WO2020092644A1

WO2020092644A1 - Biologically generated and sustainable herbicide safener

Info

Publication number: WO2020092644A1
Application number: PCT/US2019/058959
Authority: WO
Inventors: Aaron L. WALTZ
Original assignee: Concentric Ag Corporation
Priority date: 2018-10-30
Filing date: 2019-10-30
Publication date: 2020-05-07

Abstract

The present invention provides biologically generated and sustainable herbicide safeners that can protect crops from herbicide injuries. Further provided are methods of making the herbicide safeners comprising a cell-free supernatant of a microbial mixture and methods of applying the herbicide safeners to plants.

Description

BIOLOGICALLY GENERATED AND SUSTAINABLE HERBICIDE SAFENER

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

62/752,932, filed October 30, 2018, which is hereby incorporated in its entirety by reference.

BACKGROUND

[0002] Numerous herbicides are used in crop production to help control unwanted plants.

Much of the time these herbicides are benign to the crop. However, herbicides can cause crop injury particularly when applied inappropriately, by injuring non-target plants.

[0003] Herbicides can injure foliage, shoots, flowers, and fruits. If injury is severe enough, either from one or repeated exposure, it can reduce yield, produce poor crop quality, distort ornamental or nursery plants, and occasionally cause plant death. Herbicides can reduce non-target plant vigor, increase susceptibility to disease, and shorten the life cycle of a plant. Herbicide injury to non-target plants can result in illegal residues on the exposed crop. In ornamental nursery plants, even slight herbicide symptoms can affect the marketability of damaged plants.

[0004] Herbicide symptoms can be visible for a few days to several years depending on the herbicide involved, plant species, stage and rate of growth, environmental and soil conditions, and cultural practices. Thus, sometimes, herbicide symptoms can be assessed based on yield loss or marketable yields.

[0005] Several herbicide safeners have been developed and are currently on the market.

However, they cannot be effective to all the herbicides in use, because herbicides have different mechanisms of action and can affect different target molecules in the plant. There is, therefore, a need for additional herbicide safeners that can manage injuries caused by the various herbicides. Moreover, safe, environmentally friendly, and sustainable herbicide safeners are needed to minimize harms to the environment.

SUMMARY

[0006] Cell-free supernatants of microbial mixtures provided herein are effective as herbicide safeners, i.e., are effective in protecting crops, e.g., corn, soy, wheat, and carrots, from an injury caused by herbicides. Described herein are herbicide compositions or kits comprising an herbicide and an herbicide safener. In some embodiments, the herbicide and the herbicide safener are provided as a single composition, e.g., in a single vessel. In some embodiments, the herbicide and the herbicide safener are provided as a kit, e.g., in separate vessels. Also described herein are methods of using the herbicide composition or kit, comprising applying the herbicide and the herbicide safener to a target plant, either in combination or sequentially. The herbicide safener is applied in an effective amount, i.e., in an amount effective to reduce an injury to a non target plant, e.g., a crop, caused by the herbicide.

[0007] The herbicide safener comprises an isolated cell free supernatant of a

microorganism mixture culture. The microorganism mixture culture is produced by inoculating a culture media with an isolated, mixed microbial composition and incubating the inoculated media for a period of time at an incubation temperature. The isolated cell free supernatant is produced by centrifugation and filter sterilization of the microorganism mixture culture.

[0008] In some embodiments, the isolated, mixed microbial composition is the following ten microorganisms: Aspergillus oryzae, Bacillus amyloliquefaciens , Candida utilis,

Lactobacillus helveticus , Lactococcus lactis , Lactobacillus paracasei, Lactobacillus plantarum , Lactobacillus rhamnosus , Rhodopseudomonas palustris, and Saccharomyces cerevisiae. In some embodiments, the isolated, mixed microbial composition is IN-M1, deposited under ATCC Accession No. PTA-12383, or IN-M2, deposited under ATCC Accession No. PTA-121556.

[0009] In some embodiments, the herbicide comprises a PPO inhibitor, e.g., acifluorfen.

In some embodiments, the herbicide comprises an EPSP synthase inhibitor, e.g., glyphosate; and/or a carotenoid biosynthesis inhibitor, e.g., a mesotrione; and/or a long chain fatty acid inhibitor, e.g., metolachlor; and/or a photosystem II inhibitor, e.g., atrazine.

[0010] In some embodiments, the non-target plant is an agricultural crop, e.g., com, soybean, wheat, or carrot.

[0011] The methods described herein comprise applying an effective amount of the herbicide safener. In some embodiments, the method comprises applying at least 0.010-10L of the herbicide safener per a dose of herbicide for a Hectare (ha). In some embodiments, the effective amount is determined based on a yield from the non-target plant. In some

embodiments, the effective amount is determined based on a visual inspection of health of the non-target plant. The herbicide safener and herbicide can be applied to the non-target plant or a soil for the non-target plant. In some embodiments, the step of applying the herbicide composition is performed by an overhead irrigation, drip irrigation, microjet sprinkler, or direct or broadcast soil or foliar spray. In some embodiments, the herbicide composition is applied at least 50 L/ha, 75 L/ha, 100 L/ha, 150 L/ha, 200 L/ha, 300 L/ha or 500 L/ha. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a graph illustrating percentage yield differences (y-axis) between (i) soybeans treated with an herbicide composition containing an herbicide provided in the x-axis and IN-M2A (black bars) or IN-M2B (gray bars), and (ii) control soybeans treated with the herbicide alone without IN-M2A or IN-M2B.

[0013] FIG. 2 is a graph illustrating percentage yield differences (y-axis) between (i) soybeans treated with an herbicide composition containing acifluorfen and a different amount of IN-M2A (black bars) or IN-M2B (gray bars) as provided in the x-axis (0.5, 2, 8, 32, or 64 oz/Acre of IN-M2A or IN-M2B), and (ii) control soybeans treated with acifluorfen alone without IN-M2A or IN-M2B.

