WO2023245128A1 - Antimicrobial peptide - Google Patents

Abstract

An engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof comprising, consisting essentially of, or consisting of, an amino acid sequence set forth in SEQ ID NO: 1 is described, along with methods of making and using the same. The present disclosure is also related to and describes novel antimicrobial compositions, formulations, and methods of using the same, that are useful for the control of pathogenic microbes.

Description

ANTIMICROBIAL PEPTIDE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, United States Provisional Application Serial No. 63/353,508 filed on June 17, 2022, the disclosure of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[0002] This application incorporates by reference in its entirety the Sequence Listing XML entitled “225312-530339_Sequence-listing.xml” (4,505 bytes), which was created on June 15, 2023 at 11 :55 AM, and filed electronically herewith.

TECHNICAL FIELD

[0003] Engineered, non-naturally occurring antimicrobial peptides and agricultural compositions and methods containing same are described.

BACKGROUND

[0004] The lives and livelihoods of millions of people depend on several agriculturally important crops. Deleterious microbes that threaten these agriculturally important crops represent a dire threat to global food security and the economy.

[0005] Microbial infections, e.g., fungal infections, are a particular problem in damp climates, and are of major concern during crop storage; indeed, the degree and severity of fungal infections can be exacerbated by modem growing methods — as harvesting and storage systems frequently provide a favorable environment for these plant pathogens. Adding to the problems caused by microbial infections are the rich diversity of pathogenic microbes (e.g., fungi, bacteria, oomycetes, etc.) that can contribute to plant disease. World-wide international travel has aided in spreading these deleterious microbes to parts of the planet where native plants have evolved no defenses. Moreover, the emphasis on intensive monoculture practices of commercially relevant crops in concert with traditional disease-mitigation strategies has allowed pathogenic microbes to become resistant and thrive. [0006] The incidence of plant diseases has traditionally been controlled by agronomic practices that include crop rotation, the use of agrochemicals, and conventional breeding techniques. The use of chemicals to control plant pathogens, however, increases costs to farmers and causes harmful effects on the ecosystem. Accordingly, consumers and government regulators alike are becoming increasingly concerned with the environmental hazards associated with the production and use of synthetic agrochemicals for protecting plants from pathogens. Thus, there is a significant need for novel alternatives for the control of plant pathogens that possess a lower risk of pollution and environmental hazards than is characteristic of traditional agrochemical-based methods.

[0007] Accordingly, there is a need for alternative, environmentally friendly antimicrobial agents and compositions to protect economically important commodity and staple calorie crops from the threats posed by pathogenic microbes.

SUMMARY

[0008] The present disclosure describes an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof.

[0009] In addition, the present disclosure describes an antimicrobial peptide, or an agriculturally acceptable salt thereof, wherein the antimicrobial peptide consists of an amino acid sequence of: GTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 1).

[0010] In addition, the present disclosure describes antimicrobial compositions comprising: an engineered, non-naturally occurring peptide, or an agriculturally acceptable salt thereof; and an excipient.

[0011] In addition, the present disclosure describes a method of combating, controlling, killing, or inhibiting a microbe comprising: applying an antimicrobially-effective amount of an antimicrobial peptide, or an agriculturally acceptable salt thereof, as described herein, and a to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe.

[0012] In addition, the present disclosure describes a method of combating, controlling, or inhibiting a microbe comprising: applying an antimicrobially-effective amount of an antimicrobial composition comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient, to the locus of the microbe, or to a plant or animal susceptible to contact with or colonization by the microbe. [0013] In each of the aforementioned embodiments, an exemplary engineered, non- naturally occurring antimicrobial peptide consisting of an amino acid sequence as set forth in SEQ ID NO: 1, or an agriculturally acceptable salt thereof, is added to an agriculturally acceptable excipient, forming an agriculturally acceptable composition for use in the methods described herein.

[0014] In addition, the present disclosure describes an exemplary engineered, non- naturally occurring antimicrobial peptide consisting of an amino acid sequence as set forth in SEQ ID NO: 1, or an agriculturally acceptable salt thereof, added to an agriculturally acceptable excipient, forming an agriculturally acceptable composition; wherein the antimicrobial peptide is present in the composition in an amount ranging from about 10% w/w to about 0.00001% w/w, or from about 0.1% w/w to about 0.005% w/w, of the total weight of the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 shows a plasmid map for pLB602. Here, a polynucleotide operable to encode the engineered non-natural NCR13a antimicrobial peptide having the amino acid sequence of SEQ ID NO: 1, was cloned into a yeast expression vector, pKLACl, resulting in an expression vector as shown.

[0016] FIG. 2 shows a summary of a yield evaluation of WT-NCR13 and NCR13a peptides as measured via reverse phase HPLC runs using spent fermentation medium containing the secreted native WT-NCR13 or NCR13a peptides. Here, the bars on the left side of the graph (separated from the bars on the right by a gap) show the yield results for strains transformed with the pLB602 vector (encoding NCR13a). The bars on the right show the yield results for strains transformed with the pLB603 vector (encoding WT-NCR13).

[0017] FIG. 3 shows the gene productivity of the pLB602-YCT (expressing NCR31a) and pLB603-YCT (expressing WT-NCR13) strains. Gene productivity is a measurement of peptide yield from a given strain per integrated target gene copy in the strain genome, as shown as the slope of the plots. As shown here, NCR13a gene productivity was 10 mg/mL per gene copy, which significantly higher than that of WT-NCR13 (Native NCR13), which was 0.2 mg/mL per gene copy. The unexpected result indicated that the engineered non-natural NCR13a peptide can be expressed significantly more efficiently in the Kluyveromyces lactis expression system than the WT-NCR13 peptide. [0018] FIG. 4 depicts a dose response curve showing the growth of Kluyveromyces lactis as measured via relative OD600 (Relative cell density %) when incubated with WT-NCR13 (Native NCR13) or NCR13a at 30°C for 48 hours. Here, YCT306 = Kluyveromyces lactis.

[0019] FIG. 5 depicts a dose response curve showing the growth of Botrytis cinerea as measured via relative OD600 (Relative cell density %) when incubated with WT-NCR13 (Native NCR13) or NCR13a at 25°C for 72 hours.

[0020] FIG. 6 depicts a dose response curve showing the growth of Monilinia fructicola as measured via relative OD600 (Relative cell density %) when incubated with WT-NCR13 (Native NCR13) or NCR13a at 25°C for 72 hours.

DETAILED DESCRIPTION

[0021] DEFINITIONS

[0022] “5’ -end” and “3’-end” refers to the directionality, i.e., the end-to-end orientation of a nucleotide polymer (e.g., DNA). The 5 ’-end of a polynucleotide is the end of the polynucleotide that has the fifth carbon.

[0023] “Applying” or “application” or “apply” or “administering” or “administration” or “administer” means to dispense and/or otherwise provide, and refers to any method of application or route of administration. For example, applying can refer to, e.g., application of a of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, or an antimicrobial composition comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and one or more excipients, e.g., a sprayable composition, a foam; a burning formulation; a fabric treatment; a surfacetreatment; a dispersant; or a microencapsulation. By “co-application” or “co-administer” it is meant that a combination or composition described herein is applied or administered at the same time, just prior to, or just after the application of: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1; and optionally, one or more additional agents or excipients, also referred to herein as a “additional agent.” The engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1 of the present disclosure, and optionally one or more excipients can be administered alone or can be co-administered to the locus of a microbe. Co- application or co-administration is meant to include simultaneous or sequential application of the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1; and/or compositions thereof further comprising an excipient.

[0024] “Affect” refers to how a something influences another thing, e.g., how a peptide, polypeptide, protein, drug, or chemical influences a microbe.

[0025] “Alignment” refers to a method of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide, or protein sequences) for the purpose of determining their relationship to each other. Alignments are typically performed by computer programs that apply various algorithms, however, it is also possible to perform an alignment by hand. Alignment programs typically iterate through potential alignments of sequences and score the alignments using substitution tables, employing a variety of strategies to reach a potential optimal alignment score. Commonly-used alignment algorithms include, but are not limited to, CLUSTALW (see Thompson J. D., Higgins D. G., Gibson T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research 22: 4673-4680, 1994); CLUSTALV (see Larkin M. A., et al., CLUSTALW2, ClustalW and ClustalX version 2, Bioinformatics 23(21): 2947-2948, 2007); Mafft; Kalign; ProbCons; and T-Coffee (see Notredame et al., T-Coffee: A novel method for multiple sequence alignments, Journal of Molecular Biology 302: 205-217, 2000). Exemplary programs that implement one or more of the foregoing algorithms include, but are not limited to, MegAlign from DNAStar (DNAStar, Inc. 3801 Regent St. Madison, Wis. 53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio from Accelrys (Accelrys, Inc., 10188 Telesis Ct, Suite 100, San Diego, Calif. 92121). In some embodiments, an alignment will introduce “phase shifts” and/or “gaps” into one or both of the sequences being compared in order to maximize the similarity between the two sequences, and scoring refers to the process of quantitatively expressing the relatedness of the aligned sequences.

[0026] “Agent” refers to one or more chemical substances, molecules, nucleotides, polynucleotides, peptides, polypeptides, proteins, poisons, insecticides, pesticides, organic compounds, inorganic compounds, prokaryote organisms and agents produced therefrom, or eukaryote organisms and agents produced therefrom. [0027] “Agriculturally-acceptable carrier” covers all adjuvants, inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in agricultural formulation technology; these are well known to those skilled in agricultural formulations. [0028] “Agriculturally acceptable salt” is used herein synonymously with the term “pharmaceutically acceptable salt.”

