Herbicidal compositions and methods of making and using

Abstract

Bioherbicidal compositions are described, as are methods of making and using such bioherbicidal compositions.

Description

[0001] F& RRefNo.: 55877-0008W01

[0002] HERBICIDAL COMPOSITIONS AND METHODS OF MAKING AND USING

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U. S. C. § 119(e) to U. S. Application No. 63 / 764,478 filed on February 27, 2025, and U. S. Application No.

[0004] 63 / 888,561 filed on September 26, 2025, both of which are incorporated herein by reference in their entirety.

[0005] TECHNICAL FIELD

[0006] This disclosure generally relates to herbicidal compositions and methods of making and using such compositions.

[0007] BACKGROUND

[0008] Herbicidal compositions can be used to selectively control undesired vegetation (e.g., weeds) with limited to no injury or damage to nearby desirable vegetation, or herbicidal compositions can be used to control a broad spectrum of differing types of vegetation (i.e., non-selective control). Glyphosate (N-(phosphonomethyl)glycine) and paraquat (N, N’-dimethy 1-4,4’- bipyridinium dichloride) are common herbicides used throughout the world for non-selective vegetation control, however, glyphosate can take 10-14 days to see herbicidal activity and paraquat is generally considered to be toxic to humans.

[0009] Therefore, herbicide compositions are desirable that control undesired vegetation but lack evidence of human toxicity.

[0010] SUMMARY

[0011] Described herein are herbicidal compositions as well as methods of making and using such compositions.

[0012] In one aspect, methods of reducing or preventing plant growth are provided. Such methods typically include applying a composition comprising an aqueous extract of black soldier fly larvae (BSFL) to unplanted soil, to planted soil, or to a plant, thereby reducing or preventing plant growth.F& RRefNo.: 55877-0008W01

[0013] In some embodiments, the applying comprises spraying the composition on unplanted soil. In some embodiments, the applying comprises spraying the composition on planted soil. In some embodiments, the applying comprises spraying the composition on a plant.

[0014] In some embodiments, the applying is performed at least once weekly. In some embodiments, the applying is performed multiple times a week. In some embodiments, the applying is performed daily. In some embodiments, the composition is applied daily. In some embodiments, the applying is performed a single time. In some embodiments, the single application is performed shortly before planting, at the time of planting, or shortly thereafter planting.

[0015] In some embodiments, reducing or preventing plant growth comprises killing the seedling or plant. In some embodiments, reducing or preventing plant growth comprises reducing emergence. In some embodiments, reducing or preventing plant growth comprises reducing plant biomass. In some embodiments, reducing plant biomass comprises reducing total foliage biomass, total root biomass, and / or total plant biomass.

[0016] In some embodiments, the plant is a monocot or a dicot. In some embodiments, the plant is flowering.

[0017] In another aspect, compositions are provided that include an aqueous extract of black soldier fly larvae (BSFL), wherein the aqueous extract comprises herbicidal properties.

[0018] In some embodiments, the aqueous extract comprises water. In some embodiments, the aqueous extract lacks organic solvent.

[0019] In some embodiments, the BSFL is meal. In some embodiments, the BSFL is defatted BSFL.

[0020] In some embodiments, a composition further includes a carrier. In some embodiments, the carrier is plant growth media. In some embodiments, a composition further includes one or more adjuvants (e.g,, surfactants, oils, buffers, conditioners, and / or delivery modifiers (e.g., anti-foaming agents, compatibility agents, drift reduction agents)). In some embodiments, a composition further includes glyphosate. In some embodiments, a composition further includes a commercial herbicide. In some embodiments, a composition further includes a commercial fertilizer.

[0021] In another aspect, methods of making an herbicidal composition are provided. Such methods typically include the steps of providing BSFL; adding an aqueous solventF& RRefNo.: 55877-0008W01

[0022] to generate a solution of about 80% water and about 20% BSFL (w / w); mixing the solution under conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted; and filtering the solution to remove particulates from the BSFL.

[0023] In some embodiments, the aqueous solvent is water. In some embodiments, the BSFL is dried and defatted BSFL. In some embodiments, the BSFL is BSFL meal.

[0024] In some embodiments, the conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted comprise mixing at room temperature for at least about 6 hours. In some embodiments, the conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted comprise mixing at 37°C for at least about 2 hours.

[0025] In still another aspect, herbicidal composition comprising, consisting essentially of, or consisting of at least one of the following compounds are provided:

[0026] DL-β-Leucine

[0027] Caprolactam

[0028] (S)-Menthiafolic acid

[0029] Tyramine

[0030] N-Benzoyl-DL-alanine

[0031] Indole-3-acetic acid

[0032] 3 -Methylsalicylic acid

[0033] 4-Amino-3-hydroxybenzoic acid

[0034] L-659-699

[0035] Di-n-Amyl phthalate

[0036] Trimethyl citrate

[0037] 4-Methylphenol

[0038] 4-Nitrobenzaldehyde

[0039] N-Acetyl-L-phenylalanine

[0040] 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide

[0041] 3,5-dihydroxydecanoic acid

[0042] Tyrosol

[0043] Phenol

[0044] Vanillic acid

[0045] Mimosine

[0046] 4-Nitro-o-anisidine

[0047] Trimethyl(phenylimino)phosphorane

[0048] (2R, 3R,4R,5R)-2-[( 1 OS)- 10, 13 -Dihydroxytridecyl] -5 -(hydroxymethyl)-3,4- pyrrolidinediol

[0049] Phloionolic acid

[0050] Acetoacetanilide

[0051] Fast Blue RR

[0052] 2-ethyl-N-[1-[2-(1-piperidinyl)ethyl]-2-benzimidazolyl]-3-pyrazolecarboxamide 3-hydroxydodec-5-enoic acid (NP-021797)

[0053] Phe-Glu

[0054]

[0055] 8-Hydroxyoctanoic acidF& RRefNo.: 55877-0008W01

[0056] 16-Hydroxyhexadecanoic acid

[0057] Thr-Phe

[0058] 4-Chloro-N-[3-(2-oxo-l-pyrrolidinyl)propyl]-3-({2-[3-(l-pyrrolidinyl

[0059]

[0060] Pinitol diacetonide

[0061] In some embodiments, such herbicidal compositions further include one or more carriers and / or one or more adjuvants.

[0062] In yet another aspect, one or more fractions from an aqueous extract of BSFL are provided that exhibit herbicidal properties, wherein the one or more fractions elute under the LC / MS conditions described in Example 11 at about 2 mins to about 6 mins (for fractions 7-8) and at about 13 mins to about 18 mins (for fractions 14-19).

[0063] In one aspect, compositions comprising an aqueous extract of black soldier fly larvae (BSFL) are provided, wherein the aqueous extract comprises herbicidal properties.

[0064] In some embodiments, the aqueous extract comprises water. In some embodiments, the aqueous extract lacks organic solvent. In some embodiments, the BSFL is dried and defatted BSFL. in some embodiments, the BSFL is meal.

[0065] In another aspect, methods of making a herbicidal composition are provided. Such methods typically include the steps of providing BSFL; adding an aqueous solvent to generate a solution of about 80% (e.g., 60% - 99%) water and about 20% (e.g., 1% - 50%) BSFL (weight by weight); mixing the solution under conditions in which water- soluble and / or hydrophilic compounds from the BSFL are extracted; and filtering the solution to remove particulates from the BSFL.

[0066] In some embodiments, the aqueous solvent is water. In some embodiments, the BSFL is dried and defatted BSFL. In some embodiments, the BSFL is BSFL meal.

[0067] In some embodiments, the conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted comprise mixing at room temperature for at least about 6 hours (e.g., at least about 12 hours; at least about 24 hours; at least about 36 hours). In some embodiments, the conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted comprise mixing at 37°C for at least about 2 hours (e.g., at least about 8 hours; at least about 18 hours; at least about 24 hours).

[0068] In still another aspect, methods of reducing or preventing plant growth are provi ded. Such methods typically include applying any of the composition described herein to unplanted soil (e.g., pre-planting), to planted soil (i.e., containing seed), or to a plant (e.g., on the foliage of a plant), thereby reducing or preventing plant grow th.F& RRefNo.: 55877-0008W01

[0069] In some embodiments, the applying comprises spraying any of the compositions described herein on unplanted soil (e.g., pre -planting). In some embodiments, the applying comprises spraying any of the compositions described herein on planted soil (i.e., containing seed). In some embodiments, the applying comprises spraying any of the compositions described herein on a plant (e.g., on the foliage of a plant).

