Hybrid-composite biopesticides based on nanoimbibition of azadirachtin

Abstract

The present disclosure provides compositions nanocomposite biopesticides exhibiting improved efficacy, photostability, as well as controlled release and systemic delivery characteristics and methods of making and using the same. The present disclosure further provides methods for protecting a plant from a Lepidopteran pest as well as controlling a Lepidopteran pest.

Description

HYBRID-COMPOSITE BIOPESTICIDES BASED ON NANOIMBIBITION OF AZADIRACHTINREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States provisional application No. 63 / 631,083, filed April 8, 2024, herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The invention generally relates to the field of biopesticides. Novel biopesticides exhibiting insecticidal and pesticidal activity against agriculturally -relevant pests of plants and seeds are disclosed herein as well as methods of using the same. In particular, the disclosed class of biopesticides allows systemic delivery to plants and exhibits insecticidal activity against the Lepidopteran order of insect pests.BACKGROUND OF THE INVENTION

[0003] Biopesticides are described as natural, biologically occurring compounds used to control various agricultural pests. In particular, biopesticides are naturally occurring compounds obtained from animals, plants, and microorganisms such as bacteria, cyanobacteria, and microalgae. Compared to synthetic pesticides that may persist in both the soil and the environment, the use of biopesticides can be advantageous. In particular, using biopesticides can help avoid many of the human health and environmental concerns associated with synthetic pesticides. The use of biopesticides therefore represents an attractive approach toward sustainably enhancing crop protection in agriculture.

[0004] As of August 2020, there were 390 biopesticide active ingredients registered with the United States Environmental Protection Agency (EP A). These biopesticides can be further classified as biochemical pesticides, microbial pesticides, or plant- incorporated-protectants. Neem seed extract is a biochemical pesticide powder from Azadirachta indica. The active ingredient in neem seed extract, azadirachtin, is composed of several nonpolar limonoids with functional groups such as enol ether, carboxylic esters, and epoxides. Exposure to azadirachtin can adversely affect insect physiology and behavior, leading to increased mortality, reducedfeeding and growth, abnormal and delayed ecdysis, metamorphosis interference, repellence, sterility, and anatomical abnormalities owing to its direct action on insect cells and tissues. Importantly, the detrimental insecticidal effects of azadirachtin are not observed in larger animals and humans.

[0005] To mitigate environmental risks from synthetic pesticides, azadirachtin and other plant-based insecticidal components offer selective toxicity to insects with minimal off-target effects. However, effective use of azadirachtin is a challenge due to its poor water solubility caused in part by its functional terminal groups (e.g. carboxylic esters, epoxide, and enol ether). Although nanoencapsulation of neem products using polymers, metals, and biomaterials has previously been investigated, limitations with respect to efficacy, photostability, controlled release, and systemic delivery remain. Therefore, a continuing need exists in the art for the development of novel biopesticide compositions and methods that can be used to significantly improve crop protection in a variety of agricultural field environments.SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides a nanocomposite biopesticide comprising a neem seed extract, a nanocarrier, and an emulsifier, wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm and allows for systemic delivery to a plant. In some embodiments, the neem seed extract comprises azadirachtin. In other embodiments, the neem seed extract comprises about 10wt% azadirachtin to about 40wt% azadirachtin. In another embodiment, the neem seed extract comprises about 0.5wt% azadirachtin to about 4.0wt% azadirachtin. In yet another embodiment, the neem seed extract comprises about 14wt% azadirachtin, 20wt% azadirachtin, about 25wt% azadirachtin, about 30wt% azadirachtin, or about 37wt% azadirachtin. In specific embodiments, the azadirachtin is about 21wt%. In some embodiments, the nanocomposite biopesticide further comprises at least one essential oil. In certain embodiments, the essential oil comprises thyme oil, peppermint oil, rosemary oil, orange oil, lemongrass oil, clove oil, or citronella oil. In other embodiments, the nanocarrier comprises glycine, whey protein, casein, zein protein, or a combination of any thereof. The mean particle size of the nanocomposite biopesticide, in some embodiments, is between about 5 nm and about 200 nm. The mean particle size of the nanocomposite biopesticide, in another embodiment, is less than about 200 nm, about 100 nm, about 75 nm, about 50 nm, or about 20 nm. The mean particle size of the nanocomposite biopesticide, in yet another embodiment, is between about 10 nm and about 40 nm. In furtherembodiments, the emulsifier comprises Tween80, poloxamers, glucosides, or a combination of any thereof. In yet further embodiments, the nanocomposite biopesticide further comprises a radical scavenger selected from the group consisting of Vitamin E, Vitamin C, a carotenoid, terpenoids, or a combination of any thereof.

[0007] In another aspect, the present disclosure provides a nanocomposite biopesticide capable of being preparable by obtaining an oil phase solution comprising neem seed extract and an emulsifier; obtaining an aqueous phase solution comprising a nanocarrier; combining and emulsifying the aqueous phase solution and the oil phase solution to obtain an emulsified solution; freeze-drying the emulsified solution; and obtaining a freeze-dried powder comprising the nanocomposite biopesticide, wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm and allows for systemic delivery to a plant.

[0008] In yet another aspect, provided herein is a nanocomposite biopesticide capable of being preparable by obtaining an oil phase solution comprising neem seed extract and an emulsifier; obtaining an aqueous phase solution comprising a nanocarrier; combining and emulsifying the aqueous phase solution and the oil phase solution to obtain an emulsified solution; spray-drying the emulsified solution; and obtaining a spray-dried powder comprising the nanocomposite biopesticide wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm and allows for systemic delivery to a plant.

[0009] In some embodiments, the ratio of the emulsifier and the neem seed extract is about 45: 1, about 4.5:1, about 54:1, about 5.4: 1. In other embodiments, the neem seed extract comprises azadirachtin. In further embodiments, the neem seed extract comprises about 14wt% azadirachtin, 20wt% azadirachtin, about 25wt% azadirachtin, about 30wt% azadirachtin, or about 37wt% azadirachtin. In another embodiment, the nanocarrier comprises glycine, whey protein, casein, zein protein, or a combination of any thereof. In specific embodiments, the nanocarrier comprises glycine. The mean particle size of the nanocomposite biopesticide, in some embodiments, is between about 10 nm and about 40 nm. In other embodiments, the emulsifier comprises Tween80, poloxamers, glucosides, or a combination of any thereof. The nanocomposite biopesticide, in certain embodiments, further comprises a radical scavenger selected from the group consisting of Vitamin E, Vitamin C, a carotenoid, terpenoids, or a combination of any thereof. In other embodiments, the nanocarrier comprises glycine and the emulsifier comprises Tween80. In yet another embodiment, the ratio of the nanocarrier and the emulsifier is about 1:0.29, about 1:0.89, about 1:0.92, about 1: 1.11, or about 1:1.35. In other embodiments, obtaining an oil phase solution comprises sonicating theoil phase solution for at least about 5 min, at least about 10 min, at least about 15 min, or at least about 20 min. In further embodiments, obtaining the aqueous phase solution comprises sonicating the aqueous phase solution for at least about 5 min, at least about 10 min, at least about 15 min, or at least about 20 min. In still further embodiments, combining the aqueous phase solution and the oil phase solution is carried out at a volumetric ratio of about 11.85: 1, about 15:1, about 10: 1, or about 8: 1. In certain embodiments, combining the aqueous phase solution and the oil phase solution is carrier out over about 0.5 min, about 1 min, about 1.5 min, about 2 min, or about 2.5 min. In another embodiment, the nanocomposite biopesticide is dissolved in an aqueous solution. In certain embodiments, about 10 mg, about 25 mg, about 50 mg, about 75 mg, or about 100 mg of the nanocomposite is dissolved per milliliter of the aqueous solution. In some embodiments, combining and emulsifying the aqueous phase solution and the oil phase solution is carried out under ultrasonication. In other embodiments, the aqueous phase solution and the oil phase solution is carried out under tangential mixing.

[0010] In another aspect, the present disclosure provides a method of protecting a plant from a Lepidopteran pest, comprising applying a nanocomposite biopesticide described herein to a plant. In some embodiments, the method comprising contacting the Lepidopteran pest with an inhibitory amount of the nanocomposite biopesticide. In certain embodiments, contacting the Lepidopteran pest results in systemic delivery of the nanocomposite biopesticide to the pest, hi specific embodiments, the Lepidopteran pest is a .S', frugiperda larvae.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0012] FIG. 1 shows the size and structural characteristics of GNP nanoparticles including the number-averaged particle size (hydrodynamic diameter) distribution of GNP (Panel A); a transmission electron microscope image of as-prepared GNP (Panel B); and a transmission electron microscope image of resuspended GNP that was freeze-dried (Panel C).