[0029] “Ameliorate” or “amelioration” includes the arrest, prevention, decrease, or improvement in one or more the symptoms, signs, and features of the disease being treated, both temporary and long-term.

[0030] As used herein, “antimicrobial” is generally used to refer to the ability of a combination or composition of the present disclosure, to increase mortality or inhibit growth rate of microbes.

[0031] “Antimicrobial effect” refers to inhibition or stoppage of the normal metabolic processes required for continued life, or continued growth of a microbe. “Antimicrobial effect” includes killing of any individual or group of microbes.

[0032] “Antimicrobial activity” means that upon or after exposing the microbe to the combinations or compositions of the present disclosure, the microbe either dies, stops, or slows its cellular processes; stops or slows its maintenance; stops or slows its growth; fails to reproduce; and the like.

[0033] “Antimicrobial composition” refers to a composition comprising a an engineered, non-naturally occurring peptide, or an agriculturally acceptable salt thereof; and an excipient.

[0034] “Antimicrobially-effective amount” refers to an amount of (1) an engineered, non-naturally occurring peptide, or an agriculturally acceptable salt thereof, or (2) an antimicrobial composition comprising: an engineered, non-naturally occurring peptide, or an agriculturally acceptable salt thereof; and an excipient; that is sufficient to: inhibit a microbe, bring about the death of at least one microbe; noticeably reduce or decrease microbe growth, feeding, or normal physiological development; inhibit or decrease the normal microbe cellular processes, including maintenance and growth; and/or attenuate or decrease the severity of a microbial infection. This amount will vary depending on such factors including but not limited to: the specific target microbe to be controlled; the specific environment, location, plant, crop, or agricultural site to be treated; the environmental conditions, method, rate, concentration, stability, and quantity applied. Further, those having ordinary skill in the art will recognize that the antimicrobially-effective amount may also vary with respect to climatic conditions, environmental considerations, and/or frequency of application and/or severity of microbe infestation. Antimicrobially-effective amounts can be measured by use of assays that measure the reduction in growth or decline in their populations of a microbe. One measure of reduction can be to express the decrease in population in logarithmic scale typical of a specific microbial species. That is, a 1 log reduction is equivalent to a 90% reduction versus a control, a 2 log reduction is a 99% reduction, etc.

[0035] “bp” or “base pair” refers to a molecule comprising two chemical bases bonded to one another. For example, a DNA molecule consists of two winding strands, wherein each strand has a backbone made of an alternating deoxyribose and phosphate groups. Attached to each deoxyribose is one of four bases, i.e., adenine (A), cytosine (C), guanine (G), or thymine (T), wherein adenine forms a base pair with thymine, and cytosine forms a base pair with guanine. [0036] “C -terminus” or “C-terminal” refers to the free carboxyl group (i.e., -COOH) that is positioned on the terminal end of a polypeptide.

[0037] “Culture” or “cell culture” refers to the maintenance of cells in an artificial, in vitro environment.

[0038] “Culturing” refers to the propagation of organisms on or in various kinds of media. For example, the term “culturing” can mean growing a population of cells under suitable conditions in a liquid or solid medium. In some embodiments, culturing refers to fermentative recombinant production of a peptide of interest (e.g., an engineered, non-naturally occurring peptide) and/or other desired end products (typically in a vessel or reactor).

[0039] “Decreasing” or “decrease” or “decreased” or “reducing” or “reduced” or “a reduction” or “inhibiting” or “stopping” or “combatting” or “controlling” or any variation of these terms, refers to making something (e.g., the number of microbes and/or degree or severity of a microbe infection or disease) less in size, amount, intensity, or degree. For example, in some embodiments, the application of a antimicrobially-effective amount of an antimicrobial combination of the present disclosure, comprising an engineered, non-naturally occurring peptide, or an agriculturally acceptable salt thereof, and/or an antimicrobial composition comprising: an engineered, non-naturally occurring peptide, or an agriculturally acceptable salt thereof; and an excipient; to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe, can result in the following effect: a decrease or reduction in the number of microbes and/or a decrease or reduction in the degree or severity of a microbe infection or disease, relative to the number of microbes and/or degree or severity of a microbe infection or disease that has not been treated with, or had applied thereto, an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and/or an antimicrobial composition comprising: an engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient, as described herein.

[0040] In some embodiments, reducing or decreasing, e.g., the number of microbes and/or the degree or severity of a microbe infection or disease, includes any measurable decrease or complete inhibition to achieve a desired result. For example, there may be a decrease of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, in the number of microbes and/or degree or severity of a microbe infection or disease, when a plant or animal susceptible to a pathogenic microbe is treated with the antimicrobial combinations or antimicrobial compositions of the present disclosure, as compared to normal. The term “about” as used herein means within ± 10%, preferably ± 5% of a given value. Thus, in some embodiments, the terms “reduction in the number of microbes and/or degree or severity of a microbe infection or disease,” refers to a decrease or reduction in the number of microbes and/or degree or severity of a microbe infection or disease by a plant or animal susceptible to an attack by the microbe that has received an antimicrobially effective amount of a combination of the present disclosure, or an agricultural composition thereof, that is at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 2.25%, at least about 2.5%, at least about 2.75%, at least about 3%, at least about 3.25%, at least about 3.5%, at least about 3.75%, at least about 4%, at least about 4.25%, at least about 4.5%, at least about 4.75%, at least about 5%, at least about 5.25%, at least about 5.5%, at least about 5.75%, at least about 6%, at least about 6.25%, at least about 6.5%, at least about 6.75%, at least about 7%, at least about 7.25%, at least about 7.5%, at least about 7.75%, at least about 8%, at least about 8.25%, at least about 8.5%, at least about 8.75%, at least about 9%, at least about 9.25%, at least about 9.5%, at least about 9.75%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or a greater than a 100%, relative to the number of microbes and/or degree or severity of a microbe infection or disease in a plant or animal susceptible to an attack by the microbe that has not been administered an antimicrobially effective amount of an engineered, non-naturally occurring peptide of the present disclosure, or an agricultural composition thereof. [0041] “DNA” refers to deoxyribonucleic acid, comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form. For example, one or more nucleotides creates a polynucleotide.

[0042] “Excipient” refers to any agriculturally acceptable additive, carrier, surfactant, emulsifier, thickener, preservative, solvent, disintegrant, glidant, lubricant, diluent, filler, bulking agent, binder, emollient, stiffening agent, stabilizer, solubilizing agents, dispersing agent, suspending agent, antioxidant, antiseptic, wetting agent, humectant, fragrant, suspending agents, pigments, colorants, isotonic agents, viscosity enhancing agents, mucoadhesive agents, and/or any combination thereof, that can be added to a composition, preparation, and/or formulation, which may be useful in achieving a desired modification to the characteristics of the composition, preparation, and/or formulation. Such modifications include, but are not limited to, physical stability, chemical stability, therapeutic efficacy, and/or any combination thereof. In some embodiments, excipients can be formulated alongside a an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1, when preparing an antimicrobial composition, e.g., for the purpose of bulking up compositions (thus often referred to as bulking agents, fillers or diluents). In yet other embodiments, an excipient can be used to provide stability, or prevent contamination (e.g., microbial contamination). In other embodiments, an excipient can be used to confer a physical property to a composition (e.g., a composition that is a dry granular, or dry flowable powder physical form). Reference to an excipient includes both one and more than one such excipients. Suitable excipients are described in Remington's Pharmaceutical Sciences, by E.W. Martin, the disclosure of which is incorporated herein by reference in its entirety.

[0043] “IC50” or “IC50” refers to half-maximal inhibitory concentration, which is a measurement of how much of an agent is needed to inhibit a biological process by half, thus providing a measure of potency of said agent.

[0044] “Inhibiting a microbe” as used herein refers to an inhibitory effect caused as a direct result of the contact of an antimicrobial composition and/or combination described herein with the microbe, wherein the contacted microbe, for example, a fungal microbe, is incapacitated with respect to an impairment of any physiological function required for normal physiological maintenance and/or survival and/or reproduction. Inhibiting a microbe can be measured by any assay or measurement that can detect and measure a decrease in a microbe physiological function (e.g. respiration, membrane integrity, energy utilization, synthesis of vital building blocks e.g. nucleic acids, amino acids, biochemical metabolites), growth, reproduction, and/or any other parameter that is essential to the microbe’s survival and/or reproduction.

[0045] “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0046] “Inoperable” refers to the condition of a thing not functioning, malfunctioning, or no longer able to function.

[0047] “Isolated” refers to separating a thing and/or a component from its natural environment, e.g., an engineered, non-naturally occurring peptide is isolated from or separated from components, cells, recombinant peptide production systems and the like used in the synthesis of said engineered, non-naturally occurring peptide. [0048] “kb” refers to kilobase, i.e., 1000 bases. As used herein, the term “kb” means a length of nucleic acid molecules. For example, 1 kb refers to a nucleic acid molecule that is 1000 nucleotides long. A length of double-stranded DNA that is 1 kb long, contains two thousand nucleotides (i.e., one thousand on each strand). Alternatively, a length of single-stranded RNA that is 1 kb long, contains one thousand nucleotides.

[0049] “kDa” refers to kilodalton, a unit equaling 1,000 daltons; a “Dalton” is a unit of molecular weight (MW).

