Pickering emulsion for coating entomopathogenic nematodes

Abstract

Provided herein is a composition comprising a Pickering emulsion comprising a plurality of core-shell particles encapsulating an entomopathogenic organism. Further, a method for controlling pests on or within a plant or in a cultivation area is provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 358,387, filed on July 5, 2022, entitled “PICKERING EMULSION FOR COATING ENTOMOPATHOGENICNEMATODES,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of Pickering emulsions, and in particular to compositions comprising insect pathogenic organisms encapsulated by colloidosomes. Background Art

[0004] Chemical pesticides are used to eliminate, repel or mitigate arthropod pests. Broad-spectrum chemical pesticides are often harmful to the environment, humans and other non-target organisms. Compared to chemical pesticides, biological pesticides (biopesticides) are natural, biodegradable, eco-friendly and tend to have minimal impact on non-target organisms. Insect pathogenic nematodes (EPNs) are microbial control agents that have become important and play an important role in biocontrol and are incorporated into arthropod pest management as biopesticides. EPNs are considered sustainable biopesticides because they are environmentally safe and relatively harmless to mammals and other non-target organisms. EPNs are non-segmented roundworms that infect insects including larvae, pupae and adults of different insect orders such as Coleoptera, Diptera, Hemiptera, Lepidoptera, and Orthoptera. Insect pathogenic nematodes kill insects with the help of symbiotic bacteria carried in the nematode's gut and released after infection of the host. The infective larvae (IJ) of nematodes enter the host insect and enter the blood cavity through the spiracle, oral cavity, anus or in some species through the intersegmental membrane of the cuticle. The bacteria are bred in the insect hemolymph, and the infected host will usually die within 24 to 48 hours. After this, the nematode continues to feed on the host tissue and the bacteria that proliferate, mature and breed. One or more generations can be produced in the host corpse, and a large number of IJs are eventually released into the environment to infect other hosts and continue their life cycle.

[0005] Insect pathogenic nematodes have been used to control a variety of economically important pests, as disclosed in Shapiro-Ilan, DI et al., Advances in use of entomopathogenic nematodes in IPM, published in Integrated management of insect pests: Current and future developments, edited by M. Kogan and EA Heinrichs, Burleigh Dodds Science Publishing, Cambridge, UK, pp. 649-678, 2020, the contents of which are incorporated herein by reference. However, the commercialization and large-scale use of EPNs are limited because of their short shelf life in formulation and storage, resulting in low quality and reduced effectiveness against insects in the field.

[0006] The inventors speculate that encapsulation of insect pathogens (such as EPN) by Pickering emulsions may have great potential for the development of new biopesticide formulations. Summary of the invention

[0007] In one aspect, a composition is provided that includes an insect pathogen coated by a plurality of nanoparticles, wherein the plurality of nanoparticles include UV shielding nanoparticles, wherein the composition is an aqueous dispersion of the insect pathogen.

[0008] In one embodiment, the UV shielding nanoparticles are assembled within core-shell particles.

[0009] In one embodiment, a core-shell particle comprises a liquid core, the liquid core comprises oil, and wherein the liquid core is surrounded by a shell comprising a plurality of nanoparticles.

[0010] In one embodiment, the core-shell particles are characterized by an average particle size between 1 and 50 um.

[0011] In one embodiment, (i) the weight ratio between the plurality of nanoparticles in the core-shell particle or in the composition and the oil is between 1:10 and 1:100; (ii) the w / w concentration of the plurality of nanoparticles in the composition is between 0.1 and 10%.

[0012] In one embodiment, the UV shielding nanoparticles are chemically modified metal oxide particles.

[0013] In one embodiment, the metal oxide particles include a metal oxide selected from SiO2, TiO2, or both; and wherein the chemical modification includes a functional moiety covalently bonded to the metal oxide particles.

[0014] In one embodiment, the metal oxide comprises TiO2, and the functional moiety comprises any of the following: (i) an aminoalkyl group, an amino group, a hydroxyalkyl group, a thioalkyl group, an aminoalkylsilane group, a hydroxyalkylsilane group, a thioalkylsilane group, or any combination thereof; (ii) a UV absorbing group, or both (i) and (ii).

[0015] In one embodiment, the metal oxide comprises SiO2 and the functional moiety comprises a UV absorbing group.

[0016] In one embodiment, the insect pathogenic organism comprises a nematode.

[0017] In one embodiment, the nematode is an entomopathogenic infective larvae (IJ) nematode.

[0018] In one embodiment, the oil is a liquid at a temperature between 10 and 60°C.

[0019] In one embodiment, the oil is characterized by a viscosity of between 1 and 100 cP at 25°C.

[0020] In one embodiment, the oil is any of the following: (i) substantially non-toxic to insect pathogens; (ii) substantially non-phytotoxic.

[0021] In one embodiment, the oil comprises mineral oil, C10-C30 aliphatic hydrocarbons, fatty acids, monoglycerides, diglycerides, triglycerides, vegetable oils, waxes, essential oils, fragrance oils, or any combination thereof.

[0022] In one embodiment, the composition further comprises a gelling agent.

[0023] In one embodiment, the gelling agent is a polymer gelling agent including polyacrylic acid, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, poly(2-hydroxyethyl methacrylate), polyacrylamide or polysaccharide, including any salts and any combination thereof.

[0024] In one embodiment, the w / w concentration of the gelling agent in the composition is between 0.1 and 10%.

[0025] In another aspect, a pesticide composition comprising the composition of the present invention is provided; wherein the pesticide composition comprises an insecticidal effective amount of an insect pathogenic organism.

[0026] In one embodiment, the insecticide composition is formulated for application to plants, plant parts and / or to areas under cultivation, wherein the application is by spraying or coating.

[0027] In one embodiment, the insecticidally effective amount comprises an insect pathogenic organism at a concentration of at least 10 units / ml.

[0028] In another aspect, an article is provided comprising a substrate contacted with a coating comprising the insecticide composition of the present invention.

[0029] In one embodiment, the coating is in the form of a coating layer.

[0030] In one embodiment, the substrate is selected from plants or plant parts, polymer substrates, glass substrates, metal substrates, fibers, paper substrates, brick walls, sponges, textiles, woven fabrics, nonwoven fabrics or wood or any combination thereof.

[0031] In one embodiment, the coating is an insecticide coating.

[0032] In one embodiment, the coating substantially maintains its insecticidal activity when exposed to outdoor conditions for a period of time of 1 to 30 days.

[0033] In another aspect, there is provided a method for controlling pests, which comprises applying an effective amount of the pesticide composition of the present invention to at least a part of a plant, or to a cultivation area infested by pests, thereby controlling or reducing the growth of the pests.

[0034] In one embodiment, the insecticide composition is characterized by adhesion to at least a portion of a plant.

[0035] In one embodiment, applying comprises any of immersing, soaking, coating, irrigating, dipping, spraying, atomizing, scattering, brushing, injecting, or any combination thereof.

[0036] In one embodiment, the pests include arthropods.

[0037] In one embodiment, the effective amount is between 1 and 1000 L / ha.

[0038] In one embodiment, the arthropod is a plant pest.

[0039] Unless otherwise defined, all technical and / or scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art related to the present invention. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and / or materials are described below. In the event of a conflict, the patent specification (including definitions) shall prevail. In addition, the materials, methods and examples of the present invention are illustrative only and are not intended to be necessarily limited.

[0040] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram showing the saltation of APTES onto the TiO2 surface.

[0042] Figures 2A-2D are micrographs representing the following confocal microscopy images: Images of droplets in a 50:50 oil / water ratio Pickering emulsion containing 2% TiO2-NH2, (2A) droplets containing TiO2-NH2, no fluorescence; (2B) droplets containing TiO2-NH2, carboxyfluorescein staining. Cryo-SEM micrographs of a 40:60 oil / water ratio Pickering emulsion containing 1% TiO2-NH2 NPs, (2C) droplets containing 1% TiO2-NH2 NPs at a 40:60 oil / water ratio; (2D) droplets containing 1% TiO2-NH2 NPs at a 40:60 oil / water ratio.

[0043] Figures 3A-3F Images representing the oil / water ratios of TiO2-NH2 Pickering emulsions with varying NPs content in wt%: (3A) 40:60, 0.4wt%; (3B) 50:50, 0.4wt%; (3C) 40:60, 1wt%; (3D) 50:50, 1wt%; (3E) 40:60, 2wt%; (3F) 50:50, 2wt%.

[0044] Figure 4 The instability index of Pickering emulsions is plotted as a function of TiO2-NH2 content (wt%) and the percentage of oil in the emulsion (vol%). * The instability index of 40:60 and 50:50 (2%) is 0.001.

[0045] Figures 5A-5C are micrographs, representing confocal microscopic images of nematodes incorporated into the liquid phase of TiO2-NH2 Pickering emulsion oil-water volume fractions, (5A) 40:60, 0.4%; (5B) 40:60, 1%; (5C) 40:60, 2%, scale bar is 100 μm.

[0046] Figures 6A-6Dare micrographs representing confocal microscopy images of dried nematodes. Nematodes in Pickering emulsion using 1% TiO2-NH2-carboxyfluorescein in an oil-water ratio of 40:60, with (6B) and without (6A) green laser excitation at 480 nm. The blue arrow marks the dried oil droplet. (6C) Three-dimensional image of nematodes in Pickering emulsion using 1% TiO2-NH2-carboxyfluorescein in an oil-water ratio of 40:60 by volume, with (6D) and without (c) green laser excitation at 480 nm. The red arrow indicates TiO2-NH2.

[0047] Figures 7A-7D are micrographs representing SEM images of nematodes in a Pickering emulsion containing 1% TiO2-NH2 NPs at an oil / water volume fraction of 40:60. SEM micrographs of nematodes with (7B, 7D) and without (7A, 7C) Pickering emulsion. The arrows point to TiO2-APTES nanoparticles.

[0048] Figure 8 is a graph showing the survival of Steinernema carinii in 2% TiO2-NH2 emulsions with water (blue), oil-water volume fractions of 40:60 (red) and 50:50 (green) over 150 days.

[0049] Fig. 9 is a graph showing the yield of Steinernema carrionidae IJ from infected Galleria mellonella carcasses collected 15 days from emergence. Error bars represent standard deviation. One-way ANOVA was performed on the yield of infective larvae (IJ). No significance (P>0.05) was observed in the compared parameters.

[0050] Fig.10 It is a bar graph showing the mortality of infective larvae of Steinernema carinii in different formulations (titanium dioxide (TiO2), 1% and 2% Barricade (barri), and water for aqueous IJ) after ultraviolet (UV, 254nm) irradiation for 10 or 20 minutes or without UV treatment (mean ± sem). The same uppercase and lowercase letters indicate that there is no significant difference between the UV irradiation time of each formulation and between the formulations at each UV irradiation time, respectively (Tukey-Kramer test, α = 0.05).

[0051] Figures 11A-11BIt is a bar graph showing the mortality (11A) and infection rate (11B) (mean ± sem) in the carcasses of greater wax moth caused by infective larvae of Steinernema carinii in different formulations (titanium dioxide-TiO2, 1% and 2% Barricade (barri), and water for aqueous IJs) after ultraviolet (UV, 254 nm) irradiation for 10 or 20 minutes, or after UV treatment in addition to the control group (no nematodes). The same uppercase and lowercase letters indicate that there is no significant difference between the UV irradiation time of each formulation and between the formulations at each UV irradiation time, respectively (Tukey-Kramer test, α = 0.05).

[0052] Fig.12 is a bar graph showing the number of nematodes recovered from G. mellonella carcasses exposed to infective larvae of Steinernema carinii in titanium dioxide (TiO2)-based formulations or water controls after 10 or 20 minutes of ultraviolet light (UV, 254 nm) treatment or without UV treatment (mean ± sem). NS = no significant difference between water and TiO2 without UV treatment; same letters indicate no significant difference between UV exposure times.

[0053] Figures 13A-13B is a bar graph showing the comparison of (13A) survival (%) of Steinernema feltiae and Steinernema feltiae and (13B) their number of infective larvae per leaf surface area over time. The time points shown here are 0 h (immediately after application), 1, 2, 3 and 4 h. Survival (%) was subjected to ANOVA after arcsine square root transformation. When ANOVA F was significant (p<0.05), the mean values ​​were compared using Student's t test. Different letters above the bar graphs represent significant differences.

[0054] Figures 14A-14C 14A is a bar graph showing the comparison of (14A) survival (%), (14B) number of surviving infective larvae per leaf surface area, and (14C) insect mortality (%) on cotton leaves treated with water and formulations (TPE and SPEG) of Steinernema carinii over time. The time points shown are 0 hours (immediately after application), 24, 48, 72, and 96 hours after application. Insect mortality represents the mean uncorrected mortality of fourth instar cotton leafworms 48 hours after incubation at 23°C. Bars represent standard errors. Survival (%) was analyzed by ANOVA after arcsine square root transformation. When the ANOVA F was significant (p<0.05), the means were compared using the Student's t-test. Different letters represent significant differences. Insect mortality (%) was compared by Student's t-test for multiple comparisons (NS / * indicates overall significance P=0.05 for a specific time point). At the same time, means without common letters are different (Bonferroni test, P≤0.05) DETAILED DESCRIPTION

[0055] According to some embodiments, the present invention provides a composition comprising insect pathogens coated by multiple UV shielding nanoparticles. In some embodiments, the UV shielding nanoparticles are synthetic particles. In some embodiments, the nanoparticles are synthetic particles. In some embodiments, the composition is a liquid composition. In some embodiments, the composition is flowable. In some embodiments, the composition is an aqueous dispersion. In some embodiments, the composition is a liquid at a temperature between 0 and 90°C. In some embodiments, the composition is in the form of an oil-in-water emulsion (e.g., an oil-in-water Pickering emulsion). The nanoparticles according to the present invention are assembled in core-shell particles, which include a shell composed of hydrophobic nanoparticles, wherein the shell surrounds a liquid core including oil. In some embodiments, the composition is substantially non-toxic (e.g., non-phytotoxic). In some embodiments, insect pathogens can survive in the composition of the present invention. In some embodiments, the core-shell particles and / or the insect pathogens coated therewith are dispersed with an aqueous solvent. In some embodiments, the composition is an aqueous dispersion comprising multiple coated insect pathogens dispersed therein.

[0056] In some embodiments, the composition is a dry solid composition characterized by a water content of less than 5%, less than 1% or less. In some embodiments, the dry composition includes an insect pathogen coated with or contacted with a plurality of core-shell particles as described herein.

[0057] According to some embodiments, the present invention provides an insect pathogenic organism coated by a plurality of UV shielding nanoparticles. In some embodiments, the insect pathogenic organism is incorporated into a liquid composition. In some embodiments, the liquid composition is a dispersion. In some embodiments, the insect pathogenic organism is coated by a composition in the form of a water-in-oil emulsion (e.g., a water-in-oil Pickering emulsion).