[0070] In some embodiments, the applying is performed at least once weekly. In some embodiments, the applying is performed multiple times a week. In some embodiments, the applying is performed daily. In some embodiments, any of the compositions described herein is applied daily. In some embodiments, the applying is performed a single time. In some embodiments, the single application is performed shortly before planting, at the time of planting, or shortly thereafter planting.

[0071] In some embodiments, reducing or preventing plant growth comprises killing the seedling or plant. In some embodiments, reducing or preventing plant growth comprises reducing emergence. In some embodiments, reducing or preventing plant growth comprises reducing plant biomass. In some embodiments, reducing plant biomass comprises reducing total foliage biomass, total root biomass, and / or total plant biomass.

[0072] In some embodiments, the plant is a monocot or a dicot. In some embodiments, the plant is flowering.

[0073] In one aspect, compositions comprising an aqueous extract of black soldier fly larvae (BSFL) meal are provided, wherein the aqueous extract comprises herbicidal properties. In some embodiments, tire aqueous extract comprises water. In some embodiments, the aqueous extract lacks, or essentially lacks, organic solvent.

[0074] In another aspect, methods of making a herbicidal composition are provided. Such methods ty pically include the steps of providing BSFL meal; adding an aqueous solvent to generate a solution of about 80% (e.g., 60% - 99%) water and about 20% (e.g., 1% -50%) BSFL meal (weight by weight); mixing the solution under conditions in which water-soluble and / or hydrophilic compounds from the BSFL meal are extracted; and filtering the solution to remove particulates from the BSFL meal.

[0075] In some embodiments, the aqueous solvent is water. In some embodiments, the conditions in which water-soluble and / or hydrophilic compounds from the BSFL meal are extracted comprise mixing at room temperature for at least about 6 hours (e.g,, at least about 12 hours; at least about 24 hours; at least about 36 hours). In some embodiments, the conditions in which water-soluble and / or hydrophilic compounds from the BSFL mealF& RRefNo.: 55877-0008W01

[0076] are extracted comprise mixing at 37°C for at least about 2 hours (e.g., at least about 8 hours; at least about 18 hours; at least about 24 hours).

[0077] In still another aspect, methods of reducing or preventing plant growth are provided. Such methods typically include applying a composition described herein unplanted soil (e.g., pre-planting), to planted soil (i.e., containing seed), or to a plant (e.g., on the foliage of a plant), thereby reducing or preventing plant growth.

[0078] In some embodiments, the applying comprises spraying a composition described herein on unplanted soil (e.g., pre-planting). In some embodiments, the applying comprises spraying a composition described herein on planted soil (i.e., containing seed). In some embodiments, the applying comprises spraying a composition described herein on a plant (e.g., on the foliage of a plant).

[0079] In some embodiments, the applying is performed at least once weekly. In some embodiments, the applying is performed multiple times a week. In some embodiments, the applying is performed daily. In some embodiments, a composition described herein is applied daily. In some embodiments, the applying is performed a single time. In some embodiments, the single application is performed shortly before planting, at the time of planting, or shortly thereafter planting.

[0080] In some embodiments, reducing or preventing plant growth comprises killing the seedling or plant. In some embodiments, reducing or preventing plant growth comprises reducing emergence. In some embodiments, reducing or preventing plant growth comprises reducing plant biomass. In some embodiments, reducing plant biomass comprises reducing total foliage biomass, total root biomass, and / or total plant biomass.

[0081] In some embodiments, the plant is a monocot or a dicot. In some embodiments, the plant is flowering.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary’ skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.F& RRefNo.: 55877-0008W01

[0083] DESCRIPTION OF DRAWINGS FIG. 1 are photographs of plants at day 0. FIG. 1A are China Doll: 1'1= Control (no application), T2 = herbicide applied to foliage, T3 = herbicide applied to soil / base of plant, and T4 = positive control (water applied at same rate as herbicide). FIG. 1B are Pansies: 1'5 = Control (no application), T6 = Herbicide applied to foliage, T7 = Herbicide applied to soil / base of plant, and T8 = Positive control (water applied at same rate as herbicide). See, also, Example 7.

[0084] FIG. 2 are photographs of plants at day 7. FIG. 2A are China Doll: Tl= Control (no application), T2. = herbicide applied to foliage, T3 = herbicide applied to soil / base of plant, and T4 = positive control (water applied at same rate as herbicide). FIG. 2B are Pansies: T5 = Control (no application), T6 = Herbicide applied to foliage, T7 = Herbicide applied to soil / base of plant, and T8 = Positive control (water applied at same rate as herbicide). See, also, Example 7,

[0085] FIG. 3 are photographs of plants at day 14. FIG. 3A are China Doll: Tl= Control (no application), T2 = herbicide applied to foliage, T3 = herbicide applied to soil / base of plant, and T4 = positive control (water applied at same rate as herbicide). FIG. 3B are Pansies: T5 = Control (no application), T6 = Herbicide applied to foliage, T7 = Herbicide applied to soil / base of plant, and T8 = Positive control (water applied at same rate as herbicide). See, also, Example 7.

[0086] FIG. 4 are photographs of plants at day 21, FIG, 4A are China Doll: Tl= Control (no application), T2 = herbicide applied to foliage, T3 = herbicide applied to soil / base of plant, and T4 = positive control (water applied at same rate as herbicide). FIG. 4B are Pansies: T5 = Control (no application), T6 = Herbicide applied to foliage, T7 = Herbicide applied to soil / base of plant, and T8 = Positive control (water applied at same rate as herbicide). See, also, Example 7.

[0087] FIG. 5A are photographs of plants exposed to Treatment 1, which corresponds to the Negative Control, 4 weeks post-potting.

[0088] FIG. 5B are photographs of plants exposed to Treatment 2, which corresponds to Blanched Larvae Filtrate, 4 weeks post-potting.

[0089] FIG. 5C are photographs of plants exposed to Treatment 3, which corresponds to a water extract of dried meal, 4 weeks post-potting,

[0090] FIG. 6 is a representative chromatogram showing the fractions that eluted from the aqueous extract of BSFL meal under the conditions described in Example 11.F& RRefNo.: 55877-0008W01

[0091] DETAILED DESCRIPTION

[0092] Herbicidal. Compositions

[0093] Compositions that include an aqueous extract of black soldier fly larvae (BSFL) are described herein. Such compositions exhibit herbicidal properties and are referred to herein as “liquid bioherbicide.” It would be appreciated that the aqueous extract in the composition can be water-based, and organic solvents are not necessary (i,e,, the aqueous extract lacks organic solvent).

[0094] Methods of Making Liquid Bioherbicidal Compositions

[0095] Methods for making an herbicidal composition also are described herein. Initially, BSFL is provided, and an aqueous solvent is added to the BSFL. The BSFL can be intact larvae (e.g, blanched larvae) or the BSFL can be in meal form. In some instances, the BSFL can be dried and / or defatted. As used herein, meal refers to a material whose particle size has been reduced, e.g., by grinding or comparable methods. Aqueous solvents are known in the art and generally include water and water-based buffers (e.g., phosphate buffered saline (PBS)).

[0096] Solutions that include about 80% water and about 20% BSFL (weight by weight (w / w)) are described herein, but a skilled artisan would understand that essentially any amount of water (e.g., about 50% to about 99%; about 60%; about 70%; about 75%; about 85%; about 90%; about 95%) can be used with essentially any amount of BSFL (e.g., about 1% to about 50%; about 10%; about 25%; about 30%; about 40%) as long as there is enough aqueous solvent to penetrate the BSFL, dissolve the aqueous compounds and diffuse them out from the BSFL. The term “about,” as used herein, means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a number or numerical range, it modifies that number or range by extending the boundaries above and below the numerical value(s) set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20% (e.g, a variance of 15%, 10%, 5%).