[0014] FIG. 3 is a graph illustrating percentage yield differences (y-axis) between (i) soybeans treated with an herbicide composition containing glyphosate/dicamban herbicide program and a different amount of IN-M2A (black bars) or IN-M2B (gray bars), as provided in the x-axis (0.5, 2, 8, 32, or 64 oz/Acre of IN-M2A or IN-M2B) and (ii) control soybeans treated with the glyphosate/dicamban herbicide program alone without IN-M2A or IN-M2B.

[0015] FIG. 4 is a graph illustrating percentage yield differences (y-axis) between (i) soybeans treated with an herbicide composition containing glyphosate/S- metolachlor/mesotrione/atrazine herbicide program and a different amount of IN-M2A (black bars) or IN-M2B (gray bars), as provided in the x-axis (0.5, 2, 8, 32, or 64 oz/Acre of IN-M2A or IN-M2B) and (ii) control soybeans treated with the glyphosate/S-metolachlor/mesotrione/atrazine herbicide program alone without IN-M2A or IN-M2B.

[0016] FIG. 5A is a graph illustrating data from carrot fields treated with the herbicide composition at 3-5 leaf stage, and percentage yield differences of large carrots (left) and yields of total marketable carrots (right) with application of Dual Magnum® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars). FIG. 5B is a graph illustrating data from carrot fields treated with the herbicide composition at pre-leaf stage and percentage yield differences of large carrots (left) and yields of total marketable carrots (right) with application of Dual Magnum® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN- M2A (black bars) or IN-M2B (gray bars).

[0017] FIG. 6A is a graph illustrating data from carrot fields treated with the herbicide composition at 3-5 leaf stage, and illustrating percentage yield differences of large carrots (left) and total marketable carrots (right) with application of Lorox® mixed with different amounts

(0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars). FIG. 6B is a graph illustrating data from carrot fields treated with the herbicide composition at pre-leaf stage, and illustrating percentage yield differences of large carrots (left) and total marketable carrots (right) with application of Lorox® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars).

[0018] FIG. 7 is a graph illustrating percentage yield differences of large carrots (left) and marketable carrots (right) with application of Caparol® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars).

[0019] FIG. 8 is a graph illustrating percentage yield differences of large carrots (left) and marketable carrots (right) with application of Caparol® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars).

[0020] The figures depict various embodiments of the present invention for purposes of illustration only. One skilled in the art will readily recognize from the following discussion that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the invention described herein.

DETAILED DESCRIPTION

[0021] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

[0022] The term“a.i” refers to the active ingredient of an herbicide.

[0023] The term“microorganism” includes, but is not limited to, bacteria, viruses, fungi, algae, yeasts, protozoa, worms, spirochetes, single-celled, and multi-celled organisms that are included in classification schema as prokaryotes, eukaryotes, Archea, and Bacteria, and those that are known to those skilled in the art.

[0024] The term“carrier” refers to an“agriculturally acceptable carrier.” An

“agriculturally acceptable carrier” is intended to refer to any material which can be used to deliver a microbial composition as described herein, agriculturally beneficial ingredient(s), biologically active ingredient(s), etc., to a plant, a plant part (e.g., a seed), or a soil, and preferably which carrier can be added (to the plant, plant part (e.g., seed), or soil) without having an adverse effect on plant growth, soil structure, soil drainage or the like.

[0025] The term“effective amount” refers to an amount that produces the desired effect for which it is used. In the context of the present invention, an effective amount of an herbicide safener is an amount effective for reducing an injury to a non-target plant caused by a herbicide.

[0026] The term“strain” refers in general to a closed population of organisms of the same species. Accordingly, the term“strain of lactic acid bacteria” generally refers to a strain of a species of lactic acid bacteria. More particularly, the term“strain” refers to members of a microbial species, wherein such members, i.e., strains, have different genotypes and/or phenotypes. Herein, the term“genotype” encompasses both the genomic and the recombinant DNA content of a microorganism and the microorganism's proteomic and/or metabolomic profile and post translational modifications thereof. Herein, the term“phenotype” refers to observable physical characteristics dependent upon the genetic constitution of a microorganism. As one skilled in the art would recognize, microbial strains are thus composed of individual microbial cells having a common genotype and/or phenotype. Further, individual microbial cells may have specific characteristics (e.g., a specific rep-PCR pattern) which may identify them as belonging to their particular strain. A microbial strain can comprise one or more isolates of a

microorganism.

[0027] As described in more detail below, the term“herbicide” refers to a chemical or biological substance known in the art that can manage unwanted plants. The term encompasses both selective herbicides that control specific plant species while leaving desired plants, and nonselective herbicides that can be used to clear all plant material with which they come into contact.

[0028] As described in more detail below, the term“herbicide safener” refers to a chemical or biological substance that can be used in combination with herbicide to make the herbicide safer, e.g., to reduce a negative effect of the herbicide on a desired plant, such as a crop plant. The herbicide safener can work directly on the desired plant, or indirectly on a soil or environment for the desired plant to reduce the negative effect. The herbicide safener can be applied together with an herbicide or separately from an herbicide.

[0029] The term“target plant” as used herein refers to a plant that is targeted by herbicide to be removed or reduced from where the herbicide is applied.

[0030] The term“non-target plant” as used herein refers to a plant that is not targeted by herbicide. The non-target plant can be a crop plant that is desired to be protected from a target plant, or any other plant that is not intended to be removed or reduced by applying the herbicide.

[0031] The term“herbicide injury” as used herein refers to various negative symptoms of a non-target plant associated with treatment with herbicide. The symptoms include, but not limited to, symptoms on the foliage, shoots, flowers, and fruits, such as general and interveinal chlorosis, mottled chlorosis, yellow spotting, purpling of the leaves, necrosis, stem dieback, poor fruit quality, distort ornamental or nursery plants, and plant death. The symptoms may be visible or not visible. The symptoms further include reduction of plant vigor, increase of susceptibility to disease, shortened life cycle, reduction in plant size, reduction of growth of the seedlings, low yield, low marketable yield, etc. The herbicide injury can vary depending on various factors, such as the type of herbicide (e.g., herbicide’s mode of action), the amount and rate of herbicide application, methods of application, plant species, plant size, stage of growth, soil chemical and physical properties, soil moisture, temperature, relative humidity, etc.