[0013] FIG. 2 shows the zeta-potential for as-prepared nanoemulsion and for redispersion of GNP after freeze-drying. The zeta potential of as-prepared nanoemulsion was -5.8 ± 0.4 mV, while the zeta potential for redispersion of GNP after freeze-drying was -10.5 ± 1.1 mV.

[0014] FIG. 3 shows the release kinetics of azadirachtin from GNP in aqueous (water : IPA - 10 : 1 vol) solution. The release behavior followed an exponential kinetics with Cumulative release (CR)~68.2[l-eA(-t / 37.6) ] and a coefficient of variance, r2 of 0.99.

[0015] FIG. 4 shows the response of encapsulated azadirachtin in GNP composite against UV light in comparison to NSE powder using UV / Vis spectroscopy. The percent degradation measured from the absorbance intensity loss via UV / Vis spectroscopy upon irradiation with U V-AB light in dry conditions. The experiments were performed for GNP composite, a carrier composite (without NSE), NSE powder, and the corresponding controls (dark condition, no irradiation, and room temperature). All experiments were performed at a relative humidity of 50%.

[0016] FIG. 5 demonstrates S.frugiperda mortality from contact assay of S.frugiperda treated with GNP dispersion in water (Panel A); NSE dispersion in water (Panel B); and GLY + TW control dispersion in water (Panel C).

[0017] FIG. 6 shows the size analysis from the contact assay of S. frugiperda presented in FIG. 5, for GNP dispersion in water (Panel A); NSE dispersion in water (Panel B); and GLY and TW control dispersion in water (Panel C).

[0018] FIG. 7 shows SEM images of S. frugiperda . GNP (100 mg / ml) was treated on day 1 using contact assay and larva was grown for 2 days. Panel A shows control larva (day 1); Panel B shows treated larva (day 1); Panel C shows control larva (day 3); and Panel D shows treated larva (day 3).

[0019] FIG. 8 shows confocal microscope images of newly hatched .S'. frugiperda (day 1) treated with contact assay. Panels A, B, and C show newly hatched S. frugiperda treated with 100 mg / mL GNP with Nile red (50pg / mL) dispersion in water; Panels D, E, and F show newly hatched 5. frugiperda treated with Nile red (50|ig / mL) dispersion in water; and Panels G, H, and I show newly hatched S.frugiperda treated with water.

[0020] FIG. 9 shows the contact angle of GNP solutions on tomato leaves as a function of GNP concentration.

[0021] FIG. 10 shows the results of leaf feeding assay with .S’. frugiperda at varying concentrations of GNP.

[0022] FIG. 11 shows SEM images of S. frugiperda spiracle morphology, GNP (100 mg / ml) was treated on day 1 using contact assay and larva was grown for 2 days. Panel A shows control larva (day 1); Panel B shows treated larva (day 1); Panel C shows control larva (day 3); and Panel D shows treated larva (day 3).

[0023] FIG. 12 shows SEM images of control 5. frugiperda (day 1). Panel A shows upsidedown posture; Panel B shows head part; and Panel C shows mandible part.

[0024] FIG. 13 shows a schematic overview of the contact assay. Eggs are transferred to the hatching vial (Panel A); larvae emerge after 4 days (Panel B); larvae are moved and dipped in the nanopesticide solution for 2 seconds (Panel C); and larvae are moved to well tray with feed and observed for 10 days.

[0025] FIG. 14 shows a schematic overview of the leaf-feeding assay. Eggs are transferred to the hatching vial (Panel A); larvae emerge after 4 days (Panel B); leaf is dip-coated in nanopesticide solution (Panel C); leaf is dried by air (Panel D); larvae were transferred in empty well trays with no feed, get starved for 2 hours (Panel E); nanopesticide dip-coated leaf sheet is added in well trays with larvae (Panel F); feed is added to well trays after one day, and the larvae are observed over 10 days (Panel G).

[0026] FIG. 15 shows the intensity-averaged particle size (hydrodynamic diameter) distribution of as-prepared (Panel A); after freeze-drying at varying weight ratios between TW and NSE (Panels B, C, D, E, F, G, and H).

[0027] FIG. 16 shows confocal microscope images of newly hatched S. frugiperda (day 1) treated with Img / mL GNP with Nile red (0.5pg / mL) dispersion in water (100:1 dilution).DETAILED DESCRIPTION OF THE INVENTION

[0028] Plant insect pests can cause significant damage to crop plants, leading to substantial economic loss. A number of strategies are currently available and have been employed to control and limit damage caused by plant insect pests. For example, synthetic insecticides such as organophosphates, pyrethroids, and carbamates are commonly used to protect against crop damage caused by insects. However, recent estimates place the economic cost of plant insect pests at a minimum of US$70 billion annually, with associated health costs exceeding US$6.9 billion per year (e.g., Bradshaw, C. et al. Massive yet grossly underestimated global costs of invasive insects. Nat Commun 7, 12986 (2016)). Moreover, the global use of these and similar methods to control plant insect pests has created selection pressure for existing alleles that impart resistance. In light of these pressures, environmentally sustainable improvements in technology, agricultural techniques, and pest management are vital tools to expand crop production on the limited amount of arable land available for farming.

[0029] The use of biopesticides in the management of plant insect pests is an attractive alternative to using traditional synthetic insecticides. For example, biopesticides are usuallyinherently less toxic than conventional pesticides. Currently, azadirachtin, a tetranortriterpenoid derived from the neem seed of the Indian neem tree, is one of the prominent biopesticides used in agricultural worldwide. Azadirachtin is a powerful antifeedant and insect growth disruptor. However, azadirachtin exhibits poor water solubility and rapidly degrades in sunlight. These inherent characteristics of azadirachtin present challenges for its application on a large scale and are disadvantageous from an agribusiness perspective, since they result in lower efficiency and necessitate a greater number applications.

[0030] The present disclosure provides nanocomposite biopesticides exhibiting improved efficacy, photostability, as well as controlled release and systemic delivery characteristics. The combination of improved properties exhibited by the nanocomposite biopesticides described herein provides a significant advance in the art.

[0031] The present disclosure further provides novel azadirachtin-based nanocomposite biopesticides and methods of preparing and using the same. In preferred embodiments, provided herein is a nanocomposite biopesticide formulation based on freeze drying a neem seed extract emulsion with Tween 80 and glycine, with assistance of isopropanol. Such compositions are biodegradable, food-grade, and derived from the natural sources. The biodegradable characteristics of such nanocomposites are particularly advantageous toward environmental sustainability.

[0032] The compositions provided herein comprise biopesticide particles sizes in the range of about 10 nm to about 200 nm when dispersed in water. Such small sizes enable penetration into plant tissues and systematic delivery via roots and stomata. These nanocomposite formulations protect neem oil active components from UV degradation and thermal instabilities.

[0033] Additionally, the nanocomposites provided herein enable both instant release and sustained release of the biopesticides, which fulfills both short term and long term requirements for protecting crops in the agricultural field and reduces the need for frequent application. The significantly improved photostability of the nanocomposites described herein can be attributed to the light scattering effect of encapsulating the azadirachtin compound with TW, and GLY matrix, which is demonstrated to significantly reduce the degradation rate of azadirachtin.

[0034] The biological interaction between the nanoencapsulated azadirachtin and 5. frugiperda larvae is also demonstrated herein. Suspensions of freeze-dried nanocomposite biopesticides in water were shown to kill 5. frugiperda immediately after 1 day, and the efficacy of the active ingredient is increased after nanoencapsulation examined with LC50analysis. The nanoscale biopesticide described herein can promote sustainable, organic farming practices.A. Nanocomposite Neem Seed Extract Biopesticides

[0035] The present disclosure provides nanocomposite biopesticide comprising a neem seed extract, a nanocarrier, and an emulsifier. When dispersed in water the mean particle size of the nanocomposite biopesticide can be less than about 200 nm. Such small particle sizes enable penetration into plant tissues and systematic delivery via roots and stomata as well as systemic uptake inside insect larvae. Methods for calculating particle size distribution are known in the art and any such method may be used according to the embodiments of the present disclosure. In some embodiments, the mean particle size of a nanocomposite biopesticide described herein may be less than about 200 nm, less than about 175 nm, less than about 150 nm, less than about 125 nm, less than about 100 nm, less than less than about 95 nm, less than about 90 nm, less than about 85 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, less than about 24 nm, less than about 23 nm, less than about 22 nm, less than about 21 nm, less than about 20 nm, or less than about 15 nm, including all ranges and values derivable therebetween. In certain embodiments, the mean particle size of a nanocomposite biopesticide described herein may be between about 5 nm and about 200 nm, between about 5 nm and about 150 nm, between about 5 nm and about 100 nm, between about 5 nm and about 75 nm, between about 5 nm and about 50 nm, between about 5 nm and about 45 nm, between about 5 nm and about 40 nm, between about 5 nm and about 35 nm, between about 5 nm and about 30 nm, or between about 10 nm and about 40 nm, including all ranges and values derivable therebetween. In certain embodiments of the present disclosure the mean particle size of the nanocomposite biopesticide may allow for systemic delivery to a plant and or insect.