[0097] The aqueous solution including BSFL can be mixed under conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted. The BSFL in an aqueous solution can be mixed using any number of known methods including, for example, any type of stirring or mild agitation to maintain admixing of the BSFL and the aqueous solvent. It would be understood that mixing can take place at any number ofF& RRefNo.: 55877-0008W01

[0098] temperatures (e.g., about 4°C, about 22°C (e.g., room temperature), about 37°C, about 42°C or higher) for any length of time (e.g., at least about 2 hours, at least about 6 hours, at least about 8 hours, at least about 12 hours, at least about 18 hours at least about 24 hours, or at least about 36 hours), although it also would be understood that extractions under high temperatures and / or for longer periods of time can cause a degradation or decomposition of the extracted compounds, while extracts at lower temperatures and shorter periods of time can result in reduced yield.

[0099] Representative mixing conditions that can be used to bring about extraction of water-soluble and / or hydrophilic compounds can include mixing at room temperature for at least about 6 hours (e.g., at least about 12 hours; at least about 24 hours; at least about 36 hours) or mixing at 37°C for at least about 2 hours (e.g., at least about 8 hours; at least about 18 hours; at least about 24 hours).

[0100] After sufficient time has passed such that extraction has occurred, the solution can be filtered to remove the BSFL and any particulates from the aqueous phase. A skilled person would understand that the conditions in which water-soluble and / or hydrophilic compounds from tire BSFL can be extracted can vary in yield and efficiency depending upon the form of BSFL (e.g., larvae or meal), the length of time the solution is mixed, and the temperature at which the solution is mixed. See, for example, Zhang et al., 2018, “■Techniques for extraction and isolation of natural products: a comprehensive review,” Chinese Medicine, 13:20.

[0101] A composition can include tire aqueous extract described herein (i.e., the liquid bioherbicide) and also can include a carrier. It would be appreciated that a homogeneous mixture is desired when tire liquid bioherbicide described herein is combined with any type of carrier. In some instances, the carrier can be plant growth media. Plant growth media can refer to any medium in or on which plants can grow. Non-limiting examples of plant growth media include soil, potting mixtures, mulch, peat, and combinations thereof. Plant growth media also can refer to the soil in a field or other geographical location having boundaries in which plants are grown. A bioherbicidal composition as described herein can include at least 0.1% by weight (e.g., at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% by weight) of the aqueous extract. Similarly, a bioherbicidal composition as described herein can include at least 0.1% by weight (e.g., at least 0.5%. 1%, 5%, 10%, 20%, 30%. 40%, 50%, 60%. 70%, 80%, 90% or 99% by weight) of a carrier. In some instances, the carrier can be an inert material (e.g., filler), orF& RRefNo.: 55877-0008W01

[0102] a commercial herbicide (e.g., one including glyphosate) to which the liquid bioherbicide described herein is added.

[0103] Alternatively, or additionally, tire insect meal can be combined (e.g., into a homogeneous mixture) with at least 0.1% by weight (e.g., at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% by weight) of one or more adjuvants. Adjuvants improve performance by enhancing spray coverage, penetration, and stability. As used herein, adjuvants include, without limitation, surfactants (wetting agents to reduce surface tension (e.g., non-ionic or organo-silicone)), oils (crop oil concentrates (e.g., a minimum of 80% oil mixed with 15 to 20% surfactant) or methylated seed oils for cuticle penetration (e.g., a methylated vegetable or seed oil (i.e., canola, cotton, linseed, soybean) mixed with an emulsifier)), buffers (e.g., pH buffers), conditioners (to modify the water carrier properties and enhance the effectiveness of herbicides (e.g,, ammonium sulfate (AMS) or AMS replacements such as citric acid, phosphoric acid, or the product of reacting urea wi th sulfuric acid)), and / or delivery' modifiers (e.g., volatility reduction agents (VRAs), anti-foaming agents, compatibility agents, drift reduction agents).

[0104] Methods of Using Liquid Bioherbicidal Compositions

[0105] Method of using the liquid bioherbicidal compositions described herein to reduce or prevent plant growth also are provided. Methods of applying the liquid bioherbicidal compositions to unplanted soil (e.g., pre-planting), to planted soil (e.g., containing seed), and to plants (e.g., on the foliage of a plant) are known in the art. The most common method of applying a liquid bioherbicidal composition is by spraying on either the soil or the plants. Spraying can be accomplished using, without limitation, broadcasting, foliar, placement or fertigation.

[0106] It would be appreciated that the liquid bioherbicidal compositions described herein can be applied as often as necessary for the appropriate level of herbicidal activity’ that is desired (e.g., application at least once a week, multiple times a week, or daily). In some instances, the applying step can be performed a single time (e.g., shortly before planting, at tire time of planting, or shortly thereafter planting).

[0107] As demonstrated herein, applying the liquid bioherbicide described herein to soil (pre- or post-planting) or to plants (e.g., the foliage) can kill the seedling or plant, reduce emergence, and / or reduce plant biomass. As used herein, reducing plant biomass refers toF& RRefNo.: 55877-0008W01

[0108] reducing total foliage biomass, total root biomass, and / or total plant biomass. The herbicidal activity of the compositions described herein are effective against either monocots or dicots and is agnostic to whether or not the plant is flowering.

[0109] Embodiments

[0110] Embodiment 1 is a method of reducing or preventing plant growth, comprising: applying a composition comprising an aqueous extract of black soldier fly larvae (BSFL) to unplanted soil, to planted soil, or to a plant, thereby reducing or preventing plant growth

[0111] Embodiment 2 is the method of Embodiment 1, wherein the applying comprises spraying the composition on unplanted soil.

[0112] Embodiment 3 is the method of Embodiment 1 or 2, wherein the applying comprises spraying the composition on planted soil.

[0113] Embodiment 4 is the method of any of the preceding Embodiments, wherein the applying comprises spraying the composition on a plant.

[0114] Embodiment 5 is the method of any of the preceding Embodiments, wherein the applying is performed at least once weekly.

[0115] Embodiment 6 is the method of any of the preceding Embodiments, wherein the applying is performed multiple times a week.

[0116] Embodiment 7 is the method of any of the preceding Embodiments, wherein the applying is performed daily.

[0117] Embodiment 8 is the method of any of the preceding Embodiments, wherein the composition is applied daily.

[0118] Embodiment 9 is the method of any of the preceding Embodiments, wherein the applying is performed a single time.

[0119] Embodiment 10 is the method of any of the preceding Embodiments, wherein the single application is performed shortly before planting, at the time of planting, or shortly thereafter planting.

[0120] Embodiment 11 is the method of any of the preceding Embodiments, wherein reducing or preventing plant growth comprises killing the seedling or plant.

[0121] Embodiment 12 is the method of any of the preceding Embodiments, wherein reducing or preventing plant growth comprises reducing emergence.F& RRefNo.: 55877-0008W01

[0122] Embodiment 13 is the method of any of the preceding Embodiments, wherein reducing or preventing plant growth comprises reducing plant biomass.

[0123] Embodiment 14 is the method of any of the preceding Embodiments, wherein reducing plant biomass comprises reducing total foliage biomass, total root biomass, and / or total plant biomass.

[0124] Embodiment 15 is the method of any of the preceding Embodiments, wherein the plant is a monocot or a dicot.

[0125] Embodiment 16 is the method of any of the preceding Embodiments, wherein the plant is flowering.

[0126] Embodiment 17 is a composition comprising an aqueous extract of black soldier fly larvae (BSFL), wherein the aqueous extract comprises herbicidal properties.

[0127] Embodiment 18 is the composition of Embodiment 17, wherein the aqueous extract comprises water.

[0128] Embodiment 19 is the composition of Embodiment 17 or 18, wherein the aqueous extract lacks organic solvent.

[0129] Embodiment 20 is the composition of any one of Embodiments 17-19, wherein the BSFL is meal.

[0130] Embodiment 21 is the composition of any one of Embodiments 17-20, wherein the BSFL is defatted BSFL.

[0131] Embodiment 22 is the composition of any one of Embodiments 17-21, further comprising a carrier.

[0132] Embodiment 23 is the composition of any one of Embodiments 17-22, wherein the carrier is plant growth media.

[0133] Embodiment 24 is the composition of any one of Embodiments 17-23, further comprising one or more adjuvants (e.g., surfactants, oils, buffers, conditioners, and / or delivery modifiers (e.g., anti-foaming agents, compatibility agents, drift reduction agents)).

[0134] Embodiment 25 is the composition of any one of Embodiments 17-24, further comprising glyphosate.

[0135] Embodiment 26 is the composition of any one of Embodiments 17-25, further comprising a commercial herbicide.