[0032] The term“a soil for a plant” as used herein refers to a soil (1) where a plant to be treated with herbicide is currently rooted, or (2) where a plant will be planted. The soil is located, for example, in a pot, in a field, or in a greenhouse.

[0033] It must be noted that, as used in the specification and the appended claims, the singular forms“a,”“an” and“the” include plural referents unless the context clearly dictates otherwise.

[0034] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0035] For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40

41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

Herbicide compositions and kits

[0036] Described herein are herbicide compositions or kits comprising an herbicide and an herbicide safener. In some embodiments, the herbicide and the herbicide safener are provided as a single composition, e.g., in a single vessel. In some embodiments, the herbicide and the herbicide safener are provided as a kit, e.g., in separate vessels. Also described herein are methods of using the herbicide composition or kit, comprising applying the herbicide and the herbicide safener to a target plant, either in combination or sequentially. The herbicide safener is applied in an effective amount, i.e., in an amount effective to reduce an injury to a non-target plant, e.g., a crop, caused by the herbicide. Herbicides

[0037] The herbicide compositions and kits include an herbicide. Examples include a

PPO inhibitor, an EPSP synthase inhibitor, a carotenoid biosynthesis inhibitor, a long chain fatty acid inhibitor, and/or a photosystem II inhibitor. These herbicides can be used to control weeds in field crops, vegetables, tree fruits and vines, small fruits, nurseries, lawns, and industry.

[0038] The term“PPO inhibitor” refers to a class of herbicides inhibiting the function of

Protoporphyrinogen oxidase (PPO). Protoporphyrinogen oxidase (PPO) is an enzyme in the chloroplast cell that oxidizes protoporphyrinogen IX (PPGIX) to produce protoporphyrin IX (PPIX). PPO Inhibitors includes, but are not limited to, diphenylethers (e.g., Acifluorfen- sodium, Fomesafen, Lactofen, Oxyfluorfen), N-phenylphthalimides (e.g., Flumiclorac,

Flumioxazin), oxadiazoles (e.g., Oxadiazon), oxazolidinediones, phenylpyrazoles,

pyrimidindiones (e.g., Saflufenacil), thiadiazoles (e.g., Fluthiacet-methyl), and triazolinones (e.g., Carfentrazone-ethyl, sulfentrazone).

[0039] The term“EPSP synthase inhibitor” refers to a class of herbicides inhibiting the function of 5 -enolpyruvylshikimate-3 -phosphate (EPSP) synthase, a key enzyme in the shikimic acid pathway, which is involved in the synthesis of the aromatic amino acids. EPSP inhibition leads to depletion of the aromatic amino acids tryptophan, tyrosine, and phenylalanine that are needed for protein synthesis. EPSP synthase inhibitors include, but are not limited to, glycines (e.g., glyphosate).

[0040] The term“carotenoid biosynthesis inhibitor” refers to a class of herbicide interfering directly or indirectly with carotenoid production that protects chlorophyll from excessive light and photo oxidation. The carotenoid biosynthesis inhibitor includes three subgroups based on modes of action. The three subgroups are (1) herbicides inhibiting carotenoid biosynthesis at the phytoene desaturase step (PDS), including but not limited to,

Pyridiazinones (e.g., Norflurazon), Pyridinone (e.g., Fluridone), (2) herbicides inhibiting 4- hydroxyphenyl-pyruvate-dioxygenase (HPPD), including, but not limited to Callistemones (e.g.,

Mesotrione), isoxazoles (e.g., Isoxaflutole, Pyrasulfotole), and triketones (e.g., Tembotrione,

Topramezone), (3) herbicides inhibiting carotenoid biosynthesis via an unknown target, including, but not limited to, Isoxazolidinone (e.g., Clomazone) and triazole (e.g., Amitrole).

The carotenoid biosynthesis inhibitor can be mesotrione or a modification thereof.

[0041] The term“long chain fatty acid inhibitor” refers to a class of herbicide currently believed to inhibit very long chain fatty acid (VLCFA) synthesis. The long chain fatty acid inhibitor includes, but is not limited to, Acetamides (e.g., Napropamide), Chloroacetamides (e.g.,

Acetochlor, Alachlor, Dimethenamid, Metolachlor, L'-metolachlor, Propachlor), Oxyacetamides (e.g., Flufenacet). The long chain fatty acid inhibitor can be metolachlor or a modification thereof.

[0042] The term“photosystem II inhibitor” refers to a class of herbicides inhibiting photosynthesis by binding to Dl proteins of the photosystem II complex in chloroplast thylakoid membranes. Herbicide binding at Dl proteins blocks electron transport and stops C02 fixation and production of energy needed for plant growth. Photosystem II inhibitor includes, but is not limited to Phenyl-carbamates (e.g., Desmedipham, Phenmedipham), Pyridazinones (e.g.,

Pyrazon), Triazines (e.g., Ametryn, Atrazine, Prometon, Prometryn, Propazine, Simazine), Triazinones (e.g., Hexazinone, Metribuzin), Uracils (e.g., Bromacil, Terbacil), Amides (e.g., Propanil), Ureas (e.g., Diuron, Fluometuron, Linuron (e.g, Linex®, Lorox®),

Benzothiadiazinones (e.g., Bentazon), Nitrils (e.g., Bromoxynil), and Phenyl-pyridazines (e.g., Pyridate). The photosystem II inhibitor can be atrazine or a modification thereof.

Herbicide safener

[0043] The herbicide compositions and kits described herein include an herbicide safener.

The herbicide safener includes an isolated cell free supernatant of a microorganism mixture culture. The microorganism mixture culture is produced by inoculating a culture media with an isolated, mixed microbial composition and incubating the inoculated media for a period of time at an incubation temperature. The isolated cell free supernatant is produced by centrifugation and filter sterilization of the microorganism mixture culture.

[0044] In some embodiments, the isolated, mixed microbial composition includes the following ten microorganisms: Aspergillus oryzae, Bacillus amyloliquefaciens , Candida utilis, Lactobacillus helveticus , Lactococcus lactis , Lactobacillus paracasei, Lactobacillus plantarum , Lactobacillus rhamnosus , Rhodopseudomonas palustris, and Saccharomyces cerevisiae.