[0050] “Knockdown dose 50” or “KD50” refers to the median dose required to cause paralysis or cessation of movement in 50% of a population.

[0051] “Medium” (plural “media”) refers to a nutritive solution for culturing cells in cell culture.

[0052] “Microbe” refers to any microscopic organism, e.g., any multi-cellular or unicellular microorganism, or a virus, including all of the prokaryotes, namely the eubacteria and archaeabacteria, and various forms of eukaryote, comprising the protozoa, fungi (e.g., yeast). Thus, as used herein, “microbe” refers to all bacteria, all archaea, unicellular protista, unicellular animals, unicellular plants, unicellular fungi, unicellular algae, all protozoa, and all chromista. In some embodiments, a microbe can be a pathogenic microbe, wherein the microbe causes an infection or disease in a living organism when introduced into said organism; or wherein the presence of the microbe is deleterious to the organism. For example, in some embodiments, a microbe can be a pathogen to plants, e.g., a phytopathogen, such as a bacterium, a protozoan, or a fungus.

[0053] “MOA” refers to mechanism of action.

[0001] “Molecular weight (MW)” refers to the mass or weight of a molecule, and for proteins is typically measured in “daltons (Da)” or kilodaltons (kDa). In some embodiments, MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), gel chromatography, analytical ultracentrifugation, mass spectrometry, or light scattering. In some embodiments, the SDS-PAGE method is as follows: the sample of interest is separated on a gel with a set of molecular weight standards. The sample is run, and the gel is then processed with a desired stain, followed by destaining for about 2 to 14 hours. The next step is to determine the relative migration distance (Rf) of the standards and protein of interest. The migration distance can be determined using the following equation: Rf= (migration distance of the protein)/(Migration distance of the dye front). Next, the logarithm of the MW can be determined based on the values obtained for the bands in the standard; e.g., in some embodiments, the logarithm of the molecular weight of an SDS-denatured polypeptide and its relative migration distance (Rf) is plotted into a graph. After plotting the graph, interpolating the value derived will provide the molecular weight of the unknown protein band.

[0054] “Motif’ refers to a polynucleotide or polypeptide sequence that is implicated in having some biological significance and/or exerts some effect or is involved in some biological process.

[0055] “Native” refers to items found in nature in their natural, unmodified state.

[0056] “N-terminus” refers to the free amine group (i.e., -NH2) that is positioned on beginning or start of a polypeptide.

[0057] “NCBI” refers to the National Center for Biotechnology Information.

[0058] “NCR” refers to nodule-specific cysteine-rich.

[0059] “OD” refers to optical density. Typically, OD is measured using a spectrophotometer. When measuring growth over time of a cell population, OD600 is preferable to UV spectroscopy; this is because at a 600 nm wavelength, the cells will not be harmed as they would under too much UV light.

[0060] “OD600nm” or “ODeoonm” refers to optical densities of the liquid sample measured (for example, a microbial cell culture) when measured in a spectrophotometer at 600 nanometers (nm).

[0061] “One letter code” means the peptide sequence which is listed in its one letter code to distinguish the various amino acids in the primary structure of a protein: alanine=A, arginine=R, asparagine=N, aspartic acid=D, asparagine or aspartic acid=B, cysteine=C, glutamic acid=E, glutamine=Q, glutamine or glutamic acid=Z, glycine=G, histidine=H, isoleucine=I, leucine=L, lysine=K, methionine=M, phenylalanine=F, proline=P, serine=S, threonine=T, tryptophan=W, tyrosine=Y, and valine=V.

[0062] “Operable” refers to the ability to be used, the ability to do something, and/or the ability to accomplish some function or result.

[0063] “Pathogenic microbe” refers to any microbe that is deleterious or pathogenic to an organism; e.g., any microbe that causes or exacerbates an infection or disease in a living organism. For example, in some embodiments, a pathogenic microbe can be a pathogen to plants, e.g., a phytopathogen, such as a bacterium, a protozoan, or a fungus; in other embodiments, a pathogenic microbe can be a pathogen to animals. As used herein, a “plant pathogenic microbe” or “plant pathogen” can refer to a microbe that can cause disease into whole plants, plant tissues, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0064] “Pharmaceutically acceptable salt” is synonymous with agriculturally acceptable salt, and as used herein refers to a compound that is modified by making acid or base salts thereof.

[0065] “Plant” shall mean whole plants, plant tissues, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0066] “Plasmid” refers to a DNA segment that acts as a carrier for a gene of interest (e.g., NCR13a) and, when transformed or transfected into an organism, can replicate and express the DNA sequence contained within the plasmid independently of the host organism. Plasmids are a type of vector, and can be “cloning vectors” (i.e., simple plasmids used to clone a DNA fragment and/or select a host population carrying the plasmid via some selection indicator) or “expression plasmids” (i.e., plasmids used to produce large amounts of polynucleotides and/or polypeptides).

[0067] “Protein” has the same meaning as “peptide” and/or “polypeptide” in this document.

[0068] “Ratio” refers to the quantitative relation between two amounts showing the number of times one value contains or is contained within the other.

[0069] “Recombinant DNA” or “rDNA” refers to DNA that is comprised of two or more different DNA segments.

[0070] “ p.” refers to species.

[0071] “ssp.” or “subsp.” refers to subspecies.

[0072] “Susceptible to attack by a microbe (or microbes)” or “susceptible to a microbial infection” or “susceptible to microbial disease” and the like, refer to plants, or human or animal patients or subjects, susceptible to a microbe pathogen or microbial infections. [0073] “Treatment” or “treating” or “treatment of’ or “combatting” or “controlling” or “inhibiting” a pathogenic microbe; or a condition, disease, or disorder in a plant or animal susceptible to an attack by the microbe that is caused by a pathogenic microbe; or symptoms associated with a condition, disease, or disorder in a plant or animal susceptible to an attack by the microbe that is caused by a pathogenic microbe, refers to an approach for obtaining beneficial or desired results in the plant or animal susceptible to an attack by the microbe. Beneficial or desired results can include, but are not limited to, death of at least one microbe; alleviation or amelioration of one or more symptoms or conditions caused by a pathogenic microbe; diminishment of extent of condition, disorder or disease caused by a pathogenic microbe; stabilization of the state of condition, disorder or disease caused by a pathogenic microbe; prevention of development of condition, disorder or disease caused by a pathogenic microbe; prevention of spread of condition, disorder or disease caused by a pathogenic microbe; delay or slowing of condition, disorder or disease progression, delay or slowing of condition, disorder or disease onset caused by a pathogenic microbe; amelioration or palliation of the condition, disorder or disease state, and remission of a disease or disease state caused by a pathogenic microbe; whether partial or total. In some embodiments, “treating” can also mean prolonging survival of an organism beyond that expected in the absence of treatment. “Treating” can also mean inhibiting the progression of the disease or disorder, slowing the progression of disorder or disease temporarily, although in some instances, it involves halting the progression of the disorder or disease permanently. As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of one or more symptoms of a disease or condition caused by a pathogenic microbe. Thus, in some embodiments, treatment can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the number of pathogenic microbes, and/or the severity of an established disease, condition, or symptom of the disease or condition caused by a pathogenic microbe. It is understood that treatment does not necessarily refer to the death of all pathogenic microbes and/or the cure or complete ablation of the disease, condition, or symptoms of the disease or condition caused by a pathogenic microbe.

[0074] “vtzr.” refers to varietas or variety. The term “var.” is used to indicate a taxonomic category that ranks below the species level and/or subspecies (where present). In some embodiments, the term “var.” represents members differing from others of the same subspecies or species in minor but permanent or heritable characteristics.

[0075] “Vector” refers to the DNA segment that accepts a foreign gene of interest (e.g., NCR13). The gene of interest is known as an “insert” or “transgene.”

[0076] “Wild type” or “WT” refers to the phenotype and/or genotype (i.e., the appearance or sequence) of an organism, polynucleotide sequence, and/or polypeptide sequence, as it is found and/or observed in its naturally occurring state or condition.

[0077] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0078] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, solid phase and liquid nucleic acid synthesis, peptide synthesis in solution, solid phase peptide synthesis, immunology, cell culture, and formulation. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; J. F. Ramalho Ortigao, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, 3. eds.), vol. 2, pp. 1-284, Academic Press, New York. 12. Wiinsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler, E., ecL), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer- Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer- Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000); each of these references are incorporated herein by reference in their entireties.

[0079] Throughout this specification, unless the context requires otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

[0080] All patent applications, patents, and printed publications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers, or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.

[0081] ANTIMICROBIAL PEPTIDES

[0082] Biological antimicrobial agents, e.g., antimicrobial agents based on, or derived from, natural sources (such as an organism or product therefrom), may confer crop protection by suppressing disease incidence, and/or reducing the number of pathogenic microbes or the severity of symptoms caused by the same. Thus, biological antimicrobial agents represent a desirable replacement of traditional agrochemicals, and can be used to avoid or remediate the adverse effects thereof.