[0036] The present disclosure also describes neem seed extract comprising certain wt percentages (wt%) of azadirachtin. Methods for calculating the wt% of azadirachtin are known in the art. The wt% of azadirachtin may be calculated by any such method. The wt% may be calculated with respect to the nanocomposite biopesticide; or alternatively, may be calculated with respect to the neem seed extract used to prepare the nanocomposite biopesticide. For example, the nanocomposite biopesticides provided herein may comprise about 0.01wt% azadirachtin to about 20wt% azadirachtin, about 0.15wt% azadirachtin to about 15wt% azadirachtin, about 0.1 wt% azadirachtin to about 10wt% azadirachtin, about 0.2wt%azadirachtin to about 5wt% azadirachtin, or about 0.5wt% azadirachtin to about 4wt% azadirachtin, including all ranges and values derivable therebetween. In some embodiments, the nanocomposite biopesticides provided herein may comprise about 0.5wt% azadirachtin, about 0.75wt% azadirachtin, about lwt% azadirachtin, about 1.25wt% azadirachtin, or about 1.5wt% azadirachtin, including all ranges and values derivable therebetween. In other embodiments, the neem seed extract of the present disclosure may comprise about 10wt% azadirachtin to about 40wt% azadirachtin, about 14wt% azadirachtin to about 37wt% azadirachtin, or about 20wt% azadirachtin to about 25wt% azadirachtin, including all ranges and values derivable therebetween. In further embodiments, the neem seed extract comprises about 15wt% azadirachtin, about 16wt% azadirachtin, about 17wt% azadirachtin, about 18wt% azadirachtin, about 19wt% azadirachtin, about 20wt% azadirachtin, about 21wt% azadirachtin, about 22wt% azadirachtin, or about 23wt% azadirachtin, including all ranges and values derivable therebetween.

[0037] The nanocomposite biopesticides described herein comprise at least one nanocarrier and at least one emulsifier, also referred to herein as a nanostabilizer. As referred to herein, a “nanocarrier” may refer to a molecule having at least one of their dimensions within the 1-100 nm range and engaging in transport another substance, such as, small molecule. Nanocarriers provided herein include, but are not limited to, glycine, whey protein, casein, zein protein, or a combination of any thereof. A “nanostabilizer” (or “emulsifier”) refers to a surface active compound that plays a key role in the formation of nanoimbibed entities by adsorbing to the oil-water interface and thereby reducing the interfacial tension and improving stability of the nano emulsions. Nanostabilizers provided herein include, but are not limited to, Tween80, poloxamers, glucosides, or a combination of any thereof.

[0038] The nanocomposite biopesticides described herein may further comprise one of more essential oils. Essential oils having bioactivity against insects are known in the art including, but not limited to thyme oil (thymol, carvacrol), mint oil (menthol, pulegone), rosemary oil (1,8 -cineole), orange oil, lemongrass oil, clove oil (eugenol), or citronella oil (citronellal, citral), cinnamon oil (cinnamaldehyde), and oil of oregano (carvacrol). Additionally, the nanocomposite biopesticides described herein may further comprise a radical scavenger. A radical scavenger is a molecule which reacts readily with the radicals or other oxidizing agents including, but not limited to Vitamin E, Vitamin C, a carotenoid, a terpenoid, or a combination of any thereof. Thus, nanocomposite biopesticides comprising one or more essential oils, nanocarriers, emulsifiers, and / or radical scavengers are provided herein. The nanocarriers may be found alone or in combination with glycine and may include, but are not limited to,whey protein, casein, and zein protein. The emulsifiers may be found alone or in combination with Tween80 and may include, but are not limited to, poloxamers and glucosides. The radical scavengers include, but are not limited to, Vitamin E, Vitamin C, carotenoids, and terpenoids.B. Preparing Nanocomposite Neem Seed Extract Biopesticides

[0039] In certain aspects, the present disclosure provides methods of preparing a nanocomposite biopesticide as well as the nanocomposite biopesticide prepared by such methods. In some embodiments, provided herein is a nanocomposite biopesticide capable of being preparable by: obtaining an oil phase solution comprising neem seed extract and an emulsifier, obtaining an aqueous phase solution comprising a nanocarrier, combining and emulsifying the aqueous phase solution and the oil phase solution to obtain an emulsified solution, freeze-drying the emulsified solution, and obtaining a freeze-dried powder comprising the nanocomposite biopesticide, wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm and allows for systemic delivery to a plant. In other aspects, a nanocomposite biopesticide capable of being preparable by: obtaining an oil phase solution comprising neem seed extract and an emulsifier; obtaining an aqueous phase solution comprising a nanocarrier; combining and emulsifying the aqueous phase solution and the oil phase solution to obtain an emulsified solution; spray-drying the emulsified solution; and obtaining a spray-dried powder comprising the nanocomposite biopesticide wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm and allows for systemic delivery to a plant.

[0040] In some embodiments, the amount of emulsifier and neem seed extract may be expressed as a ratio. For example, in certain embodiments the ratio of the emulsifier and the neem seed extract is about 60:1, about 55:1, about 54:1, about 45:1, about 40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1, about 10: 1, about 9:1, about 8:1, about 7: 1, about 6:1, about 5.5: 1, about 5.4:1, about 5:1, about 4.9: 1, about 4.8: 1, about 4.7:1, about 4.6: 1, about 4.5:1, about 4.25:1, about 4: 1, about 3: 1, about 2:1, or about 1: 1, , including all ranges and values derivable therebetween.

[0041] In other embodiments, the amount of nanocarrier and emulsifier may be expressed as a ratio. For example, in certain embodiments the ratio of nanocarrier and emulsifier is about 1:0.1, about 1:0.15, about 1:0.2, about 1:0.25, about 1:0.28, about 1:0.29, about 1:0.3, about 1 :0.4, about 1 :0.5, about 1 :0.6, about 1 :0.7, about 1 :0.8, about 1 :0.9, about 1 :1 , about 1 :1.1 , about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, or about 1:2, including all ranges and values derivable therebetween.

[0042] In certain embodiments, obtaining an oil phase solution comprising neem seed extract and an emulsifier, and obtaining an aqueous phase solution comprising a nanocarrier, may be carried out under specific conditions. For example, obtaining an oil phase solution and / or an aqueous phase solution may comprise sonicating the oil phase solution and / or aqueous phase solution for at least about 1 min, at least about 2 min, at least about 3 min, at least about 4 min, at least about 5 min, at least about 6 min, at least about 7 min, at least about 8 min, at least about 9 min, at least about 10 min, at least about 11 min, at least about 12 min, at least about 13 min, at least about 14 min, at least about 15 min, at least about 20 min, at least about 25 min, at least about 30 min, at least about 35 min, at least about 40 min, or at least about 45 min. Similarly, combining the aqueous phase solution and the oil phase solution may be carried out under specific conditions, e.g. a specific volumetric ratio. In such embodiments, combining the aqueous phase solution and the oil phase solution may be carried out at a volumetric ratio of about 20:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, or about 5:1, including all ranges and values derivable therebetween. In further embodiments, combining the aqueous phase solution and the oil phase solution is carrier out over about 0. 1 min, about 0.25 min, about 0.5 min, about 0.75 min, about 1 min, about 1.25 min, about 1.5 min, about 1.75 min, about 2 min, about 2.5 min, or about 5 min, including all ranges and values derivable therebetween.

[0043] In another aspect, after freeze-drying or spray-draying the nanocomposite biopesticide, the resulting powder may be dissolved in in an aqueous solution. In some embodiments, about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, or about 100 mg of the nanocomposite is dissolved per milliliter of the aqueous solution, including all ranges and values derivable therebetween.

