9

9 Technical powder — 0 94.0 10 Technical powder 2 (5 cm) 27.7 29.5

11 Untreated — 0 1.0

Example 4

Upon the addition of flour, the casein formulation retains insecticidal activity when exposed to simulated rain. Casein formulations were prepared as described previously and the flour used was 961, Illinois Cereal Mills, Paris, IL.

Several formulations of casein were admixed with flour and exposed to simulated rain (Tables 4 and 5) were compared. The formulations with casein resisted wash-off better than formulations without casein. A formulation with flour alone was not better than technical powder for the resistance to wash-off, and casein and flour with no B. thuringiensis had no insecticidal activity against European corn borer (Table 4).

Table 4. Mortality and original activity remaining (% OAR) after two inches of simulated rain for casein formulations of B. thuringiensis (Bt).

Trt# % Casein % Flour Rain, In. % Mortality % OAR

1 Untreated (No Bt) — 0 0 --

2 0.25% (No Bt) 0.5% 0 5 3 0.25% (No Bt) 0.5% 2 (5 cm) 1

4 0.25% — 0 90 5 0.25% 2 (5 cm) 65 72.2

6 0.25% 1.0% 0 86

7 0.25% 1.0% 2 (5 cm) 44 64.7

8 0.5% 0 82 9 0.5% 2 (5 cm) 73 89.0

10 0.5% 0.5% 0 90 11 0.5% 0.5% 2 (5 cm) 74 82.2

12 0.5% 1.0% 0 86 13 0.5% 1.0% 2 (5 cm) 59 68.6

14 — 1.0% 0 92 15 1.0% 2 (5 cm) 10 10.9

16 (Bt only) 0 62 17 (Bt only) .... 2 (5 cm) 23 37.1

Table 5. Mortality and original activity remaining (% OAR) after two inches of simulated rain for formulations of B. thuringiensis (Bt).

Trt# % Casein % Flour Rain, In. % Mortality % OAR

1 0.25% — 0 86

2 0.25% 2 (5 cm) 51 59.3

3 0.25% 0.5% 0 90 4 0.25% 0.5% 2 (5 cm) 64 71.1

5 0.25% 1.0% 0 96 6 0.25% 1.0% 2 (5 cm) 78 81.2

7 0.5% — 0 95 8 0.5% 2 (5 cm) 71 74.7

9 0.5% 0.5% 0 93 10 0.5% 0.5% 2 (5 cm) 68 73.1

11 0.5% 1.0% 0 96 12 0.5% 1.0% 2 (5 cm) 54 56.2

13 .... 1.0% 0 92 14 1.0% 2 (5 cm) 28 30.4

15 Technical powder — 0 92 16 Technical powder 2 (5 cm) 35 38.0

17 Untreated (No Bt) — 0 0 —

Example 5

The previously described casein formulation was compared with a second casein formulation made with a water soluble casein (casein salt) and a cross-linking agent.

Different concentrations of casein and the cross-linking agent were compared for their ability to provide resistance to wash-off. As shown below, the soluble form of casein with the cross-linking agent was effective at resisting wash-off (Tables 6 and 7). Neither, the casein-salt or the cross-linking agent alone provided resistance to wash-off. Likewise, neither casein-salt or the cross-linking agent provided insecticidal activity as expressed by formulations made without B. thuringiensis (Table 6, treatments 2 and 3, Table 7, treatment 1). In the absence of simulated rain, none of the ingredients reduced the insecticidal activity of the B. thuringiensis technical powder. Table 6. Mortality and original activity remaining (% OAR) of casein formulations made with Bacillus thuringiensis as the active agent.

Cross- Bt Trt # Casein (% w/v) link (% w/v) Rain, In. Mortality % OAR

1 None 0 None 0 3 —

2 Casein salt (0.5) 0.03 None 0 2 —

3 None 0.03 None 0 0

4 None 0 Bt 0 88 5 None 0 Bt 2 (5 cm) 23 26

6 None 0.03 Bt 0 95

7 None 0.03 Bt 2 (5 cm) 26 27

8 Casein (0.5) 0 Bt 0 92 9 Casein (0.5) 0 Bt 2 (5 cm) 77 83

10 Casein salt (0.5) 0 Bt 0 91 11 Casein salt (0.5) 0 Bt 2 (5 cm) 22 24

12 Casein salt (0.25) 0.03 Bt 0 92 13 Casein salt (0.25) 0.03 Bt 2 (5 cm) 39 42

14 Casein salt (0.5) 0.03 Bt 0 88 15 Casein salt (0.5) 0.03 Bt 2 (5 cm) 39 44

Table 7. Mortality and original activity remaining (%OAR) of casein formulations made with B. thuringiensis (Bt) as the active agent.