[0136] Embodiment 27 is tire composition of any one of Embodiments 17-26, further comprising a commercial fertilizer.F& RRefNo.: 55877-0008W01

[0137] Embodiment 28 is a method of making an herbicidal composition, comprising the steps of: providing BSFL; adding an aqueous solvent to generate a solution of about 80% water and about 20% BSFL (w / w); mixing the solution under conditions in which water- soluble and / or hydrophilic compounds from the BSFL are extracted; and filtering the solution to remove particulates from the BSFL.

[0138] Embodiment 29 is the method of Embodiment 28, wherein the aqueous solvent is water.

[0139] Embodiment 30 is the method of Embodiment 28 or 29, wherein the BSFL is dried and defatted BSFL.

[0140] Embodiment 31 is the method of any one of Embodiments 28-30, wherein the BSFL is BSFL meal.

[0141] Embodiment 32 is the method of any one of Embodiments 28-31, wherein the conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted comprise mixing at room temperature for at least about 6 hours.

[0142] Embodiment 33 is the method of any one of Embodiments 28-32, wherein the conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted comprise mixing at 37°C for at least about 2 hours.

[0143] Embodiment 34 is an herbicidal composition comprising, consisting essentially of, or consisting of at least one of the following compounds:

[0144] DL-β-Leucine

[0145] Caprolactam

[0146] (S)-Menthiafolic acid

[0147] Tyramine

[0148] N-Benzoyl-DL-alanine

[0149] Indole-3-acetic acid

[0150] 3 -Methylsalicylic acid

[0151] 4-Amino-3-hydroxybenzoic acid

[0152] L-659-699

[0153] Di-n-Amyl phthalate

[0154] Trimethyl citrate

[0155] 4-Methylphenol

[0156] 4-Nitrobenzaldehyde

[0157] N-Acetyl-L-phenylalanine

[0158] 1 -ethyl-3 -[3 -(dimethylamino)propyl] carbodiimide

[0159] 3,5-dihydroxydecanoic acid

[0160] Tyrosol

[0161] Phenol

[0162] Vanillic acid

[0163] Mimosine

[0164]

[0165] 4-Nitro-o-anisidineF& RRefNo.: 55877-0008W01

[0166] Trimethyl(phenylimino)phosphorane

[0167] (2R,3R,4R,5R)-2-[(10S)-10,13-Dihydroxytridecyl]-5-(hydroxymethyl)-3,4-pyrrolidinediol

[0168] Phloionolic acid

[0169] Acetoacetanilide

[0170] Fast Blue RR

[0171] 2-ethyl-N-[1-[2-(1-piperidinyl)ethyl]-2-benzimidazolyl]-3-pyrazolecarboxamide 3 -hydroxy dodec-5-enoic acid (NP-021797)

[0172] Phe-Glu

[0173] 8-Hydroxyoctanoic acid

[0174] 16-Hydroxyhexadecanoic acid

[0175] Thr-Phe

[0176] 4-Chloro-N-[3-(2-oxo-l-pyrrolidinyl)propyl]-3-({2-[3-(l-pyrrolidinyl

[0177] sulfonyl)benzoyl]hydrazino}carbonyl)benzenesulfonamide

[0178]

[0179] Pinitol diacetonide

[0180] Embodiment 35 is the herbicidal composition of Embodiment 34, further comprises one or more carriers and / or one or more adjuvants.

[0181] Embodiment 36 is one or more fractions from an aqueous extract of BSFL that exhibit herbicidal properties, wherein the one or more fractions elute under LC / MS conditions at about 2 mins to about 6 mins (for fractions 7-8) and at about 13 mins to about 18 mins (for fractions 14-19).

[0182] In accordance with the present invention, there may be employed molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. Tire invention will be further described in the follow ing examples, w hich do not limit the scope of the methods and compositions of matter described in the claims.

[0183] EXAMPLES

[0184] Example 1 ---Product Manufacturing

[0185] Water extract from BSFL meal (referred to herein as ‘liquid bioherbicide”) is made using a solution of 20% BSFL meal and 80% water (weight by weight). This mixture is stirred at room temperature for at least 24 hours, followed by a filtration step to remove any remaining particulate matter. Resulting material can then be sprayed onto soil to prevent plant emergence from soil.F& RRefNo.: 55877-0008W01

[0186] Example 2 ---Liquid Application of BSFL Meal -----Daily Application Replacing Water Purpose

[0187] To determine if water extract from BSFL meal can be used as a liquid bioherbicide by replacing daily watering.

[0188] Methods & Results

[0189] This trial examined the herbicide effect of the water extract from BSFL meal. The water extract from BSFL meal was tested at an inclusion rate of 12 grams per day added to each 4” pot. N=6 replicate pots per treatment, with 2 experimental treatments, for a total of 12 pots on study. Primary response variables were measured at 22 days (approximately 3 weeks) and include seedling emergence (% of plants emerged).

[0190] All soil was steam sterilized using a benchtop autoclave to reduce risk of mold and bacterial contamination. Three cucumber seeds were planted at an appropriate depth in each 4’’ pot and then grown in a GEN1000 plant growth chamber (Conviron, Model #: GEN 1000). Light conditions were set at 10 h dark and 14 h light. Temperature conditions were set at 22.8°C. Plants were watered daily to maintain soil moisture.

[0191] Table 1: Treatment Description

[0192] Treatment # Description N

[0193] 1 Control - water daily 6 2 12g liquid bioherbicide applied daily 6

[0194]

[0195] TOTAL, POTS 12

[0196] Table 2: Emergence at Harvest (% of Plants)

[0197] Treatment T1 T2

[0198] Emergence # at Harvest. 94% 0%

[0199]

[0200] SEM23% 0%

[0201] 2SEM:::Standard Error of the Mean

[0202] Overall Conclusions

[0203] Plants in the control group emerged more readily than those in the treatment group. Treatment group drastically reduced emergence (50% effect) over the 3 -week period. At 3 weeks, no plants were visible in any of the treatment group pots.F& RRefNo.: 55877-0008W01

[0204] Example 3 - --Liquid Application of BSFL Meal - --Water Equalization

[0205] Purpose

[0206] To determine if water extract from BSFL meal can be used as a liquid bioherbicide by replacing daily watering. The total water amount provided to each pot was equalized to account for the different volumes of water in the various treatments.

[0207] Methods & Results

[0208] This trial examined the herbicide effect of a water extract derived from BSFL meal. BSFL meal water extract was tested at an inclusion rate of 12 grams per day added to each 4” pot. In pots provided with the water extract from BSFL meal, additional water was added to ensure plants received the same amount of total moisture regardless of treatment. N=6 replicate pots per treatment, with 2 experimental treatments, for a total of 12 pots in the study. Primary response variables were measured at 26 days (approximately 4 weeks) and include seedling emergence (% of plants emerged), total plant biomass, total foliage biomass, and total root biomass.

[0209] All soil was steam sterilized using a benchtop autoclave to reduce risk of mold and bacterial contamination. Three cucumber seeds and 3 onion seeds were planted at an appropriate depth in each 4” pot and then grown in a GEN 1000 plant growth chamber (Conviron, Model # GEN 1000). Light conditions were set at 10 h dark and 14 h light. Temperature conditions were set at 22.8°C. Plants were watered daily to maintain soil moisture.

[0210] Table 3: Treatment Description

[0211] Treatment # Description N

[0212] 1 Control – water daily 6 2 12g liquid bioherbicide applied daily + water 6

[0213]

[0214] TOTAL POTS 12

[0215] Table 4: Emergence at Harvest (% of Plants)

[0216] Treatment T1 T2

[0217] Emergence % at Harvest 100% 0%

[0218]

[0219] SEM24.4% 0%

[0220] 2SEM = Standard Error of the MeanF& RRefNo.: 55877-0008W01

[0221] Table 5: Plant Weights at Harvest

[0222] Treatment T1 T2 SEM2Total Plant Biomass (g) 5.63 0 0.53 Total Foliar Biomass (g) 4.08 0 0.32

[0223]

[0224] Total Root Biomass (g) 1.49 0 0.24

[0225] 2SEM = Standard Error of the Mean

[0226] Overall Conclusions

[0227] Plants in the control group emerged more readily than those in the treatment group. At 3 weeks, no plants were visible in any of the treatment group pots. Using the water extract from BSFL meal completely reduced all plant weights measured at harvest. Therefore, the water extract from BSFL meal is effective at reducing seedling emergence and associated harvest weights.