[0058] Alternatively, the composition comprises core-shell particles and oil, wherein the core-shell particles comprise a shell surrounding an aqueous core. The nanoparticle according to the present invention is a hydrophobic nanoparticle, and is assembled in the shell of the core-shell particles. In some embodiments, the core-shell particles and / or the insect pathogenic organisms coated therein are dispersed with an oil solution. In some embodiments, the composition is an oil dispersion, which comprises a plurality of coated insect pathogenic organisms dispersed therein.

[0059] In some embodiments, the water-in-oil emulsion substantially maintains its water content for about 24 hours after application to the plant.

[0060] According to one aspect of the present invention, a composition (e.g., a dispersion or emulsion) is provided, comprising an insect pathogen coated by or in contact with a plurality of nanoparticles, wherein the plurality of nanoparticles comprises hydrophobic nanoparticles; wherein the plurality of nanoparticles are assembled in a core-shell particle, the core-shell particle comprises a liquid aqueous core, and the plurality of nanoparticles are arranged in a shell that stabilizes the liquid core. Optionally, the insect pathogen is dispersed in an oil-based main phase. In some embodiments, the composition is a liquid or fluid (e.g., a free-flowing liquid composition). In some embodiments, the composition comprises a gelling agent. In some embodiments, the composition is formulated for application by, for example, spraying, atomizing, brushing, etc. In some embodiments, the composition and / or all of its components are / are non-phytotoxic.

[0061] In some embodiments, the shell is a single layer shell. In some embodiments, the particle is in an interface between an aqueous phase and an oil phase. In some embodiments, the insect pathogenic organism is configured to infect and / or kill insects. In some embodiments, the insect pathogenic organism is viable within the composition, so that when it is exposed to plants and / or habitats, the insect pathogenic organism is characterized by anti-pathogenic activity.

[0062] According to one aspect of the present invention, there is provided an aqueous dispersion comprising an insect pathogenic organism coated with or in contact with a plurality of nanoparticles, wherein the plurality of nanoparticles comprises UV shielding nanoparticles; wherein the plurality of nanoparticles are assembled in a core-shell particle, the core-shell particle comprising a liquid oil as a core and a plurality of nanoparticles arranged in a shell of a stable liquid core. In some embodiments, the composition is a liquid or fluid (e.g., a free-flowing liquid composition). In some embodiments, the composition is substantially free of gel (e.g., hydrogel) or any non-Newtonian fluid. In some embodiments, the composition is formulated for application by, for example, spraying, atomization, brushing, etc. In some embodiments, the composition and / or all of its components are / are non-phytotoxic.

[0063] In some embodiments, the shell is a single layer shell. In some embodiments, the particle is in an interface between a core comprising oil (e.g., a secondary phase) and an aqueous solvent (e.g., a primary phase). In some embodiments, the insect pathogenic organism is configured to infect and / or kill insects. In some embodiments, the insect pathogenic organism is viable.

[0064] The present invention, in some embodiments thereof, is based on the surprising discovery that Pickering emulsions (i.e., oil-in-water and oil-in-water emulsions) comprising from about 1% w / w to about 5% w / w of surface-modified metal oxide nanoparticles (e.g., titanium dioxide or silica-based particles) excel in encapsulating insect pathogenic nematodes, thereby maintaining the viability of the nematodes within the composition of the invention and under ambient conditions (i.e., when the composition is applied to plants and / or areas of cultivation). In addition, the compositions of the invention comprising encapsulated insect pathogenic nematodes (EPNs) are characterized by having enhanced or prolonged viability and / or insecticidal activity under open air conditions compared to unencapsulated insect pathogenic nematodes (e.g., compositions consisting solely of EPNs and an aqueous solvent).

[0065] Oil-in-water compositions

[0066] In one aspect of the invention, a composition (e.g., a liquid composition) comprising a plurality of particles is provided, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% of the particles in the composition are coated or covered with at least one insect pathogen. In some embodiments, the particles are micron-sized particles in the form of droplets stabilized by a shell. In some embodiments, the particles are in the form of core-shell particles (e.g., each particle includes a shell and a core). In some embodiments, the composition is in the form of an emulsion or dispersion. In some embodiments, the particles are in the form of a colloid. In some embodiments, the liquid composition is an O / W Pickering emulsion. In some embodiments, the liquid composition is a flowable composition (or fluid) at a temperature between 0 and 90°C. In some embodiments, the liquid composition is a liquid at a temperature between 0 and 90°C.

[0067] As used herein, the term "Pickering emulsion" refers to an emulsion that utilizes solid particles as stabilizers to stabilize droplets of a substance that are dispersed as droplets in the dispersed phase throughout the continuous phase.

[0068] As used herein, the term "emulsion" refers to a combination of at least two fluids, wherein one fluid is present in the form of droplets in another fluid. The term "emulsion" includes microemulsions.

[0069] As used herein, the term "fluid" refers to a material that tends to flow and conforms to its container profile, i.e., liquid, gas, viscoelastic fluid, etc. Typically, a fluid is a material that cannot withstand static shear stress, and when shear stress is applied, a continuous permanent deformation occurs in the fluid. The fluid can have any suitable viscosity that allows flow. In some embodiments, the fluid is characterized in that the viscosity is enough to be applied (for example, applied to a plant or plant part and / or applied to a cultivation area), such as by spraying or fumigation. If there are two or more fluids, then those of ordinary skill in the art can independently select each fluid in substantially any fluid (liquid, gas, etc.) by considering the relationship between the fluids. In some cases, a droplet can be included in a carrier fluid (for example, a liquid).

[0070] According to one aspect of the present invention, the composition of the present invention is a fluid (or flowable composition) comprising an aqueous solvent and core-shell particles dispersed in the aqueous solvent, wherein each core-shell particle comprises a liquid core surrounded by a shell comprising nanoparticles; the liquid core comprises oil; the nanoparticles are chemically surface-modified metal oxide nanoparticles; the w / w ratio of the aqueous solvent and the oil in the composition is between 70:30 and 40:60, or between about 70:30 and about 50:50, wherein the w / w concentration of the nanoparticles in the composition is between 0.1 and 10%; and the nanoparticles are in contact with, coated with, and / or encapsulated by insect pathogens. In some embodiments, each insect pathogen in the composition of the present invention is encapsulated or coated by the core-shell particles. In some embodiments, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of the entire surface of the insect pathogen is surrounded, encapsulated, and / or in contact with the core-shell particles.

[0071] In some embodiments, the compositions of the present invention include an aqueous solvent (also referred to herein as a "major phase") and a plurality of core-shell particles of the present invention dispersed therein.

[0072] In some embodiments, the aqueous solvent is an agriculturally acceptable carrier.

[0073] In some embodiments, the oil is water insoluble and substantially free of insect pathogen toxicity. In some embodiments, the oil is characterized by a water solubility of less than 1 g / 100 L, less than 0.1 g / 100 L, less than 0.01 g / 100 L, less than 0.001 g / 100 L, including any ranges therebetween.

[0074] In some embodiments, the oil is characterized by a water solubility between 0.0001 and 0.1 g / 100L, between 0.0001 and 0.001 g / 100L, between 0.001 and 0.005 g / 100L, between 0.005 and 0.01 g / 100L, between 0.01 and 0.05 g / 100L, between 0.05 and 0.1 g / 100L, including any ranges therebetween.

[0075] In some embodiments, the oil is a hydrophobic solvent compatible with insect pathogens. In some embodiments, the term "compatible" as used herein refers to a solvent that does not have a substantial effect on the vitality of insect pathogens. In some embodiments, the term "compatible" as used herein refers to a solvent that does not have a substantial effect on the viability of plants. In some embodiments, when the oil reduces the vitality by no more than 20%, no more than 15%, no more than 10%, no more than 5%, no more than 1% of the initial insect pathogenic protozoan population, wherein the reduction in vitality is less than 20min, less than 15min, less than 10min, less than 1min, including any range between the two, it is called compatible. In some embodiments, insect pathogens are compatible with oil when exposed to oil under the treatment conditions disclosed herein. In some embodiments, when the oil is substantially nontoxic to insect pathogens and / or substantially nontoxic to plants, it is called compatible.

[0076] In some embodiments, the oil is characterized by a boiling point of at least 80°C, at least 90°C, at least 100°C, at least 120°C, at least 150°C, at least 170°C, at least 200°C, including any ranges therebetween.

[0077] In some embodiments, the oil is characterized by a viscosity at 25°C of between 1 and 100 cP, between 1 and 5 cP, between 5 and 10 cP, between 10 and 15 cP, between 15 and 20 cP, between 20 and 100 cP, between 10 and 100 cP, between 10 and 50 cP, between 50 and 100 cP, between 15 and 30 cP, between 15 and 50 cP, between 20 and 50 cP, between 10 and 30 cP, between 10 and 40 cP, between 10 and 60 cP, between 10 and 70 cP, between 10 and 80 cP, including any ranges in between.

[0078] In some embodiments, the oil includes or is selected from mineral oil, C10-C30 aliphatic hydrocarbons, fatty acids, monoglycerides, diglycerides, triglycerides, vegetable oils, waxes, essential oils, fragrance oils, or any combination thereof.

[0079] In an embodiment, the oil is selected from the group consisting of mineral oils, essential oils, vegetable oils, organic oils, lipids, and any water-immiscible liquid that is compatible with insect pathogenic organisms.

[0080] As used herein, the term "mineral oil" refers to oil obtained from a mineral source. In some embodiments, mineral oil refers to a liquid byproduct of refining crude oil to make gasoline and other petroleum products. Mineral oil is any of a variety of colorless, odorless, light alkane mixtures ranging from C-10 to C-40 or C-15 to C-40. Mineral oils are available in light and heavy grades. In some embodiments, mineral oil refers to a crude and / or purified fraction obtained from a mineral source. In some embodiments, mineral oil is chemically modified. Mineral oils are known in the art and are used herein in the same manner as is commonly used in the art. Such oils are readily available from commercial chemical suppliers around the world. Methods for preparing mineral oils are also known in the art.

[0081] In some embodiments, the oil is or includes a C10-40, C10-20, C10-30, C10-15, C15-30, C15-40, C15-20, C20-30, C20-40 hydrocarbon chain, including any ranges in between.

[0082] According to the present invention, the non-limiting examples of suitable oils include mineral oils, paraffin oils (based on normal alkanes), naphthenic oils (based on naphthenes), hydrocarbon oils (based on hydrocarbons), fatty acids (including short-chain fatty acids and / or long-chain fatty acids) and / or any esters thereof, vegetable oils (including oils of fatty acids and / or their esters extracted from seeds or other plant parts), waxes, essential oils (based on plant extracts) and aromatic oils (based on aromatic hydrocarbons, and are distinct from essential oils). These oils are all well known in the art.

[0083] In some embodiments, oil is or comprises single oil species. In some embodiments, oil is the fluid that is basically made up of mineral oil, C10-C30 aliphatic hydrocarbons, fatty acid, monoglyceride, diglyceride, triglyceride, vegetable oil, wax, essential oil, aromatic oil or its any combination. In some embodiments, oil is substantially free of additional liquid.

[0084] In some embodiments, the core-shell particles of the present invention include an oil core and an amphoteric shell. In some embodiments, the core-shell particles are in the form of colloids.

[0085] In some embodiments, the core-shell particles have a substantially spherical geometry or shape. In some embodiments, a plurality of core-shell particles do not have any characteristic geometry or shape. In some embodiments, the core-shell particles have a spherical, quasi-spherical, quasi-ellipsoidal, concave, irregular shape or any combination thereof. It will be appreciated by those skilled in the art that the exact shape of each of the plurality of core-shell particles may be different. In addition, the exact shape of the core-shell particles may come from any of the above-mentioned geometric forms, so that the shape of the particles does not fully conform to a specific geometric form. It will be appreciated by those skilled in the art that the exact shape of the core-shell particles may deviate greatly (e.g., a deviation of at least 5%, at least 10%, at least 20%) from a specific geometry (e.g., a sphere or ellipse).

[0086] In some embodiments, the average particle size of the core-shell particles is between 0.5 μm and 100 μm, 1 μm and 100 μm, 1 μm and 50 μm, 10 μm and 50 μm, 1 μm and 10 μm, 10 μm and 50 μm, 10 μm and 20 μm, 20 μm and 50 μm, 20 μm and 30 μm, 30 μm and 50 μm, 50 μm and 80 μm, 1 μm and 20 μm, 1 μm and 30 μm, 5 μm and 30 μm, 10 μm and 30 μm, 10 μm and 50 μm, 1 μm and 40 μm, 1 μm and 5 μm, 1 μm and 8 μm, 5 μm and 10 μm, 5 μm and 50 μm, 5 μm and 20 μm, 5 μm and 30 μm, including any range or value therebetween.

[0087] In some embodiments, the size of the core-shell particles described herein represents the average size (eg, arithmetic mean) of a plurality of particles within a composition of the present invention.

[0088] In some embodiments, the average particle size refers to the particle size of at least 50%, 60%, 70%, 80%, 90%, 95% or 99% of the particles, including any range therebetween. In some embodiments, the range of the average particle size is: 5 μm to 50 μm, 1 μm to 50 μm, 5 μm to 30 μm, including any range therebetween. In some embodiments, the average particle size of the core-shell particles described herein is a wet diameter (i.e., based on commonly used particle size measurement techniques, such as microscopy, DLS or other methods known in the art to measure the particle diameter in the liquid composition). In some embodiments, a plurality of core-shell particles have a uniform size. "Uniform" or "homogenous" refers to a particle size distribution having a variation range of less than, for example, ± 60%, ± 50%, ± 40%, ± 30%, ± 20% or ± 10%, including any value therebetween.

[0089] In some embodiments, the core-shell particles are in the form of droplets.

[0090] As used herein, the term "droplet" refers to an isolated portion of a first fluid surrounded by a second fluid. It should be noted that the droplets are not necessarily spherical and may be other shapes (e.g., depending on the external environment). The droplets of fluid may have any shape and / or size. Typically, monodisperse droplets are substantially the same size. In some embodiments, the average particle size of the droplets refers to the cross-sectional dimension (e.g., diameter) of the droplets. In some embodiments, the cross-sectional dimension refers to the maximum distance between two points on the outer portion of the particle shell. In non-spherical droplets, the average particle size of a single droplet is the diameter of a perfect sphere having the same volume as the non-spherical droplet.

[0091] In some embodiments, the core-shell particles comprise 0.5% to 20%, 0.5% to 1%, 1% to 10%, 1% to 5%, 2% to 3%, 3% to 5%, 5% to 10%, 10% to 20% (w / w) nanoparticles (i.e., hydrophilic nanoparticles) by weight of the core-shell particles, including any ranges therebetween.

[0092] In some embodiments, the weight ratio between the plurality of nanoparticles and the oil within the core-shell particle or within the composition of the present invention is between 1:10 and 1:100, between 1:10 and 1:80, between 1:10 and 1:50, between 1:30 and 1:50, between 1:20 and 1:50, between 1:30 and 1:100, between 1:30 and 1:80, between 1:50 and 1:100, including any ranges therebetween.

[0093] In some embodiments, the shell is in the form of a layer. In some embodiments, the shell is in the form of a uniform layer. In some embodiments, the shell is in the form of a homogeneous layer. In some embodiments, the nanoparticles are uniformly distributed throughout the volume of the shell. In some embodiments, the nanoparticles are uniformly distributed on the liquid core. In some embodiments, the nanoparticles are uniformly distributed on the surface of the liquid core (e.g., in an intermediate phase between the primary phase and the secondary phase).