[0044] In further embodiments, combining and emulsifying the aqueous phase solution and the oil phase solution, may be carried out under specific conditions. The conditions and processes used to combine and emulsify the aqueous phase solution and the oil phase solution may yield beneficial technical characteristics to the resulting nanocomposite biopesticide. For example, combining and emulsifying the aqueous phase solution and the oil phase solution is carried out under ultrasonication, tangential mixing, or a combination thereof. In particular, ultrasonication, using a critical energy intensity (also referred to as power density) of at least 10 W / mL, was shown to result in a particle size below 300-400 nm. Absent ultrasonication, much larger particle sizes may be obtained. In some aspects, ultrasonication may be carried out using a critical energy intensity (or power density) of at least 10 W / mL, at least 11 W / mL,at least 12 W / mL, at least 13 W / mL, at least 14 W / mL, at least 15 W / mL, or at least 20 W / mL, including all ranges and values derivable therebetween.

[0045] Furthermore, the tangential mixing was shown to result in uniform particle size distribution. Specifically, tangential impinging jets provide a micro-turbulent region with a very high turbulent and mixed region ensuring the formation of uniform particle size distribution and energetically forcing nanoparticles into well-defined shapes.C. Method of protecting a plant from a Lepidopteran pest and controlling a Lepidopteran pest

[0046] In some aspects, the present disclosure provided methods for protecting a plant from a Lepidopteran pest; and controlling a Lepidopteran pest using nanocomposite biopesticides of the present disclosure. Such methods may include applying a nanocomposite biopesticide provided herein to a plant; and contacting the Lepidopteran pest with an inhibitory amount of the nanocomposite biopesticide provided herein, respectively. “Contacting,” “Contacted,” or like term is intended to refer to being present in the vicinity of the target pest, or the delivery of a pesticidally effective amount of the nanocomposite biopesticide to the target pest through exterior contact with the pest or through ingestion by the pest.

[0047] Reference in this application to the terms “active” or “activity”, “insecticidal” or “insecticidal activity”, “pesticidal” or “pesticidal activity”, and similar terms, refer to efficacy of a biopesticide, such as a nanocomposite biopesticide disclosed herein, in inhibiting (inhibiting growth, feeding, fecundity, or viability of a plant pest), suppressing (suppressing growth, feeding, fecundity, or viability of a plant pest), controlling (controlling the pest infestation, controlling the pest feeding activities on a particular crop) or killing (causing the morbidity, mortality, or reduced fecundity of a plant pest) a pest. These terms are intended to include the result of providing an effective amount of a nanocomposite biopesticide to a pest where the exposure of the pest to the nanocomposite biopesticide results in inhibiting, suppressing, controlling, or killing. These terms also include repulsion of the pest from the plant, a tissue of the plant, a plant part, seed, plant cells, or from the particular geographic location where the plant may be growing, as a result of providing an effective amount of the nanocomposite biopesticide on the plant. In some embodiments, the nanocomposite biopesticide can be applied to the plant or to the environment within the location where the plant is located. The terms “bioactivity”, “effective”, “efficacious” or variations thereof are also terms interchangeably utilized in this application to describe the effects of nanocomposite biopesticides of the present invention on plant pests. In certain embodiments, such termsdescribe, for example, a decrease in the growth of a insect pest, a decrease in the ability of the insect to survive, grow, feed, and / or reproduce, a decrease in the infectivity of a insect plant pest, a decrease in the infestation of a plant by a insect plant pest, as compared to an appropriate control.

[0048] In any of the methods described herein, plants grown in the presence of the nanocomposite biopesticide or formulation thereof can exhibit decreased susceptibility to a pest, such as S. frugiperda , as compared to plants grown in the absence of the nanocomposite biopesticide, under the same conditions. In any of the methods described herein, plants grown in the presence of the nanocomposite biopesticide or formulation thereof can exhibit decreased insect damage, including reduced defoliation, and / or reduced S. frugiperda larvae, as compared to plants grown in the absence of the nanocomposite biopesticide, under the same conditions. In any of the methods described herein, plants or the locus in which the plant is grown, such as soil, to which the nanocomposite biopesticide or formulation thereof has been applied can exhibit reduced S. frugiperda larvae and / or reduced .S'. frugiperda larvae per volume of soil, as compared to plants grown in the absence of the nanocomposite biopesticide, under the same conditions. In any of the methods described herein, an nanocomposite biopesticide or a formulation applied to S. frugiperda can result in direct killing, degradation, behavioral modification, or prevention of reproduction.

[0049] In one embodiment, the nanocomposite biopesticide, composition, or formulation of the present invention decreases damage caused by a plant insect pest by at least about 0.5%, or by at least about 1%, or by at least about 2%, or by at least about 3%, or by at least about 5%, or by at least about 6%, or by at least about 7%, or by at least about 8%, or by at least about 9%, or by at least about 10%, or by at least about 11%, or by at least about 12% when compared to plants produced under the same conditions but without treatment.

[0050] In any of the methods described herein, plants grown in the presence of the nanocomposite biopesticide or formulation thereof can exhibit decreased susceptibility to a insect pest as compared to plants grown in the absence of the nanocomposite biopesticide, under the same conditions

[0051] The term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more,” unless specifically noted otherwise. The terms “comprise,” “have,” and “include” areopen-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” are also open- ended. For example, any method that “comprises,” “has,” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any system or method that “comprises,” “has,” or “includes” one or more components is not limited to possessing only those components and covers other unlisted components.

[0052] Other objects, features, and advantages of the present disclosure are apparent from detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Any embodiment of the present disclosure may be used in combination with any other embodiment described herein.

[0053] All references herein are incorporated herein by reference in their entirety.EXAMPLES

[0054] The following examples are included to illustrate embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result 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.Example 1: Size, structural, and colloidal characteristics of nanocomposite biopesticide before and after freeze-drying.

[0055] The size, structure, and zeta potential of the particles are the core properties for characterizing a nanoemulsion and their delivery mechanism in the nanopesticide studies. The size and structural characteristics of GNP nanoparticles are provided herein. FIG. 1 presentsthe particle size distribution, and TEM micrograph of as-prepared nanoemulsion and freeze- dried cake redispersion of GNP. The particle size distribution averaged by number is monodispersed for both as-prepared and resuspended freeze-dried cake can be observed (FIG. 1; Panel A). After the particle was freeze-dried, the hydrodynamic size measured from DLS decreased to 39.6 ± 29. Inm, decreased from as-prepared nanoemulsion particle size of 47.6 + 24.8nm with presence of IPA, by Z-average. The structure of nanoparticles shown in TEM micrograph images were spherical for both as-prepared nanoemulsion and freeze-dried cake redispersed case and their size dimension range were comparable to hydrodynamic sizes measured by DLS (FIG. 1; Panels B and C). Additional details of the size distributions plots averaged by the intensity, and corresponding data with different mixing ratio between emulsifier and neem seed extract are reported in FIG. 15 and Table 1. The stability of the nanoparticle dispersion is determined by the ratio between emulsifier and the non-polar ingredient, which is TW and NSE for this case, respectively. By increasing the TW up to 10 more times than NSE by weight, the multi-dispersity with two peaks due to an aggregation has been mostly eliminated. From the comparison from 4:1 to as-prepared (10: 1, and GLY added), the Polydispersity index (PDI) has continuously decreased from 0.39 ± 0.03 to 0.22 ± 0.02, which indicates that the uniformity of the nanoparticles have been improved by adding the portion of emulsifier TW, and co-emulsifier GLY. The ratio lower than 4:1 did not have a peak near lOnm scale.Table 1. Particle characterization data of different mixing ratios for the emulsion, between TW and NSEOrganic phase Water phaseAzadirachtin Mixing ratio (Isopropanol 10ml) (Water 100ml)Z- Average (nm) PDI wt% after(TW:NSE) Tween 80 Neem seed Glycine freeze-drying (TW) extract (NSE) (GLY)1:1 0.05g 0.05g - 105.7 ± 3.7 0.18 ± 0.01 10.52:1 0.10g 0.05g - 85.6 ± 2.8 0.26 ± 0.02 7.04:1 0.20g 0.05g - 115.3 ± 43.3 0.39 ± 0.03 4.26:1 0.30g 0.05g - 140.0 ± 70.4 0.36 ± 0.10 3.08:1 0.40g 0.05g - 72.6 ± 15.3 0.27 ± 0.03 2.310: 1 0.50g 0.05g - 13.6 ± 0.7 0.25 ± 0.04 1.910:10.50g 0.05g 0.45g 47.6 ± 24.8 0.22 ± 0.04 1.1(As-prepared)10:1(After freeze- 0.50g 0.05g 0.45g 39.6 ± 29.1 0.22 ± 0.02 1.1 drying)* NSE has pure azadirachtin 21wt% and the rest are other limonoid derivatives, polyphenolics, lignins, fatty acid esters, and lipids