[0228] Example 4 — Titrated Application of Liquid Bioherbicide

[0229] Purpose

[0230] To determine how frequently liquid bioherbicide derived from BSFL meal should be applied in order to decrease seedling emergence and plant harvest weights.

[0231] Methods & Results

[0232] This trial examined the herbicide effect of a water extract derived from BSFL meal. BSFL meal water extract was applied at an inclusion rate of 12 grams per application to each 4” pot. liquid bioherbicide was applied at various frequencies to determine the frequency needed to suppress growth (applied once, weekly, 3x weekly, 5x weekly). N=6 replicate pots per treatment, with 5 experimental treatments, for a total of 30 pots in the study. Primary response variables were measured at 21 days (approximately 3 weeks) and include seedling emergence (% of plants emerged), total plant biomass, total foliage biomass, and total root biomass.

[0233] All soil was steam sterilized using a benchtop autoclave to reduce risk of mold and bacterial contamination, 3 cucumber seeds and 3 onion seeds were planted at an appropriate depth in each 4” pot and then grown in a GEN1000 plant growth chamber (Conviron, Model #: GEN1000). Light conditions were set at 10 h dark and 14 h light. Temperature conditions were set at 22.8°C. Plants were watered daily to maintain soil moisture.

[0234]

[0235] F& RRefNo.: 55877-0008W01

[0236] Table 6: Treatment Description

[0237] Treatment # Description N

[0238] 1 Control - water daily 6 2 12g liquid bioherbicide applied once at planting 6 3 12g liquid bioherbicide applied weekly 6 4 12g liquid bioherbicide applied 3x weekly 6 5 12g liquid bioherbicide applied 5x weekly 6

[0239]

[0240] TO TAL POTS 30

[0241] Table 7: Emergence at Harvest (% of Plants) Treatment Ti T2 T3 T4 T5 Emergence % at Harvest 89% 78% 61% 0% 0%

[0242]

[0243] SEM21.38% 1.25% 0.91% 0% 0%

[0244] 2SEM = Standard Error of the Mean

[0245] Table 8: Plant Weights at Harvest

[0246] Treatment Tl T2 T3 T4 T5 Total Plant Biomass (g) 5.815 7.737 4.317 0.072 0.05 Total Foliar Biomass (g) 2.703 4.245 3.213 0 0

[0247]

[0248] Total Root Biomass (g) 3.098 3.18 1.12 0.072 0.05

[0249] Overall Conclusions

[0250] Plants in the control group emerged more readily than those in the treatment groups. At 3 weeks, no plants were visible in pots applied with the liquid bioherbicide 3x or 5x weekly. Using liquid bioherbicide once at planting had minimal impact on all parameters measured. Using liquid bioherbicide weekly decreased seedling emergence, total plant biomass, and total root biomass at harvest. Therefore, water extract from BSFL meal is effective at reducing seedling emergence and associated harvest weights. Minor effects can be seen with a single application or weekly applications, while major effects are noted when applied more frequently (daily or every -other day).

[0251] Example 5 - - -Effective Dose When Applied at Planting

[0252] Purpose

[0253] To determine if frequent application of liquid bioherbicide derived from BSFL meal is necessary to reduce plant grow th, or if a higher dose could be used with only a single application.F& RRefNo.: 55877-0008W01

[0254] Methods & Results

[0255] This trial examined the herbicide effect of a water extract derived from BSFL meal. BSFL meal water extract was applied at an inclusion rate of either 0 g, 12 g, 36 g, or 72 g per 4” pot. N=6 replicate pots per treatment, with 4 experimental treatments, for a total of 24 pots on study. Primary response variables were measured at 24 days (approximately 3 weeks) and include seedling emergence (% of plants emerged), total plant biomass, total foliage biomass, and total root biomass.

[0256] All soil was steam sterilized using a benchtop autoclave to reduce risk of mold and bacterial contamination. Three cucumber seeds and 3 onion seeds were planted at an appropriate depth in each 4” pot and then grown in a GEN 1000 plant growth chamber (Conviron, Model #: GEN 1000). Light conditions were set at 10 h dark and 14 h light. Temperature conditions were set at 22.8°C. Plants were watered daily to maintain soil moisture.

[0257] Table 9: Treatment Description

[0258] Treatment # Description N

[0259] 1 Control - 0 g liquid bioherbicide applied 6 2 12 g liquid bioherbicide applied once at planting 6 36 g liquid bioherbicide applied once at planting 6

[0260] 4 72 g liquid bioherbicide applied once at planting 6

[0261]

[0262] TOTAL POTS 24

[0263] Table 10: Emergence at Harvest (% of Plants)

[0264] Treatment T1 T2 T3 T4

[0265] Emergence % at Harvest 92% 89% 39% 11%

[0266] SEM24% 4% 6% 6%

[0267]

[0268] 2SEM = Standard Error of the Mean

[0269] Table 11: Plant Weights at Harvest

[0270] Treatment T1 T2 T3 T4 Total Plant Biomass (g) 2 2.487 0.99 0.038 Total Foliar Biomass (g) 1.488 1.77 0.738 0.033

[0271]

[0272] Total Root Biomass (g) 0.498 0.685 0.255 0.008

[0273] Overall Conclusions

[0274] Liquid bioherbicide application of 36 g and 72 g once at planting had a major impact on seedling emergence, total plant biomass, total foliage biomass, and total rootF& RRefNo.: 55877-0008W01

[0275] biomass. Application rate of 12 g did not have a major impact on any parameters measured. Therefore, we can conclude that a minimum of 36 g of liquid bioherbicide is needed to impact plant growth in a 4” pot.

[0276] Example 6 — Efficacy of Liquid Bioherbicide on Mature Plants

[0277] Purpose

[0278] To determine if the liquid bioherbicide described herein can be used as an herbicide on mature pansies.

[0279] Methods & Results

[0280] This trial examined the herbicide effect of a w ater extract derived from BSFL meal. BSFL meal water extract was applied at an inclusion rate of 72 grams per 4” pot. N=6 replicate pots per treatment, with 4 experimental treatments, for a total of 24 pots on study. Photos were taken throughout the trial to analyze herbicide effect. Total foliage biomass was measured at 27 days (approximately 4 weeks).

[0281] All plants were reared in a GEN 1000 plant growth chamber (Conviron, Model #: GEN1000), Light conditions were set at 10 h dark and 14 h light. Temperature conditions were set at 22.8°C. Plants were watered daily to maintain soil moisture.

[0282] Table 12: Treatment Description

[0283] Treatment # Description N

[0284] 1 Control - 0 g liquid bioherbicide applied 6 4 72 g liquid bioherbicide applied 6

[0285]

[0286] TOTAL POTS 12

[0287] Table 13: Total Foliage Biomass at Harvest

[0288] Treatment T1 T2

[0289] Total Foliar Biomass (g) 6.89 1.01

[0290]

[0291] SEM 0.88 0.14

[0292] SEM = Standard Error of the Mean

[0293] Overall Conclusions

[0294] Liquid bioherbicide application of 72 g had a major impact on mature plants, causing visible stunting and death.F& RRefNo.: 55877-0008W01

[0295] Example 7 - -Efficacy of Liquid Bioherbicide on China Doll and Pansies

[0296] Purpose

[0297] To determine if liquid bioherbicide can be used as an herbicide on mature plants (flowering and non-flowering).

[0298] Methods & Results

[0299] This trial examined the herbicide effect of a water extract derived from BSFL meal. BSFL meal water extract was applied at an inclusion rate of 72 grams per 4” pot. N=6 replicate pots per treatment, with 4 experimental treatments, for a total of 24 pots on study. Photos were taken throughout the trial to analyze herbicide effect. Total foliage biomass was measured at 22 days (approximately 3 weeks).

[0300] All plants were reared in a GEN 1000 plant growth chamber (Conviron, Model #: GEN1000). Light conditions were set at 10 h dark and 14 h light. Temperature conditions were set at 22.8°C. Plants were watered daily to maintain soil moisture.