[0094] In some embodiments, the shell includes an inner portion toward the core (e.g., the core of the particle) and an outer portion toward the aqueous solvent. In some embodiments, the shell is located in the mesophase. In some embodiments, the composition is an o / w emulsion, wherein the secondary oil phase forms the core, and the shell of the core-shell particle of the present invention is located in the mesophase.

[0095] In some embodiments, the shell stabilizes the core. In some embodiments, the shell encapsulates the core.

[0096] In some embodiments, the shell has a thickness of 5 nm to 50 nm, 15 nm to 50 nm, 30 nm to 50 nm, 1 nm to 50 nm, 2 nm to 50 nm, 5 μm to 10 nm, 10 nm to 50 nm, 5 nm to 30 nm, 15 nm to 30 nm, 1 nm to 20 nm, 2 nm to 20 nm, 5 nm to 20 nm, or 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 40 nm to 80 nm, 80 nm to 100 nm, 100 nm to 200 nm, 200 nm to 300 nm, 300 nm to 500 nm, including any range therebetween. In some embodiments, the shell thickness is quantified using a scanning electron microscope (SEM).

[0097] In some embodiments, the nanoparticles include UV shielding nanoparticles. In some embodiments, the nanoparticles include UV shielding metal / metalloid oxide nanoparticles. In some embodiments, the nanoparticles include a metal / metalloid oxide as a core and UV shielding surface groups covalently bonded thereto. In some embodiments, the metal / metalloid oxide includes a divalent metal oxide or a tetravalent metal oxide selected from silicon oxide, titanium dioxide, zirconium oxide, or any combination thereof.

[0098] In some embodiments, the term "UV-shielding nanoparticle" refers to a particle configured to absorb, reflect, or dissipate incoming UV radiation. In some embodiments, the core of the UV-shielding nanoparticle (e.g., a tetravalent metal oxide) is UV-shielding, i.e., capable of absorbing, reflecting, or dissipating (e.g., by scattering) incoming UV radiation. An exemplary UV-shielding tetravalent metal oxide particle is a titanium dioxide nanoparticle.

[0099] In some embodiments, each nanoparticle is a chemically modified metal / metalloid oxide nanoparticle. In some embodiments, the chemically modified metal / metalloid oxide nanoparticle includes a functional portion covalently bonded to the metal / metalloid oxide nanoparticle. In some embodiments, the chemically modified metal oxide nanoparticle is a hydrophilic particle. In some embodiments, the hydrophilic particle includes a hydrophilic functional portion.

[0100] In some embodiments, each nanoparticle is a chemically modified titanium dioxide (e.g., including Ti(IV) and optionally further including a Ti(III) element) particle that includes a functional portion covalently bonded to the nanoparticle. In some embodiments, the functional portion (i.e., the hydrophilic functional portion) includes an aminoalkyl group, an amino group, a hydroxyl group, a carboxyl group, a thio, a hydroxyalkyl group, a thioalkyl group, an aminoalkylsilane group, a hydroxyalkylsilane group, a thioalkylsilane group, or any combination thereof.

[0101] In some embodiments, the functional moiety is represented by the formula:

[0102] wherein the dashed bond represents the point of attachment to the particle (e.g., to an oxygen atom of the particle); n is an integer from 1 to 30; Y is H or includes a heteroatom; X is optionally substituted C1-C10 alkyl, Si(R1)2, or is absent; R is H or a substituent; and each R1 is independently H, a bond, -O-, a substituent, optionally substituted C1-C6 alkyl, -O(C1-C6 alkyl), -OH, or a combination thereof.

[0103] In some embodiments, each substituent is independently selected from -NO2, -CN, -OH, -CONH2, -CONR'2, -CNNR'2, -CSNR'2, -CONH-OH, -CONH-NH2, -NHCOR', -NHCSR', -NHCNR', -NC(=O)R', -NC(=O)OR', -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-SO2R', -SOR', -SR', -SO2OR', -SO2N(R')2, -NHNR'2, -NNR', carbonyl, C1-C 10 haloalkyl, optionally substituted C1-C 10 Alkyl, -NH2, -N(R')2, -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)2, C1-C 10 Halogenated alkoxy, hydroxyl (C1-C 10 Alkyl), hydroxyl (C1-C 10 Alkoxy), alkoxy (C1-C 10 Alkyl), alkoxy (C1-C 10 Alkoxy), amino (C1-C 10 Alkyl), -CONH(C1-C 10 Alkyl), -CON(C1-C 10 alkyl)2, -CO2H, -CO2R', -OCOR', -C(=O)R', -OC(=O)OR', -OC(=O)NR', ​​-OC(=S)OR', -OC(=S)NR', ​​heteroatom, cycloalkyl, heteroaryl, heteroaryl, (C1-C 10 alkyl)alkyl-cycloalkyl, (C1-C 10 alkyl)alkyl-aryl, (C1-C 10alkyl)alkyl-heteroaryl, or any combination thereof, wherein cycloalkyl, heteroaryl, heteroaryl are each substituted or unsubstituted; and each R' is independently H, or includes optionally substituted C1-C 10 Alkyl, C1-C 10 Alkyl aryl, C1-C 10 Alkylcycloalkyl, optionally substituted C3-C 10 Cycloalkyl, optionally substituted C3-C 10 Heterocycloalkyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof. In some embodiments, the heteroatom is or includes S, O, SR', OR', or N(R')2.

[0104] In some embodiments, Y is N(R')2, and X is Si(R1)2.

[0105] In some embodiments, the functional moiety is represented by the formula:

[0106] wherein the dashed bond represents the point of attachment to the particle (e.g., to an oxygen atom of the particle); n is an integer ranging from 1 to 30, 1 to 10, 1 to 20, 3 to 5, 3 to 30, 3 to 20, 3 to 10, and any ranges therebetween; R is as described herein; Y is N(R')2; and X is Si(R1)2, wherein each R1 is independently -O(C1-C6 alkyl), -O-, -OH, H, optionally substituted C1-C6 alkyl, or a combination thereof.

[0107] In some embodiments, the functional moieties and / or nanoparticles of the invention are positively charged (e.g., have an intrinsic positive charge or undergo protonation in an aqueous solvent, such as at a pH between 0 and 9). In some embodiments, the nanoparticles of the invention are characterized by a positive zeta potential.

[0108] In some embodiments, the functional moiety comprises wherein each R1 is as described herein, and wherein at least one R1 is a point of attachment to the particle (eg, to an oxygen atom of the particle).

[0109] In some embodiments, the functional moiety is substantially free of halogen (such as fluorine and / or chlorine moieties). In some embodiments, the functional moiety is substantially free of haloalkyl moieties. In some embodiments, the functional moiety is substantially free of haloalkylsilane moieties. In some embodiments, the functional moiety is free of fluorine atoms.

[0110] In some embodiments, nanoparticles characterized by contact angles less than 90°, 85°, 80°, 75°, including any ranges or values ​​therebetween, are referred to as hydrophilic nanoparticles.

[0111] In some embodiments, the nanoparticles include metal oxide particles (e.g., UV shielding or non-UV shielding particles) and functional moieties associated therewith, wherein the functional moieties include UV shielding groups. As used herein, the term "UV shielding" refers to compounds or chemical groups that absorb, reflect and / or dissipate ultraviolet light. UV absorbing compounds or chemical groups include functional moieties (chromophores) containing low excitation energy valence electrons. In some embodiments, the UV absorbing groups include multiple aromatic (and / or heteroaromatic) rings (fused rings, bicyclic or polycyclic).

[0112] In some embodiments, the UV shielding group or UV shielding nanoparticle is characterized by at least one of: (i) UV absorbance, (ii) UV reflectivity, and (iii) UV scattering, which is between 60% and 100%, between 65% and 100%, between 70% and 100%, between 80% and 100%, between 90% and 100%, between 95% and 100%, between 60% and 99%, between 65% and 99%, between 70% and 99%, between 80% and 9 ...90% and 100%, between 95% and 100%, between 90% and 100%, between 95% and 100%, between 90% and 100%, between 95% and 100%, between 90% and 100%, between 95% and 100%, between 95% and 100%, between 95% and 100%, between 95% and 100%, between 95% and 100%, between 95% and 100%, between 95% 99%, between 95% and 99%, between 60% and 98%, between 65% and 98%, between 70% and 98%, between 80% and 98%, between 90% and 98%, between 95% and 98%, between 60% and 95%, between 65% and 95%, between 70% and 95%, between 80% and 95%, between 90% and 95%, between 60% and 80%, between 65% and 80%, or between 70% and 80%, including any ranges therebetween, relative to the intensity of incoming UV light. In some embodiments, UV absorbance is measured according to ASTM D1003. As used herein, the term "UV absorbance" refers to the percentage of incoming UV light intensity absorbed by a UV shielding group and / or UV shielding nanoparticle.

[0113] The term "silica" as used herein refers to a structure containing at least the following elements: silicon and oxygen. Silica may have a basic formula of SiO2, or may have a basic formula including Si x O y Another structure of (wherein x and y may each independently be about 1 to 10).

[0114] In some embodiments, the metal oxide comprises nanoclay, SiO2, TiO2, Al2O3, Fe2O3, ZnO and ZrO or any combination thereof. In some embodiments, the nanoparticles are substantially non-porous particles. In some embodiments, the nanoparticles are substantially porous particles (e.g., mesoporous particles).

[0115] In some embodiments, the nanoparticles are surface-modified metal oxide nanoparticles that include (i) TiO 2 -based particles and / or (ii) SiO 2 particles modified with functional moieties, as described above.

[0116] In some embodiments, the oil constitutes between 50% and 90%, between 50% and 70%, between 70% and 90%, between 70% and 99%, between 80% and 99%, between 90% and 99%, including any range therebetween, by weight of the core of the core-shell particles (e.g., droplets) of the present invention. In some embodiments, the terms "core shell particle" and "particle" are used interchangeably herein.

[0117] In some embodiments, the nanoparticles are characterized in that the average (e.g., arithmetic mean) particle size is 1nm to 900nm. In some embodiments, the average particle size of the nanoparticles is between 2nm to 600nm, 2nm to 550nm, 2nm to 520nm, 2nm to 500nm, 2nm to 480nm, 2nm to 450nm, 2nm to 400nm, 2nm to 350nm, 2nm to 300nm, 2nm to 250nm, 2nm to 200nm, 2nm to 150nm, 2nm to 100nm, 5nm to 600nm, 10nm to 600nm, 15nm to 600nm, 20nm to 600nm, 40nm to 600nm, 50nm to 600nm. In some embodiments, the size of at least 90% of the nanoparticles varies within a range of less than ± 25%, ± 20%, ± 15%, ± 19%, ± 5%, including any value therebetween.

[0118] As used herein, the terms "nanoparticle", "nano", "nanosized" and any grammatical derivatives thereof (used interchangeably herein) describe particles having at least one dimension (e.g., diameter, length) ranging in size from about 1 nm to 100 nm. As used herein, "NP(s)" refers to nanoparticles.

[0119] In some embodiments, the term "average" particle size refers to the physical diameter of the nanoparticles (also referred to as the "dry diameter"). In some embodiments, according to some embodiments of the present invention, the dry diameter of the particles can be assessed using transmission electron microscopy (TEM) or scanning electron microscopy (SEM) imaging.

[0120] The shape of the nanoparticles can be roughly spherical, incompletely spherical (especially the size attached to the substrate), rod-shaped, columnar, ribbon-shaped, sponge-shaped and any other shape, or a cluster form of any of these shapes, or a mixed shape of one or more shapes. In some embodiments, the hydrophobic particles have a spherical, quasi-spherical, quasi-ellipsoidal, irregular shape or any combination thereof.

[0121] In some embodiments, nanoparticles are in the interface between the main phase and the secondary phase. In some embodiments, the main phase is a continuous phase. In some embodiments, the secondary phase is a dispersed phase. In some embodiments, hydrophobic particles are in the interface of the core (e.g., aqueous core) of the main phase (e.g., a hydrophobic solvent as described herein) and the core-shell particles (e.g., a colloid) as described herein.

[0122] In some embodiments, the w / w concentration of the nanoparticles in the composition is between 0.5 and 10%, between 0.5 and 5%, between 0.8 and 10%, between 0.8 and 5%, between 0.9 and 10%, between 0.9 and 5%, between about 1 and 10%, between about 1 and 5%, between about 1 and 3%, between about 3 and 10%, between about 2 and 5%, between 5 and 10%, including any ranges therebetween. In some embodiments, the w / w concentration of the nanoparticles affects the viscosity of the composition. In some embodiments, the w / w concentration of the nanoparticles predetermines the viscosity of the composition and further predetermines the particle size of the core-shell particles of the present invention.

[0123] In some embodiments, the w / w ratio between the aqueous solvent and the oil in the composition is between 70:30 and 40:60, between 70:30 and 45:65, between 70:30 and about 50:50, including any ranges therebetween.

[0124] In some embodiments, the composition of the invention (o / w emulsion) comprises a w / w concentration of nanoparticles between 0.5 and 10%, or between 1 and 3%, and the w / w ratio between the aqueous solvent and the oil described herein is stable (e.g., no phase separation, etc.) over the time periods described herein.

[0125] In some embodiments, the composition is a stable liquid composition. In some embodiments, the liquid composition is stable for at least 6 hours (h), at least 12h, at least 24h, at least 48h, at least 72h, at least 96h, at least 10 days (d), at least one month (m), at least 6m, at least 12m%, including any range therebetween.

[0126] As used herein, the term "stable" refers to the ability of a liquid composition to substantially maintain its integrity, such as substantially no aggregation, precipitation, and / or phase separation. In some embodiments, a stable composition (e.g., a composition or liquid composition of the present invention) is substantially free of aggregates. In some embodiments, the aggregates include multiple core-shell particles that adhere or bind to each other. In some embodiments, a stable composition is substantially free of free (e.g., non-encapsulated) insect pathogens. In some embodiments, when at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, including any range of initial insect pathogenic bioloads in between, is still encapsulated in the core-shell particles under suitable storage conditions and within the time period described herein, the composition of the present invention is referred to as stable. In some embodiments, when at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, including any range of initial insect pathogenic bioloads in between, is still alive when stored under suitable storage conditions, the composition of the present invention is referred to as stable.

[0127] In some embodiments, the o / w emulsion further comprises a gelling agent. In some embodiments, the gelling agent is as described below (for w / o emulsion). In some embodiments, the w / w concentration of the gelling agent in the o / w emulsion is between 0.5 and 5%, between 0.5 and 10%, between 0.2 and 10%, between 0.8 and 5%, between 0.9 and 5%, between about 1 and 5%, between about 1 and 3%, between about 3 and 5%, between about 2 and 5%, between 0.5 and 4%, including any ranges therebetween.