[0056] The zeta potential of as-prepared nanoemulsion was -5.8 ± 0.4 mV, while it was -10.5 ± 1.1 mV for redispersion of GNP after freeze-drying (FIG. 2). After the formulation is freeze-dried, the arrangement of TW surrounding the non-polar compounds can get much tighter so that the surface charge becomes slightly more negative and it reduces the chance for particles to aggregate, also the average size of the nanoparticles can get smaller. With the absence of IPA droplet after freeze-dried redispersion, there are two modes of mechanism that neem seed extract does not aggregate and precipitate: First, the non-polar compounds are stressed to the inner-core when freeze-drying exerts the Laplace pressure of TW molecules to squeeze NSE inside. Second, the glycine molecules create the hydrogen bonding network after the composite is dissolved in a water, and their interactive attractions between carboxylic and amine groups act as matrix to prevent the collision between the nanomicelles. Nanoparticles are highly soluble in water, since TW+NSE particle has spherical structure with hydrophilic groups at the outside surface owing to oxygen elements on TW head part, also the freeze dried GLY matrix is highly water-soluble.Example 2: Freeze-drying Composite Biopesticide Powder and Characterization of the SameRelease kinetics of azadirachtin from Nanocomposite Biopesticide

[0057] It is important to understand the release kinetics of encapsulated pesticides to correlate their response rate with mortality test results and predicting the natural prolonging portions by prolonged encapsulation without losing their pesticidal functionality. Since natural pesticide compounds can degrade and decompose by exposure to the sunlight, microbes, and the water, biopesticide formulations with sustaining and gradually releasing profiles are advantageous. FIG. 3 demonstrates that freeze-dried GNP redispersion has gradual and slower release compared to freely diffused NSE, up to day 7 reaching 68.2 ± 2.1% of the release while most of NSE diffuses by day 2-3, and up to day 7 reaching 102.8 ± 1.5%calculated from the fitted curve. While 68.2 % of the azadirachtin compounds are released, the rest of 31.8 % are remaining in the TW emulsions over 7 days. The release was slow and gradual, sustained for 5+ days to reach a plateau (over 90% of the saturated release level) and has followed an exponential profile and was close to a first-order kinetics. In correspondence with equation (1) and (2) shown in Example 5, the drug loading efficiency is 5%, and the encapsulation efficiency is 31.8%. This sustained release profile, remaining 31.8% encapsulated is advantageous for killing the insects for longer periods, not only an instant action when the pesticide was directly fed by the insects or delivered systemically inside. This synergistic collaboration of instant release and sustained release can both cover the short and longer term for protecting the crops in the agricultural fields, since instantly released azadirachtin can be hydrolyzed, oxidized with pH alteration, digested by microbes with enzymatic mechanisms to lose their functionality within 4 days.Stability of Nanocomposite Biopesticide Exposed to UV irradiation

[0058] The photostability of the pesticide under field conditions is crucial. For instance, the essential oils can decompose when exposed to heat, light, and oxidation. Likewise, azadirachtin under heat and sunlight has been reported to lose 50% of its bioactivity in at least 1.84-5.54 days over 45-54 °C (J. B. Stokes et al. (1982) Effect of sunlight on azadirachtin: Antifeeding potency, Journal of Environmental Science and Health. Part A: Environmental Science and Engineering, 17:1, 57-65). In FIG. 4, the response of encapsulated azadirachtin in GNP composite against the UV light simulating sunlight in comparison to NSE powder using UV / Vis spectroscopy is reported. On a given condition, 47.0 ± 3.0 % of NSE powder has degraded within four days. On the other hand, 41.3 + 2.0 % of GNP composite, and 29.5 ± 3.2 % of GNP composite without NSE have degraded for the same irradiation duration. The biodegradable characteristics of those particles are advantageous for the aspect of environmental sustainability. Overall, the improvement of photostability between GNP composite and NSE powder has been +27.7% for 4 days. The change in degradation rate is believed to be attributable to the light scattering effect by encapsulating the azadirachtin compound with TW, and GLY matrix. For example, by Mie and Rayleigh scattering, some portions of the photonic energy exerted to the particle core comprising azadirachtin have been distributed to the other encapsulant molecules. If there has been no UV light and heat applied to the samples, as the untreated control data of these three samples have been unchanging for four days, the degradation of azadirachtin is not likely to happen.Example 3: Mortality of S. frugiperda Exposed to Nanocomposite Biopesticide and Bulk Neem Seed Extract

[0059] The contact assay with varying concentrations over time is reported in FIG. 5. Panel A shows the GNP mortality S. frugiperda for contact assay for 11 days with 6 different dilution concentrations including water (Omg / mL) while Panels B and C show the data for NSE powder with 4 different dilution concentrations, and GLY+TW control with 3 different dilutions for a same period. For each concentration from the highest in Panels B and C corresponds to GNP composition in Panel A, respectively. To compare the efficacy and response time between GNP and other controls, the statistical analysis with p-value in Table 2.Tabic 2. Difference report (p-value) for (a) contact assay mortality (b) size analysis from contact assay (c) between two assays.(a) (p-value) for contact assay mortality* Day 1 are all 0 mortality in the beginning(b) (p-value) for size analysis from contact assay(c) (p-value) between two assays* Day 1 are all 0 mortality in tire beginning for both assays

[0060] The LC50 value by day is reported in Table 4. From the LC50 table, the efficacy of NSE was always higher and had quicker response when incorporated into the GNP. Up to day 2, there was significant statistical different between GNP compared to bulk NSE (Table 2(a); p < 0.05), since GNP had superior instant killing effect owing to its much higher solubility in water and mobility of 50 nm scale nanoparticle dispersion. This corresponds to the first order kinetics trend shown from FIG. 3 that in the beginning, the release rate is much quicker than after day 2~4. Since bulk NSE is poorly soluble in water, the size of the particle is severely poly dispersed and aggregated, hence majority of the particles are not effectively impact the pests compared to nano-sized particles with no aggregation. The carrier compounds with GNP or bulk NSE had significant statistical difference all the time (Table 2(a); p < 0.05) and have not shown mortality except the highest concentration (950mg / mL) with maximum of 30% death.

[0061] It was remarkable GNP has been more effective at lower concentrations compared to bulk NSE. After 7 days, lOmg / mL GNP (0.5mg / mL NSE incorporated) showed mortality around 80 ± 5.8 %, while 0.5mg / mL of bulk NSE showed mortality of 40.6 ± 6.7 - 68.8 ± 12.0 %. The pesticidal reaction has increased owing to reduced size and increased mobility compared to bulk NSE and this may facilitate the delivery of azadirachtin into the insect organs and tissues. The lethal effect of carrier compounds on particular S. frugiperda have not been investigated so far, since GLY is considered as building blocks for the cells as amino acid, and TW is food additive. From the past research, TW with 1 % (w / v) concentration have shown 0% mortality to Musca domestica and Chrysomya albiceps. The highest concentration of carrier compound has been nearly 100% w / v scale, but from second dilutions (10% w / v), it did not show any mortality.Table 3. LCso interpolation for contact mortality* When the average mortality of the highest concentration is <50%, interpolation of the LC50 value is not possible* LC50 of the second row was derived from dividing the first row by 20 since NSE is 5wt% of GNP assuming that other compounds generally have no lethality to S. frugiperda .* This calculation program is available at www.aatbio.c m / t ols / lc50-calculator

[0062] FIG. 6 shows the increase of length by growth of the S. frugiperda corresponding to the contact assay in FIG. 5. This supporting information demonstrates whether the insect has been killed as well as the antifeeding and growing deterrent effects of the pesticide by comparing the length change with water control (Omg / mL). From FIG. 6, Panels A and B, the length increase has been inversely proportional to concentration level. The statistical analysis for difference (p-value) is provided in Table 2(b) and there has been no significant statistical difference between GNP and bulk NSE regarding the length increase trend (p > 0.05). From FIG. 6, Panel C, the length between GLY and TW controls and water had no difference overlapped with error bars. There has been significant statistical difference between the GLY and TW controls and GNP or bulk NSE (p < 0.05 except day 3 near 0.05). Azadirachtin is known as causing delayed ecdysis and feeding deterrence against pests like S'. frugiperda . In brief, the development time from 1st to 6th instar larvae can conclude before 14 days and initiate pupating if the growth is completed, and the length data can provide the continuous response of pesticide against the pest, and the growth curve follows the linear equation model for 11 days.