[0301] Table 14: Treatment Description

[0302] Treatment # Species Description N 1 China Doll Control - 0 g liquid bioherbicide applied 6 2 China Doll 72 g liquid bioherbicide applied to foliage 6 3 China Doll 72 g liquid bioherbicide applied to soil 6 4 China Doll Control - 72 g Water applied 6 5 Pansies Control - 0 g liquid bioherbicide applied 6 6 Pansies 72 g liquid bioherbicide applied to foliage 6 7 Pansies 72 g liquid bioherbicide applied to soil 6 8 Pansies Control - 72 g Water applied 6

[0303]

[0304] TOTAL POTS 48

[0305] Table 15: Total Foliage Biomass* at harvest Treatment T1 T2 T3 T4 T5 T6 T7 T8 Total Foliar Biomass (g) 9.56 7.45 1.99 11.04 8.88 12.36 1.97 11.67

[0306]

[0307] SEM 0.25 0.49 0.52 1.55 0.62 1.36 0.25 1.54 SEM = Standard Error of the Mean

[0308] * includes live and dead biomass (e.g., T2 killed much of the plant material but at a slower rate, so there was more biomass than T3 but it was dead / dying biomass).F& RRefNo.: 55877-0008W01

[0309] Overall Conclusions

[0310] Liquid bioherbicide application of 72 g had a major impact on mature flowering and non-flowering plants (pansies and China doll, respectively), causing visible stunting and death. See FIGs. 1 - 4.

[0311] Example 8 — Blanched Larvae has Herbicidal Activity

[0312] The purpose of this trial was to examine whether blanched larvae that is not dried and defatted has herbicidal activity as previously seen with the dried and defatted product. A total of three (3) treatments were used for this trial:

[0313] • Treatment 1 - Negative Control (water only).

[0314] • Treatment 2 - Blanched larvae filtrate. Blanched larvae were produced by placing frozen larvae in boiling water for 3-5 minutes. The blanched material was then homogenized in a commercial blender for 1 min, and 800 ml of water added to achieve a fluid solution. The solution was mixed at 24 hours, then filtered through cheesecloth to remove large particle. The resulting filtrate was used for Treatment 2.

[0315] • Treatment 3 - Water extract of dried and partially defatted larvae (standard treatment).

[0316] Catharanthus roseus (Pink periwinkle) were potted in 4-inch pots with 3 -inch depth potting soil. Each plant was randomly assigned to one of the treatments, with 5 replicate pots per treatment.

[0317] Initially, plants had their assigned treatment applied at 75 ml initial dosing. For Treatment 1 and 3, water was provided periodically to maintain soil moisture (3-4 times per week, 15 ml per application). For Treatment 2, blanched larvae filtrate was applied 3- 4 times per week at 30 ml per application, at the same frequency as water provisioning for Treatment 1 and 3.

[0318] After about 4 weeks, it was evident that Treatment 3 resulted in plant death (see FIG. 5C), as previously demonstrated (compare to FIGs. 3 and 4), while Treatment 1 resulted in no change or even a slight growth of plants (FIG. 5A). Treatment 2, blanched larvae filtrate, did not impact plant health as severely as Treatment 3, but it was clear that there was some negative impact on the growth and health of plants treated with this material (FIG. 5B).F& RRefNo.: 55877-0008W01

[0319] Example 9 ---Agar Assay with BSFL Meal

[0320] Summary

[0321] Liquid bioherbicide inhibited growth and germination of cucumbers grown on agar. The previous experiments were confirmed that the full-strength (unseparated) liquid bioherbicide was effective at inhibiting, as was the same fractions previously shown to inhibit germination and affect root growth. Commercial compounds IAA (indole-3-acetic acid) and mimosine also inhibited germination and showed similar root architecture defects, both alone and in combination.

[0322] Cucumber plants were used in these experiments. Seeds were prepared by surface sterilizing seeds in 1 % dilute bleach for 4 minutes under a laminar flow hood. Seeds were rinsed in sterile, deionized water and set aside for plating on agar.

[0323] 0.6% w / v agar was used in sterile, deionized water. Agar (3.6 g) was weighed and placed in 1 L Erlenmeyer flasks containing 600 mL DI water, 1% liquid bioherbicide (6 mL) was added before or after autoclaving. Flasks were covered with aluminum foil and autoclaved for 30 minutes.

[0324] Procedure

[0325] Autoclaved agar media was placed in a water bath at 60°C to prevent the agar from solidifying. A flask was removed from the water bath and placed in the laminar flow hood. Filter-sterilized liquid bioherbicide was added to the media and swirled to mix. Equal portions of the liquid were poured into sterile petri dishes and the media allowed to solidify (~ 10 mins). Using sterile forceps, 4 cucumber seeds were placed onto the surface of the solidified agar in Pyrex dishes. The radicle end of the cucumber seed should be slightly pushed into the top 0.25 inches of agar to allow roots to grow into the agar media. Petri dishes were wrapped in breathable tape and placed in a growth chamber under the following conditions: 23°C day temp, 20°C night temp; 65% humidity; 14 hour days (0800 - 2200); and 300 μmol light intensity.

[0326] Cucumbers germinated at about 7 days after treatment (DAT) and images were obtained at 7 DAT. Alternatively, the seeds can be treated with the liquid bioherbicide at 7 DAT and images obtained.

[0327]

[0328] F& RRefNo.: 55877-0008W01

[0329] Example 10 - --Evaluate BSFL Meal Activity

[0330] Experiment Description Overview

[0331] Cucumber plants were tested in a controlled growth chamber for the effect of BSFL meal on plant growth using image analysis.

[0332] BSFL meal was tested with standard potting soil with or without sterilization via autoclaving.

[0333] Four (4) application rates of the BSFL meal, plus a control, were tested -with five (5) biological replicates per treatment.

[0334] Su mmary of Conclusions

[0335] Insect meal inhibits germination in a dose-dependent fashion.

[0336] When plants do germinate, insect meal reduces plant health. Specifically, plants are smaller and more yellow with increasing amounts of insect meal.

[0337] Autoclaving the BSFL meal did not have a statistically significant effect on germination or growth compared to non-autoclaved BSFL meal.

[0338] Description of Materials and Methods

[0339] Cucumber seeds Cucumis sativus, variety Marketmore 76; Johnny’s Seeds), were planted ~1 cm deep in standard potting soil in standard pots. Plants were grown in a Conviron walk-in growth chamber with standard conditions (23°C day / 20°C night, 65% humidity, 300 μmol light intensity, 14 hr day length) and watered daily with a hose and standard industrial water (i.e., not water containing nutrients). All plants were allowed to drain thoroughly and independently so that no active compounds could be absorbed by neighboring pots.

[0340] Plants were imaged from above once per hour during daylight hours using a Raspberry Pi camera. Images were analyzed using high-throughput image analysis software PlantCV, Briefly, images were color corrected and size standardized using a color card. Plants were separated from the background using computer vision. Each plant was then measured for germination date, leaf area (plant size), and plant color (hue circular mean), which is a representation of plant health. Outputs include quantitative measurements for each plant.

[0341] Both frequentist (traditional) and Bayesian statistics was used to determine significant differences due to product, autoclaving status, and soil nutrient status.F& RRefNo.: 55877-0008W01

[0342] Experiment 1

[0343] Experiment 1 used only standard potting soil (BM7-35%) which includes a “starter charge” or nutrients; Experiment 1 did not include a “no starter charge” soil type. Insect meal was autoclaved or not autoclaved.

[0344] Table 16. Inclusion Rates

[0345] Treatment ID Soil type Autoclave status Meal loading

[0346] T01 BM7 not autoclaved 0 lb / cubic foot

[0347] T03 BM7 not autoclaved 0.25 lb / cubic foot

[0348] T04 BM7 not autoclaved 0.5 lb / cubic foot

[0349] T05 BM7 not autoclaved 1 lb / cubic foot

[0350] T10 BM7 not autoclaved 1.5 lb / cubic foot

[0351] T07 BM7 autoclaved Autoclaved 0.25 lb / cubic foot T08 BM7 autoclaved Autoclaved 0.5 lb / cubic foot T09 BM7 autoclaved Autoclaved 1 lb / cubic foot

[0352]

[0353] T11 BM7 autoclaved Autoclaved 1.5 lb / cubic foot

[0354] Results

[0355] Treatment with autoclaved meal reduced germination rate. Treatment 7 (T07), 8 (T08), and 9 (T09) resulted in significantly delayed germination. Treatment with autoclaved meal at the highest concentrations reduced the hue of the plants (decreased greenness). Treatment had little to no effect on plant dry weight but had minor, but statistically significant, effects on plant wet weight.

[0356] Experiment 2

[0357] Experiment 2 used standard potting soil (BM7-35%). Meal was autoclaved or not autoclaved.