[0128] In some embodiments, the insect pathogenic organism is viable within the composition of the present invention. In some embodiments, the insect pathogenic organism is viable within the composition of the present invention and / or is viable when the composition of the present invention is applied to a plant / plant part; and / or applied to a cultivation area. As used herein, the term "viable" includes being able to: replicate genome or DNA, infect or invade a host (e.g., an insect), reproduce, cell proliferation, RNA synthesis, protein translation, energy production process, bacterial secretion, or any combination thereof.

[0129] In some embodiments, the composition includes a single genus or different genera of insect pathogenic organisms. In some embodiments, the insect pathogenic organism is or includes a biopesticide. In some embodiments, the insect pathogenic organism is characterized by having insecticidal (e.g., insecticide) activity. In some embodiments, the insect pathogenic organism can reduce or control the growth (e.g., activity, reproduction, etc.) of pests. In some embodiments, the insect pathogenic organism can reduce or control insect pests in plants and / or cultivated areas (e.g., infested by pests). In some embodiments, the insect pathogenic organism can prevent the infestation of plants and / or soil by pests. In some embodiments, the insect pathogenic organism is characterized by being toxic to pests. In some embodiments, the insect pathogenic organism is characterized by being able to initiate systemic acquired resistance (SAR) in plants.

[0130] The term "SAR" as used herein is well known to those skilled in the art and refers primarily to the systemic response of plants or plant parts to pathogen infection. SAR can be assessed by well-known procedures, such as described by Ruisheng An et al. in Biological Control, 93 (2016) 24-29.

[0131] In some embodiments, the entomopathogen is capable of reducing the activity or load of pests, wherein the activity and / or load refers to plants (or plant parts) and / or cultivated areas (e.g., soil). In some embodiments, the plant part is selected from stems, leaves, inflorescences, roots, fruits, seeds, or any combination thereof.

[0132] In some embodiments, the insect pathogenic organism comprises a nematode. In some embodiments, the nematode is a nematode characterized by insecticidal activity. In some embodiments, the nematode is an insect pathogenic nematode (EPN). In some embodiments, the nematode (e.g., EPN) is an infective larva (IJ) nematode.

[0133] In some embodiments, the nematode (e.g., EPN) is selected from the genus Heterorhabditis and Steinernema, including any combination thereof. In some embodiments, the EPN is selected from the group consisting of Steinernema carpocapsae (Sc), Heterorhabditis bacteriophora (Hb), Heterorhabditis indica (Hi), Steinernemafeltiae (Sf), Steinernema kraussei (Sk), Steinernemaglaseri (Sg), Phasmarhabditis hermaphrodita (Ph), Phasmarhabditis spp. neopapillosa (Pn)), Phasmarhabditis californica (Pc)), S.riobrave, S.scapterisci, and S.longicaudum, including any combination thereof. In some embodiments, the EPN includes the species of Steinernema scapteris (Sc). In some embodiments, the EPN includes the species of Heterorhabditis bacteriophilus (Hb). In some embodiments, the EPN includes the species of Heterorhabditis indica (Hi). In some embodiments, the EPN includes the species of Steinernema noctuidae (Sf). In some embodiments, the EPN includes the species of Steinernema cruzi (Sk). In some embodiments, the EPN includes the species of Steinernema grisea (Sg). In some embodiments, the EPN includes the species of hermaphroditic mucophagous (Ph). In some embodiments, the EPN includes the species of neopapillosa (Pn). In some embodiments, the EPN comprises a species of Myxophagus californica (Pc). In some embodiments, the EPN comprises a species of Heterorhabditis zealandica (Hz). In some embodiments, the EPN comprises a species of Heterorhabditis megidis (Hm).

[0134] In some embodiments, the EPN is in the third stage, the infective larvae (IJ) stage. In some embodiments, the EPN is in the third stage stage. In certain embodiments, the EPN is in the infective larvae (IJ) stage.

[0135] In some embodiments, 50-100%, 50-99%, 70-99%, 90-99%, 90-100% of the nematodes in the composition are in the IJ stage. In some embodiments, the insect pathogenic organisms in the compositions of the present invention consist essentially of IJ nematodes. In some embodiments, the insect pathogenic organisms or compositions of the present invention are active against plant pathogenic arthropods. In some embodiments, the compositions of the present invention are characterized by specific insecticidal activity, wherein the specific insecticidal activity comprises the insect pathogenic organism and / or the composition comprising the same is specifically active against plant pathogenic insects.

[0136] In some embodiments, the pest is selected from agricultural pathogens, plant pathogens, veterinary pests, household pests, soil pathogens, or both. In some embodiments, the pathogen is selected from aphids, insects, or any combination thereof. In some embodiments, the pathogen (e.g., agricultural pathogen, plant pathogen, and / or soil pathogen) is selected from, but not limited to, Lepidoptera, Coleoptera, Hemiptera, Diptera, Orthoptera, Acari, and Gastropoda.

[0137] In some embodiments, the pathogen belongs to the order Lepidoptera. In some embodiments, the pest belongs to the suborder Anapsids. In some embodiments, the pathogen belongs to the suborder Proboscis. In some embodiments, the pathogen belongs to the suborder Heteroptera. In some embodiments, the pathogen belongs to the suborder Ophioptera.

[0138] In some embodiments, the pathogen belongs to the order Coleoptera. In some embodiments, the pathogen belongs to the suborder Carnivora. In some embodiments, the pathogen belongs to the suborder Protocoleoptera. In some embodiments, the pathogen belongs to the suborder Mycovora. In some embodiments, the pathogen belongs to the suborder Polyphaga. In some embodiments, the pest belongs to the suborder Protocoleoptera.

[0139] In some embodiments, the pathogen is of the order Hemiptera. In some embodiments, the pathogen is of the suborder Cephalocereus. In some embodiments, the pathogen is of the suborder Hemiptera. In some embodiments, the pathogen is of the suborder Hemiptera. In some embodiments, the pathogen is of the suborder Heteroptera. In some embodiments, the pathogen is of the suborder Sternorhynchus.

[0140] In certain embodiments, the term "pathogen" and the term "pest" are used interchangeably herein. In certain embodiments, the pest belongs to the order Diptera. In certain embodiments, the pest belongs to the order Orthoptera. In certain embodiments, the pest belongs to the suborder Xiphophora. In certain embodiments, the pest belongs to the suborder Conura. In certain embodiments, the pest belongs to the suborder Acari. In certain embodiments, the pest belongs to the suborder True Acari. In certain embodiments, the pest belongs to the suborder Parasitoides.

[0141] In some embodiments, the pest belongs to the class Gastropoda. In some embodiments, the pest belongs to the family of Halospermidae. In some embodiments, the pest belongs to the family of Microslugidae. In some embodiments, the pest belongs to the family of Ophiopodidae. In some embodiments, the pest belongs to the family of Slugidae. In some embodiments, the pest belongs to the family of Ophiopodidae. In some embodiments, the pest belongs to the family of Thripidae. In some embodiments, the pest is a plant parasitic nematode. In some embodiments, the pest belongs to the order Ixodidae.

[0142] Water in Oil

[0143] In another aspect of the invention, the liquid composition is in the form of a W / O emulsion (eg, a W / O Pickering emulsion).

[0144] According to one aspect of the present invention, a composition (e.g., a liquid composition or a flowable composition) is provided, which includes oil, a gelling agent, and a plurality of nanoparticles, wherein the composition is in the form of core-shell particles dispersed in the oil, wherein each core-shell particle includes a liquid core surrounded by a shell including nanoparticles; the liquid core includes an aqueous solvent; the nanoparticles are surface-modified hydrophobic metal oxide nanoparticles; the w / w ratio of the oil solution and water in the composition is between 70:30 and 40:60, or between about 70:30 and about 50:50; wherein the w / w concentration of the nanoparticles in the composition is between 0.1 and 10%; and the nanoparticles are in contact with, coated with, and / or encapsulated by insect pathogens. In some embodiments, each insect pathogen in the composition of the present invention is encapsulated or coated by the core-shell particles. In some embodiments, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of the entire surface of the insect pathogen is surrounded, encapsulated, and / or in contact with the core-shell particles.

[0145] In some embodiments, the core-shell particles include an aqueous core and an amphiphilic shell. In some embodiments, as disclosed above, the core-shell particles are in the form of colloids.

[0146] In some embodiments, the shape of the core-shell particles (ie, aqueous core particles) is as disclosed above. In some embodiments, the average particle size of the core-shell particles (ie, aqueous core particles) is as disclosed above, such as between 0.5μ and 100μm, including any range or value therebetween.

[0147] In some embodiments, the core-shell particles are in the form of droplets. In some embodiments, the physical properties (shape, size) of the core-shell particles and the chemical composition (e.g., ratio and composition of the primary phase / secondary phase) of the w / o emulsion are the same as disclosed herein for the o / w emulsion.

[0148] In some embodiments, the weight ratio between the plurality of nanoparticles within the core-shell particles or w / o emulsions of the present invention and the aqueous solvent is between 1:10 and 1:100, between 1:10 and 1:80, between 1:10 and 1:50, between 1:30 and 1:50, between 1:20 and 1:50, between 1:30 and 1:100, between 1:30 and 1:80, between 1:50 and 1:100, including any ranges therebetween.

[0149] In some embodiments, each hydrophobic nanoparticle is a surface-modified metal oxide particle.In some embodiments, the metal oxide comprises a divalent metal / metalloid oxide or a tetravalent metal / metalloid oxide selected from silicon oxide, titanium dioxide, zirconium oxide, or any combination thereof.

[0150] In some embodiments, each nanoparticle is a chemically modified metal oxide particle comprising a hydrophobic functional moiety covalently bonded to the metal / metalloid oxide particle. In some embodiments, the hydrophobic functional moiety comprises a (C1-C20) alkyl group, a (C1-C20) alkylsilyl group, a (C1-C4) alkylsilyl group, a phenyl group, a thiol group, a vinyl group, a fluoroalkyl group, a haloalkyl group, a halogen, an epoxy group, a cycloalkane, an olefin, a haloolefin, an alkyne, an ether, a silyl group, a siloxane group, and a thioether, or any combination thereof.

[0151] In some embodiments, the hydrophobic nanoparticles are characterized by a contact angle greater than 120°, 125°, 130°, 135°, including any range or value therebetween.

[0152] In some embodiments, the hydrophobic functional moiety includes a UV shielding group, wherein the UV shielding group is as described above.

[0153] In some embodiments, the hydrophobic nanoparticles are characterized by an average (e.g., arithmetic mean) particle size of 1 nm to 900 nm. In some embodiments, the average particle size of the hydrophobic nanoparticles is between 2 nm and 600 nm, 2 nm to 550 nm, 2 nm to 520 nm, 2 nm to 500 nm, 2 nm to 480 nm, 2 nm to 450 nm, 2 nm to 400 nm, 2 nm to 350 nm, 2 nm to 300 nm, 2 nm to 250 nm, 2 nm to 200 nm, 2 nm to 150 nm, 2 nm to 100 nm, 5 nm to 600 nm, 10 nm to 600 nm, 15 nm to 600 nm, 20 nm to 600 nm, 40 nm to 600 nm, 50 nm to 600 nm. In some embodiments, the average particle size is determined by SEM.

[0154] In some embodiments, the w / w concentration of the hydrophobic nanoparticles in the w / o emulsion is between 0.5 and 10%, between 0.5 and 5%, between 0.8 and 10%, between 0.8 and 5%, between 0.9 and 10%, between 0.9 and 5%, between about 1 and 10%, between about 1 and 5%, between about 1 and 3%, between about 3 and 10%, between about 2 and 5%, between 5 and 10%, including any ranges therebetween. In some embodiments, the w / w concentration of the nanoparticles affects the viscosity of the composition.

[0155] In some embodiments, the w / w ratio between oil and aqueous solvent in the w / o emulsion is between 70:30 and 40:60, between 70:30 and 45:65, between 70:30 and about 50:50, including any ranges therebetween.

[0156] In some embodiments, the gelling agent comprises a natural or synthetic polymer (optionally cross-linked by a covalent cross-linking agent or a non-covalent cross-linking agent) configured to form a gel in the presence of water. Non-limiting examples of gelling agents include, but are not limited to, polyacrylic acid, polyvinyl alcohol, polyethylene glycol, polysaccharides (e.g., glue, alginate, chitosan, pectin, hyaluronic acid, etc.), poly (2-hydroxyethyl methacrylate), polyacrylamide, including any salt thereof or any combination thereof.

[0157] In some embodiments, the w / w concentration of the gelling agent in the composition is between 0.5 and 5%, between 0.5 and 10%, between 0.2 and 10%, between 0.8 and 5%, between 0.9 and 5%, between about 1 and 5%, between about 1 and 3%, between about 3 and 5%, between about 2 and 5%, between 0.5 and 4%, including any ranges therebetween.

[0158] In some embodiments, the w / w concentration of the gelling agent affects the viscosity of the composition. In some embodiments, the w / w concentration of the gelling agent predetermines the viscosity of the composition and further predetermines the water evaporation rate within the core-shell particles of the present invention. In some embodiments, the presence of the gelling agent substantially maintains the water content encapsulated within the core-shell particles (e.g., when the o / w composition is applied to a plant / habitat).

[0159] In some embodiments, the w / o emulsion of the present invention comprising 0.5 to 10% w / w, or 1 to 3% w / w of hydrophobic nanoparticles; 0.2 to 10% w / w of gelling agent, and a w / w ratio between oil and aqueous solvent as described herein is stable (e.g., no phase separation, etc.) over the time periods described herein. In some embodiments, the composition stability is as described above.

[0160] In some embodiments, the insect pathogenic organism is viable in the w / o emulsion of the present invention. In some embodiments, the insect pathogenic organism is viable in the w / o emulsion of the present invention and / or when the w / o emulsion of the present invention is applied to the plant / plant part; and / or applied to the cultivation area, the insect pathogenic organism is viable. The term "viable" is as described above.

[0161] In some embodiments, the insect pathogen is as described above.

[0162] Insecticide composition

[0163] In some embodiments, the compositions of the present invention are insecticide compositions.

[0164] In some embodiments, the insecticide composition of the present invention (i.e., o / w, w / o emulsion) is characterized by a large amount of retention time on plants and / or parts thereof. In some embodiments, the retention time is sufficient to maintain at least 30%, at least 50%, at least 70%, at least 90% of the initial insecticidal activity of the composition (referring to the activity on the day of application), including any range therebetween. In some embodiments, the retention time is at least 1 day (d), at least 2d, at least 5d, at least 7d, at least 10d, at least 15d, at least 30d, including any range therebetween. In some embodiments, the composition of the present invention remains stably combined (physically stable) or in contact with plants and / or parts of plants when exposed to environmental conditions in the cultivation area (e.g., exposed to UV light, rain, moisture, temperatures between 0-50°C, etc.). In some embodiments, when applied to plants or in habitats, the composition of the present invention forms a coating on plants and / or parts thereof.

[0165] In some embodiments, the coating layer that comprises the present composition and / or the insecticide composition and contacts with plant and / or its part or is attached on plant and / or its part is stable to climate change. In some embodiments, the coating layer substantially prevents the damage of the environment to the insect pathogenic organism (for example, due to environmental conditions such as temperature change, heat, cold, UV radiation and atmospheric gases). In some embodiments, the coating layer, after being exposed to environmental damage, substantially retains the insecticidal activity of the insect pathogenic organism in the time period described herein. In some embodiments, the coating layer is stable to temperature change, heat, cold, UV radiation and atmospheric gases. In some embodiments, the insecticidal performance and / or stability of the coating layer are not affected or changed by climate change as described herein.