[0045] In some embodiments, the isolated, mixed microbial composition includes one or more of the following: Aspergillus oryzae , e.g., strain IN-AOl, deposited September 4, 2014 with ATCC, PTA-121551; Bacillus spp., for example, Bacillus amyloliquefaciens , e.g., strain IN- BSI, deposited January 11, 2012 with ATCC, PTA-12385; Rhodopseudomonas spp., for example, Rhodopseudomonas palustris, e.g., strain IN-RPI, deposited January 11, 2012 with ATCC, PTA-12387; Rhodopseudomonas palustris, e.g., strain IN-RP2, deposited September 4, 2014 with ATCC, PTA-121553; Candida spp., for example, Candida utilis, e.g., strain IN-CU1, deposited September 4, 2014 with ATCC, PTA-121550; Lactobacillus spp., for example, Lactobacillus helveticus, e.g., strain IN-LHI, deposited January 11, 2012, with ATCC, PTA- 12386; Lactobacillus rhamnosus, e.g., strain IN-LR1, deposited September 4, 2014 with ATCC, PTA-121554; Lactobacillus paracasei, e.g., strain IN-LC1, deposited September 4, 2014 with ATCC, PTA-121549; Lactobacillus plantarum , e.g., strain IN-LP1, deposited September 4, 2014 with ATCC, PTA-121555; Lactococcus spp ., for example, Lactococcus lactis , e.g., strain IN- LL1, deposited September 4, 2014 with ATCC, PTA-121552; Pseudomonas spp., for example, Pseudomonas aeruginosa or Pseudomonas fluorescens ; Saccharomyces spp., for example, Saccharomyces cerevisiae, e.g., strain IN- SCI, deposited on January 11, 2012 with ATCC, PTA- 12384; or Streptococcus spp., for example, Streptococcus lactis, or combinations thereof.

[0046] In some embodiments, the isolated, mixed microbial composition is IN-M1, deposited January 11, 2012 with ATCC, PTA-12383 and/or IN-M2, deposited September 4, 2014 with ATCC, PTA-121556.

[0047] IN-BSI, ATCC Deposit No. PTA-12385, was previously identified to be Bacillus subtilis in US Publication Nos. 20160100587 and 20160102251, and US Patent No. 9175258 based on 16S rRNA sequence and API testing, but later identified to be Bacillus

amyloliquefaciens by full genome sequencing. IN-LC1, ATCC Deposit No. PTA-121549, was previously identified to be Lactobacillus casei in US Publication Nos. 20160100587 and

20160102251, and US Patent No. 9175258 based on 16S rRNA sequence and API testing, but later identified to be Lactobacillus paracasei by full genome sequencing.

[0048] The microorganisms used in the present invention were selected for their capability to grow and live in a cooperative fashion, and to provide or synthesize compounds that are beneficial to their growth and survival. For example, the sources of cell-free supernatant compositions disclosed in the present disclosure can, during fermentation (culture), produce metabolites that are reactive in a cooperative manner.

[0049] The cell free supernatant of the microorganism mixture can be generated by a method known in the art. Relevant methods are described in US Publication Nos. 20160100587 and 20160102251, and US Patent No. 9175258, which are incorporated by reference in their entireties herein.

[0050] A culture media is inoculated with the isolated, mixed microbial composition; the inoculated media is incubated for a period of time at an incubation temperature. The culture media can be a fermentation, nutritive or culture broth well known to one of skill in the art. In one embodiment, the culture media includes water, a carbohydrate source, e.g., molasses or dextrose, a micronutrient source, e.g., calcium, magnesium, iron, zinc, e.g., a mineral power, sea salt, and wheat bran. In some embodiments, the culture media further includes kelp, bentonite clay, fish emulsion, and/or soy flour. [0051] The period of time can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or more. In some embodiments, the period of time is 6 weeks. The incubation temperature is 24-37, 30-32, 35-37, 32-37, or 30-37 degrees Celsius. In some embodiments, the incubation temperature is 30, 31, 32, 33, 34, 35, 36, or 37 degrees Celsius. In some embodiments, the incubation temperature is 30 degrees Celsius.

[0052] In one embodiment, the culture media is as follows, and the inoculated culture media is incubated at 30 degrees Celsius for 1 week:

[0053] The isolated cell free supernatant is produced by centrifugation and/or filter sterilization of the microorganism mixture culture. Centrifugation can be performed using any method well known to one of skill in the art, e.g., centrifuged at a centrifugal force of at least l0,000Xg for at least 10 minutes.

[0054] In some embodiments, filtration is performed and the filter membrane has a pore size of 0.1 pm, 0.2 pm, 0.3 pm, 0.4 pm, 0.5 pm, 1 pm, 10 pm, or 20 pm. In some embodiments, the filter membrane has a pore size between 0.1 and 5 pm. In some embodiments , the filter membrane has a pore size of 0.2 pm.

[0055] In some embodiments, the isolated cell free supernatant is further filtered with a filter membrane capable of passing through components smaller than lOkDa, 20kDa, 30kDa, 40kDa, 50kDa or lOOkDa. The filtration can be performed with a filter membrane capable of filtering out components larger than lOkDa, 20kDa, 30kDa, 40kDa, 50kDa or lOOkDa. The cell free supernatant obtained from the filtration can comprise components same as or smaller than lOkDa, 20kDa, 30kDa, 40kDa, 50kDa or lOOkDa.

[0056] The cell-free supernatant can be chemically characterized. In some embodiments, the cell-free supernatant is pH 3.7-4.0 and/or comprises 2000 - 3000 micrograms potassium per gram, and/or 600 - 1000 microgram sodium per gram, and/or 500-1000 microgram potassium per gram, and/or 300-350 microgram magnesium per gram, and/or 600 ppm sulphur, and/or 30- 130 ppm phosphorus.

Other components

[0057] The herbicide composition or kit of the present invention can contain other components. For example, it can comprise a nutrient component. The nutrient component can be powders, granules, or pellets, or a liquid, including solutions or suspensions, which contains nutrients in the solution or in the mixture.