Cross-link Trt# Casein (% w/v) (% w/v) bt Rain, In. Mortality %OAR

1. None 0 none 0 2 —

2. None 0 Bt 0 90

3. None 0 Bt 2 (5 cm) 32 35

4. None 0.75 Bt 0 91 5. None 0.75 Bt* 2 (5 cm) 20 22

6. Casein salt (0.5) 0 Bt 0 76

7. Casein salt (0.5) 0 Bt 2 (5 cm) 32 42

8. Casein salt (0.5) 0.75 Bt 0 87

9. Casein salt (0.5) 0.75 Bt 2 (5 cm) 62 71

Example 6 The formulations resisted wash-off when applied under a variety of conditions, such as different volumes of application. Several formulations of B. thuringiensis kurstaki including flour/sucrose, and casein were compared. The application rates were 7 and 35 mL/pass for the low and high rates, respectively. All treatments were mixed with technical powder as described previously to be assayed against European corn borer. The low application sprayed 7 mL total spray volume per pass for the equivalent field rate of 5 gal/Acre (47 L/ha). The high application rate applied made 5 passes for 35-36 mL total volume, equivalent to about 25 gal/ Acre (234 L/ha) field rate. The initial concentration of B. thuringiensis was mixed at the rate of 10 mg per 50 mL for the 25 gal Acre (234 L/ha) spray. A higher concentration of B. thuringiensis was required for the 5 gal/ Acre (47 L/ha) sprays = 50 mg per 50 mL so that spray volume treatments delivered similar amounts of B. thuringiensis. Casein-salt with the cross-linking agent (0.25% w/v) was used for the casein formulation. The volume of application by the spray chamber was adjusted by using an 8001 nozzle, 30 psi (2.1 Kg/cm²), and 4 mph (6.4 km/h) track speed.

Casein-salt formulations provided improved resistance to wash-off when compared to the flour/sucrose and technical powder formulations (Table 7). In general, the volume of the application did not affect the initial insecticidal activity and had only a small affect on resistance to wash-off For example, casein-salt formulations with

XenTari applied at 47 l/ha had <40% OAR compared with >70% OAR for the 235 l/ha application.

Table 8. Mortality and original activity remaining (% OAR) of casein formulations made with B. thuringiensis kurstaki (bt), applied at different spray volumes and assayed against the European corn borer.

bt Volume Rain in.

Trt# Formulation mg/50 mL gal/A Mortality % OAR

1 None None - - 4 ~

2 None 10 25 (233 L/ha) 0 75

3 None 10 25 (233 L/ha) 2 (5 cm) 37 49

4 None 50 5 (47 L/ha) 0 63

5 None 50 5 (47 L ha) 2 (5 cm) 15 24

6 Casein-salt 0.5% 10 25 (233 L/ha) 0 87

7 Casein-salt 0.5% 10 25 (233 L/ha) 2 (5 cm) 49 56

8 Casein-salt 0.5% 50 5 (47 L ha) 0 82

9 Casein-salt 0.5% 50 5 (47 L/ha) 2 (5 cm) 62 76

10 Flour/sucrose 2% 10 25 (233 L/ha) 0 100

11 Flour/sucrose 2% 10 25 (233 L ha) 2 (5 cm) 37 37

12 Flour/sucrose 2% 50 5 (47 L /ha) 0 98 bt Volume Rain in.

Trt# Formulation mg/50 mL gal/A Mortality % OAR

13 Flour/sucrose 2% 50 5 (47 L ha) 2 (5 cm) 36 37

Example 7

Formulations provided similar responses when made with different preparations of B. thuringiensis and assayed against different insects. The same formulations as in Example 5 were compared, except that the active ingredient was XenTari, B. thuringiensis subsp. aizawai, and the formulations were assayed against the beet armyworm. Several formulations were compared including flour/sucrose, and casein formulations. As discussed above, two rates of application were compared to show that resistance to wash-off was not lost by application at lower volumes. The application rates were 7 and 35 mL/pass for the low and high rates, respectively.