[0166] In some embodiments, the insecticide composition or the composition of the present invention (ie, o / w, w / o emulsion) is characterized by a viscosity of 10 2 Up to 10 5 Between 10 2 Up to 10 4 Between 10 2 Up to 10 3 Between 10 3 Up to 10 4 Between 10 4 Up to 10 5 cP, including any range therebetween.

[0167] In some embodiments, the viscosity of the pesticide composition is sufficient to stabilize it, thereby producing a composition characterized as being stable for at least 1 month (m), at least 2 m, at least 6 m, at least 10 m, at least 12 m, at least 24 m, at least 36 m, including any ranges therebetween.

[0168] In some embodiments, the stability of the composition refers to long-term storage under appropriate storage conditions, which include: ambient atmosphere and temperature below 30°C, below 20°C, below 15°C, below 10°C, below 5°C, below 2°C, below 0°C, including any ranges therebetween, etc. In some embodiments, suitable storage conditions include low storage temperatures sufficient to substantially prevent microbial spore germination. In some embodiments, the term "stable" is as described herein.

[0169] In some embodiments, the insecticide composition is in the form of (i) an oil-in-water emulsion, and / or an oil-in-water Pickering emulsion; (ii) a water-in-oil emulsion, and / or a water-in-oil Pickering emulsion, which includes an insect pathogenic organism. In some embodiments, the composition of the present invention is characterized in that the composition of the present invention has prolonged insecticidal activity and / or prolonged residence time in plants (or parts thereof) and / or in cultivation areas compared to unencapsulated insect pathogenic organisms (e.g., similar insect pathogenic organisms dispersed in an aqueous solvent, without the core-shell particles of the present invention).

[0170] In some embodiments, the term "prolonged" as used herein is extended by at least 1d, at least 5d, at least 10d, at least 15d, at least 20d, at least 30d, including any ranges therebetween. In some embodiments, the compositions of the present invention substantially prevent damage by insect pathogens for a period of between 1d and 60d, between 1d and 10d, between 1d and 5d, between 10d and 20d, between 20d and 30d, between 30d and 40d, between 40d and 60d, including any ranges therebetween (e.g., when exposed to outdoor conditions).

[0171] In some embodiments, the pesticide composition includes an agriculturally acceptable carrier. In some embodiments, the aqueous solvent of the pesticide composition is an agriculturally acceptable solvent.

[0172] In some embodiments, the insecticide compositions of the present invention include an insecticide effective amount of an insect pathogenic organism, wherein the insect pathogenic organism is characterized by an insecticide activity as described herein. In some embodiments, the insecticide effective amount is sufficient to reduce plant damage associated with pests, reduce growth and / or reduce the pest load on plants or in a cultivation area, wherein the reduction includes any statistically significant reduction, such as a reduction of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, including any range therebetween.

[0173] In some embodiments, the insecticidal effective amount comprises an insect pathogen concentration within the insecticide composition of the present invention of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40% v / v, including any range therebetween.

[0174] In some embodiments, the insecticide effective amount includes the concentration of insect pathogens in the insecticide composition of the present invention between 10 and 1.000.000, between 10 and 100.000, between 100 and 100.000, between 100 and 10.000, between 100 and 5000, between 500 and 5000, at least 100, at least 500, at least 1000 units / ml, including any ranges therebetween. The term "units / ml" refers to the number of insect pathogens per milliliter of composition.

[0175] In some embodiments, the insecticide composition of the present invention is a liquid composition formulated for application to an infected plant or part thereof or to an infected cultivation area (such as an infected soil). In some embodiments, the plant and / or soil is infested by pests (such as plant pathogens, soil pathogens, or both). In some embodiments, the insecticide composition of the present invention is formulated (e.g., as described herein, characterized by a suitable viscosity) for application by spraying, atomizing, fumigating, and coating, including any combination thereof.

[0176] In some embodiments, the insecticide composition of the present invention is used as: a UV protective coating, an insect repellent composition, a plant protection composition, or any combination thereof.

[0177] thing

[0178] According to some embodiments, the present invention provides an article comprising a substrate, which is contacted with a composition of the present invention, or with an insect pathogenic organism coated by or in contact with the core-shell particles of the present invention. In some embodiments, the insect pathogenic organism coated by or in contact with the core-shell particles of the present invention is also referred to herein as an "encapsulated insect pathogenic organism".

[0179] In some embodiments, the encapsulated insect pathogenic organism is combined with the substrate. In some embodiments, the encapsulated insect pathogenic organism is mixed with the substrate. In some embodiments, the encapsulated insect pathogenic organism is present as a coating on the substrate. In some embodiments, the coating is a substantially uniform layer. In some embodiments, the coating is uniformly distributed on at least a portion of the substrate.

[0180] In some embodiments, the coating layer is characterized by an average thickness between 1 and 100 um, between 1 and 10 um, between 10 and 20 um, between 2 and 50 um, between 50 and 100 um, including any ranges therebetween.

[0181] According to some embodiments, the present invention provides an article comprising a liquid composition of the present invention. In some embodiments, the article comprises a liquid composition (e.g., an oil-in-water emulsion) within a package. In some embodiments, the package is or comprises a container suitable for holding a liquid volume.

[0182] In some embodiments, the substrate is selected from plants or plant parts (e.g., leaves, stems, fruits, etc.), soil, polymer substrates, glass substrates, metal substrates, paper substrates, carton substrates, polystyrene substrates, tissue-like substrates, brick walls, sponges, textiles, nonwovens, or wood. In some embodiments, the substrate is or includes edible substances.

[0183] In some embodiments, the article or coating is stable. In some embodiments, the coating is characterized by an average thickness of 100 nm to 500 μm, 100 nm to 400 μm, 100 nm to 300 nm, 300 nm to 500 nm, 500 nm to 1000 nm, 250 nm to 400 μm, 500 nm to 400 μm, 900 nm to 400 μm, 1 μm to 400 μm, 10 μm to 400 μm, 50 μm to 400 μm, 100 μm to 400 μm, 250 μm to 400 μm, 10 nm to 100 μm, 25 nm to 400 μm, 10 nm to 100 μm, 25 nm to 400 μm, 10 nm to 100 μm, 25 nm to 400 μm, 10 nm to 200 μm, 2 ... 100μm, 50nm to 100μm, 100nm to 100μm, 250nm to 100μm, 500nm to 100μm, 900nm to 100μm, 1μm to 100μm, 10μm to 100μm, 50μm to 100μm, 10nm to 10μm, 25nm to 10μm, 50nm to 10μm, 100nm to 10μm, 250nm to 10μm, 500nm to 10μm, 900nm to 10μm, or 1μm to 10μm, including any range in between.

[0184] The term "stable" as used herein refers to the ability of an article (e.g., a coated substrate) to maintain its structural and / or mechanical integrity, such as the absence of cracks and / or deformation. In some embodiments, an article is referred to as stable if it substantially maintains its structural and / or mechanical integrity under outdoor conditions (e.g., temperatures of -25 to 75°C, UV and / or visible light exposure). In some embodiments, a stable article is rigid under outdoor conditions. In some embodiments, a stable article may maintain its tensile strength and / or elasticity. In some embodiments, substantially as described below.

[0185] In some embodiments, the coating is stable over a temperature range of -100°C to 100°C, -50°C to 100°C, -10°C to 100°C, -10°C to 50°C, 0°C to 100°C, 0°C to 50°C, 5°C to 60°C, 0°C to 10°C, 10°C to 100°C, 50°C to 100°C, 10°C to 50°C, 10°C to 70°C, 5°C to 60°C, including any ranges therebetween.

[0186] In some embodiments, an article comprises an outer surface and an inner surface, wherein the outer surface is in contact with or bonded to a coating described herein.

[0187] In some embodiments, the compositions of the present invention are characterized by adhesion to a substrate.

[0188] In some embodiments, the coating is an insecticide coating. In some embodiments, the coating has at least one characteristic selected from the group consisting of an antifungal coating, an antimicrobial coating, an anti-insect coating, an antiviral coating, an anti-mold coating, a plant protection coating, and an insecticide coating.

[0189] In some embodiments, the articles or pesticide compositions of the present invention can reduce at least one of the following: (i) growth of pests, and (ii) pest load on or in a substrate by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 100%, including any ranges therebetween, compared to a control. In some embodiments, the articles or pesticide compositions of the present invention can reduce at least one of (i) growth of pests, and (ii) pest load in an application area (e.g., an area under cultivation and / or a substrate located near the application site). In some embodiments, the control is a similar substrate without the pesticide composition of the present invention (e.g., a substrate with pests that has not been treated with any pesticide composition). In some embodiments, the pesticide coating can reduce pest load and / or pest growth by a factor of between 2 and 20, between 2 and 5, between 5 and 7, between 7 and 10, between 10 and 12, between 12 and 15, between 15 and 20, including any ranges therebetween. In some embodiments, the reduction is compared to a control.

[0190] In some embodiments, the coating according to the present invention is stable under environmental conditions. In some embodiments, the coating is stable when exposed to: ambient temperature and / or temperature changes, heat, cold, UV radiation, and atmospheric gases. In some embodiments, the properties of the coating are not affected or changed by climate change as described herein. In some embodiments, the articles according to the present invention are stable to climate change. In some embodiments, the articles are stable to temperature changes, heat, cold, UV radiation, and atmospheric corrosive elements. In some embodiments, the properties of the articles of the present invention (e.g., insecticide activity) are not substantially affected or changed by climate change as described herein.

[0191] In some embodiments, the article (e.g., a substrate coated with a pesticide composition) is stable for at least 1 d, at least 5 d, at least 7 d, at least 10 d, at least 15 d, at least 30 d, at least 60 d, at least 100 d, at least 300 d, including any ranges therebetween.

[0192] In some embodiments, the article is in the form of a kit (e.g., a pesticide kit or an agricultural kit) comprising a first compartment comprising nanoparticles and oil, as described herein. In some embodiments, the w / w concentration of the nanoparticles in the oil is between 0.5 and 10%.

[0193] In some embodiments, the kit comprises a second compartment comprising an insect pathogen. In some embodiments, the second compartment further comprises an aqueous solvent (e.g., an agriculturally acceptable carrier). In some embodiments, the w / w concentration of the insect pathogen in the aqueous solvent is up to 80%, up to 60%, up to 50%, up to 20%, including any ranges therebetween.

[0194] In some embodiments, the kit further comprises instructions for mixing the second compartment (eg, which comprises an EPN organism, or a composition comprising the same) with the first compartment in a predetermined ratio to obtain a composition of the invention.

[0195] method

[0196] According to some embodiments, the present invention provides a method for controlling pests or reducing their growth, comprising contacting an effective amount of a pesticidal composition with a locus infested by the pests.

[0197] As used herein, the term "locus" refers to a habitat, plant, seed, material, or environment where a pest is growing, may grow, or may pass through. For example, a locus can be: a crop, tree, fruit, grain, forage species, vines and / or ornamental plants, plant parts, interior or exterior surfaces of a building (such as a grain storage location); building materials used in a building (such as impregnated wood). In some embodiments, the terms "locus" and "area under cultivation" are used interchangeably herein. In some embodiments, the term "locus" refers to a plant and / or a part of a plant (e.g., a leaf).

[0198] In some embodiments, there is a method for preventing pest infestation, the method comprising applying an effective amount of the pesticide composition of the present invention to at least a portion of a plant, and / or to a cultivation area. In some embodiments, there is a method for controlling pests or reducing their growth, the method comprising applying an effective amount of the pesticide composition of the present invention to at least a portion of a plant infested by pests, or to a cultivation area, thereby controlling or reducing the growth of pests.

[0199] In some embodiments, the method is used to kill pathogens or reduce pathogen load. In some embodiments, the method kills pathogens or reduces their growth by applying an effective amount of a pesticide composition described herein to a plant. As used herein, the terms "pest" and "pathogen" are used interchangeably herein.

[0200] In some embodiments, the term "reducing" or any grammatical derivative thereof means at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in growth, or even complete inhibition of growth, compared to growth of a pathogen not exposed to a treatment described herein during that given time. In some embodiments, the term "completely inhibited" or any grammatical derivative thereof means 100% inhibition of growth at a given time, compared to growth of a pathogen not exposed to a treatment described herein during that given time. In some embodiments, the terms "completely inhibited" and "eradicated" (including any grammatical forms thereof) are used interchangeably herein.

[0201] In some embodiments, the pesticidal compositions of the present invention are characterized by adhesion to at least a portion of a plant.

[0202] In some embodiments, the plant includes a cultivated plant or a part thereof.

[0203] In some embodiments, the pesticide composition of the present invention is applied to plants or plant parts by a method comprising: soaking, coating, irrigating, dipping, spraying, atomizing, scattering, brushing, injecting, or any combination thereof.

[0204] In some embodiments, the pesticide composition of the present invention is applied at any plant cultivation stage, such as sowing, before sowing, before harvest, after harvest, storage, etc.

[0205] In some embodiments, the effective amount is sufficient to control pests, kill pests, reduce pest loads, reduce pest growth, reduce pest-associated plant damage, prevent pests from infesting plants and / or areas under cultivation, or any combination thereof.

[0206] In some embodiments, the insecticidally effective amount of the liquid composition disclosed herein is at least 1 L / ha, at least 10 L / ha, at least 50 L / ha, at least 100 L / ha, at least 500 L / ha, at least 1000 L / ha, at least 5000 L / ha, including any ranges therebetween.

[0207] On the other hand, a method for obtaining a liquid preparation including living organisms is provided, the method comprising providing a dispersion including living organisms and an aqueous solvent (e.g., an agriculturally acceptable carrier), and adding or contacting the dispersion with a mixture including UV shielding nanoparticles and oil, thereby obtaining a liquid preparation. In some embodiments, the living organism is an invertebrate (e.g., arthropod, insect, aphid, nematode or any combination thereof). In some embodiments, the method is to coat the living organism with the core-shell particles of the present invention. In some embodiments, the method is used to combine the core-shell particles of the present invention with living organisms. In some embodiments, the method is used to disperse or be formulated in an emulsion (e.g., w / o or o / w emulsion) with living organisms.

[0208] In some embodiments, the method is as described herein, wherein the w / w concentration of the UV shielding nanoparticles in the oil is between 0.5 and 10%. In some embodiments, the method is as described herein, wherein the w / w concentration of the insect pathogen in the aqueous solvent is up to 80%, up to 60%, up to 50%, up to 20%, including any ranges therebetween.

[0209] In some embodiments, the method is used to extend the shelf life of an aqueous composition comprising a living organism.

[0210] General Description

[0211] As used herein, the term "about" refers to ± 10%.

[0212] The terms "comprises," "comprising," "includes," "including," "having" and their cognates mean "including but not limited to."

[0213] The term "consisting of means" means "including and limited to".