[0063] Overall, the mortality of the larvae with contact assay and corresponding length, LC50, also the statistical differences with p-value have revealed the enhanced efficacy of nanoencapsulated azadirachtin with GLY and TW. Compared to the past studies, the contact assay result has shown the same trend which mortality and concentration of the drug has positive correlation. By exposure to azadirachtin, delayed ecdysis, interference of the growth, feeding deterrence, sterility, anatomical abberations were reported attributed from the physiological impact on cells, tissues, nerves, and endocrine systems.

[0064] To investigate further direct feeding effect in the simulated field environment, the leaffeeding assay has been tested for the same period and same concentration with GNP in FIG. 10. The leaf-feeding assay has multiple mode of actions: First, the insects can die from starving owing to the feeding deterrence effect. Second, the insects can feed the nanopesticideand the response happens from the midgut by the digestion. Third, the insects can interface the leaf skins coated with nanopesticide, and the particles can transport via intrusion to the skins. From FIG. 10, the leaf-feeding assay has responded much quicker compared to contact assay with smaller amounts of the nanocomposite biopesticide. From Table 2(c), the effect of GNP by contact assay has had significant statistical difference up to day 6 compared to leaf-feeding assay result (p-value; < 0.05). Table 4 reports the LC50 interpolation for leaf feeding mortality.Table 4. LC50 interpolation for leaf feeding mortalityDay 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11LC50GNP 71.3 5.5 0.88 0.21 0.21 0.21 0.21 0.21 0.21 0.21[mg / mL]* This calculation program is available at www.aatbio.com / tools / lc50-calculator

[0065] The efficacy difference can be directly compared by LC50 values between Table 3 and Table 4 by day, the leaf-feeding assay was more detrimental than contact assay results. This difference may be attributable to the distinct major pathways between two assays: Contact assay surrounds the insects with the drug for 2 seconds and the major pathways are the mandible, spiracles, and the skin whereas leaf-feeding assay focuses on the digestive pathway (little inhalation through spiracle and skin intrusion of the nanoparticles touching on the leaf). Additionally, feeding can be deterred greatly by antifeeding effect by azadirachtin coated on the leaf, but contact assay lets the insect to freely feed the diet.Morphological and mechanistic analysis of interactions between S.frugiperda and nanocomposite biopesticide

[0066] To investigate the possible uptake pathway for nanoparticles into the insect organs and nerves, 5. frugiperda larvae were prepared in prior to SEM during contact assay. In FIG. 7, the morphological overview of the insect on the day 1 control and day 3 control and treated with GNP are shown. Also, by focusing on the mandible and spiracles (FIGS. 11 and 12), the two main possible uptake pathways are confirmed. The mandible is an oral digesting pathway which is connected to the digestive system of the larva, and the spiracle is a respiratory pathway which is connected to the trachea and inner tissues. There are a pair of spiracles in each body segments of the larva, and total 10-11 body segments are seen in the SEM image. The size of mandible and spiracles measured from the SEM images is reported in Table 5.Table 5. The structural and surface characteristics of mandible and spiracle of .S', frugiperda used in this study. (*The average length of filtering protrusion shown in the spiracle images is 2.3 ± 0.5) _S. frugiperda Mandible SpiracleLong diameter (pm) 82.6 ± 9.0 6.7 ± 1.4Short diameter (pm) 22.2 ± 9.6 4.7 ± 1.0Long x Short dimension (pm) 1833.7 ± 86.4 31.5 ± 1.4Anatomical function Digestion Respiration* ± values indicate the standard deviation.* The length of mandible is measured from the distance between the large teeth from the larva mouth

[0067] As number of spiracles are 20-22, it is calculated that the uptake pathway area of mandible is far larger than the summation of area from the spiracles, and the hydrodynamic size of the nanoparticles reported in Table 1 is much smaller than both of the uptake pathways. During the contact assay in 2 seconds, it is assumed that the uptake volume of nanoparticles will be much larger to the digestive organs via mandible compared to other inner tissues via spiracles or penetrations through the coatings.

[0068] From FIG. 7, the scrawniness and severely wrinkled morphology is shown from treated larvae for day 1 and 3. Correspondingly, FIG. 11 shows the deposition of nanoparticles and more wrinkles near the spiracles from treated larvae. Azadirachtin may have caused the loss of appetite toward moist diets and have led to their severe dehydration, which have increased the surface rugosity and caused the imbalance of the osmotic pressures of the cells. To further examine the delivery pathway of the nanoparticles, Nile red fluorescent dye has been tagged during the preparation of initial composite biopesticides and tested through the same contact assay procedure in FIG. 8. In Panels A, B, and C of FIG. 8, the Nile red has been intensively accumulated throughout the path starting from the mandible part. There has been widely spread fluorescence everywhere, possibly due to the spiracle pathway and nanoparticle intrusions through the skins, but the intensity over mandible to digestion pathway was much more obvious with the comparative brightness. In comparison, the 1 / 100 diluted sample compared to Panels A, B, and C of FIG. 8 has been tested and is shown in FIG. 16 to show the less accumulation of nanoparticles when the concentration of the solution is much lower. In Panels D, E, and F of FIG. 8, Nile red in water has been tested as control. Since Nile red is not soluble in water, the mobility of the fluorescence itself has been much lower than nanopesticide tagged fluorescence for the same amount. Panels G, H, and I of FIG. 8 shows the water as another control and the contrast intensity was much lower. It is assumed that uptaken nanoparticles can be translocated into any organs such as endocrinal,respiratory, neural, cardiovascular, digestive, renal, and reproductive tissues, and the GNP nanoparticles can be delivered to all body parts confirmed with confocal microscopy.Example 4: Wetting and adhesion characteristics of composite biopesticide solutions on tomato leaves

[0069] In FIG. 9, the static angle of 5 different concentrations of GNP suspension on tomato leaves used for leaf-feeding assay is reported. The coating behavior corresponds to the hydrophilicity and the static angle profiles between the substrate and the solutes in water. It is known that leaf surfaces are naturally hydrophobic owing to the natural wax layers and microroughness. The static angle of water droplet on the tomato leaf surface was measured as 99.0 ± 1.6°. The static angle from the lowest concentration from FIG. 9 overlaps in the range of water droplet with standard error, but it greatly starts to drop from Img / mL scale. In the simulated field, as the solvent dries by time, the solutes can either be coated or moved away from the surface depending on the hydrophilicity. As the static angle from highest concentration has been near 60°, most of the nanoparticles are assumed to be coated on the leaf surface. The biopesticides can either directly sprayed to the insects or coat the plant surfaces to protect against the pests. The contact assay and the leaf-feeding assay are correlated with those cases respectively. GLY is highly water-soluble and hydrophilic, and TW is amphiphilic and strongly bound to NSE compounds and can stick to the leaf surface simultaneously. GLY and TW both have contributed for increasing the wettability to make GNP nanoparticles to adhere on the tomato leaf substrate.Example 5: Materials and Methods

[0070] Nanocomposite Biopesticide Materials: Glycine powder (>99.5%, cell culture reagent, Alfa Aesar, Haverhill, MA, USA, CAS# 56-40-6) was obtained to establish the composite matrix for co-emulsifying and encapsulating the neem seed extract (natural limonoids powder, Fortune Aza Technical, India) powder during freeze-drying process. Neem seed extract consists of 21wt% azadirachtin, 24 wt% of other pesticidal limonoid compounds, and the rest are polyphenolics, lignins, fatty acid esters, and lipids. Tween 80 (Polyoxyethylene Sorbitan Monooleate, TCI, Portland, OR, USA, CAS# 9005-65-6) was prepared to emulsify the nonpolar compounds from neem seed extract. Isopropyl alcohol (99%, VWR, Radnor, PA, USA, CAS# 67-63-0) was arranged for initially solubilizing and stabilizing the emulsion of the neem seed extract in water (o / w) with Tween 80 and after freeze drying, it is eliminated. Deionized (DI) water was used for the aqueous solvent preparation.