Casein formulations resisted wash-off better than the flour/sucrose and XenTari formulations for both application rates (Table 9). In general, the lower volume of application did not resist wash-off as well a high volume applications for each of the formulations. This may be the result of ingredients already in the XenTari formulation. The casein formulation expressed good insecticidal activity against the beet armyworm.

Table 9. Mortality and original activity remaining (% OAR) of casein formulations made with Bacillus thuringiensis subsp. aizawai (XenTari) (bt), applied at different spray volumes and assayed against the beet armyworm.

Bt Trt# Formulation mg/50mL Volume gal/A Rain In. Mortality % OAR

1 None one - — 2 ~

2 None 100 25 (233 L/ha) 0 75

3 None 100 25 (233 L/ha) 2 (5 cm) 9 12

4 None 500 5 (47 L/ha) 0 80 5 None 500 5 (47 L ha) 2 (5 cm) 7 9

6 Casein-salt 0.5% 100 25 (233 L/ha) 0 76

7 Casein-salt 0.5% 100 25 (233 L/ha) 2 (5 cm) 54 71

8 Casein-salt 0.5% 500 5 (47 L/ha) 0 85

9 Casein-salt 0.5% 500 5 (47 L/ha) 2 (5 cm) 34 40

10 Flour/sucrose 2% 100 25 (233 L ha) 0 100 1 1 Flour/sucrose 2% 100 25 (233 L/ha) 2 (5 cm) 20 20

12 Flour/sucrose 2% 500 5 (47 L ha) 0 93 Bt

Trt# Formulation mg/50mL Volume gal A Rain In. Mortality % OAR

13 Flour/sucrose 2% | 500 5 (47 L/ha) | 2 (5 cm) 1 16

Example 8

The following two experiments compare different commercial preparations of B. thuringiensis in a casein formulation for insecticidal activity against the European corn borer and for their resistance to wash-off. (Tables 10 and 11) B. thuringiensis preparations included technical powder, Dipel 2X, MNP stabilized cream (Mycogen Corp., 4980 Carroll Canyon Road, San Diego, CA) and MVP formulated product. Formulations were mixed at the same activity level for each of the preparations. All formulations were mixed with Mycogen's MVP cream (at 2 g of liquid cream / 50 mL which is equivalent to about 40 mg dry powder/50 mL) and Abbott's technical powder (at 10 mg/50 mL) except for Mycogen's formulated product and Abbott's Dipel 2X. The application used 35-36 mL total volume for about 25 gal/A (234 L/ha). Casein-salt with the cross-linking agent (0.25% w/v) was used for the casein formulation in the first experiment, and both formulations were used in the second experiment. The first experiment contained unusually high larval mortality in the control group and the treatment mortality means were corrected using Abbott's formula before the determination of the % OAR (Table 10). The casein formulation provided the best resistance to wash-off as indicated by the % OAR.

In the second experiment, Dipel 2X and MVP formulations were used for the active preparations, not technical powder (Table 11). Casein and casein-salt formulations made with Abbott's' technical powder and Mycogen's technical cream were compared with flour/sucrose, and commercial formulations (Dipel 2X and MVP) (Table 11). The main effects tested in this experiment were B. thuringiensis preparation (Abbott's versus Mycogen's B. thuringiensis), simulated rain (rain versus no rain) and formulation (KOH, azcote, flour/sucrose, casein-salt and casein). Formulations made of azcote represented ingredients used to make the casein-salt formulation. Treatments with Abbott's technical powder averaged 62% larval mortality and treatments with Mycogen's cream averaged 61% larval mortality. Analysis of variance indicated that the B. thuringiensis preparation had no significant (P>0.05) affect on the mortality of European corn borer. Also, interactions with active agent were not significant (_P>0.05). Simulated rain and formulation significantly ( <0.0001) affected larval mortality. The application of 5 cm simulated rain reduced the observed larval mortality from 91% to 33% over all the other factors. Among the formulations tested, casein-salt formulation provided the best resistance to wash-off as indicated by the 79% OAR. The experiment showed that casein- salt (79% OAR), and casein (58% OAR) provided resistance to wash-off that was superior than that of commercial formulations available from Abbott (Dipel 2X, 21% OAR) and Mycogen (MPV, 1% OAR). All formulations provided >86% mortality for larvae in the absence of simulated rain with the exception of the KOH solution which averaged 68% larval mortality.