Methods of using herbicide safeners

[0058] Also described herein are methods of using the herbicide composition or kit for protecting a non-target plant from an herbicide injury, comprising applying the herbicide and the herbicide safener to a target plant, either concurrently or sequentially. If applied concurrently, the herbicide and the herbicide safener can be applied as a single composition or as two compositions. The herbicide safener is applied in an effective amount, i.e., in an amount effective to reduce an injury to a non-target plant, e.g., a crop, caused by the herbicide.

[0059] The methods can be used to protect various non-target plants. The non-target plants can be a crop, a landscape plant, an ornamental plant, a nursery plant, or others. In particular embodiments, the crop is com, soybean, wheat or carrot.

[0060] The herbicide and the herbicide safener can be applied directly to a non-target plant, or to a soil currently planted with a non-target plant or a soil to be planted with a non-target plant in the future. In some embodiments, the herbicide and the herbicide safener is applied to the soil close to another soil planted or to be planted with the non-target plant. In some embodiment, a field to be treated with the herbicide and the herbicide safener has both a target plant and a non-target plant, only a target plant, or only a non-target plant.

[0061] The herbicide and the herbicide safener can be applied to all or some parts of the plant. For example, the herbicide and the herbicide safener can be applied to the stems, roots, leaves, and/or propagules (e.g., cuttings). The plant may be treated once or more than once during developmental stages.

[0062] The herbicide and the herbicide safener can be applied using a method currently used for application of a corresponding herbicide. For example, the herbicide and the herbicide safener is applied as a suspension, as wettable powders, or impregnated on a dry material (such as a fertilizer granule or peat pellet, or an emulsifiable concentrate. In some embodiments, the herbicide and the herbicide safener is applied through overhead irrigation, drip irrigation, microjet sprinklers, or as a directed or broadcast soil or foliar spray (bare ground application or to aboveground plant parts) with any standard application equipment typically used in crop production. Suitable application methods include, but are not limited to, high or low pressure spraying, drenching, coating, immersion, and soil injection. In various aspects, the herbicide and the herbicide safener can be applied to soil or other plant growth media and/or can be applied to seeds prior to or during planting. In some embodiments, the herbicide and the herbicide safener can be applied to a polymer as a wetting agent and/or gel that releases water as needed. In some embodiments, the herbicide and the herbicide safener can be applied to a delivery system for actives that affect solubility to concentrate actives for seed coatings.

[0063] The herbicide and the herbicide safener can be applied before emergence of a target plant or after emergence of a target plant. In some embodiments, the herbicide and the herbicide safener is applied before planting a non-target plant, or after planting a non-target plant. In some embodiments, the herbicide and the herbicide safener is applied at least 1 week, 2 weeks, 3 weeks, 1 months, 2 months, 3 months, 4 months, 5 months, or 6 months before planting the non-target plant. In some embodiments, the herbicide and the herbicide safener is applied at pre-leaf stage, at 1-2 leaf stage, at 3-4 leaf stage, at 3-5 leaf stage, or later. In some

embodiments, the herbicide and the herbicide safener is applied at least 1 week, 2 weeks, 3 weeks, 1 months, 2 months, 3 months, 4 months, 5 months, or 6 months after planting the non target plant. In some embodiments, the herbicide and the herbicide safener is applied 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 5-20 weeks, 10-15 weeks, 10-14 weeks, 11 weeks, 12 weeks, 13 weeks, or 14 weeks before harvesting a crop from the non-target plant. In some

embodiments, the herbicide and the herbicide safener is applied as needed, for example, when unwanted plants emerge. In some embodiments, the herbicide and the herbicide safener is applied regularly, once every week, once in 2 weeks, once in 3 weeks, once in 4 weeks, once in 5 weeks, once in 6 weeks, once in 8 weeks, once in 9 weeks, once in 10 weeks, once in 6 months, or once every year.

[0064] This technique can be useful in a variety of plant environments such as fields, greenhouse facilities, vertical farms, urban greening systems, and hydroponic systems.

[0065] In typical embodiments, an herbicide composition is generated by mixing an herbicide and an herbicide safener before applying it to a non-target plant or to a soil for a non target plant. Therefore, the method of protecting a non-target plant from an herbicide injury as provided herein can further comprise a step of generating an herbicide composition by mixing an herbicide and an herbicide safener. In some embodiments, the step of mixing an herbicide and an herbicide safener is performed by a person or an agent of a person who applies the herbicide composition to a non-target plant or to a soil for a non-target plant. In other embodiments, the step of mixing an herbicide and an herbicide safener is performed by a provider or a

manufacturer of an herbicide composition.

[0066] An herbicide composition is applied to a non-target plant or a soil for a non-target plant at an effective amount. An effective amount of an herbicide composition comprises an effective amount of herbicide and an effective amount of an herbicide safener. In some embodiments, an effective amount of an herbicide composition comprises an herbicide and an herbicide safener at an effective ratio.

Effective amount

[0067] The herbicide safener is applied at an effective amount to reduce an injury to a non-target plant caused by the herbicide. The effective amount can vary depending on the target plant, the non-target plant, and/or the herbicide.

[0068] Effective amounts can be identified by applying various amounts of the herbicide safener mixed with herbicide and testing their effects on plants. The effective amounts can be identified based on their effects on growth, yield, or survival of the non-target plant when applied to the non-target plant and/or the target plant together with the herbicide. In some embodiments, effective amounts can be identified based on the visual inspection of the plant health after application of the herbicide composition. In some embodiments, effective amounts can be identified based on chemical analysis of the non-target plant after application of the herbicide composition. The effective amounts can vary depending on whether the composition is applied to a field or to a pot.