[0358] Table 17. Inclusion Rates

[0359] Treatment ID Soil type Autoclave status Meal loading

[0360] T01 BM7 not autoclaved 0 lb / cubic foot

[0361]

[0362] T03 BM7 not autoclaved 0.25 lb / cubic footF& RRefNo.: 55877-0008W01

[0363] T04 BM7 not autoclaved 0.5 lb / cubic foot

[0364] T05 BM7 not autoclaved 1 lb / cubic foot

[0365] T10 BM7 not autoclaved 1.5 Ib / cubic foot

[0366] T07 BM7 autoclaved Autoclaved 0.25 lb / cubic foot T08 BM7 autoclaved Autoclaved 0.5 lb / cubic foot T09 BM7 autoclaved Autoclaved 1 lb / cubic foot

[0367]

[0368] T11 BM7 autoclaved Autoclaved 1.5 lb / cubic foot

[0369] Results

[0370] The effect of autoclaved meal on plants was not statistically significantly different from the effect of non-autoclaved meal. 0.5 lb of meal per cubic yard of soil as well as the higher amounts significantly reduced germination rates. Those plants that did germinate had reduced leaf area and reduced hue circular mean (i.e., reduced greenness). These are measures of plant health and suggest that, even when the plants are able to germinate, they are smaller and less healthy than the control plants.

[0371] Example 11 — Fractionating Liquid Bioherbicide and Evaluating the Fractions Experimental Overview

[0372] The liquid bioherbicide described herein was separated into fractions using liquid chromatography (LC) to test individual fractions for plant growth effects. Liquid bioherbicide and the subsequent fractions were evaluated using LC-MS / MS to identify putative metabolites present. Two different plant species (cucumber and onion) were used in the evaluation, with only cucumber being progressed after the first experiment. A petri plate assay was developed and validated for evaluating liquid bioherbicide on plant germination and growth.

[0373] Germination rate was not a useful method for evaluating the liquid bioherbicide and the subsequent fractions because the seeds germinate (root emerges from seed) in the presence of the liquid bioherbicide and the subsequent fractions, then arrest growth. The liquid bioherbicide and the subsequent fractions caused reduction in green tissue emergence and a distinct root phenotype of thicker primary root and stubby secondary roots. Plant phenotype at the end of the plate assay was a successful evaluation method.F& RRefNo.: 55877-0008W01

[0374] LC-MS / MS Method

[0375] Pooled samples for each treatment condition were generated for data-dependent MS2 analyses by combining 20 pL of each sample in a specific treatment together. LC-MS data was acquired using a Thermo QE Orbitrap mass spectrometer coupled to a Thermo Dionex UltiMate 3000 UHPLC system. The QE was operated with an ESI voltage of +4.2 kV (positive ESI) and - 3,9 kV (negative ESI), transfer tube temperature of 250°C, sheath / aux / sweep gas settings of 10 / 5 / 1 respectively, and a vaporizer gas temperature of 60 °C. Full-scan MS1 spectra were acquired using polarity switching with a resolution of 70,000 @ m / z 250, an AGC target of 1e6, max inject time of 100 ms, and a scan range from m / z 120 to 1700. Data-dependent MS2 spectra were acquired for the top 12 ions in a scan with a resolution of 17,500 @ m / z 250, an AGC target of 1e5, max inject time of 50 ms, and stepped NCE of 25, 45, and 55. To increase profiling depth of the data-dependent scans, the scan range was divided into three sections (m / z 100 to 300, 300 to 500, and 500 to 1200), and MS2 spectra were acquired separately for each section in both positive and negative mode, for a total of six MS2 raw files per pooled sample.

[0376] Petri Dish Bioassay

[0377] Petri dishes provide a fast, simple screening method for evaluating compound effects on plant growth. In addition, they provide a clear view of plant roots as they are growing. Plates are prepared in a sterile environment (biological flow hood). Liquid, autoclaved agar is mixed with specified amounts of the liquid bioherbicide or the subsequent fractions and poured into plastic petri dishes, then allowed to solidify.

[0378] Cucumber and / or onion seeds are bleached for surface sterilization and placed onto the plates. Plates are grown undisturbed for 7-10 days in a controlled growth chamber (23°C day / 20°C night, 65% humidity, 300 μmol light intensity, 14 hr day length). Four seeds were placed on each plate, with three plates per treatment.

[0379] Plates were initially evaluated by recording date of germination, and using images of the plants upon completion, as well as by measuring the weight of the seedlings. In subsequent experiments, only plant images were collected upon completion, and only in cucumber, given the quality of each experimental method. Active fractions were evaluated as most similar to the positive control (full-strength liquid bioherbicide) with phenotypes of reduced green tissue, reduced germination, and abnormal root systems.F& RRefNo.: 55877-0008W01

[0380] HPLC Method

[0381] Filter sterilized liquid bioherbicide was concentrated in a vacuum and redissolved in 50% MeOH at double the original concentration. The sample was fractionated using a HIL1C method with a 4.6 x 250 mm amino column (Supelco). Solvent was A: 10 mM ammonium bicarb in H2O B: 95:5 Acetonitrile with 10 mM ammonium bicarb 1.5 mL / min. Gradient was 100% B (0-1 min), decreased to 30% B over 19 min (1-20 min), held at 30% B for 5 min (20-25 min), then re-equilibrated to 100% B (25-30 min) to prepare for the next injection.

[0382] Results

[0383] 757 putative metabolites were found in the crude liquid bioherbicide; 137 putative metabolites were identified based on a library search. Fractionation and LC-MS / MS of each fraction as described herein resulted in 2,981 putative metabolites. Chromatographic fraction 7-8 (eluting at about 2 mins to about 6 mins) and fraction 14-19 (eluting at about 13 mins to about 18 mins) of the liquid bioherbicide were identified as active, while other fractions exhibited little to no activity. See the chromatogram in FIG. 6.

[0384] Thirty-four unique metabolites were found in fractions 7-8. Twenty-seven metabolites were putatively identified based on a library search (see Table 18 below, listed in order of most to least abundant). (S)-menthiafolic acid, L-659-699, and mimosine are representative candidates for the observed activity in fractions 7-8. Twenty-four unique metabolites were found in fractions 14-19. Seven metabolites were putatively identified based on a library search (see Table 19 below', listed in order of most to least abundant). Phe-Glu is a representative candidate for the observed activity from fractions 14-19.

[0385] Table 18. Putative metabolites identified from Fractions 7-8 DL-β-Leucine _ Caprolactam _

[0386] (S)-Menthiafolic acid _ Tyramine _

[0387] N-Benzoyl-DL-alanine _

[0388] Indole-3 -acetic acid _

[0389] 3 -Methylsalicylic acid _

[0390] 4-Amino-3-hydroxybenzoic acid _ C659-699

[0391] Di-n-Amyl phthalate _

[0392]

[0393] F& RRefNo.: 55877-0008W01

[0394] Trimethyl citrate

[0395] 4-Methylphenol

[0396] 4-Nitrobenzaldehyde

[0397] N-Acetyl-L-phenylalanine

[0398] 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide

[0399] 3,5-dihydroxydecanoic acid

[0400] Tyrosol

[0401] Phenol

[0402] Vanillic acid

[0403] Mimosine

[0404] 4-Nitro-o-anisidine

[0405] Trimethyl(phenylimino)phosphorane

[0406] (2R,3R,4R,5R)-2-[( 1 OS)- 10, 13 -Dihydroxytridecyl] -5- (hydroxymethyl)-3,4-pyrrolidinediol

[0407] Phloionolic acid

[0408] Acetoacetanilide

[0409] Fast Blue RR

[0410] 2-ethyl-N-[l-[2-(l-piperidinyl)ethyl]-2-benzimidazolyl]-3-

[0411]

[0412] pyrazolecarboxamide

[0413] Table 19. Putative metabolites identified from Fractions 14-19 3-hydroxydodec-5-enoic acid (NP-021797) _

[0414] Phe-Glu _

[0415] 8-Hydroxyoctanoic acid _

[0416] 16-Hydroxyhexadecanoic acid _

[0417] Thr-Phe _

[0418] 4-Chloro-N-[3-(2-oxo-1-pyrrolidinyl)propyl]-3-({2-[3-(1-pyrrolidinylsulfonyl)benzoyl]hydrazino}carbonyl)benzenesulfonamide _

[0419]

[0420] Pinitol diacetonide

[0421] Fractions 7-8 (pooled) were subfractionated to further identify activity. Briefly, fifteen treatments (and a control) were designed and are described below in Table 20. For each treatment, three plates were used with 6 mL media in each (20 mL of media prepared per treatment). The method described above was used with the following modifications. Treatments (0.2 mL) were performed in 50 mL falcon tubes. Media was autoclaved in 500 mL containers. Two separate bottles containing 175 mL DI water and 1.05 g of agar were prepared and autoclaved. In the laminar flow hood, 20 mL of agar was added to the respective treatment containers and swirled to mix.