[0083] Legume plants employ a symbiotic relationship with soil rhizobia that allows these plants to take up biologically usable nitrogen, which is fixed by the bacteria in exchange for nutrients and carbon resources. Endosymbiotic rhizobia induce changes in the host plant, which results in the formation of a specialized root organ called a nodule. See Roy et al., Symbiotic Outcome Modified by the Diversification from 7 to over 700 Nodule-Specific Cysteine- Rich Peptides. GENES (BASEL). 2020 Apr; 11(4): 348. Within nodules, the rhizobia undergo distinct morphological and metabolic changes, and differentiate into nitrogen-fixing bacteroids. Id. Bacteroid differentiation and activity is regulated by a group of peptides called nodulespecific cysteine-rich (NCR) peptides. See Mergaert et al., Gene Expression in Nitrogen-Fixing Symbiotic Nodule Cells in Medicago truncatula and Other Nodulating Plants . PLANT CELL. 2020 Jan; 32(1): 42-68; Kereszt et al., Impact of Plant Peptides on Symbiotic Nodule Development and Functioning. FRONT PLANT SCI. 2018; 9: 1026.

[0084] Members of the NCR peptide gene family have been identified in legumes including, but not limited to, Vicia faba, Medicago sativa, Trifolium repens, Galega orientalis, Pisum sativum, Astragalus sinicus, Cicer arietinum and Glycyrrhiza lepidota.

[0085] NCR peptides have a structure that resembles antimicrobial defensin peptides, which are effectors of innate immunity in plants and animals, including humans. See Kereszt et al., Impact of Plant Peptides on Symbiotic Nodule Development and Functioning. FRONT PLANT SCI. 2018; 9: 1026. And, NCRs have been shown to exhibit antimicrobial activity, e.g., against gram-negative and gram-positive bacteria as well as unicellular and filamentous fungi. See Maroti and Kondorosi, Nitrogen-fixing Rhizobium-Zegw/ne symbiosis: are polyploidy and host peptide-governed symbiont differentiation general principles of endosymbiosis? FRONT MICROBIOL. 2014; 5: 326; Maroti et al., Natural roles of antimicrobial peptides in microbes, plants and animals. RES MICROBIOL. 2011 May;162(4):363-74.

[0086] The present disclosure provides an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; agricultural compositions thereof, further comprising an excipient; and methods of making and using the same.

Table 1. Amino acid and polynucleotide sequence of the engineered, non-naturally occurring antimicrobial peptide NCR13a.

[0087] In some embodiments, an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, wherein the antimicrobial peptide has an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0088] In some embodiments, an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof has an amino acid sequence consisting of: GTKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 1).

[0089] In some embodiments, the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof of the present disclosure do not comprise any: mutations, amino acid substitutions, amino acid additions, or amino acid deletions. In some embodiments, the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof amino sequence consists of the amino acid sequence of SEQ ID NO: 1, and does not comprise any: mutations, amino acid substitutions, amino acid additions, or amino acid deletions relative to the amino acid sequence of SEQ ID NO: 1.

[0090] METHODS FOR PRODUCING ANTIMICROBIAL PEPTIDES

[0091] In some illustrative embodiments, the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof of the present disclosure can be produced and/or obtained via any method well known in the art of peptide synthesis.

[0092] Recombinant Antimicrobial Peptides

[0093] In some embodiments, the engineered, non-naturally occurring antimicrobial peptide of the present disclosure can be produced using recombinant methods.

[0094] In some embodiments, an engineered, non-naturally occurring antimicrobial peptide of the present disclosure can be created using any known method for producing a peptide or protein. For example, in some embodiments, and without limitation, an engineered, non- naturally occurring antimicrobial peptide of the present disclosure can be created using a recombinant expression system, such as yeast expression system or a bacterial expression system. However, those having ordinary skill in the art will recognize that other methods of protein production are available.

[0095] In some embodiments, an engineered, non-naturally occurring antimicrobial peptide of the present disclosure can be produced using a recombinant expression system.

[0096] The recombinant expression of an engineered, non-naturally occurring antimicrobial peptide of the present disclosure is practicable in a wide variety of host cells. In some embodiments, the host cell can be any host cell that satisfies the requirements of the enduser. For example, in some embodiments, a user may desire to use one specific type of host cell (e.g., a yeast cell or a bacteria cell) as opposed to another; the preference of a given host cell can range from desired codon usage, availability and cost, among other considerations.

[0097] In some embodiments, a polynucleotide operable to encode an engineered, non- naturally occurring antimicrobial peptide of the present disclosure as provided in Table 1 (SEQ ID NO: 2) can be cloned into a vector using a variety of cloning strategies, and commercial cloning kits and materials readily available to those having ordinary skill in the art. For example, a polynucleotide which encodes the engineered, non-naturally occurring antimicrobial peptide can be cloned into a vector using such strategies as the SnapFast; Gateway; TOPO; Gibson; LIC; InFusionHD; or Electra strategies. There are numerous commercially available vectors that can be used to produce an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof of the present disclosure. See Janke et al., A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004 Aug; 21(11):947-62; see also, Adams et al. Methods in Yeast Genetics. Cold Spring Harbor, NY, 1997, the disclosures of which are incorporated herein by reference in its entireties.

[0098] In some embodiments, a polynucleotide encoding an engineered, non-naturally occurring antimicrobial peptide of the present disclosure can be cloned into a vector such as a plasmid, cosmid, virus (bacteriophage, animal viruses, and plant viruses), and/or artificial chromosome (e.g., YACs).

[0099] In some embodiments, a polynucleotide encoding an engineered, non-naturally occurring antimicrobial peptide of the present disclosure can be inserted into other commercially available plasmids and/or vectors that are readily available to those having skill in the art, e.g., plasmids are available from Addgene (a non-profit plasmid repository); GenScript®; Takara®; Qiagen®; and Promega™.

[0100] Additional exemplary methods of producing recombinant peptides are provided in PCT Application Nos. PCT/US2013/030042, PCT/US2017/055596, PCT/US2019/051093, PCT/US2021/028254, PCT/US2021/030277, the disclosures of which is incorporated herein by reference in their entireties. [0101] Chemically synthesizing polynucleotides

[0102] In some embodiments, the polynucleotide sequence encoding an engineered, non- naturally occurring antimicrobial peptide of the present disclosure can be chemically synthesized using commercially available polynucleotide synthesis services, such as those offered by GENEWIZ® (e.g., TurboGENE™; PriorityGENE; and FragmentGENE), or SIGMA- ALDRICH® (e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos). Exemplary method for generating DNA and or custom chemically synthesized polynucleotides are well known in the art, and are illustratively provided in U.S. Patent No. 5,736,135, Serial No. 08/389,615, filed on Feb. 13, 1995, the disclosure of which is incorporated herein by reference in its entirety. See also Agarwal, et al., Chemical synthesis of polynucleotides. Angew Chem Int Ed Engl. 1972 Jun; 11(6):451-9; Ohtsuka et al., Recent developments in the chemical synthesis of polynucleotides. Nucleic Acids Res. 1982 Nov 11; 10(21): 6553-6570; Sondek & Shortle. A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphoramidites. Proc Natl Acad Sei U S A. 1992 Apr 15; 89(8): 3581-3585; Beaucage S. L., et al., Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach. Tetrahedron, Elsevier Science Publishers, Amsterdam, NL, vol. 48, No. 12, 1992, pp. 2223-2311; Agrawal (1993) Protocols for Oligonucleotides and Analogs: Synthesis and Properties; Methods in Molecular Biology Vol. 20, the disclosures of which are incorporated herein by reference in their entirety.

[0103] Chemically synthesizing polynucleotides allows for a DNA sequence to be generated that is tailored to produce a desired peptide based on the arrangement of nucleotides within said sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A] or thymine [T] molecules); the mRNA sequence that is transcribed from the chemically synthesized DNA polynucleotide can be translated to a sequence of amino acids, each amino acid corresponding to a codon in the mRNA sequence.

[0104] Obtaining an engineered, non-naturally occurring antimicrobial peptide of the present disclosure from a chemically synthesized DNA polynucleotide sequence and/or a wildtype DNA polynucleotide sequence can be achieved by cloning the DNA sequence into an appropriate vector. There are a variety of expression vectors available, host organisms, and cloning strategies known to those having ordinary skill in the art. For example, the vector can be a plasmid, which can introduce a heterologous gene and/or expression cassette into yeast cells to be transcribed and translated. The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A vector may contain “vector elements” such as an origin of replication (ORI); a gene that confers antibiotic resistance to allow for selection; multiple cloning sites; a promoter region; a selection marker for non-bacterial transfection; and a primer binding site. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al., 1989 and Ausubel et al., 1996, both incorporated herein by reference. In addition to synthesizing a polynucleotide operable to encode an engineered, non-naturally occurring antimicrobial peptide of the present disclosure, a vector may also encode a targeting molecule. A targeting molecule is one that directs the desired nucleic acid to a particular tissue, cell, or other location.

[0105] Chemically synthesizing peptides

[0106] Peptide synthesis or the chemical synthesis of peptides and/or polypeptides can be used to produce or synthesize an engineered, non-naturally occurring antimicrobial peptide of the present disclosure. These methods can be performed by those having ordinary skill in the art, and/or through the use of commercial vendors (e.g., GenScript®; Piscataway, New Jersey). For example, in some embodiments, chemical peptide synthesis can be achieved using Liquid phase peptide synthesis (LPPS), or solid phase peptide synthesis (SPPS).

[0107] In some embodiments, peptide synthesis can generally be achieved by using a strategy wherein the coupling the carboxyl group of a subsequent amino acid to the N-terminus of a preceding amino acid generates the nascent polypeptide chain — a process that is opposite to the type of polypeptide synthesis that occurs in nature.