[0071] Oil-in-water emulsification of azadirachtin with Tween 80 and glycine: A 0.45 g of glycine (GLY) powder in lOOmL of water has been sonicated in a water bath for 10 min at room temperature (22°C). Tween 80 (TW) (0.50 g) and neem seed extract (NSE) powder (0.05g) have been dissolved in 10 mL of isopropyl alcohol (IP A) and sonicated in a water bath for 10 min at room temperature (22°C). Next, the lOmL of IPA solution (oil phase) was added to lOOmL of GLY solution (aqueous phase) slowly for Imin drop- wise, while the GLY solution beaker has been sonicated in a water bath for 10 more minutes. A dynamic light scattering (DLS) (Malvern Instruments, Ltd., Malvern, UK) was run to measure the hydrodynamic size and zeta-potential of as-prepared nanoemulsion sample at 25 °C. TEM (FEI TECNAI G2 F20 ST FE-TEM, FEI Company, Hillsboro, OR, USA) was used for visualizing the size distribution characteristics and surface morphology of as-prepared sample. Samples were transferred on copper grids with 300 meshes by using negative staining procedures and using uranyl acetate as a staining solution at room temperature. After freeze- drying the as-prepared solution to eliminate the IPA, the composite powders were resuspended to DI water for measuring the TEM to compare with images the as-prepared status.

[0072] Freeze-drying process for composite biopesticide powder and characterization: As- prepared nanoemulsion with water and IPA 10 to 1 volume ratio was frozen with liquid nitrogen rapidly to stabilize the crystalline network formed by glycine molecules under glass transition temperature to effectively disperse the emulsion droplets, and eliminate the IPA.28 The frozen solid in a centrifuge tube were placed in a vacuum freeze-drier (FreeZone 4.5 Liter Benchtop Freeze Dry System, Labconco Corporation, Kansas City, MO, USA) at -45~50°C for 48 hours. The glycine nanopesticide (GNP) has structured a foam cake after freeze-drying.

[0073] Release Kinetics of the azadirachtin from the glycine matrix: To investigate the release kinetics of the GNP, the freeze-dried powder (GLY 0.045g + TW 0.050g + NSE 0.005g) was resuspended in 10 mL water and ImL IPA mixture and transferred into a dialysis membrane (3.5 kD, Fisher Scientific, Waltham, MA, USA, S 132725) and closed tight with clamps. 49 times more volume of water and IPA mixture (10: 1 volume ratio) were prepared to finally dilute the dialysis membrane with volumetric ratio of 50: 1 at room temperature (22°C). The release of active ingredient, azadirachtin from GLY matrix was analyzed in terms of intensity measured by UV / Vis spectroscopy (UV-1800, Shimadzu, Japan) from the outside of the dialysis membrane. 3 mL of solutions were placed in quartz cuvette to measure the UV / Vis spectra between 200 - 800nm, and the absorbance intensity at 220nm was collected to summarize the concentration increase by time after the release of the active ingredient. In parallel, the freeze-dried powder with absence of NSE (GLY 0.045g + TW 0.050g) and thebulk NSE (0.005g) samples were measured with a same protocol. To characterize only azadirachtin release from GNP, the free GNP (freeze-dried powder with absence of NSE) was subtracted from the GNP intensity at 220nm. The standard calibration was measured in advance of the release test at 5 - 6 different concentrations (220nm, GNP R2 = 0.9963, free GNP R2 = 0.9913, NSE R2 = 0.9997). Dialysis membrane assay has been repeated for three times. Drug loading and encapsulation efficiency was calculated using the equations as below:

[0074] UV light stability test: The photostability of GNP powder was investigated by exposing the sample to the simulated sunlight, a UV-AB lamp (160W, REPTI ZOO, Miami, FL, USA). The distance between the bulb and the sample was 15cm and the temperature was adjusted to 45°C. The flux was adjusted near 2000 pW / cm2 (1850 + 306 pW / cm2), measured by a digital UV-AB light meter (UV513AB, General tools, Secaucus, NJ USA, spectrum range 290-370 nm, calibration point 365 nm, temperature between 0~50°C). The GNP powder, and also a carrier powder was suspended in water and coated on a quartz block with 3 drops separately. NSE powder was dissolved in 1PA and coated on a quartz block with 3 drops. The coated quartz block was completely dried in a biological safety cabinet overnight and characterized by UV / Vis spectroscopy to measure the degradation of absorbance intensity at 220nm by time. As a control, the same replicas were placed with no light irradiation in the room temperature. Photodegradation assay has been repeated for at least 3-5 times.

[0075] Larvae preparation and characterization: S. frugiperda were obtained from Benzon Research (Carlisle, PA, USA). The eggs were laid on the damp paper towels in a zipper storage bag and shipped in refrigerated condition with humid towels inside the zipper storage bag for maintaining the humidity. A sectional paper towel with eggs were separated and placed inside the glass vial with 3 cm3 block of Benzon Research insect diet. This ingredient was sourced from Southland Products and consists of soy flour and wheat germ in an agar base containing vitamins and minerals. A glass vial was sealed with fine mesh fabric with tape and rubber bands to prevent the escape of the larvae. A glass vial was placed under fluorescent light for the growing for 12 hours with a constant room temperature. The larvae has emerged after 4 days in a hatching set-up. This hatching day was set as day 0 for mortality experiments and imaging analysis. The first instar larvae of day 1 were transferred to theexperimental dish to take a scanning electron microscope (JSM-7500F; Jeol USA, Peabody, MA, USA) image (SEM). Larvae were dipped inside the GNP dissolved in a water (lOOmg / mL) for 2 seconds and observed after 2 days with SEM. Corresponding control larvae without dipping was measured on day 1 and day 3. To immobilize the larvae, they were refrigerated for 1 more day in 4 °C so that the tissues are not frozen, and they were stick on the carbon tape. To examine the delivery result of the GNP uptake systemically inside the larvae, Nile red (TCI, USA, CAS# 7385-67-3) fluorescence dye was tagged to the as-prepared step of GNP formulation. Nile red was 1 / 100 weight amount of NSE in the formulation. The larvae on day 1 was dipped into the GNP tagged with Nile red dissolved in a water (lOOmg / mL) for 2 seconds and transferred into the trays with a 1 cm3 block of feed for 1 hours, then immobilized in the refrigerator with 4 °C for 1 day, and the image was taken by confocal microscope (ZEISS LSM 780 NLO Multiphoton Microscope, Jena, Germany). As a control, 1 / 100 concentration of treatment, corresponding amount of Nile red with water, and pure water dipped larvae samples were contrasted with the brightness of the fluorescence intensity.

[0076] Mortality and growth analysis between the larvae and the composite biopesticide: S. frugiperda larvae were tested with nanopesticide with contact assay, which dip-coats the larvae inside the pesticide solution for 2 seconds using forcep and monitor their mortality and growth on the tray with feeds. To investigate the efficacy of GNP dissolved in water, 6 different concentrations were tested with 30 larvae, and 10 larvae were grouped as one each to score the mortality percentage. There were three groups with mortality percentage and the data was averaged with standard error. In comparison, the bulk NSE powder samples with 4 different concentrations, and the carrier (GLY + TW) compounds with 3 different concentrations, water dipped samples were tested the same way to analyze further with LC50 interpolation and statistical analysis. On day 1 , larvae were transferred with paintbrush and forcep to dip them inside the samples for 2 seconds and then placed in a white polystyrene bio-assay tray. Initial viability of the larvae after placed in trays was checked in response to gentle touching with forceps. Well trays were covered with bioassay tray lids to clearly ventilate and seal simultaneously under fluorescent grow lights for 12 hours with 24 °C. The mortality was scored daily by the direct observation of their movements by stimulating with forceps for 5 seconds, also the color, turgidity, and morphologies were examined. To obtain the growth data, the length of larva was measured by photographing the larvae inside the bioassay tray using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Until the mortality results are unchanging for 3 days, the survival and the length data werecontinuously gathered. At the end of the instar stages, the surviving larvae started to pupae. The 50% lethal concentration at a certain time point, LC50 was calculated using AAT Bioquest LC50 calculator (AAT Bioquest, Sunnyvale, CA, USA).29, 30 The mortality comparison between other treatments were analyzed by two-way ANOVA using JMP (JMP 16.1.0, JMP Statistical Discovery LLC., Cary, NC, USA) statistical analysis software and the p-values per each day were gathered. To further evaluate the performance of GNP under simulated field conditions, a leaf-feeding assay was tested. The first instar larvae were moved from the hatching vials to the bioassay trays without feeding cake to make them starve for 2 hours. A sectioned tomato leaflets were quickly dip-coated with samples and air dried, and then incorporated into the bioassay trays with starved day 1 larvae. The larvae were captivated inside the sealed well and 24 hours have been passed. On day 2, the leaflets were wilted and the feeding cakes of 1 cm3 were distributed into the wells since otherwise larvae would all die because of starvation. The daily mortality was checked the same way as contact assay. The illustration of schematic process is uploaded in FIGS. 13 and 14.