[0214] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially change the essential and novel characteristics of the claimed composition, method or structure.

[0215] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments, and / or to exclude the incorporation of features of other embodiments.

[0216] The term “optionally” is used herein to mean “provided in some embodiments and not provided in other embodiments.” Any particular embodiment of the present invention may include a number of “optional” features, unless such features conflict with one another.

[0217] As used herein, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0218] Throughout this specification, various embodiments of the present invention may be presented in the format of a range. It should be understood that the description in the range format is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present invention. Therefore, the description of a range should be deemed to have specifically disclosed all possible subranges and individual values ​​within the range. For example, a description such as a range from 1 to 6 should be deemed to specifically disclose subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and individual values ​​within the range, such as 1, 2, 3, 4, 5, and 6. This applies no matter how large the range is.

[0219] Whenever a numerical range is indicated herein, its meaning includes any reference numeral (decimal or integer) within the indicated range. The phrases "range between" a first indicated numeral and a second indicated numeral and "range from a first indicated numeral to a second indicated numeral" are used interchangeably herein to include the first and second indicated numerals and all decimals and integers therebetween.

[0220] As used herein, the term "substantially" means at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, including any range or value therebetween.

[0221] The term "method" as used herein refers to manners, means, techniques and procedures for accomplishing a given task, including but not limited to manners, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine or readily developed from known manners, means, techniques and procedures.

[0222] As used herein, the term "treating" includes eliminating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating the clinical or visual symptoms of a condition, or substantially preventing the appearance of clinical or visual symptoms of a condition.

[0223] It will be appreciated that certain features of the invention described in the context of different embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination, or as appropriate in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments unless the embodiment cannot be implemented without those elements.

[0224] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0225] Example

[0226] Materials and methods

[0227] Titanium dioxide (AEROXIDE TiO2 P25, primary particle size about 21 nm, obtained from Evonik, Germany), (3-aminopropyl)triethoxysilane (99% Sigma-Aldrich, USA), mineral oil (RTM-10, Sigma-Aldrich, USA), ultrapure deionized water (ULS / MS grade), Triton X-100 (laboratory grade, Sigma-Aldrich, USA), anhydrous ethanol and 96% ethanol (Bio Lab, Israel).

[0228] Titanium dioxide (TiO2) nanoparticles were silanized with APTES(NH2).

[0229] TiO2 (5.63 g) was added to 130 mL of ethanol and stirred until fully dispersed. Then 11 mL of APTES was slowly added to the solution. The reaction was carried out at ambient temperature for 1.5 h. After silanization, the particles were collected by centrifugation (9000 rpm, 10 min) and rinsed four times with ethanol. Finally, the TiO2-NH2 NPs were dried in vacuum at 35 °C for 3 h.

[0230] Preparation of TiO2-NH2 Pickering Emulsion

[0231] Pickering emulsions were prepared from amine-functionalized TiO2 in water and mineral oil. First, TiO2-NH2 NPs were dispersed in deionized water by sonication for 5 min (Sonics Vibra-Cell 750W, amplitude 25%) with increasing TiO2 contents: 0.4, 1, and 2 wt%. Mineral oil was then added with oil-water ratios of 10:90, 30:70, 40:60, and 50:50, respectively. The mixture was sonicated for 10 min (Sonics Vibra-Cell 750W, amplitude 35%) for emulsification.

[0232] Labeling TiO2-NH2 particles with carboxyfluorescein

[0233] To label the TiO2-NH2 particles, a MES buffer (pH 3) at a concentration of 0.5 M was used. 5(6)-carboxyfluorescein was added to the MES buffer at a concentration of 0.1 mg / ml to prepare a dye solution. EDC at a concentration of 15 mg / ml was added to the MES buffer to prepare a reaction catalyst solution. To obtain an amide reaction, 100 mg of TiO2-NH2 particles (powder) were added to 0.1 ml of dye solution, 0.3 ml of catalyst solution, and 0.6 ml of MES buffer. The powder was stirred thoroughly at room temperature for one hour using a magnetic stirrer until the particles were completely dispersed. After the amide reaction was completed, the particles were collected by centrifugation (9500 rpm, 20 min), rinsed 5 times with MES buffer, and finally rinsed with distilled water. The labeled TiO2-NH2 particles were then dried at 35°C under vacuum (70 mmHg) for three days.

[0234] High-resolution cryogenic scanning electron microscopy

[0235] The Pickering emulsion samples were analyzed using a high-resolution cryo-SEM JSM-7800F Schottky field emission scanning electron microscope (Jeol, Ltd.; Tokyo, Japan) equipped with a cryo system (Quorum PP3010, Quorum Technologies Ltd.; Laughton, UK). All heat exchange units of the cryo system use liquid nitrogen. 2 μl of the emulsion was placed on a sample holder between two rivets and vitrified using liquid nitrogen. The sample was then transferred to the preparation chamber where it was fractured (-140°C). The exposed fracture surface was sublimated at -90°C for 10 min to eliminate any condensed ice and then plated with platinum. The sample temperature was maintained at -140°C. Images were collected using a low electron detector (LED) at an accelerating voltage of 5.0 kV and a working distance of 3.9 mm.

[0236] use Software (LUM GmbH, Berlin, Germany) analyzes the instability index of emulsification separation (creamingseparation) and calculates it with the attached software (SepView 6.0; LUM). 400 μL vol% 2wt% TiO2-NH2 emulsions of 40:60 and 50:50 were loaded into polycarbonate cuvettes with an optical path length of 2 mm, and centrifuged at 600 rpm at 25 ° C. in triplicate. Transmission curves were captured at a wavelength of 865 nm for 6 hours in the whole cell (200 curves were captured every 5 s, 100 curves were captured every 10 s, 100 curves were captured every 30 s, and 600 curves were captured every 60 s).

[0237] 2. Nematode Preparation

[0238] Steinernema carinata (Weiser) from the commercial product Nemastar (e-nema, GMBH, Schwentinental, Germany) was routinely propagated on the last instar larvae of Galleria mellonella (L.) and reared in the laboratory of the Volcani Center. Newly born Steinernema carinata IJs were collected from modified White traps and stored at 8°C and used within 20 days after birth. The nematodes were vacuum filtered through a filter paper through a 30 μm filter, collected in distilled water, and labeled as nematode stock solution. The number of IJs in 20 μl of the stock solution was counted twice to determine the nematode concentration. The stock solution was diluted to a nematode concentration of 1000 ± 100 nematodes / ml.

[0239] Adding nematodes to TiO2-NH2 emulsion

[0240] The IJ of Steinernema carinii extracted from the stock solution was centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded. The required volume of emulsion (10 ml) was added and mixed gently. The IJ concentration was maintained at 1000 ± 100 nematodes / ml. The IJ was stored in falcon tubes for shelf life studies and infectivity studies, where it was used immediately.

[0241] Scanning electron microscopy (SEM)

[0242] SEM measurements were performed using a MIRA3 field emission SEM microscope (TESCAN, Brno / Czech Republic) with an accelerating voltage of 1 to 3 kV and equipped with a secondary electron detector. The Pickering emulsion samples were drop-cast on a conductive double-adhesive carbon tape and dried under ambient conditions. Before imaging, a thin layer of palladium / gold was evaporated on the sample to make it conductive and to avoid charging of the sample surface by the electron beam.

[0243] Shelf life of nematodes

[0244] Two TiO2-NH2 emulsions (4:6 and 5:5) with the addition of Steinernema carinii and a control group (nematodes in water) were stored at 8°C. The IJ concentration used for all treatments was 1000±100 IJ / ml in a volume of 10 ml. The survival rate of IJ in TiO2 emulsions and controls was measured every 15 days. The samples (20 μl) were diluted with 7 ml of distilled water. The samples were observed and counted under a stereomicroscope (Olympus, SZH10). The IJs were probed with a needle and were judged to be dead if no movement was found. Each treatment contained five technical replicates, and the experiment was repeated twice. 25-125 IJs were counted for each treatment and time point.

[0245] Insect bioassay of IJ in TiO2-NH2 emulsion

[0246] A G. mellonella colony was continuously maintained at the laboratory of the Volcani Centre. For the bioassay, the 5th instar was collected from the colony. TiO2-NH2 emulsions (4:6 and 5:5) containing Steinernema carinella IJ (500 μl) were filled in petri dishes (90 mm) covered with a single layer of filter paper (Whatman No. 1). Other treatments included Steinernema carinella in water, emulsions only (40:60 and 50:50) and a negative control of water only. For each treatment, 5 G. mellonella were added to each plate and sealed with plastic wrap. The experimental set-up was incubated at 23 °C for 48 h. Insect mortality was recorded after an additional 48 h of incubation. Each treatment contained five replicates and the experiment was repeated three times.

[0247] The dead larvae of each treatment were transferred to a modified White trap and cultured at 23°C to assess the IJ production of each greater wax moth. New IJs were collected weekly. The collected IJ solution (20 μl) was added to 7 ml of water in persipex and counted under a stereomicroscope. The total number of newly emerged IJs was calculated based on the volume of solution collected, and only live IJs were considered when counting. Each treatment contained five replicates, and the experiment was repeated twice. The data represented are the cumulative averages of two biological replicates.

[0248] Preparation of nematodes for confocal microscopy analysis

[0249] Samples were analyzed by laser scanning confocal microscopy. To obtain 3D images, a Leica SP8 laser scanning microscope (Leica, Wetzlar, Germany) equipped with a solid-state laser for 488 nm light, a HC PL APOCS 20× / 0.75 objective (Leica, Wetzlar, Germany) and Leica Application Suite X software (LAS X, Leica, Wetzlar, Germany) was used. The GFP signal was imaged using an argon laser and its emission was detected in the range of 500-520 nm. For the small moth Steinernema carboxyfluorescein, the image stack was first projected using a Z projection (maximum intensity) to find all the fluorescent nematodes, and then the images were analyzed using Fiji software. Fluorescence emission of the carboxyfluorescein emission signal in the range of 500-530 was recorded with a PMT and HyD (hybrid) detector.

[0250] Statistical analysis

[0251] The percentage data obtained were arcsine transformed before statistical analysis using SPS software (JMP Professional 15.0). ANOVA was used to determine general effects and interactions. Tukey's HSD (honestly significant difference) test was performed for multiple comparisons (P < 0.05). The effect on IJ production / larvae was analyzed using one-way ANOVA, and the means were compared using Tukey-kramer HSD test.

[0252] Example 1

[0253] Preparation and characterization of Pickering emulsions

[0254] like Figure 1 As shown, TiO2 NPs were functionalized with NH2 by introducing amine groups on the NP surface via saltation (or silanization).

[0255] Oil-in-water Pickering emulsions stabilized by amine-functionalized TiO2 NPs were prepared. Different amine-functionalized TiO2-NH2 contents and oil-water ratios were used to determine the optimal conditions for a stable Pickering emulsion system that met the requirements for nematode formulations. TiO2-NH2 contents were varied (0.4, 1, and 2 wt%) at oil / water ratios of 10:90, 30:70, 40:60, and 50:50, respectively.

[0256] The structure of the emulsion was characterized using confocal microscopy and cryo-HRSEM (Figure 2). To investigate the location of the TiO2-NH2 particles in the emulsion, we used TiO2-NH2 particles covalently labeled with the fluorescent dye carboxyfluorescein. The dye was covalently fixed to the TiO2-NH2 particles via amidation (EDC-NHS) via the amine end of the APTES molecule. The carboxyfluorescein dye has an excitation wavelength of 488 nm and an emission wavelength of 500-530 nm, giving it a green color. Confocal microscopy analysis ( Figure 1 A-1B) confirms the self-assembly of TiO2-NH2 particles at the oil / water interface, as the green fluorescence signal forms a ring around the oil droplet, indicating that the particles are indeed located at the interface as Pickering stabilizers.

[0257] Adjusting the emulsion composition helps fine-tune the resulting droplet size, which is critical for proper coating of nematodes. Thus, the relatively high stability of Pickering emulsions can be attributed to the low rate of droplet aggregation.

[0258] The stability of the Pickering emulsions over time was evaluated by LUMisizer (LUM GmbH, Germany), confirming the visual observations. Most of the prepared emulsions were stable for 7 days. The highest stability was achieved for emulsions with TiO2-NH2 contents of 0.4, 1, and 2 wt% at oil / water volume fractions of 40:60 and 50:50, which lasted for more than 5 months ( Figures 3A-3F ).

[0259] The stability of the emulsion was quantitatively characterized by calculating the instability index using the LUMiSizer. The transmittance was measured when the emulsion was centrifuged in a cuvette at 25°C. The results showed a positive correlation between particle content and stability, where increasing TiO2-NH2 content increased stability ( Figure 4 ). The emulsion with 2% TiO2-NH2 content was the most stable at different oil / water ratios. There was no significant difference in the instability index at oil / water ratios of 50:50 and 40:60 for any TiO2-NH2 content. The emulsion with 2% TiO2 was the most stable at oil / water volume fractions of 40:60 and 50:50, with the smallest droplet size and the lowest instability index. It was found that the emulsion with 2wt% TiO2-NH2 content was the most stable at different oil / water ratios and was suitable for individual formulation of nematodes because of the small droplet volume.

[0260] Example 2

[0261] Formulating EPN in Pickering Emulsion

[0262] 200 μl of the suspension of Steinernema carinii was allowed to settle in an Eppendorf tube (1.5 ml) for 15 minutes. Pickering emulsions (400 μl) containing 0.4%, 1% and 2% TiO2-NH2 with an oil-water volume fraction of 40:60 were added to the nematode residue. Confocal microscopy micrographs showed that successful binding of nematodes was observed between the droplets in all preparations (Figure 5). In addition, the confocal microscopy images also showed that smaller droplets produced better coating ability due to the larger contact area with the nematodes (Figure 6). As mentioned above, the 2% TiO2-NH2 emulsions with oil-water ratios of 40:60 and 50:50 were the most stable and had the smallest droplet size ( Figure 4 and 5). Therefore, Pickering emulsions containing 2% TiO2-NH2 and oil-water volume ratios of 40:60 and 50:50 were considered to be the most suitable formulations.

[0263] Confocal analysis of dried preparations was performed using Pickering emulsions of green labeled particles of carboxyfluorescein. An equal amount of 200 μl of the suspension of Steinernema carinii was allowed to settle in an Eppendorf tube (1.5 ml) for 15 minutes. Then, 400 μl of the emulsion was added to the nematode residue. 30 μl of the nematode preparation was then dried at 25°C on a glass slide for analysis. Figure 6A-6B Demonstrating the successful binding of dried nematodes between oil droplets. The green color marks the TiO2-NH2-carboxyfluorescein NPs at the interface. Figure 6B-6C3D images of the dried preparations are shown. These images show that the nematodes are completely coated with the labeled NPs. During the drying process, the oil droplets stick to each other due to the evaporation of water. This phenomenon promotes the diffusion of TiO2 on the surface of the nematodes.

[0264] SEM analysis of EPN formulated in Pickering emulsion showed that the nematodes were successfully coated by the mineral oil of Pickering emulsion (Figure 7). It was observed that the TiO2 nanoparticles were deposited mainly along the stripes or lines on the body of the nematodes after drying. Based on our preliminary analysis, this area shrank with drying. This may provide a hypothesis for the potential protection of nematodes by this formulation under stress conditions.