[0108] Peptide deprotection is an important first step in the chemical synthesis of polypeptides. Peptide deprotection is the process in which the reactive groups of amino acids are blocked through the use of chemicals in order to prevent said amino acid’s functional group from taking part in an unwanted or non-specific reaction or side reaction; in other words, the amino acids are “protected” from taking part in these undesirable reactions. [0109] Prior to synthesizing the peptide chain, the amino acids must be “deprotected” to allow the chain to form (i.e., amino acids to bind). Chemicals used to protect the N-termini include 9-fhiorenylmethoxycarbonyl (Fmoc), and tert-butoxycarbonyl (Boe), each of which can be removed via the use of a mild base (e.g., piperidine) and a moderately strong acid (e.g., trifluoracetic acid (TFA)), respectively.

[0110] The C-terminus protectant required is dependent on the type of chemical peptide synthesis strategy used: e.g., LPPS requires protection of the C-terminal amino acid, whereas SPPS does not owing to the solid support which acts as the protecting group. Side chain amino acids require the use of several different protecting groups that vary based on the individual peptide sequence and N-terminal protection strategy; typically, however, the protecting group used for side chain amino acids are based on the tert-butyl (tBu) or benzyl (Bzl) protecting groups.

[0111] Amino acid coupling is the next step in a peptide synthesis procedure. To effectuate amino acid coupling, the incoming amino acid’s C-terminal carboxylic acid must be activated: this can be accomplished using carbodiimides such as diisopropylcarbodiimide (DIC), or dicyclohexylcarbodiimide (DCC), which react with the incoming amino acid’s carboxyl group to form an O-acylisourea intermediate. The O-acylisourea intermediate is subsequently displaced via nucleophilic attack via the primary amino group on the N-terminus of the growing peptide chain. The reactive intermediate generated by carbodiimides can result in the racemization of amino acids. To avoid racemization of the amino acids, reagents such as 1 -hydroxybenzotriazole (HOBt) are added in order to react with the O-acylisourea intermediate. Other couple agents that may be used include 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU), and benzotriazol- l-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), with the additional activating bases. Finally, amino acid deprotection and coupling.

[0112] At the end of the synthesis process, removal of the protecting groups from the polypeptide must occur — a process that usually occurs through acidolysis. Determining which reagent is required for peptide cleavage is a function of the protection scheme used and overall synthesis method. For example, in some embodiments, hydrogen bromide (HBr); hydrogen fluoride (HF); or trifluoromethane sulfonic acid (TFMSA) can be used to cleave Bzl and Boe groups. Alternatively, in other embodiments, a less strong acid such as TFA can effectuate acidolysis of tBut and Fmoc groups. Finally, peptides can be purified based on the peptide’s physiochemical characteristics (e.g., charge, size, hydrophobicity, etc.). Techniques that can be used to purify peptides include Purification techniques include Reverse-phase chromatography (RPC); Size-exclusion chromatography; Partition chromatography; High-performance liquid chromatography (HPLC); and Ion exchange chromatography (IEC).

[0113] Exemplary methods of peptide synthesis can be found in Anderson G. W. and McGregor A. C. (1957) T-butyloxycarbonylamino acids and their use in peptide synthesis. Journal of the American Chemical Society. 79, 6180-3; Carpino L. A. (1957) Oxidative reactions of hydrazines. Iv. Elimination of nitrogen from 1, l-disubstituted-2-arenesulfonhydrazidesl-4. Journal of the American Chemical Society. 79, 4427-31; McKay F. C. and Albertson N. F. (1957) New amine-masking groups for peptide synthesis. Journal of the American Chemical Society. 79, 4686-90; Merrifield R. B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society. 85, 2149-54; Carpino L. A. and Han G. Y. (1972) 9-fluorenylmethoxycarbonyl amino-protecting group. The Journal of Organic Chemistry. 37, 3404-9; and A Lloyd- Williams P. et al. (1997) Chemical approaches to the synthesis of peptides and proteins. Boca Raton: CRC Press. 278; U.S. Patent Nos: 3,714,140 (filed Mar. 16, 1971); 4,411,994 (filed June 8, 1978); 7,785,832 (filed Jan. 20, 2006); 8,314,208 (filed Feb. 10, 2006); and 10,442,834 (filed Oct., 2, 2015); and United States Patent Application 2005/0165215 (filed Dec. 23, 2004), the disclosures of which are incorporated herein by reference in their entireties.

[0114] Any of the methods described herein can be used to generate an engineered, non- naturally occurring antimicrobial peptide described herein, e.g., an engineered, non-naturally occurring antimicrobial peptide consisting of an amino acid sequence set forth in SEQ ID NO: 1, or encoded by the nucleotide sequence of SEQ ID NO: 2.

[0115] ANTIMICROBIAL FORMULATIONS AND COMPOSITIONS

[0116] Excipients; generally

[0117] In some embodiments, an engineered, non-naturally occurring antimicrobial peptide of the present disclosure, or an agriculturally acceptable salt thereof, can further comprise one or more excipients to make an agriculturally useful composition.

[0118] For example, in some embodiments, the excipient can be any agriculturally acceptable additive, carrier, surfactant, emulsifier, thickener, preservative, solvent, disintegrant, glidant, lubricant, diluent, filler, bulking agent, binder, emollient, stiffening agent, stabilizer, solubilizing agents, dispersing agent, suspending agent, antioxidant, antiseptic, wetting agent, humectant, fragrant, suspending agents, pigments, colorants, isotonic agents, viscosity enhancing agents, mucoadhesive agents, and/or any combination thereof.

[0119] The use of excipients for the formulation of agricultural compositions is known to those having ordinary skill in the art.

[0120] In some embodiments, the excipient can be added to an antimicrobial composition, preparation, and/or formulation of the present disclosure, which may aid in achieving a desired modification to the characteristics of the antimicrobial composition, preparation, and/or formulation. Such modifications include, but are not limited to, physical stability, chemical stability, therapeutic efficacy, and/or any combination thereof.

[0121] In some embodiments, excipients can be formulated alongside an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, when preparing an antimicrobial composition, e.g., for the purpose of bulking up compositions (thus often referred to as bulking agents, fillers or diluents).

[0122] In yet other embodiments, an excipient can be used to provide stability, or prevent contamination. In other embodiments, an excipient can be used to confer a physical property to a composition (e.g., a composition that is a dry granular, or dry flowable powder physical form). Reference to an excipient includes both one and more than one such excipients. Suitable excipients are described in Remington's Pharmaceutical Sciences, by E.W. Martin, the disclosure of which is incorporated herein by reference in its entirety.

[0123] In some embodiments, e.g., the excipient can be independently selected from thickeners, viscosity enhancing agents, bulking agents, penetration enhancers, buffers, preservatives, diluents, binders, lubricants, glidants, disintegrants, fillers, solubilizing agents, pH modifying agents, preservatives, stabilizing agents, anti-oxidants, wetting or emulsifying agents, suspending agents, pigments, colorants, isotonic agents, emulsifiers, and diagnostic agents.

[0002] In some embodiments, an antimicrobial composition of the present disclosure comprising an excipient may contain an amount of excipient ranging from about 0.005 wt% to about 99 wt%.

[0124] Formulations; generally

[0125] Any of the antimicrobial compositions of the present disclosure can be formulated according to the methods described herein, and used to inhibit microbial growth and/or proliferation, and/or kill the microbes, and/or control the damage caused by their actions, especially their damage to plants.

[0126] Antimicrobial compositions of the present disclosure can be formulated for use as agrochemical compositions. For example, in some embodiments, agrochemical compositions can include, but is not limited to, aerosols and/or aerosolized products (e.g., sprays, fumigants, powders, dusts, and/or gases); granules, dusts, powders, or seed dressings.

[0127] In some embodiments, the antimicrobial compositions may be formulated as a powder, dust, pellet, granule, spray, emulsion, colloid, solution, or such like, and may be prepared by such conventional means as desiccation, lyophilization, homogenization, extraction, filtration, centrifugation, or sedimentation. In all such compositions, the engineered, non- naturally occurring antimicrobial peptide may be present in said antimicrobial composition in a concentration of from about 0.0001% to about 99% by weight.

[0128] The compositions may be formulated prior to administration or application in the field, in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline and/or other buffer. In some embodiments, the formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable carriers can be solid or liquid and are well known in the art. In some embodiments, the compositions may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal composition with suitable adjuvants using conventional compositions techniques. Suitable compositions and application methods are described in U.S. Pat. No. 6,468,523, the disclosure of which is incorporated by reference herein in its entirety.

[0129] In some embodiments, the active ingredients of the present disclosure can be applied in the form of compositions and can be applied to the crop area or plant to be treated, simultaneously or in succession, with other non-active compounds. These compounds can be fertilizers, weed killers, cryoprotectants, surfactants, detergents, soaps, dormant oils, polymers, and/or time-release or biodegradable carriers that permit long-term dosing of a target area following a single application of the composition. One or more of these non-active compounds can be prepared, if desired, together with further agriculturally acceptable carriers, surfactants or application-promoting adjuvants customarily employed in the art of composition. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in composition technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

[0130] Methods of applying an active ingredients of the present disclosure or an agrochemical composition of the present disclosure that has an engineered, non-naturally occurring antimicrobial peptide, as produced by the methods described herein of the present disclosure, include leaf application, seed coating and soil application. In some embodiments, the number of applications and the rate of application may depend on the intensity of infestation by the corresponding pest.