[0077] Plant germination for foliar adhesion and wetting test: To provide the tomato leaves, red robin tomato plant seeds were sowed in a potting mix under fluorescent grow lights (Agrobrite T5, Hydrofarm, Petaluma, CA, USA) in 22-28 °C for 14 hours and the rest of the day remained with no light. The plants were irrigated and fertilized with care and grown over 5 weeks. The static contact angle of the GNP dissolved in a water with 5 different concentrations was measured on the tomato leaf detached from the plants. The digital photo of the static angle was analyzed with ImageJ software, using LBADSA plug-in. The tomato leaves used for the leaf-feeding assay is same as for the foliar adhesion and wetting test.* *

[0078] All of the 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 or aspects, it will be apparent to those of skill in the art that variations may be applied to the 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.T1

Claims

CLAIMSWhat is claimed is:

1. A nanocomposite biopesticide comprising: a) a neem seed extract; b) a nanocarrier; and c) an emulsifier; wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm and allows for systemic delivery to a plant.

2. The nanocomposite biopesticide of claim 1, wherein the neem seed extract comprises azadirachtin.

3. The nanocomposite biopesticide of claim 2, wherein the nanocomposite biopesticide comprises about 0.1wt% azadirachtin to about 10wt% azadirachtin.

4. The nanocomposite biopesticide of claim 3, wherein the nanocomposite biopesticide comprises about 0.25wt% azadirachtin to about 6wt% azadirachtin.

5. The nanocomposite biopesticide of claim 4, wherein the nanocomposite biopesticide comprises about 0.5wt% azadirachtin to about 4wt% azadirachtin.

6. The nanocomposite biopesticide of claim 3, wherein the nanocomposite biopesticide comprises lwt% azadirachtin.

7. The nanocomposite biopesticide of claim 1, further comprising at least one essential oil.

8. The nanocomposite biopesticide of claim 7, wherein the essential oil comprises thyme oil, peppermint oil, rosemary oil, orange oil, lemongrass oil, clove oil, or citronella oil.

9. The nanocomposite biopesticide of claim 1 , wherein the nanocarrier comprises glycine, whey protein, casein, zein protein, or a combination of any thereof.

10. The nanocomposite biopesticide of claim 1, wherein the mean particle size of the nanocomposite biopesticide is between about 5 nm and about 200 nm.

11. The nanocomposite biopesticide of claim 10, wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm, about 100 nm, about 75 nm, about 50 nm, or about 20 nm.

12. The nanocomposite biopesticide of claim 10, wherein the mean particle size of the nanocomposite biopesticide is between about 10 nm and about 40 nm.

13. The nanocomposite biopesticide of claim 1, wherein the emulsifier comprises Tween80, poloxamers, glucosides, or a combination of any thereof.

14. The nanocomposite biopesticide of claim 1, further comprising a radical scavenger selected from the group consisting of Vitamin E, Vitamin C, a carotenoid, terpenoids, or a combination of any thereof.

15. A nanocomposite biopesticide capable of being preparable by: obtaining an oil phase solution comprising neem seed extract and an emulsifier; obtaining an aqueous phase solution comprising a nanocarrier; combining and emulsifying the aqueous phase solution and the oil phase solution to obtain an emulsified solution; freeze-drying the emulsified solution; and obtaining a freeze-dried powder comprising the nanocomposite biopesticide wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm and allows for systemic delivery to a plant.

16. A nanocomposite biopesticide capable of being preparable by: obtaining an oil phase solution comprising neem seed extract and an emulsifier; obtaining an aqueous phase solution comprising a nanocarrier; combining and emulsifying the aqueous phase solution and the oil phase solution to obtain an emulsified solution; spray-drying the emulsified solution; and obtaining a spray-dried powder comprising the nanocomposite biopesticide wherein the mean particle size of the nanocomposite biopesticide is less than about 200 nm and allows for systemic delivery to a plant.

17. The nanocomposite biopesticide of claim 15 or 16, wherein the ratio of the emulsifier and the neem seed extract is about 45: 1, about 4.5: 1, about 54:1, about 5.4: 1.

18. The nanocomposite biopesticide of claim 15 or 16, wherein the neem seed extract comprises azadirachtin.

19. The nanocomposite biopesticide of claim 18, wherein the neem seed extract comprises about 10wt% azadirachtin to about 40wt% azadirachtin.

20. The nanocomposite biopesticide of claim 19, wherein the neem seed extract comprises about 14wt% azadirachtin, 20wt% azadirachtin, about 25wt% azadirachtin, about 30wt% azadirachtin, or about 37wt% azadirachtin.

21. The nanocomposite biopesticide of claim 16, wherein the neem seed extract comprises about 21wt% azadirachtin.

22. The nanocomposite biopesticide of claim 15 or 16, wherein the nanocarrier comprises glycine, whey protein, casein, zein protein, or a combination of any thereof.

23. The nanocomposite biopesticide of claim 15 or 16, wherein the nanocarrier comprises glycine.

24. The nanocomposite biopesticide of claim 15 or 16, wherein the mean particle size of the nanocomposite biopesticide is between about 10 nm and about 40 nm.

25. The nanocomposite biopesticide of claim 15 or 16, wherein the emulsifier comprises Tween80, poloxamers, glucosides, or a combination of any thereof.

26. The nanocomposite biopesticide of claim 15 or 16, further comprising a radical scavenger selected from the group consisting of Vitamin E, Vitamin C, a carotenoid, terpenoids, or a combination of any thereof.

27. The nanocomposite biopesticide of claim 15 or 16, wherein the nanocarrier comprises glycine and the emulsifier comprises Tween80.

28. The nanocomposite biopesticide of claim 27, wherein the ratio of the nanocarrier and the emulsifier is about 1:0.29, about 1:0.89, about 1:0.92, about 1: 1.11, or about 1: 1.35.

29. The nanocomposite biopesticide of claim 15 or 16, wherein obtaining an oil phase solution comprises sonicating the oil phase solution for at least about 5 min, at least about 10 min, at least about 15 min, or at least about 20 min.

30. The nanocomposite biopesticide of claim 15 or 16, wherein obtaining the aqueous phase solution comprises sonicating the aqueous phase solution for at least about 5 min, at least about 10 min, at least about 15 min, or at least about 20 min.

31. The nanocomposite biopesticide of claim 15 or 16, wherein combining the aqueous phase solution and the oil phase solution is carried out at a volumetric ratio of about 15:1, about 11.85:1, about 10:1, or about 8:1.

32. The nanocomposite biopesticide of claim 15 or 16, wherein combining the aqueous phase solution and the oil phase solution is carrier out over about 0.5 min, about 1 min, about 1.5 min, about 2 min, or about 2.5 min.

33. The nanocomposite biopesticide of claim 15 or 16, wherein the nanocomposite biopesticide is dissolved in an aqueous solution.

34. The nanocomposite biopesticide of claim 33, wherein about 10 mg, about 25 mg, about 50 mg, about 75 mg, or about 100 mg of the nanocomposite is dissolved per milliliter of the aqueous solution.

35. The nanocomposite biopesticide of claim 15 or 16, wherein combining and emulsifying the aqueous phase solution and the oil phase solution is carried out under ultrasonication.

36. The nanocomposite biopesticide of claim 15 or 16, wherein combining and emulsifying the aqueous phase solution and the oil phase solution is carried out under tangential mixing.

37. A method of protecting a plant from a Lepidopteran pest, the method comprising applying the nanocomposite biopesticide of claim 1; or claim 15 or 16 to a plant.

38. A method of controlling a Lepidopteran pest, the method comprising contacting the Lepidopteran pest with an inhibitory amount of the nanocomposite biopesticide of claim 1; or claim 15 or 16.

39. The method of claim 38, wherein contacting the Lepidopteran pest results in systemic delivery of the nanocomposite biopesticide to the pest.

40. The method of claim 39, wherein the Lepidopteran pest is a .S’, frugiperda larvae.

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