[0069] In some embodiments, the effective amount is at least 0.010, 0.020, 0.030, 0.035,

0.050, 0.100, 0.140, 0.140, 0.200, 0.250, 0.500, 0.560, 1, 2, 2.240, 2.250, 3, 3.380, 3.500, 4, 5, or 10L of herbicide safener, i.e., cell free supernatant of the microorganism mixture per a dose of herbicide for a Hectare (ha). The total dose of the herbicide composition for a Hectare can be at least 50, 100, 200, 500 or 1000L. In some embodiments, the cell free supernatant of the microorganism mixture is 0.01%, 0.015%, 0.0175%, 0.03%, 0.05%, 0.07%, 0.1%, 0.15%, 0.2%, 0.25%, 0.28%, 0.3%, 0.4%, 0.5%, 0.75%, 1%, 1.1%, 1.12%, 1.25%, 1.5%, 1.6%, 1.69%, 1.75%, 2%, 2.5%, or 3% (v/v) of the herbicide composition.

[0070] In some embodiments, the herbicide comprises acifluorfen and the effective amount is at least 0.035L of the cell free supernatant of the microorganism mixture per l800g a.i. (active ingredient) of the herbicide. In some embodiments, the herbicide comprises acifluorfen and the effective amount is at least 0.140L, 0.560L, 2.240L, or 3.380L of the cell free supernatant of the microorganism mixture per l800g a.i. of the herbicide.

[0071] In some embodiments, the herbicide comprises glyphosate, dicamba, or glyphosate/dicambe and the effective amount is at least 2.240L of the cell free supernatant of the microorganism mixture per 5400g a.i. of the herbicide.

[0072] In some embodiments, the herbicide comprises glyphosate, mesotrione, metolachlor, atrazine, or glyphosate/mesotrione/metolachlor/atrazine, and the effective amount is at least 0.035L of the cell free supernatant of the microorganism mixture per 7455g a.i. of the herbicide. In some embodiments, the effective amount is at least 0.140L, 0.560L, 2.249L, or 3.380L of the cell free supernatant of the microorganism mixture per 7455g a.i. of the herbicide.

[0073] In some embodiments, the herbicide comprises linuron (e.g., Lorox®) and the effective amount is at least 0.035L of the cell free supernatant of the microorganism mixture per 1964 g a.i. of the herbicide. In some embodiments, the effective amount is at least 0.280L or 2.240L of the cell free supernatant of the microorganism mixture per l964g a.i. of the herbicide.

[0074] In some embodiments, the herbicide comprises triazinones (e.g., metribuzin) and the effective amount is at least 0.035L of the cell free supernatant of the microorganism mixture per 2l0g a.i. of the herbicide. In some embodiments, the effective amount is at least 0.280L or 2.240L of the cell free supernatant of the microorganism mixture per 2l0g a.i. of the herbicide.

[0075] Effective amounts can be identified by applying various amounts of the herbicide safener together with relevant herbicide and testing their effects on plants. The effective amounts can be identified based on health, growth, yield, or survival of the non-target plant treated with the composition. Therefore, in some embodiments, the method of protecting a non-target plant from an herbicide injury can further comprise a step of identifying an effective amount.

[0076] In some embodiments, an herbicide composition applied per ha includes at least

0.02L, 0.03L, 0.1L, 0.15L, 0.1L, 0.25L, 0.5L, 1L, 2L, 3L or 4L of a cell free supernatant of a microbial mixture. In some embodiments, an herbicide composition applied per Acre includes at least 0.25 Oz, 0.5 Oz, 1 Oz, 2 Oz, 3 Oz, 3 Oz, 8 Oz, 10 Oz, 20 Oz, 30 Oz, 32 Oz, 40 Oz, 50 Oz, 60 Oz, 64 Oz, 100 Oz, 200 Oz, 500 Oz, 1000 Oz or 2000 Oz of a cell free supernatant of a microbial mixture.

[0077] In some embodiments, an herbicide composition applied per ha includes between

0.01 and 20 L, between 0.01 and 10 L, between 0.02 and 5, or between 0.03 and 5 L of the cell free supernatant of a microbial mixture. In some embodiments, an herbicide composition applied per Acre includes between 0.1 and 500, between 0.1 and 250, between 0.1 and 100, between 0.2 and 80, or between 0.5 and 64 of the cell free supernatant of a microbial mixture. [0078] An effective amount of herbicide can be determined based on information available in the art, for example, as described in MP44 Recommended Chemicals for weed and brush control (Arkansas 2018). An effective amount of herbicide safener can be determined relative to the activity, concentration, or amount of herbicide that the herbicide safener is mixed with, or plant or soil that the herbicide safener is applied to.

Examples

[0079] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

[0080] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art.

Example 1: Production of an isolated cell free supernatant of a microorganism mixture culture

Production of an isolated, mixed microbial composition IN-MI

[0081] A microorganism mixture, IN-M1, deposited January 11, 2012 with ATCC Patent

Deposit Designation No. PTA-12383, was produced as described in International Pub. No.

WO2016178086, which is incorporated in its entirety herein.

Production of an isolated, mixed microbial composition IN-M2

[0082] A microorganism mixture, IN-M2, deposited September 4, 2014 with ATCC

Patent Deposit Designation No. PTA-121556, was produced as described in International Pub. No. WO2016178086, which is incorporated in its entirety herein.

Production of an isolated, mixed microbial composition IN-M2A and IN-M2B

[0083] A microorganism mixture comprising Aspergillus oryzae, Bacillus

amyloliquefaciens , Candida utilis, Lactobacillus helveticus , Lactococcus lactis , Lactobacillus paracasei, Lactobacillus plantarum , Lactobacillus rhamnosus , Rhodopseudomonas palustris, and Saccharomyces cerevisiae was produced by combining microorganisms deposited with ATCC as summarized below in TABLE 1.

[0084] The microorganisms were grown in 1000 liter batches in the following culture media:

[0085] In some embodiments an initial seed culture is generated with the microorganism mixture described in Table 1, and the seed culture is used to inoculate the culture media. In some embodiments, the culture media was inoculated with 50 liters of the seed culture.

[0086] The total volume of the inoculated culture media was brought up to 1000 liters with non-chlorinated warm water. The inoculated media was incubated for 1 week at 30 degrees Celsius.