Table 10. Raw mortality, Abbott's mortality (corrected for the control mortality) and original activity remaining (% OAR) for control of European com borer by formulations of B. thuringiensis (B.t.).

Formulation Corrected %

Trt# w/v Bt Preparation Rain, In. Mortality Mortality OAR

1 None None — 67 — ~

2 None MVP 0 98 94 3 None MVP 2 (5 cm) 56 0 0

4 None Technical powder 0 89 67

5 None Technical powder 2 (5 cm) 68 3 4

6 Casein 0.5% MVP 0 97 91

7 Casein 0.5% MVP 2 (5 cm) 90 70 77

8 Flour/sue 2% MVP 0 100 100 9 Flour/sue 2% MVP 2 (5 cm) 52 0 0

Table 11. Mortality for control of European com borer by formulations made with Mycogen's and Abbott's preparations of Bacillus thuringiensis as the active agents.

Trt# Preparation Formulation Rain, In. Mortality % OAR

1 Untreated None 0 2 —

2 Mycogen None 2 (5 cm) 0 0 3 Mycogen None 0 97

4 Mycogen KOH 2 (5 cm) 1 1.5

5 Mycogen KOH 0 68 Trt # Preparation Formulation Rain, In. Mortality % OAR

6 Mycogen Casein 2 (5 cm) 76 78 salt/AZCote

7 Mycogen Casein 0 97 salt/AZCote

8 Mycogen AZCote 2 (5 cm) 1 1

9 Mycogen AZCote 0 99

10 Mycogen Casein 2 (5 cm) 54 57

11 Mycogen Casein 0 94

12 Mycogen Flour/sucrose 2 (5 cm) 1 1

13 Mycogen Flour/sucrose 0 100

14 Abbott None 2 (5 cm) 36 41

15 Abbott None 0 87

16 Abbott KOH 2 (5 cm) 14 23

17 Abbott KOH 0 64

18 Abbott Casein salt 2 (5 cm) 71 79

19 Abbott Casein salt 0 90

20 Abbott AZCote 2 (5 cm) 14 16

21 Abbott AZCote 0 86

22 Abbott Casein 2 (5 cm) 58 58

23 Abbott Casein 0 100

24 Abbott Flour/sucrose 2 (5 cm) 21 21

25 Abbott Flour/sucrose 0 100

26 Mycogen (Formulation) 2 (5 cm) 1 1

27 Mycogen (Formulation) 0 100

28 Abbott Dipel 2X 2 (5 cm) 21 21

29 Abbott Dipel 2X 0 98

Example 9

Casein can protect B. thuringiensis from degradation by light (Table 13). When formulated and unformulated B. thuringiensis was applied to cotton plants and exposed to the Suntest CPS light for 8 hr, casein formulated B. thuringiensis retained 70 % OAR compared with 40 % OAR for unformulated B. thuringiensis. Based on a paired T test, the OAR for casein is significantly greater then the OAR for technical powder (t=3.96; n=3, P<0.03). Table 13. Mortality of neonate European com borer when fed on cotton leaves treated with casein formulated or technical powder Bacillus thuringiensis and exposed to light from the Suntest CPS machine for 8 hours.

No Exposure 8 hr Exposure

Form Rep N Mortalitv SD N Mortalitv SD OAR

Bt 1 10 85.4 13.9 10 34.5 31.4 40

Bt 2 8 65.6 21.8 8 28.8 8.5 44

Bt 3 10 67.9 21.0 10 24.3 15.9 36

Casein 1 10 83.6 18.1 8 46.3 23.1 55

Casein 2 8 64.1 18.1 8 51.1 20.6 80

Casein 3 10 52.8 33.4 10 39.4 21.9 75

Example 10

This field experiment showed the relative effects of rain and sunlight on the residual insecticidal activity of the casein formulation using B. thuringiensis as the active agent. Cabbage plants in field plots were treated with casein, flour/sucrose, and Dipel 2X formulations, plus an untreated control for a total of four formulations. The field was arranged in a split plot design for rain and no rain treatments. Also, three levels of shade were included in the design for a total of 24 treatment combinations (4 formulations x 2 rain treatments x 3 levels of shade = 24). Formulations were applied with a CO₂ charged backpack sprayer through a 3 nozzle spray boom that directed the spray at the plants. The equivalent of 33 gallons/ Acre ((308 L/ha (band application directed at the row at 19.3 mL/m of row)) were applied at 4 mph (6.4 km h) and 50 psi (3.5 Kg/cm). Formulations were allowed to dry, and then 1.25 inches (3.2 cm) of simulated rain were applied to half the plots via a rainbird irrigation system. After simulating rain, plastic tents were erected over plots to provide various levels of shading. Black plastic was used to impose complete shade. Clear plastic allowed light to penetrate but prevented natural rain from falling on the plants. No cover was used on the third treatments, which represented natural unshaded condition. This experiment was replicated three times.