[0131] In some embodiments, antimicrobial compositions of the present disclosure may be prophylactically applied to an environmental area to prevent infection by a pathogenic microbe, which may be killed or reduced in numbers in a given area by the methods of the present disclosure. In some embodiments, the microbe comes into contact with, an antimicrobially-effective amount of the antimicrobial compositions of the present disclosure as described herein.

[0132] In some embodiments, the antimicrobial compositions of the present disclosure may be made by formulating the engineered, non-naturally occurring antimicrobial peptide with the desired agriculturally-acceptable carrier. The combinations and/or compositions may be formulated prior to administration in an appropriate means such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or suitable diluent, such as saline and/or other buffer. In some embodiments, the formulated compositions may be in the form of a dust or granular material, or a suspension in oil (vegetable or mineral), or water or oil/water emulsions, or as a wettable powder, or in combination with any other carrier material suitable for agricultural application. Suitable agricultural carriers can be solid or liquid and are well known in the art. In some embodiments, the composition may be mixed with one or more solid or liquid adjuvants and prepared by various means, e.g., by homogeneously mixing, blending and/or grinding the pesticidal composition with suitable adjuvants using conventional composition techniques. Suitable composition and application methods are described in U.S. Pat. No. 6,468,523, herein incorporated by reference in its entirety. [0133] In some embodiments an antimicrobial composition of the present disclosure can be formulated at a pH ranging from about 5 to about 11 ; from about 5.5 to about 11 ; from about 6 to about 11 ; from about 6.5 to about 11 ; from about 7 to about 11 ; from about 7.5 to about 11 ; from about 8 to about 11 ; from about 8.5 to about 11 ; from about 9 to about 11 ; from about 9.5 to about 11; from about 10 to about 11; or from about 10.5 to about 11.

[0134] In some embodiments an antimicrobial composition of the present disclosure can be formulated at a pH ranging from about 5 to about 11 ; from about 5 to about 10.5; from about 5 to about 10; from about 5 to about 9.5; from about 5 to about 9; from about 5 to about 8.5; from about 5 to about 8; from about 5 to about 7.5; from about 5 to about 7; from about 5 to about 6.5; from about 5 to about 6; or from about 5 to about 5.5.

[0135] In some embodiments an antimicrobial composition of the present disclosure can be formulated into a granule form (granular formulation). Methods of generating a granular formulation are well known in the art, and include: crystallization, precipitation, pan-coating, fluid bed coating, agglomeration (e.g., fluid bed agglomeration), rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation, and the like.

[0136] In some embodiments, the granular formulation can be generated via agglomeration, e.g., spray-drying agglomeration; rewet agglomeration; fluid bed agglomeration; and the like.

[0137] In some embodiments, the type of agglomeration can be fluid bed agglomeration. Exemplary methods of fluid bed agglomeration are provided in U.S. Patent No. 7,582,147; the disclosure of which is incorporated herein by reference in its entirety.

[0138] In some embodiments, the granular formulation can be generated via fluid bed agglomeration.

[0139] In some embodiments, the granular formulation can be generated by spraying the active and inert ingredients onto a blank carrier in a fluid bed.

[0140] In some embodiments, the granular formulation can be generated by spraying the active and inert ingredients (excipients) onto a blank carrier and granulated in pan granulator. [0141] In some embodiments, the granular formulation can be generated by mixing the active and inert powders (i.e., one or more excipients described herein) and water, and subsequently granulated by passing the ingredients through an extruder. [0142] In some embodiments, the granular formulation can be generated by mixing the active and inert powders (i.e., one or more excipients described herein) with water, and granulated by roll compaction.

[0143] Antimicrobial compositions comprising an engineered, non-naturally occurring antimicrobial peptide , or an agriculturally acceptable salt thereof; and an excipient, said compositions can include, but are not limited to, aerosols and/or aerosolized products, e.g., sprays, fumigants, powders, dusts, and/or gases; and/or seed dressings.

[0144] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient.

[0145] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient.

[0146] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, wherein the engineered, non-naturally occurring antimicrobial peptide consists of an amino acid sequence set forth in SEQ ID NO: 1.

[0147] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient, wherein the engineered, non-naturally occurring antimicrobial peptide consists of an amino acid sequence as set forth in SEQ ID NO: 1.

[0148] Sprayable compositions

[0149] Examples of spray products of the present disclosure can include field sprayable compositions for agricultural usage and indoor sprays for use in interior spaces in a residential or commercial space. In some embodiments, residual sprays or space sprays having an engineered, non-naturally occurring antimicrobial peptide and an excipient can be used to reduce or eliminate microbes in an interior space.

[0150] Surface spraying indoors (SSI) is the technique of applying a variable volume sprayable volume of a composition onto indoor surfaces where vectors rest, such as on walls, windows, floors and ceilings. The primary goal of variable volume sprayable volume is to reduce the lifespan of the microbe and thereby reduce or interrupt disease transmission. As its name implies, SSI involves applying the composition onto the walls and other surfaces of a house with a residual antimicrobial (e.g., an engineered, non-naturally occurring antimicrobial peptide).

[0151] In one embodiment, the composition having an engineered, non-naturally occurring antimicrobial peptide and an excipient will inhibit growth and/or proliferation of microbes that come in contact with these surfaces.

[0152] In contrast to SSI, which requires that the an engineered, non-naturally occurring antimicrobial peptide or an agriculturally acceptable salt thereof, be bound to surfaces of dwellings, such as walls or ceilings, as with a paint, for example, space spray products of the disclosure rely on the production of a large number of small droplets intended to be distributed through a volume of air over a given period of time. When these droplets impact on a target microbe, they deliver an effective dose of an engineered, non-naturally occurring antimicrobial peptide, effective to inhibit or kill the microbe. The traditional methods for generating a spacespray include thermal fogging (whereby a dense cloud of a composition having an engineered, non-naturally occurring antimicrobial peptide is produced giving the appearance of a thick fog) and Ultra Low Volume (ULV), whereby droplets are produced by a cold, mechanical aerosolgenerating machine. Ready-to-use aerosols such as aerosol cans may also be used.

[0153] In some embodiments, a sprayable composition may contain an amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, ranging from about 0.0001 wt% to about 99.9999 wt%, based on the total weight of the sprayable composition.

[0154] In some embodiments, a sprayable composition may contain an amount of an excipient, or a plurality of excipients in the aggregate, ranging from about 0.0001 wt% to about 99.9999 wt%.

[0155] Foams

[0156] The antimicrobial compositions of the present disclosure comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient may be made available in a spray product as an aerosol-based application, including aerosolized foam applications. Pressurized cans are the typical vehicle for the formation of aerosols. An aerosol propellant that is compatible with the composition having an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient are used. Preferably, a liquefied-gas type propellant is used. [0157] Suitable propellants include compressed air, carbon dioxide, butane and nitrogen. The concentration of the propellant in the active compound composition is from about 5 percent to about 40 percent by weight of the pyridine composition, preferably from about 15 percent to about 30 percent by weight of the composition an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient.

[0158] In one embodiment, antimicrobial compositions of the present disclosure comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient can also include one or more foaming agents. Foaming agents that can be used include sodium laureth sulfate, cocamide DEA, and cocamidopropyl betaine. Preferably, the sodium laureth sulfate, cocamide DEA and cocamidopropyl are used in combination. The concentration of the foaming agent(s) in the active compound composition is from about 10 percent to about 25 percent by weight, more preferably 15 percent to 20 percent by weight of the composition.

[0159] When such compositions are used in an aerosol application not containing foaming agents, the active compositions of the present disclosure can be used without the need for mixing directly prior to use. However, aerosol compositions containing the foaming agents do require mixing (i.e., shaking) immediately prior to use. In addition, if the compositions containing foaming agents are used for an extended time, they may require additional mixing at periodic intervals during use.

[0160] In some embodiments, a foam may contain an amount of an engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, ranging from about 0.0001 wt% to about 99.9999 wt%.

[0161] In some embodiments, a foam may contain an amount of an excipient or a plurality of excipients, in the aggregate, ranging from about 0.005 wt% to about 99 wt%.

[0162] Fabric treatments

[0163] In some embodiments, fabrics and garments may be made that contain an antimicrobially-effective composition comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient. In some embodiments, the concentration of the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof in the polymeric material, fiber, yam, weave, net, or substrate described herein, can be varied within a relatively wide concentration range from, for example, 0.05 to 15 percent by weight, preferably 0.2 to 10 percent by weight, more preferably 0.4 to 8 percent by weight, especially 0.5 to 5, such as 1 to 3, percent by weight.

[0164] Similarly, the concentration of the antimicrobial composition of the present disclosure comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient (whether for treating surfaces or for coating a fiber, yam, net, weave) can be varied within a relatively wide concentration range from, for example 0.1 to 70 percent by weight, such as 0.5 to 50 percent by weight, preferably 1 to 40 percent by weight, more preferably 5 to 30 percent by weight, especially 10 to 20 percent by weight.

[0165] The concentration of the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof may be chosen according to the field of application such that the requirements concerning inhibitory or lethal efficacy, durability and toxicity are met. Adapting the properties of the material can also be accomplished and so custom-tailored textile fabrics are obtainable in this way.

[0166] Accordingly, an effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof can depend on the specific use pattern, the microbe against which control is most desired and the environment in which the an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof will be used. Therefore, an effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof is sufficient that inhibition or killing or one or more targeted microbes are achieved.