Preparation of a cell free supernatant of the microorganism mixture

[0087] A cell-free supernatant (“CFS”) was obtained by centrifuging the inoculated microbial culture prepared using the methods as described above for at least 10 minutes at a centrifugal force of at least 10,000 x g. The supernatant was then filter sterilized with a 0.2 micron filter (Pall Centrastak C-100 unit/Pall microfiltration cassettes-Centrasette™ II Cassettes and plated to confirm the absence of cells.

[0088] The cell-free supernatant collected from the filtration step was stored and named as“IN-M2A.” In some cases, the IN-M2A cell free supernatant was further processed by filtering out components larger than 30kDa, following similar steps. The sample obtained from the additional filtration is named as“IN-M2B”

Example 2: Field experiment for testing herbicide safening effects of microbial

compositions

[0089] The two cell-free supernatants IN-M2A and IN-M2B generated as described above were tested for their safening effects on various herbicides. TABLE 2 provides a list of herbicide compositions comprising the cell-free supernatant and herbicide tested on corn.

TABLE 3 provides herbicide compositions tested on soybeans, TABLE 4 provide herbicide compositions tested on spring wheat, and TABLE 5 provides herbicide compositions tested on carrots.

Table 2: Herbicide compositions for corns

Table 3: Herbicide compositions for soybeans

[0090] The herbicide compositions (herbicide plus herbicide safener, i.e., cell free supernatants, described in Tables 2-5 were tested in a total of 13 field trials - 3 with com, 4 with soybeans, 2 with spring wheat, and 4 with carrots. Each of the herbicide programs was applied to the crops using higher than normal rates (about 3 times of the labeled, standard rates) to amplify the potential herbicide damage and to make it easy to identify herbicide safening effects. The herbicides were applied once using a broadcast spray boom as a post-emergence spray to crop plants typically between V2 and V6 growth stage.

[0091] FIG. 1 provides overall yields of soybeans with application of each herbicide composition containing herbicide identified on the x-axis and IN-M2A (black bars) or IN-M2B

(gray bars). The safening effects were measured and presented as % difference of yields compared to control crops treated with each herbicide alone without IN-M2A or IN-M2B.

Among the eight herbicides (i.e., acifluorfen; bromoxynil/MCPA; glyphosate;

glyphosate/dicamba; trifensulfuron; pyrasulf/bromox; bromoxynil/atrazine; glyphosate/ mesotrione/metolachlor/atrazine), IN-M2A and/or IN-M2B provided safening effects only for acifluorfen, glyphosate/dicamba, and glyphosate/ mesotrione/metolachlor/atrazine herbicide programs.

[0092] In soybeans treated with acifluorfen, both IN-M2A and IN-M2B provided positive yield responses across all the tested amounts from 0.5 to 64 oz/Acre (FIG. 2). In soybeans treated with glyphosate/dicamba, IN-M2A and IN-M2B provided mixed results in low concentrations, but they provided positive yield responses at high concentrations - 32 and 64 oz/Acre (FIG. 3). In soybeans treated with glyphosate/mesotrione/metolachlor/atrazine herbicide, IN-M2A resulted in positive yield response across all the tested amounts from 0.5 to 64 oz/Acre (FIG. 4). However, IN-M2B provided mixed results.

[0093] FIGs. 5A-B provide yields of large carrots (left) and yields of total marketable carrots (right) with application of Dual Magnum® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars). The safening effects were measured and presented as % difference of yields compared to control crops treated with each herbicide alone without IN-M2A or IN-M2B. FIG. 5A provides data from carrot fields treated with the herbicide composition at 3-5 leaf stage, and FIG. 5B provides data from carrot fields treated with the herbicide composition at pre-leaf stage. There was no significant yield differences in carrots treated with Dual Magnum® alone or Dual Magnum® mixed with IN-M2A or IN-M2B in either developmental stage.

[0094] FIGs. 6A-B provide percentage yield differences of large carrots (left) and total marketable carrots (right) with application of Lorox® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars) as compared to carrots with application of Lorox® alone. FIG. 6A provides data from carrot fields treated with the herbicide

composition at 3-5 leaf stage, and FIG. 6B provides data from carrot fields treated with the herbicide composition at pre-leaf stage. Both IN-M2A and IN-M2B provided positive yield responses across most of the tested amounts from 0.5 to 32 oz/Acre when applied either 3-5 leaf stage or pre-leaf stage. 32 oz/Acre of IN-M2B, however, provided negative yield responses, particularly when applied at 3-5 leaf stage.

[0095] FIG. 7 provides percentage yield differences of large carrots (left) and total marketable carrots (right) with application of Caparol® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars) as compared to carrots with application of Caparol® alone. IN-M2A provided negative yield responses and IN-M2B provided no significant yield responses across all the tested amounts from 0.5 to 32 oz/Acre.

[0096] FIG. 8 provides percentage yield differences of large carrots (left) and total marketable carrots (right) with application of Caparol® mixed with different amounts (0.5, 4 or 32 oz/Acre) of IN-M2A (black bars) or IN-M2B (gray bars) as compared to carrots with application of Caparol® alone. Both IN-M2A and IN-M2B provided positive yield responses across most of the tested amounts from 0.5 to 32 oz/Acre.

[0097] The results demonstrate that the cell free supernatant (IN-M2A and IN-M2B) has herbicide safening effects on some, not all, herbicides at optimal concentrations. This suggests that an effective amount of herbicide compositions containing certain herbicide and IN-M2A or IN-M2B will cause less herbicide injury to crops. Thus, IN-M2A and IN-M2B can be used as a safe, environmentally friendly, and sustainable herbicide safener that minimize harms to the environment.

[0098] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0099] While various specific embodiments have been illustrated and described, the above specification is not restrictive. It will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). Many variations will become apparent to those skilled in the art upon review of this specification.

Claims

WHAT IS CLAIMED IS:

1. A composition or kit comprising an herbicide and an herbicide safener, the herbicide safener comprising an isolated cell free supernatant of a microorganism mixture culture produced by inoculating a culture media with an isolated, mixed microbial composition and incubating the inoculated media for a period of time at an incubation temperature, the isolated, mixed microbial composition compri sing Aspergillus oryzae, Bacillus

amyloliquefaciens, Candida utilis, Lactobacillus helveticus, Lactobacillus paracasei,

Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Rhodopseudomonas palustris and Saccharomyces cerevisiae.