Treatments were evaluated based on mortality of cabbage looper larvae assayed on excised cabbage leaves. Ten leaf samples per plot were collected 1,2,4 and 7 days after formulation application. A 33 cm² leaf disk was cut from each leaf, placed in a petri dish with 10 neonate larvae and incubated for 3 days at 28° C. After incubation, the number of live and dead larvae were counted and the mortality was calculated. Casein extended residual insecticidal activity of B. thuringiensis when compared with the commercial formulation, Dipel 2X. The average mortality of cabbage looper exposed to the casein formulation over all treatments was 81% compared with 74%, and

64% for flour/sucrose and Dipel formulations, respectively. Casein extended residual activity by resisting wash-off and by reduced degradation in sunlight. Ability of the formulations to resist wash-off is best expressed by comparing rain vs. no rain treatments for the 1-day evaluation. Percentage of insecticidal activity remaining averaged 94%,

77%, and 81%, for casein, Dipel, and flour/sucrose, respectively. In comparing formulations for protection from sunlight, the percentage original activity remaining

(comparing no cover with black plastic, for no rain treatments, 7-day evaluation), of casein, Dipel, and flour/sucrose formulations were 67%, 30%, and 54% respectively.

Table 14. Mortality of cabbage looper exposed to field grown cabbage leaves treated with formulations of Bacillus thuringiensis and exposed to simulated rain and shade.

Formulation Rain, In. Shade 1-day 2-day 4-day 7-day

Casein 0 Black plastic 94 96 93 85

Casein 1.25 (3.2 cm) Black plastic 88 93 79 82

Casein 0 Clear plastic 94 84 79 42

Casein 1.25 (3.2 cm) Clear plastic 85 87 69 44

Casein 0 None 94 92 78 57

Casein 1.25 (3.2 cm) None 93 89 73 55

Dipel 2X 0 Black plastic 95 96 81 78

Dipel 2X 1.25 (3.2 cm) Black plastic 72 76 61 55

Dipel 2X 0 Clear plastic 90 86 61 36

Dipel 2X 1.25 (3.2 cm) Clear plastic 73 60 35 19

Dipel 2X 0 None 92 79 53 24

Dipel 2X 1.25 (3.2 cm) None 68 59 42 41

Flour/sucrose 0 Black plastic 98 98 89 95

Flour/sucrose 1.25 (3.2 cm) Black plastic 89 88 79 62

Flour/sucrose 0 Clear plastic 98 96 88 47

Flour/sucrose 1.25 (3.2 cm) Clear plastic 80 55 56 25

Flour/sucrose 0 None 98 97 78 51 Formulation Rain, In. Shade 1-day 2-day 4-day 7-day

Flour/sucrose 1.25 (3.2 cm) None 69 59 38 28

Untreated 0 Black plastic 22 23 7 10 Untreated 1.25 (3.2 cm) Black plastic 8 6 13 10

Untreated 0 Clear plastic 30 36 23 21 Untreated 1.25 (3.2 cm) Clear plastic 26 29 16 4

Untreated 0 None 34 29 18 18 Untreated 1.25 (3.2 cm) None 32 18 23 21

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

WHAT is CLAIMED IS:

1. A casein-based formulation for pest control comprising a pesticidally effective amount of a pest control agent, casein, and an alkalinizing agent.

2. The casein-based formulation of claim 0 further comprising an aqueous solvent.

3. The casein-based formulation of claim 0 wherein the pH of said sprayable pest control formulation is from about 7.5 to about 12.0.

4. The casein-based formulation of claim 0 wherein the pH of said sprayable pest control formulation is from about 8.0 to about 10.0.

5. The casein-based formulation of claim 0 wherein the amount of said casein in said casein-based formulation in weight/volume percent is from about 0.1 percent to about 1.5 percent.

6. The casein-based formulation of claim 0 wherein the amout of said casein in said casein-based formulation in weight/volume percent is from about 0.2 percent to about 1.0 percent.