[0265] Example 3

[0266] Nematode shelf life

[0267] The nematode Steinernema carinii was suspended in Pickering emulsion containing 2% TiO2-NH2 at oil-water ratios of 40:60 and 50:50. The survival of IJs at 8°C was monitored every 15 days for one month and then monthly for 5 months to evaluate the shelf life of the formulations. On the first day, the survival of the 50:50 Pickering emulsion was slightly different, but not statistically different. 60 days after treatment, both formulations showed similar IJ survival of Steinernema carinii in water as the control sample (P>0.05)( Figure 8 ). The survival rates of nematodes in all treatments were higher than 98%, indicating that the environment in the TiO2-NH2 Pickering emulsion was similar to that in water.

[0268] Effect of UV radiation on the survival rate of IJ

[0269] The inventors further tested the effect of UV radiation on IJ viability in water and in the formulations of the invention by exposing each test sample to ultraviolet light (254 nm) for 10 or 20 minutes in a Labconco Purifier Class II Biosafety Cabinet (Model 36209; Labconco, Kansas City, MI).

[0270] The preparations used in the trials included 1% or 2% II. Flame retardant gel and exemplary compositions of the invention (see Example 1). The nanoparticle formulations were produced by the Institute of Post-Harvest and Food Sciences, Volcani Center, Israel Agricultural Research Organization.

[0271] To prepare the IJ suspension used in various treatments, 5 ml of IJ suspension in distilled water was placed in a 15 ml centrifuge tube and centrifuged at 2000 rpm for 2 min. Carefully remove the water, and the IJ precipitate of about 100 μl at the bottom of the tube was used to prepare an IJ preparation with a concentration of 1000 IJ / ml. For the IJ in 1% and 2% Barricade, first 100 μl of IJ precipitate was mixed with distilled water, shaken, and then 0.1 and 0.2 g of Barricade gel were added, respectively, and the final volume of each replicate test tube was 10 ml. About 2 ml of IJ suspension was moved to the center of a 60 mm culture dish lid with a pipette. Because the Barricade preparation was thicker in texture, a spatula was used to transfer the mixture and weigh about 2 g. The dish was randomly placed under UV light and exposed to UV light for 10 or 20 min.

[0272] After UV treatment, the dishes were incubated for 24 h in a growth chamber at 25 °C and LD 14:10. Water loss was compensated by weighing the dishes before and after incubation. The IJ suspensions were mixed; 1 ml of the solution was used to assess the toxicity of the IJs, and the rest was used to assess the mortality of the IJs. The number of live and dead IJs was counted under a microscope, and IJs that did not respond to the probe were considered dead. Approximately 100 IJs were examined in each sample. There were three replicate dishes for each treatment, and the entire experiment was performed three times.

[0273] To evaluate the toxicity of IJ after UV exposure, control formulations (without IJ) included: water, 1% and 2% Barricade and TiO2 formulations. After UV treatment and incubation at 25°C for 24h, 1ml of IJ suspension (or 1g of IJ in Barricade) was taken from each culture dish and transferred to a 100mm culture dish equipped with a single layer of filter paper, and 10 larvae of the tail stage of greater wax moth were added to each plate. The plate was placed on a tray, covered with a plastic bag, and kept moist with a wet paper towel. Insect mortality was assessed 48h after incubation at 25°C. Insect carcasses were dissected under a microscope (15x) to check for nematode infection. Each treatment consisted of three plates as a repeat, and the experiment was repeated twice.

[0274] To further evaluate the effect of UV radiation on IJ virulence, the number of nematodes invading G. mellonella in TiO2-based formulations and water controls was determined after IJs were exposed to UV treatment for 0, 10, or 20 min. IJ suspensions in water or TiO2 formulations were prepared according to the same procedure as above at a concentration of 1000 IJ / ml. Approximately 1 ml of the IJ suspension was transferred to the center of a 60 mm Petri dish lid and placed under UV light for 10 or 20 min, except that no UV treatment was performed. The dishes were then incubated at 25 °C for 24 h and then inoculated with G. mellonella. Ten insects were used in each of six dishes as experimental units for replicate treatments. Insect mortality was assessed 3 days after inoculation, and dead insects were dissected to calculate the number of nematodes invading each of the 10 insects per dish; samples that were not dissected in time were frozen and stored temporarily and thawed at a later time for dissection. A total of 60 insects were dissected for each treatment.

[0275] In the effect of UV radiation on IJ survival, IJ mortality varied significantly with the agent (F = 22.65, df = 3, 96, P < 0.0001) and UV exposure time (F = 90.33, df = 2, 96, P < 0.0001); there was a significant interaction between the agent and the length of UV treatment time (F = 14.86, df = 6, 96, P < 0.0001) ( Fig.10 ). Except for 2% Barricade and the TiO2-based preparation of the present invention, the IJ mortality rate increased significantly after 10 minutes of UV irradiation; except for the TiO2-based preparation of the present invention, the IJ mortality rate increased further after 20 minutes of UV irradiation, and even after 20 minutes of UV irradiation, the survival rate of the TiO2-based preparation of the present invention was still 99%; UV irradiation for 20 minutes was more damaging to IJ than UV irradiation for 10 minutes. During the 10 minutes of UV irradiation, the mortality rate of aqueous IJ was the highest, followed by 1% Barricade, 2% Barricade and the TiO2-based preparation of the present invention, the latter of which had almost no dead IJs (mortality rate less than 1%). After 20 minutes of UV irradiation, the mortality rate of IJs mixed with 1% Barricade was the highest, with no significant difference from aqueous IJs, while the mortality rate of IJs encapsulated in the TiO2-based preparation of the present invention was still the lowest (1%).

[0276] UV radiation significantly affected the virulence of IJ (F = 85.94, df = 3, 80, P < 0.0001), and the degree of influence varied with the formulation (F = 48.21, df = 3, 80, P < 0.0001), resulting in insect mortality. There was a significant interaction between UV exposure time and formulation (F = 18.11, df = 9, 80, P < 0.0001) ( Fig.11A). All formulations of IJ had 100% insect mortality without UV treatment. After 10 or 20 min of UV treatment, the insect mortality of all formulations, except for the TiO2-based formulations of the present invention, decreased significantly; regardless of the length of UV exposure time (except for no UV treatment), the IJ mortality in these formulations was no higher than that of the formulation control, while the IJ in the TiO2-based formulations of the present invention still caused 100% insect mortality even after 20 min UV. Comparing the formulations, the insect mortality of the TiO2-based formulations of the present invention was higher than that of the other formulations at both 10 and 20 min UV, while there was no difference between the other formulations (including water-based IJ). Compared with water, the formulation control did not cause significant insect mortality.

[0277] Upon autopsy, most dead insects showed nematode infection. Consistent with mortality, IJ infection levels also varied significantly with preparation (F = 31.17, df = 3, 80, P < 0.0001) and UV exposure time (F = 60.75, df = 3, 80, P < 0.0001), with a significant interaction between the two (F = 12.12, df = 9, 80, P < 0.0001) ( Fig. 11B ). In the absence of UV radiation, all formulations mixed with IJ caused almost 100% infection. The TiO2-based formulation of the present invention still had an infection rate of 88% after 10 or 20 min UV, which was not reduced compared to no UV treatment (90%). All other formulations did not show IJ infection after UV exposure.

[0278] Effects of UV radiation on nematode-invading insects

[0279] Similar to previous experiments, in the TiO2-based formulations of the present invention, insect mortality (F=0.00, df=2,30, P=1.0000) and infection rate (F=0.11, df=2,30, P=0.8986) did not decrease after 10 or 20 min of UV irradiation; regardless of UV treatment, all insects died and the infection rate was ≥95%.

[0280] When counting the number of invading nematodes after insect dissection, there was no difference between the water control and the IJ in the TiO2-based formulation of the present invention without UV treatment (F=0.18, df=1,118, P=0.6738).

[0281] However, after inoculation of IJ in water, no insects died and no nematodes successfully invaded the host after 10 and 20 min UV. Surprisingly, for the TiO2-based formulation of the present invention, consistent with infection, it was found that the number of invading nematodes did not decrease after 10 or 20 min UV compared to no UV treatment; on the contrary, after 20 min UV, the number of nematodes recovered was higher than no UV (F = 3.18, df = 2,177, P = 0.0439) ( Fig.12 ).

[0282] Activity of EPN against Galleria mellonella in TiO2-NH2 emulsion

[0283] The mortality of insects infected with G. mellonella by IJ of Steinernema carinii in TiO2-NH2 Pickering emulsion was recorded within 48 hours (Table 1). The results showed that the mortality of nematodes against G. mellonella in the fifth instar in water and TiO2-NH2 Pickering emulsion (4:6 and 5:5) was significantly different (P<0.001) within 48 hours. However, there were no statistically significant differences between the treatments (P>0.05), indicating that the treatments had no adverse effects on IJ activity. In addition, there was no difference in mortality between the control group (water only) and the emulsion only treatment. This suggests that the mortality in the emulsion treatment was due to the penetration of IJ.

[0284] Table 1: Insect mortality (%) of 5th instar larvae of G. mellonella by Steinernema carinella in water, 2% TiO2-NH2 emulsion (oil:water concentration of 4:6 and 5:5) at 48 hours post inoculation. Numbers represent cumulative means of 3 biological replicates. Insect mortality (%) (n=120) was subjected to two-way ANOVA after arcsine square root transformation. Means were compared when ANOVA F was significant (P<0.05). Means were compared using Tukey HSD test.

[0285]

[0286] Based on the production of G. mellonella larvae

[0287] The newly formed IJs were collected and the number of newly formed IJs was determined based on the survival rate. The volume ratio of the emulsion had no effect on the number of newly formed IJs, and there was no significant difference compared with the control (P>0.05). When the emulsion with an oil-water volume ratio of 40:60 and 50:50 and the control group (nematodes in water) were used, the number of IJs emerging per larva was 64,000, 66,000, and 50,000, respectively ( Fig. 9 The preparation showed extremely high stability and maintained the activity of EPN after 120 days of storage.

[0288] EPN was coated with TiO2 nanoparticles alone via an oil-in-water Pickering emulsion. Mineral oil-in-water Pickering emulsions were prepared under ultrasound-induced shear forces. The droplets were stabilized by amine-functionalized titanium dioxide (TiO2-NH2) NPs. The emulsions with 2% TiO2-NH2 content were the most stable with the smallest droplet size at oil-water ratios of 40:60 and 50:50, which were suitable for nematode preparations. Oil and titanium dioxide NP aggregates were detected on the nematode surface before and after the drying process at room temperature of 25°C.

[0289] Exemplary formulations of the present invention allow oil droplets and TiO2-NH2 NPs to coat nematodes individually. Shear forces increase the surface area of ​​particles occupying the surface of each oil droplet, thereby forming significant contact with the EPN. Comprehensive coverage of nematodes is necessary to prevent dehydration and reduce prolonged UV exposure. We expect that this coating will protect nematodes more effectively from other external climatic conditions in the field (temperature, humidity, UV, dryness) than other formulations. The stability and droplet properties of the formulation can be easily adjusted by appropriately changing the NP concentration and water-in-oil ratio. The formulation we developed is low-cost, easy to prepare and apply, environmentally sustainable, and suitable for large-scale EPN operations. According to our research results, nematodes can be stored for a long time at temperatures of 4°C and 23°C. It was found that the formulation was biocompatible with the nematodes studied, and the survival time, infectivity, and speed of emergence from the carcass of the nematodes were comparable to those of aqueous solvents.

[0290] Example 4

[0291] Formulation and survival of EPN in Pickering emulsion after foliar application

[0292] The inventors studied the survival of IJ in cotton leaves and water, and the TiO2-based and SiO2-based formulations of the present invention extended the survival time of IJ from 14h to>40h and>80h, respectively. Under field conditions, the SiO2-based formulation of the present invention extended the survival time of IJ from 2h to 8h.

[0293] Materials and methods:

[0294] Nematode source and culture

[0295] Steinernema carinella was cultured on late instar G. mellonella. The carcasses were transferred to White traps and IJs that emerged 10 days after infection were collected and filtered for further evaluation. The EPN species used for the initial screening were Steinernema carinella and Steinernema noctuidae.

[0296] Insect source and culture

[0297] Greater wax moth community:

[0298] G. mellonella larvae were reared in sterilized glass bottles at ARO (Volcani Center). Larvae were fed every 2 days and kept separately to avoid overcrowding and the occurrence of diseases. Bottles were placed in a room at 25 ± 2 °C with a 12:12 h photoperiod and low humidity was maintained using an air dryer.

[0299] Cotton leafworm (Spodoptera littoralis) community:

[0300] The cotton leafworm B insect colony was continuously reared at ARO (Volcani Center). The cotton leafworm was chosen as a foliar pest model because it is a polyphagous lepidopteran foliar pest and is relevant to agriculture. The colony was reared in an insect incubator at a temperature of 25 ± 2 °C and a photoperiod of 12:12 (light:dark). To ensure uniform development, 4th instar larvae, 16-19 days after hatching, were used.

[0301] Preparation of titanium dioxide-NH2 Pickering emulsion (TPE):

[0302] The preparation method of Pickering emulsion is as described herein. Specifically, when the content of titanium dioxide-NH2 is 1wt%, titanium dioxide-NH2 NPs are ultrasonically dispersed in deionized water for 5min (Sonics VibraCell 750W, amplitude 25%). Then mineral oil is added, and the volume ratio of oil / water is 4:6. The mixture is ultrasonicated for 10min to achieve emulsification (Sonics Vibra-Cell 750W, amplitude 35%).

[0303] Preparation of Silica Pickering Emulsion Gel (SPEG):

[0304] Pickering emulsions were prepared with commercial hydrophobic silica (Aerosil R972, fumed silica treated with dimethyldichlorosilane, primary particle size estimated to be 16 nm, obtained from Evonik, Germany) in paraffin oil and water (Sigma-Aldrich, analytical grade). First, silica NPs were dispersed in paraffin oil by ultrasound for 5 min (Sonics Vibra-Cell 750W, amplitude 25%) with a silica content of 0.5 wt%. 0.5% potassium polyacrylate (PPA) was added to water with a W / O volume ratio of 40:60. The mixture was then ultrasounded for 10 min to emulsify. For the 0.5% potassium polyacrylate (PPA) polymer solution, 0.5 g of PPA was added to 99.5 mL of water and stirred for 1-2 h to completely dissolve it.

[0305] Formulating EPN IJ with Pickering Emulsion:

[0306] IJs of Steinernema carinata were collected from modified White traps. Newly emerged IJs were vacuum filtered, suspended in distilled water, and adjusted to a concentration of 1000 nematodes / ml. IJs were suspended in various formulations by centrifugation at 4000 g for 2 min, and the supernatant was discarded. An equal volume of formulation was added to the nematodes and gently mixed to form a uniform suspension. Two formulations were evaluated: titanium Pickering emulsion (TPE) and a novel silica Pickering emulsion gel (SPEG).