2. The composition or kit of claim 1, wherein the herbicide and the herbicide safener are in separate vessels.

3. The composition or kit of claim 1, wherein the herbicide and the herbicide safener are in a single vessel.

4. The composition or kit of claims 1-3, wherein the herbicide comprises a PPO inhibitor, an EPSP synthase inhibitor, a carotenoid biosynthesis inhibitor, a long chain fatty acid inhibitor, and/or a photosystem II inhibitor.

5. The composition or kit of claims 1-3, wherein the herbicide comprises metribuzin or linuron or glyphosate or mesotrione or metolachlor or atrazine.

6. The composition or kit of claims 1-5, wherein the isolated, mixed microbial composition consists of Aspergillus oryzae, Bacillus amyloliquefaciens, Candida utilis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus,

Lactococcus lactis, Rhodopseudomonas palustris and Saccharomyces cerevisiae.

7. The composition or kit of claims 1-5, wherein the isolated, mixed microbial composition comprises Aspergillus oryzae , IN-AOl, deposited September 4, 2014 with ATCC, PTA-121551; Bacillus amyloliquefaciens , IN-BSI, deposited January 11, 2012 with ATCC, PTA-12385;

Candida utilis , IN-CU1, deposited September 4, 2014 with ATCC, PTA-121550; Lactobacillus helveticus , IN-LHI, deposited January 11, 2012, with ATCC, PTA-12386; Lactobacillus paracasei , IN-LC1, deposited September 4, 2014 with ATCC, PTA-121549; Lactobacillus plantarum , IN-LP1, deposited September 4, 2014 with ATCC, PTA-121555; Lactobacillus rhamnosus , IN-LR1, deposited September 4, 2014 with ATCC, PTA-121554; Lactococcus lactis , IN-LL1, deposited September 4, 2014 with ATCC, PTA-121552; Rhodopseudomonas palustris , IN-RPI, deposited January 11, 2012 with ATCC, PTA-12387 or Rhodopseudomonas palustris , IN-RP2, deposited September 4, 2014 with ATCC, PTA-121553; and Saccharomyces cerevisiae , IN- SCI, deposited on January 11, 2012 with ATCC, PTA-12384.

8. The composition or kit of claims 1-5, wherein the isolated, mixed microbial composition consists of Aspergillus oryzae , IN-AOl, deposited September 4, 2014 with ATCC, PTA-121551; Bacillus amyloliquefaciens , IN-BSI, deposited January 11, 2012 with ATCC, PTA-12385;

9. The composition or kit of any of the above claims, wherein the culture media comprises water, a carbon source, a micronutrient source, a sea salt, and a wheat bran.

10. The composition or kit of claim 9, wherein the culture media comprises water, a molasses, a mineral power, a sea salt, a wheat bran, a kelp, a bentonite clay, a fish emulsion, and/or soy flour.

11. The composition or kit of claim 9, wherein the culture media consists of the following:

12. The composition or kit of any of the above claims, wherein the inoculated media is incubated for 1-6 weeks at 24-37 degrees Celsius.

13. The composition or kit of claim 12, wherein the inoculated media is incubated for 1 week at 30 degrees Celsius.

14. The composition or kit of any of the above claims, wherein the microorganism mixture culture is centrifuged and filter sterilized to produce the isolated cell free supernatant.

15. The composition or kit of claim 14, wherein the microorganism mixture culture is centrifuged at l0,000g or more for 10 minutes and filter sterilized with a 0.2 micron filter to produce the isolated cell free supernatant.

16. The composition or kit of claim 15, wherein the isolated cell free supernatant is passed through a 30kD filter.

17. The composition or kit of any of the above claims, wherein the cell-free supernatant is pH 3.7-40 and comprises 2000 - 3000 micrograms potassium per gram, 600 - 1000 micrograms sodium per gram, 500-1000 micrograms potassium per gram, 300-350 micrograms magnesium per gram, 600 ppm sulphur, and 30-130 ppm phosphorus.

18. A method of protecting a non-target plant from an herbicide injury, comprising applying the herbicide composition or kit of any of the above claims to the non-target plant or a soil for the non-target plant.

19. The method of claim 18, wherein the non-target plant is a com, soybean, wheat, or carrot plant.

20. The method of claim 18, wherein the non-target plant is soybean, the herbicide comprises acifluorfen and the effective amount of the herbicide safener is at least 0.035L, 0.140L, 0.560L, 2.240L or 3.380L per l800g a.i. of the herbicide.

21. The method of claim 18, wherein the non-target plant is soybean, the herbicide comprises glyphosate and dicamba and the effective amount of the herbicide safener is at least 2.240L per 5400g a.i. of the herbicide.

22. The method of claim 18, wherein the non-target plant is corn, the herbicide comprises glyphosate, mesotrione, metolachlor, and atrazine and the effective amount of the herbicide safener is at least 0.035L, 0.140L, 0.560L, 2.240L, or 3.380L per 7455g a.i. of the herbicide.

23. The method of claim 18, wherein the non-target plant is carrot, the herbicide comprises linuron and the effective amount of the herbicide safener is at least 0.035L, 0.560L, 0.280L, or 2.240L per 1964 g a.i. of the herbicide.

24. The method of claim 18, wherein the non-target plant is carrot, the herbicide comprises metribuzin and the effective amount of the herbicide safener is at least 0.035L, 0.280L, or 2.240L per 2l0g a.i. of the herbicide.

25. The method of claims 15-24, wherein the herbicide and the herbicide safener are applied concurrently.

26. The method of claims 15-24, wherein the herbicide and the herbicide safener are applied sequentially.

27. The method of claims 15-26, wherein the step of applying the herbicide composition or kit is performed by an overhead irrigation, a drip irrigation, a microjet sprinkler, or a direct or broadcast soil or foliar spray.

28. The method of claims 15-27, wherein the herbicide composition is applied at least 50 L/ha, 75 L/ha, 100 L/ha, 150 L/ha, 200 L/ha, 300 L/ha or 500 L/ha.