[0167] In some embodiments, a fabric treatment may contain an amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof ranging from about 0.0001 wt% to about 99.9999 wt%.

[0168] In some embodiments, a fabric treatment may contain an amount of an excipient, or a plurality of excipients, in the aggregate ranging from about 0.0001 wt% to about 99.9999 wt%.

[0169] Surface-treatment compositions

[0170] In some embodiments, the present disclosure provides compositions having an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient, for coating walls, floors and ceilings inside of buildings, and for coating a substrate or non-living material. The inventive compositions comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient, can be prepared using known techniques for the purpose in mind. Preparations of antimicrobial compositions of the present disclosure comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient could be so formulated to also contain a binder to facilitate the binding of the compound to the surface or other substrate. Agents useful for binding are known in the art and tend to be polymeric in form. The type of binder suitable for a compositions to be applied to a wall surface having particular porosities and/or binding characteristics would be different compared to a fiber, yam, weave or net — thus, a skilled person, based on known teachings, would select a suitable binder based on the desired surface and/or substrate.

[0171] Typical binders include but are not limited to poly vinyl alcohol, modified starch, poly vinyl acrylate, polyacrylic, polyvinyl acetate co polymer, polyurethane, and modified vegetable oils. Suitable binders can include latex dispersions derived from a wide variety of polymers and co-polymers and combinations thereof. Suitable latexes for use as binders in the inventive compositions comprise polymers and copolymers of styrene, alkyl styrenes, isoprene, butadiene, acrylonitrile lower alkyl acrylates, vinyl chloride, vinylidene chloride, vinyl esters of lower carboxylic acids and alpha, beta-ethylenically unsaturated carboxylic acids, including polymers containing three or more different monomer species copolymerized therein, as well as post-dispersed suspensions of silicones or polyurethanes. Also suitable may be a polytetrafluoroethylene (PTFE) polymer for binding the active ingredient to other surfaces.

[0172] In some embodiments, a surface-treatment composition may contain an amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof ranging from about 0.0001 wt% to about 99.9999 wt%.

[0173] In some embodiments, a surface-treatment composition may contain an amount of an excipient, or a plurality of excipients, in the aggregate, ranging from about 0.0001 wt% to about 99.9999 wt%.

[0174] Dispersants

[0175] In some exemplary embodiments, an antimicrobial composition of the present disclosure comprises an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient, wherein the excipient can be a diluent or carrier (e.g., such as water), a polymeric binder, and/or additional components such as a dispersing agent, a polymerizing agent, an emulsifying agent, a thickener, an alcohol, a fragrance, or any other inert excipients used in the preparation of sprayable actives known in the art.

[0176] In some embodiments, a dispersant may contain an amount of an engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, ranging from about 0.0001 wt% to about 99.9999 wt%.

[0177] In some embodiments, a dispersant may contain an amount of an excipient, or a plurality of excipients, in the aggregate, ranging from about 0.0001 wt% to about 99.9999 wt%.

[0178] Suspensions

[0179] In some embodiments, an antimicrobial composition comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient, can be prepared in a number of different forms or formulation types, such as suspensions or capsules suspensions. And a person skilled in the art can prepare the relevant composition based on the properties of the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, its uses, and also its application type.

[0180] For example, in some embodiments, the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, used in the methods, embodiments, and other aspects of the present disclosure, may be encapsulated in a suspension or capsule suspension formulation. An encapsulated engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, of the present disclosure can provide improved wash-fastness, and also a longer period of activity. The formulation can be organic based or aqueous based, preferably aqueous based.

[0181] In some embodiments, a suspension may contain an amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, ranging from about 0.0001 wt% to about 99.9999 wt%.

[0182] In some embodiments, a suspension may contain an amount of an excipient, or a plurality of excipients, in the aggregate, ranging from about 0.0001 wt% to about 99.9999 wt%.

[0183] Microencapsulation

[0184] Microencapsulated engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, suitable for use in the compositions and methods according to the present disclosure may be prepared with any suitable technique known in the art. For example, various processes for microencapsulating material have been previously developed. These processes can be divided into three categories: physical methods, phase separation, and interfacial reaction.

[0185] In the physical methods category, microcapsule wall material and core particles are physically brought together and the wall material flows around the core particle to form the microcapsule. In the phase separation category, microcapsules are formed by emulsifying or dispersing the core material in an immiscible continuous phase in which the wall material is dissolved and caused to physically separate from the continuous phase, such as by coacervation, and deposit around the core particles. In the interfacial reaction category, microcapsules are formed by emulsifying or dispersing the core material in an immiscible continuous phase and then an interfacial polymerization reaction is caused to take place at the surface of the core particles. The concentration of the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof present in the microcapsules can vary from about 0.0001% to about 99.9999% by weight of the microcapsule.

[0186] In some embodiments, a microencapsulation may contain an amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof ranging from about 0.0001 wt% to about 99.9999 wt%.

[0187] In some embodiments, a microencapsulation may contain an amount of an excipient, or a plurality of excipients, in the aggregate, ranging from about 0.0001 wt% to about 99.9999 wt%.

[0188] Any of the compositions or formulations described herein can be made using an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof described herein (e.g., an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1), and any of the excipients described herein.

[0189] Any of the compositions or formulations described herein can additionally contain other known biologically active agents, such as, for example, a fungicide, herbicide, or insecticide.

[0190] Kits, compositions, dispersants, and the ingredients thereof [0191] The combinations used in the antimicrobial compositions of the present disclosure comprising an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient, methods, embodiments and other aspects according to the present disclosure, may be formed by mixing all ingredients together, optionally with water, and optionally using suitable mixing and/or dispersing aggregates. In general, such a combination is formed at a temperature of from 10°C to 70°C, preferably 15 °C to 50°C, more preferably 20°C to 40°C.

[0192] Generally, an antimicrobial composition of the present disclosure comprising one or more of (Al and A2), (B), (C), and/or (D) is possible, wherein it is possible to use: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof (Al) and an excipient (A2); solid polymer (B); optional additional additives (D); and to disperse them in the aqueous component (C). If a binder is present in an antimicrobial composition of the present disclosure, it is preferred to use dispersions of the polymeric binder (B) in water as well as aqueous compositions of the an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof (Al) and excipient (A2) in water which have been separately prepared before. Such separate compositions may contain additional additives for stabilizing (Al and A2) and/or (B) in the respective antimicrobial compositions and are commercially available. In a second process step, such raw compositions and optionally additional water (component (C)) are added. Also, combinations of the abovementioned ingredients based on the foregoing scheme are likewise possible, e.g., using a pre-formed dispersion of (Al and A2) and/or (B) and mixing it with solid (Al and A2) and/or (B). A dispersion of the polymeric binder (B) may be a pre-manufactured dispersion already made by a chemicals manufacturer.

[0193] Moreover, it is also within the scope of the present disclosure to use “hand-made” dispersions, i.e., dispersions made in small-scale by an end-user. Such dispersions may be made by providing a mixture of about 20 percent of the binder (B) in water, heating the mixture to temperature of 90°C to 100°C and intensively stirring the mixture for several hours. It is possible to manufacture the antimicrobial composition as a final product so that it can be readily used by the end-user for the process according to the present disclosure. It is of course similarly possible to manufacture a concentrate, which may be diluted by the end-user with additional water (C) to the desired concentration for use. [0194] In an embodiment, a composition (having an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient) suitable for SSI application or a coating formulation (having an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient), contains the active ingredient and a carrier, such as water, and may also one or more co-formulants selected from a dispersant, a wetter, an anti-freeze, a thickener, a preservative, an emulsifier and a binder or sticker.

[0195] In some embodiments, an exemplary solid combination of an engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, is generally milled to a desired particle size, such as the particle size distribution d(0.5) is generally from 3 to 20, preferably 5 to 15, especially 7 to 12, pm.

[0196] Furthermore, it may be possible to ship an antimicrobial composition to the enduser as a kit, e.g., comprising at least a first component of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof (Al) and an excipient (A2) (either separately or as a mixture); and a second component comprising at least one polymeric binder (B). Further additives (D) may be a third separate component of the kit, or may be already mixed with components (Al and A2) and/or (B). The end-user may prepare the antimicrobial composition for use by mixing (Al) and (A2) and mixing. Alternatively, the end-user may prepare the antimicrobial composition for use by just adding water (C) to the components of the kit and mixing. The components of the kit may also be compositions in water. Of course it is possible to combine an aqueous composition of one of the components with a dry composition of the other component(s). As an example, the kit can consist of one composition of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof (Al) and an excipient (A2) and optionally water (C); and a second, separate composition of at least one polymeric binder (B), water as component (C) and optionally one or more components (D). [0197] The concentrations of the components (Al and A2), (B), (C) and optionally (D) will be selected by the skilled artisan depending of the technique to be used for coating/treating. In some embodiments, the amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof can be up to 50, preferably 1 to 50, such as 10 to 40, especially 15 to 30, percent by weight, based on weight of the composition. The amount of polymeric binder (B) may be in the range of 0.01 to 30, preferably 0.5 to 15, more preferably 1 to 10, especially 1 to 5, percent by weight, based on weight of the composition. If present, in general the amount of additional components (D) is from 0.1 to 20, preferably 0.5 to 15, percent by weight, based on weight of the composition. If present, suitable amounts of pigments and/or dyestuffs and/or fragrances are in general 0.01 to 5, preferably 0.1 to 3, more preferably 0.2 to 2, percent by weight, based on weight of the composition. In some embodiments, a composition ready for use comprises 0.1 to 40, preferably 1 to 30, percent of components (Al and A2), (B), and optionally (D), the residual amount being water (C). In some embodiments, a concentration of a concentrate to be diluted by the end-user may comprise 5 to 70, preferably 10 to 60, percent of components (Al and A2), (B), and optionally (D), the residual amount being water (C).