[0422] The results of these experiments are shown in Table 21, Briefly, subfraction #8 (from original fractions 7-8) was the only subfraction that exhibited bioherbicidal activity,F& RRefNo.: 55877-0008W01

[0423] at a level that was similar to the herbicidal activity exhibited by IAA and mimosine (separately or together).

[0424] Table 20. Experimental Design

[0425] Tube

[0426] Number Content

[0427] 0 Negative control - media only

[0428] 1 Subfraction 1

[0429] 2 Subfraction 2

[0430] 3 Subfraction 3

[0431] 4 Subfraction 4

[0432] 5 Subfraction 5

[0433] 6 Subfraction 6

[0434] 7 Subfraction 7

[0435] 8 Subfraction 8

[0436] 9 Subfraction 9

[0437] 10 IAA (50 pM)

[0438] 11 Mimosine (125 pM)

[0439] 12 IAA and Mimosine (50 pM and 125 pM, respectively) 13 Original HPLC Fractions 7-8

[0440] 14 Filtered liquid bioherbicide

[0441]

[0442] 15 Unfiltered liquid bioherbicide

[0443] Table 21. Experimental Results

[0444] Tube Number Results

[0445] 0 Germination and growth as expected

[0446] 1 No herbicidal activity observed

[0447] 2 No herbicidal activity observed

[0448] 3 No herbicidal activity observed

[0449] 4 No herbicidal activity observed

[0450] 5 No herbicidal activity observed

[0451] 6 No herbicidal activity observed

[0452] 7 No herbicidal activity observed

[0453] 8 Germination inhibited and stubby root growth

[0454] 9 No herbicidal activity observed

[0455] 10 Germination inhibited and stubby root growth with many branches 11 Germination inhibited

[0456] 12 Germination inhibited and stubby root growth

[0457] 13 Exhibited bioherbicidal activity

[0458] 14 Exhibited bioherbicidal activity

[0459]

[0460] 15 Exhibited bioherbicidal activityF& RRefNo.: 55877-0008W01

[0461] Example 12 - -Evaluating Metabolites Identified in the liquid bioherbicide

[0462] Hie environmental stability and safety of each identified metabolite is assessed. For example, how long tire identified metabolite remains in the environment and other safety measures that may be necessary for EPA registration are determined. In addition, the activity of an identified metabolite can be tested, for example, against other plant species, in different soil types, under different environmental conditions, and application methods (e.g., spray vs. broadcasting).

[0463] It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0464] Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

Claims

F& RRefNo.: 55877-0008W01WHAT IS CLAIMED IS:

1. A method of reducing or preventing plant growth, comprising:applying a composition comprising an aqueous extract of black soldier fly larvae (BSFL) to unplanted soil, to planted soil, or to a plant,thereby reducing or preventing plant growth.

2. The method of claim 1, wherein the applying comprises spraying the composition on unplanted soil.

3. The method of claim 1, wherein the applying comprises spraying the composition on planted soil.

4. The method of claim 1, wherein the applying comprises spraying the composition on a plant.

5. Tire method of claim 1, wherein the applying is performed at least once ■weekly.

6. The method of claim 1, wherein the applying is performed multiple times a week.

7. lire method of claim 1, wherein the applying is performed daily.

8. The method of claim 1, wherein the composition is applied daily.

9. The method of claim 1, wherein the applying is performed a single time,10. The method of claim 9, wherein the single application is performed shortly before planting, at the time of planting, or shortly thereafter planting.

11. The method of claim 1, wherein reducing or preventing plant growth comprises killing the seedling or plant.F& RRefNo.: 55877-0008W0112. The method of claim 1, wherein reducing or preventing plant growth comprises reducing emergence.

13. The method of claim 1, wherein reducing or preventing plant growth comprises reducing plant biomass.

14. The method of claim 13, wherein reducing plant biomass comprises reducing total foliage biomass, total root biomass, and / or total plant biomass.

15. The method of claim 1, wherein the plant is a monocot or a dicot.

16. The method of claim 1, wherein the plant is flowering.

17. A composition comprising an aqueous extract of black soldier fly larvae (BSFL), wherein the aqueous extract comprises herbicidal properties.

18. The composition of claim 17, wherein the aqueous extract comprises water.

19. The composition of claim 17, w herein the aqueous extract lacks organic solvent.

20. The composition of claim 17, w herein the BSFL is meal.

21. The composition of claim 17, wherein the BSFL is defatted BSFL.

22. The composition of claim 17, further comprising a carrier.

23. lire composition of claim 17, wherein the carrier is plant growth media.F& RRefNo.: 55877-0008W0124. The composition of claim 17, further comprising one or more adjuvants (e.g., surfactants, oils, buffers, conditioners, and / or delivery modifiers (e.g., anti-foaming agents, compatibility agents, drift reduction agents)).

25. The composition of claim 17, further comprising glyphosate.

26. The composition of claim 17, further comprising a commercial herbicide.

27. lire composition of claim 17, further comprising a commercial fertilizer.

28. A method of making an herbicidal composition, comprising the steps of:providing BSFL;adding an aqueous solvent to generate a solution of about 80% water and about 20% BSFL (w / w);mixing the solution under conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted; andfiltering the solution to remove particulates from the BSFL,29. The method of claim 28, wherein the aqueous solvent is water.

30. The method of claim 28, wherein the BSFL is dried and defatted BSFL.

31. lire method of any claim 28, wherein the BSFL is BSFL meal.

32. The method of claim 28, wherein the conditions in which w'ater-soluble and / or hydrophilic compounds from the BSFL are extracted comprise mixing at room temperature for at least about 6 hours.

33. The method of claim 28, wherein the conditions in which water-soluble and / or hydrophilic compounds from the BSFL are extracted comprise mixing at 37°C for at least about 2 hours.F& RRefNo.: 55877-0008W0134. An herbicidal composition comprising, consisting essentially of, or consisting of at least one of tire following compounds:DL-β-LeucineCaprolactam(S)-Menthiafolic acidTyramineN-Benzoyl-DL-alaninelndole-3-acetic acid3 -Methyl salicylic acid4-Amino-3-hydroxybenzoic acidL-659-699Di -n- Amyl phthalateTrimethyl citrate4-Methylphenol4-NitrobenzaldehydeN -Acety 1-L-phenylalanine1 -ethyl-3 -[3 -(dimethylamino)propyl] carbodi imide3,5-dihydroxydecanoic acidTyrosolPhenolVanillic acidMimosine4-Nitro-o-anisidineTrimethyl(phenylimino)phosphorane(2R,3R,4R,5R)-2-[(10S)-l 0, 13 -Dihydroxytridecyl] -5 -(hydroxymethyl )-3, 4- pyrrolidinediolPhloionolic acidAcetoacetanilideFast Blue RR2-e thyl-N- [ 1 - [2-( 1 -piperidiny 1 ) ethyl] -2-benzimidazoly 1] -3 -py razolecarboxamide 3-hydroxydodec-5-enoic acid (NP-021797)Phe-Glu8 -Hydroxy octanoic acid16-Hydroxyhexadecanoic acidThr-Phe4-Chloro-N-[ 3 -(2-oxo- 1 -py rrolidinyl)propyT| -3 -( { 2-[3 -( 1 -pyrrolidinylsulfonyl)benzoyT|hydrazino}carbonyl)benzenesulfonamidePinitol diacetonide35. The herbicidal composition further comprises one or more carriers and / or one or more adjuvants.36, One or more fractions from an aqueous extract of BSFL that exhibit herbicidal properties, wherein the one or more fractions elute under LC / MS conditions at about 2 mins to about 6 mins (for fractions 7-8) and at about 13 mins to about 18 mins (for fractions 14-19) under the conditions generally described in Example 11.

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