7. The casein-based formulation of claim 0 wherein said pest control agent is dimilin, malathion, carbaryl, diazinon, 2,4-D, metolachlor, glyphosate, paraquat, trifluralin, B. thuringiensis or recombinant B. thuringiensis toxin.

8. The sprayable pest control formulation of claim 0 wherein said recombinant

B. thuringiensis toxin is encapsulated in substantially intact cells.

9. The sprayable pest control formulation of claim 0 further comprising a pest phagostimulant or a pest attractant.

10. The sprayable pest control formulation of claim 0 wherein said pest attractant is a pheromone.

11. The sprayable pest control formulation of claim 0 wherein said phagostimulant is cucurbitacin.

12. A method of delivering the casein-based formulation of claim 0 comprising spraying said pest control formulation onto an external surface of an organism.

13. A casein-based formulation for pest control comprising a pesticidally effective amount of a pest control agent, casein salt, and a cross-linking agent.

14. The casein-based formulation of claim 0 further comprising an aqueous solvent.

15. The casein-based formulation of claim 0 wherein said cross-linking agent is the salt of zirconium carbonate.

16. The casein-based formulation of claim 0 wherein said salt of zirconium carbonate is ammonium zirconium carbonate.

17. The casein-based formulation of claim 0 wherein the amount of said ammonium zirconium carbonate in weight/volume percent is from about 0.01 percent to about 0.1 percent.

18. The casein-based formulation of claim 0 wherein the amount of said casein salt in said casein-based formulation in weight/volume percent is from about 0.1 percent to about 1.5 percent.

19. The casein-based formulation of claim 0 wherein the amount of said casein salt in said casein-based formulation in weight/volume percent is from about 0.2 percent to about 1.0 percent.

20. The casein-based formulation of claim 0 wherein said pest control agent is dimilin, malathion, carbaryl, diazinon, 2,4-D, metolachlor, glyphosate, paraquat, trifluralin, B. thuringiensis or recombinant B. thuringiensis toxin.

21. The sprayable pest control formulation of claim 0 wherein said recombinant B. thuringiensis toxin is encapsulated in substantially intact cells.

22. The sprayable pest control formulation of claim 0 further comprising a pest phagostimulant or a pest attractant.

23. The sprayable pest control formulation of claim 0 wherein said pest attractant is a pheromone.

24. The sprayable pest control formulation of claim 0 wherein said phagostimulant is cucurbitacin.

25. A method of delivering the casein-based formulation of claim 0 comprising spraying said pest control formulation onto an external surface of an organism.

26. A method of protecting a pest control agent from environmental degradation comprising admixing said pest control agent with a protective amount of casein, an aqueous solvent, and an alkalinizing agent to form a casein-based formulation for pest control and exposing said casein-based formulation for pest control to the environment.

27. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein the pH of said casein-based formulation for pest control is from about 7.5 to about 12.0.

28. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein the pH of said casein-based formulation for pest control is from about 8.0 to about 10.0.

29. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein said pest control agent is protected against radiation inactivation.

30. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein said pest control agent is protected against washoff.

31. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein the protective amount of said casein in said casein-based formulation for pest control in weight/volume percent is from about 0.1 percent to about 1.5 percent.

32. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein the protective amount of said casein in said casein-based formulation for pest control in weight/volume percent is from about 0.2 percent to about 1.0 percent.

33. A method of protecting a pest control agent from environmental degradation comprising admixing said pest control agent with a protective amount of casein salt, an aqueous solvent, and a cross-linking agent to form a casein-based formulation for pest control and exposing said casein-based formulation for pest control to the environment.

34. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein said cross-linking agent is the salt of zirconium carbonate.

35. The casein-based formulation of claim 0 wherein said salt of zirconium carbonate is ammonium zirconium carbonate.

36. The casein-based formulation of claim 0. wherein the amount of said ammonium zirconium carbonate in weight/volume percent is from about 0.01 percent to about 0.1 percent.

37. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein said pest control agent is protected against radiation inactivation.

38. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein said pest control agent is protected against washoff.

39. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein the protective amount of said casein salt in said casein-based formulation for pest control in weight volume percent is from about 0.1 percent to about 1.5 percent.

40. The method of protecting a pest control agent from environmental degradation according to claim 0 wherein the protective amount of said casein salt in said casein-based formulation for pest control in weight/volume percent is from about 0.2 percent to about 1.0 percent.