[0307] Plant experiments were conducted on cotton plants. Cotton plants of Akala variety were grown in pots and maintained under ambient conditions in a greenhouse for 3 months for nematode survival and efficacy experiments.

[0308] Screening of EPN species on cotton plants:

[0309] Infectious larvae of Steinernema carinii and Steinernema noctuidae were vacuum filtered, suspended in distilled water, and adjusted to a concentration of 1000 nematodes / ml. IJs were sprayed on cotton leaves at a temperature of 24-26°C and different humidity levels (50-70% RH). Temperature and humidity were monitored using a data logger (SSN23E). Samples (leaves; one leaf / technical replicate) were collected every hour, placed in a Petri dish containing 7 ml of tap water, cultured overnight (to allow IJs to fully recover), and the survival rate was estimated. The cultured leaves were washed to remove IJs from the leaves. Under a binocular (Olympus, SZH10, 30x magnification), a Petri dish containing water was used to count the total number of surviving IJs. Nematodes that were active in movement or responded to the probe were scored as surviving nematodes. The surface area of ​​the leaves was calculated using ImageJ software. The total number of live nematodes was normalized to the surface area of ​​the leaves. The experiment was repeated at least twice, with five technical replicates for each experiment. After the initial screening of EPN species, Steinernema carinata was used for further experiments.

[0310] The formulated EPNs were evaluated under controlled conditions:

[0311] 15 ml of nematodes in water (control) and nematodes in TPE and SPEG formulations were applied on cotton leaves using a manual sprayer. All treatments were cultured in a temperature and humidity controlled box, with a temperature of 25.4 ± 0.7 ° C and a relative humidity of 82 ± 6.2% for experiment 1; and a temperature of 24.3 ± 3 ° C and a relative humidity of 72.5 ± 6% for experiment 2. Since no significant differences between the experiments were observed (wald p value = 0.88), these experiments were merged. Samples of each treatment (leaf; one leaf / technical repeat) were collected within 24h intervals (0h (immediately after application), 24h after application, 48h after application, 72h after application, and 96h after application). As described above, the leaves were evaluated for nematode survival. Nematodes were active in movement or responded to the probe and were rated as alive. Temperature and humidity were monitored using a data logger (SSN23E). The experiment was repeated twice, with five replicates for each experiment.

[0312] Insect bioassay:

[0313] Nematode activity was detected against B. cotton leafworm in the first instar by spraying nematodes on cotton plants. Cotton plants were treated with prepared nematodes and compared with cotton plants treated with nematodes, water, TPE and SPEG. Leaf samples (leaves) were collected at 0h (immediately after application), 24h, 48h, 72h and 96h after application. The samples were placed in a 90mm culture dish and covered with a moistened Whatman filter paper. Five larvae were added, the lid was closed, sealed with plastic wrap, and cultivated at 23 ± 1°C. Mortality was assessed after 48h. Each treatment was repeated five times, and the entire experiment was repeated twice.

[0314] Relationship between EPN deposition and persistence:

[0315] Deposition was determined as the coverage / retention of the EPN IJ on the leaves after application. It will be appreciated by those skilled in the art that enhanced adhesion and deposition are prerequisites for improving the efficacy of EPN foliar applications. 2 The IJ deposition pattern of the function was calculated, and the ratio of the number of surviving IJs to the leaf surface area was obtained to obtain the surviving IJ / cm 2 number.

[0316] Evaluation of formulated EPN under greenhouse conditions:

[0317] EPN (Steinernema carinata) was prepared, applied to cotton leaves, and nematode survival and efficacy were evaluated in an uncontrolled greenhouse where temperature and humidity were continuously monitored using a data logger (SSN23E). The temperature ranges for Experiments 1 and 2 were 25.7-34.4°C and 31.6-37.5°C, respectively. The relative humidity ranges for Experiments 1 and 2 were 44-66.8% RH and 46.5-63.1% RH, respectively. The experiment was repeated twice, with five replicates per treatment, to determine survival and distribution.

[0318] Statistical analysis

[0319] All statistical analyses were performed using JMP 16 Professional (SAS Institute Inc.). Comparisons of survival (%) (n = 150) were based on the arcsine-transformed proportion of live nematodes. Standard least squares analysis was performed using restricted maximum likelihood (REML), followed by Student's t-test for multiple comparisons between means. Means were compared when F in ANOVA was significant (p < 0.05). Insect mortality (%) data were compared using standard least squares, using REML, followed by Student's t-test for multiple comparisons between means. Means at each time point were further compared and Bonferroni corrections were performed (α = 0.003). In addition, efficacy results under uncontrolled greenhouse conditions are expressed as replicate means of a single experiment. Insect mortality (%) data were compared using REML, using standard least squares, followed by Student's t-test for multiple comparisons between means. For IJs surviving on leaf area, the number of live IJs was normalized to leaf surface area. Normalized data were subjected to analysis of variance and multiple comparisons between means were performed by Student's t test, where leaf surface area was considered as a random measurement within biological replicates. LT analysis of survival was performed by Probit 4P model and back prediction of % survival data. 50 . Treating time as a continuous factor, the slopes are compared.

[0320] Assessment of IJ survival on cotton leaf surfaces over time

[0321] The survival rate and live nematode distribution of water-suspended Steinernema carinella IJ on cotton plants were significantly different (F (1,138) =345.5660; P<0.0001 and P=0.0084, respectively). The survival rate of Steinernema carinii was about 6 times that of Steinernema noctuidae (F (4,138) =99.0602; P<0.0001)( Fig.13A ). Two hours after application, the survival rate of S. carinii was less than 5%, while after 4 hours, the survival rate of S. cercariae was about 50%.

[0322] Although nematode deposition on leaves generally decreased over time for both species, the number of live nematodes on leaves was significantly higher for S. carinella compared with S. noctuidae (F (1,133.9) =40.8889; P<0.0001)( Fig. 13B LT of Steinernema carinella and Steinernema argentipes 50 3.08 ± 0.31 hours and 0.26 ± 0.34 hours, respectively. Comparison of the nematode survival slopes showed that the survival rate of Steinernema carinii was significantly lower than that of S. noctuidae over time (t(144) = 3.92; P = 0.0001). The survival rate of S. noctuidae IJ showed a sudden drop to below 50% in the first hour after application. However, the survival rate of Steinernema carinii gradually decreased.

[0323] Screening of agents for survival and efficacy under mild conditions

[0324] Formulated and unformulated EPN IJ were applied by spraying on cotton leaves to test their survival and efficacy under mild conditions. Fig.14A Figure 3 shows the survival of IJs in water (control group) and EPN IJs formulated in SPEG and TPE, where the survival of the control group after 24 h and longer was significantly lower than that of IJs formulated in TPE and SPEG (F (2,134) =215.8536; P<0.0001). In addition, the SPEG formulation was very effective in maintaining IJ survival for 96 hours compared with TPE (F (1,134) =14.9131; P = 0.0002). Nevertheless, within 48-96 hours, the number of surviving IJs / cm 2 Significantly higher than the control group (F (3,129.8) =15.0720; P<0.0001). At the same time point, TPE and water were not statistically significant (F (3,129.4) =1.7006; P>0.05)( Fig. 14B ). In particular, SPEG showed a significantly higher number of surviving IJs / cm at the measured time points compared with the control group and TPE. 2About 3 times higher. The LT50 of water, TPE and SPEG were 14±2.41h, 49±3.37h and 85±7.05h, respectively. The survival rates of IJ in SPEG and TPE were 6 and 3 times that of the control group, respectively. Comparison of slopes showed that the slopes of water and IJ in TPE were not statistically significant (t(143)=0.40; P=0.6933), while the slopes of water and IJ in SPEG were statistically significant (t(143)=-4.63; P=<0.0001). However, the slopes of IJ in TPE and IJ in SPEG were not statistically significant (t(143)=0.40; P=0.6933), indicating that the rate of change of nematodes over time in SPEG was slower than that in TPE and water. The infectivity of EPN IJ was maintained for 96h in SPEG, which was significantly increased compared with the control (F (2,134) =5.2478; P=0.0064)( Fig. 14C ). Infectivity of EPN protected in SPEG increased by about 3.5-4 times compared to water within 48-96 h. Thus, the efficacy of IJ was maintained across environmental conditions.

[0325] Effects of extreme greenhouse conditions on the survival rate and efficacy of formulations

[0326] Formulated and unformulated EPN IJs were sprayed on cotton leaves and their survival and efficacy under extreme greenhouse conditions were studied. The survival rates of IJs in water (control), IJs in SPEG, and IJs in TPE were significantly lower than those of the formulated IJ samples (F (2,107) =106.6484; P < 0.0001). IJs formulated in SPEG and TPE maintained IJ survival over the 8 h experiment. SPEG was more effective than TPE; therefore, IJ survival in SPEG was significantly different from that in TPE (F (4,107) =17.3201; P<0.0001). After 2 h, the survival rates of IJ in water, IJ in SPEG, and IJ in TPE were approximately 30%, approximately 90%, and approximately 70%, respectively. Considering the extreme environmental conditions, both formulations modestly protected EPN IJs, however, the SPEG formulation had a significantly lower survival rate per cm within 4 h. 2 The number of live nematodes retained was at least 2-fold higher than that of the control group and TPE. 50 The values ​​were 1.53±0.26, 3.13±0.22, and 6.54±1.33 hours, respectively.

[0327] The inventors have successfully integrated EPN (i.e., Steinernema carinella and Steinernema noctuidae) with target foliar pests (i.e., greater wax moth) by formulating the nematodes in a protective wrapper (Pickering's emulsion), thereby improving the survival and efficacy of EPN on leaves. Under mild conditions, SPEG and TPE formulations extended the survival and efficacy of IJs from less than 2 hours after application to approximately 8 hours.

[0328] Although the present invention has been described in conjunction with its specific embodiments, many alternatives, modifications and variations are apparent to those skilled in the art. Therefore, the present invention is intended to encompass all such alternatives, modifications and variations within the spirit and broad scope of the appended claims.

[0329] All publications, patents and patent applications mentioned in this specification are incorporated in their entirety by reference into this specification to the same extent as if each individual publication, patent or patent application were specifically and individually incorporated by reference into this specification. In addition, citing or indicating any reference in this application should not be construed as an admission that the reference is available as prior art for the present invention. Where section headings are used, they should not be construed as necessarily limiting.

Claims

1. A composition comprising an insect pathogenic organism coated by a plurality of nanoparticles, wherein the plurality of nanoparticles comprises UV shielding nanoparticles, wherein the composition is an aqueous dispersion of the insect pathogenic organism.

2. The composition of claim 1, wherein the UV shielding nanoparticles are assembled within core-shell particles.

3. The composition of claim 2, wherein the core-shell particle comprises a liquid core comprising oil, and wherein the liquid core is surrounded by a shell comprising the plurality of nanoparticles.

4. The composition of claim 2 or 3, wherein the core-shell particles are characterized by an average particle size between 1 and 50 um.

5. The composition of any one of claims 2 to 4, wherein (i) the weight ratio of the multiple nanoparticles within the core-shell particles or within the composition to the oil is between 1:10 and 1:100; (ii) the w / w concentration of the multiple nanoparticles within the composition is between 0.1 and 10%.

6. The composition of claim 1, wherein the UV shielding nanoparticles are chemically modified metal oxide particles.

7. The composition of claim 6, wherein the metal oxide particles comprise a metal oxide selected from SiO2, TiO2, or both; and wherein the chemical modification comprises a functional moiety covalently bonded to the metal oxide particles.

8. The composition of claim 7, wherein the metal oxide comprises TiO2, and the functional moiety comprises any one of: (i) an aminoalkyl group, an amino group, a hydroxyalkyl group, a thioalkyl group, an aminoalkylsilane group, a hydroxyalkylsilane group, a thioalkylsilane group, or any combination thereof; (ii) a UV absorbing group, or both (i) and (ii).

9. The composition of claim 7, wherein the metal oxide is selected from SiO2 and TiO2.

10. The composition of any one of claims 1 to 9, wherein the insect pathogenic organism comprises a nematode.

11. The composition of claim 10, wherein the nematode is an entomopathogenic infective juvenile nematode.

12. The composition of claim 3, wherein the oil is a liquid at a temperature between 10 and 60°C.

13. The composition of claim 3 or 12, wherein the oil is characterized by a viscosity at 25°C between 1 and 100 cP.

14. The composition of any one of claims 3 and 12-13, wherein the oil is any of the following: (i) substantially non-toxic to the insect pathogenic organism; (ii) substantially non-phytotoxic.

15. The composition of any one of claims 3 and 12-14, wherein the oil comprises mineral oil, C10-C30 aliphatic hydrocarbons, fatty acids, monoglycerides, diglycerides, triglycerides, vegetable oils, waxes, essential oils or fragrance oils, including any combination thereof.

16. The composition according to any one of claims 1 to 15, wherein the composition is in the form of an emulsion.

17. The composition according to any one of claims 1 to 16, wherein the composition further comprises a gelling agent.

18. The composition of claim 17, wherein the gelling agent is a polymeric gelling agent comprising polyacrylic acid, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, poly(2-hydroxyethyl methacrylate), polyacrylamide or a polysaccharide, including any salt and any combination thereof.

19. An insecticide composition comprising the composition of any one of claims 1 to 18; wherein the insecticide composition comprises an insecticidally effective amount of the insect pathogenic organism.

20. The insecticide composition according to claim 19, which is formulated for spraying or coating.

21. The insecticide composition of claim 19 or 20, wherein the insecticidally effective amount comprises a concentration of the insect pathogenic organism in the insecticide composition of at least 10 units / ml.

22. An article comprising: A substrate contacted with a coating comprising a pesticide composition according to any one of claims 19 to 21.

23. The article of claim 22, wherein the coating is in the form of a coating layer.

24. The article of claim 22 or 23, wherein the substrate is selected from plants or plant parts, polymer substrates, glass substrates, metal substrates, fibers, paper substrates, brick walls, sponges, textiles, woven fabrics, nonwoven fabrics or wood, or any combination thereof.

25. The article of any one of claims 22 to 24, wherein the coating is a pesticide coating.

26. The article of claim 25, wherein the coating substantially retains its insecticide activity over a period of time of 1 to 30 days of outdoor exposure.

27. A method for controlling pests, comprising applying an effective amount of the pesticidal composition according to any one of claims 19 to 21 to at least part of a plant, or to a cultivation area infested by the pests, thereby controlling or reducing the growth of the pests.

28. The method of claim 27, wherein the insecticide composition is characterized by adherence to at least a portion of the plant.

29. The method of claim 27 or 28, wherein the applying comprises immersing, soaking, coating, irrigating, dipping, spraying, atomizing, scattering, brushing, injecting, or any combination thereof.

30. A method according to any one of claims 27 to 29, wherein the pests comprise arthropods.

31. The method of any one of claims 27 to 29, wherein the effective amount is 1 to 1000 L / ha.

32. The method of any one of claims 27 to 31 , wherein the arthropod is a plant pest.

33. The composition of claim 17 or 18, wherein the concentration of the gelling agent in the composition is between 0.1 and 10% w / w.

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