[0198] RANGES AND DESCRIPTIONS OF COMPOSITION COMPONENTS [0199] % v/v; % w/w; and % w/v

[0200] As used herein, ^UN!N” or “% v/v” or “volume per volume” refers to the volume concentration of a solution (“v/v” stands for volume per volume). Here, v/v can be used when both components of a solution are liquids. For example, when 50 mL of ingredient X is diluted with 50 mL of water, there will be 50 mL of ingredient X in a total volume of 100 mL; therefore, this can be expressed as “ingredient X 50% v/v.” Percent volume per volume (% v/v) is calculated as follows: (volume of solute (mL)/ volume of solution (100 mL)); e.g., % v/v = mL of solute/ 100 mL of solution.

[0201] As used herein, “w/w” or “% w/w” or “weight per weight” or “wt/wt” or “% wt/wt” refers to the weight concentration of a composition or solution, i.e., percent weight in weight (“w/w” stands for weight per weight). Here, w/w expresses the number of grams (g) of a constituent in 100 g of solution or mixture. For example, a mixture consisting of 30 g of ingredient X, and 70 g of water would be expressed as “ingredient X 30% w/w.” Percent weight per weight (% w/w) is calculated as follows: (weight of solute (g)/ weight of solution (g)) x 100; or (mass of solute (g)/ mass of solution (g)) x 100.

[0202] As used herein, “"WIN” or “% w/v” or “weight per volume” refers to the mass concentration of a solution, i.e., percent weight in volume (“"WIN” stands for weight per volume). Here, w/v expresses the number of grams (g) of a constituent in 100 mL of solution. For example, if 1 g of ingredient X is used to make up a total volume of 100 mL, then a “1% w/v solution of ingredient X” has been made. Percent weight per volume (% w/v) is calculated as follows: (Mass of solute (g)/ Volume of solution (mL)) x 100. [0203] In some embodiments, an antimicrobial composition of the present disclosure can comprise: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient; wherein the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof is in an amount ranging from about 0.00001% w/w to about 99.9% w/w of the total composition, or from about 0.01% to about 99.9%; from about 0.02% to about 99.9%; from about 0.03% to about 99.9%; from about 0.04% to about 99.9%; from about 0.05% to about 99.9%; from about 0.06% to about 99.9%; from about 0.07% to about 99.9%; from about 0.08% to about 99.9%; from about 0.09% to about 99.9%; from about 0.1% to about 99.9%; from about 0.2% to about 99.9%; from about 0.3% to about 99.9%; from about 0.4% to about 99.9%; from about 0.5% to about 99.9%; from about 0.6% to about 99.9%; from about 0.7% to about 99.9%; from about 0.8% to about 99.9%; from about 0.9% to about 99.9%; from about 1% to about 99.9%; from about 2% to about 99.9%; from about 3% to about 99.9%; from about 4% to about 99.9%; from about 5% to about 99.9%; from about 6% to about 99.9%; from about 7% to about 99.9%; from about 8% to about 99.9%; from about 9% to about 99.9%; from about 10% to about 99.9%; from about 11% to about 99.9%; from about 12% to about 99.9%; from about 13% to about 99.9%; from about 14% to about 99.9%; from about 15% to about 99.9%; from about 16% to about 99.9%; from about 17% to about 99.9%; from about 18% to about 99.9%; from about 19% to about 99.9%; from about 20% to about 99.9%; from about 21% to about 99.9%; from about 22% to about 99.9%; from about 23% to about 99.9%; from about 24% to about 99.9%; from about 25% to about 99.9%; from about 26% to about 99.9%; from about 27% to about 99.9%; from about 28% to about 99.9%; from about 29% to about 99.9%; from about 30% to about 99.9%; from about 31% to about 99.9%; from about 32% to about 99.9%; from about 33% to about 99.9%; from about 34% to about 99.9%; from about 35% to about 99.9%; from about 36% to about 99.9%; from about 37% to about 99.9%; from about 38% to about 99.9%; from about 39% to about 99.9%; from about 40% to about 99.9%; from about 41% to about 99.9%; from about 42% to about 99.9%; from about 43% to about 99.9%; from about 44% to about 99.9%; from about 45% to about 99.9%; from about 46% to about 99.9%; from about 47% to about 99.9%; from about 48% to about 99.9%; from about 49% to about 99.9%; from about 50% to about 99.9%; from about 51% to about 99.9%; from about 52% to about 99.9%; from about 53% to about 99.9%; from about 54% to about 99.9%; from about 55% to about 99.9%; from about 56% to about 99.9%; from about 57% to about 99.9%; from about 58% to about 99.9%; from about 59% to about 99.9%; from about 60% to about 99.9%; from about 61% to about 99.9%; from about 62% to about 99.9%; from about 63% to about 99.9%; from about 64% to about 99.9%; from about 65% to about 99.9%; from about 66% to about 99.9%; from about 67% to about 99.9%; from about 68% to about 99.9%; from about 69% to about 99.9%; from about 70% to about 99.9%; from about 71% to about 99.9%; from about 72% to about 99.9%; from about 73% to about 99.9%; from about 74% to about 99.9%; from about 75% to about 99.9%; from about 76% to about 99.9%; from about 77% to about 99.9%; from about 78% to about 99.9%; from about 79% to about 99.9%; from about 80% to about 99.9%; from about 81% to about 99.9%; from about 82% to about 99.9%; from about 83% to about 99.9%; from about 84% to about 99.9%; from about 85% to about 99.9%; from about 86% to about 99.9%; from about 87% to about 99.9%; from about 88% to about 99.9%; from about 89% to about 99.9%; from about 90% to about 99.9%; from about 91% to about 99.9%; from about 92% to about 99.9%; from about 93% to about 99.9%; from about 94% to about 99.9%; from about 95% to about 99.9%; from about 96% to about 99.9%; from about 97% to about 99.9%; from about 98% to about 99.9%; or from about 99% to about 99.9%, w/w of the total composition.

[0204] In some embodiments, an antimicrobial composition of the present disclosure can comprise: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient.

[0205] In some embodiments, an antimicrobial composition of the present disclosure can comprise: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; wherein the excipient is in an amount ranging from about 0.01% to about 99.9%; from about 0.02% to about 99.9%; from about 0.03% to about 99.9%; from about 0.04% to about 99.9%; from about 0.05% to about 99.9%; from about 0.06% to about 99.9%; from about 0.07% to about 99.9%; from about 0.08% to about 99.9%; from about 0.09% to about 99.9%; from about 0.1% to about 99.9%; from about 0.2% to about 99.9%; from about 0.3% to about 99.9%; from about 0.4% to about 99.9%; from about 0.5% to about 99.9%; from about 0.6% to about 99.9%; from about 0.7% to about 99.9%; from about 0.8% to about 99.9%; from about 0.9% to about 99.9%; from about 1% to about 99.9%; from about 2% to about 99.9%; from about 3% to about 99.9%; from about 4% to about 99.9%; from about 5% to about 99.9%; from about 6% to about 99.9%; from about 7% to about 99.9%; from about 8% to about 99.9%; from about 9% to about 99.9%; from about 10% to about 99.9%; from about 11% to about 99.9%; from about 12% to about 99.9%; from about 13% to about 99.9%; from about 14% to about 99.9%; from about 15% to about 99.9%; from about 16% to about 99.9%; from about 17% to about 99.9%; from about 18% to about 99.9%; from about 19% to about 99.9%; from about 20% to about 99.9%; from about 21% to about 99.9%; from about 22% to about 99.9%; from about 23% to about 99.9%; from about 24% to about 99.9%; from about 25% to about 99.9%; from about 26% to about 99.9%; from about 27% to about 99.9%; from about 28% to about 99.9%; from about 29% to about 99.9%; from about 30% to about 99.9%; from about 31% to about 99.9%; from about 32% to about 99.9%; from about 33% to about 99.9%; from about 34% to about 99.9%; from about 35% to about 99.9%; from about 36% to about 99.9%; from about 37% to about 99.9%; from about 38% to about 99.9%; from about 39% to about 99.9%; from about 40% to about 99.9%; from about 41% to about 99.9%; from about 42% to about 99.9%; from about 43% to about 99.9%; from about 44% to about 99.9%; from about 45% to about 99.9%; from about 46% to about 99.9%; from about 47% to about 99.9%; from about 48% to about 99.9%; from about 49% to about 99.9%; from about 50% to about 99.9%; from about 51% to about 99.9%; from about 52% to about 99.9%; from about 53% to about 99.9%; from about 54% to about 99.9%; from about 55% to about 99.9%; from about 56% to about 99.9%; from about 57% to about 99.9%; from about 58% to about 99.9%; from about 59% to about 99.9%; from about 60% to about 99.9%; from about 61% to about 99.9%; from about 62% to about 99.9%; from about 63% to about 99.9%; from about 64% to about 99.9%; from about 65% to about 99.9%; from about 66% to about 99.9%; from about 67% to about 99.9%; from about 68% to about 99.9%; from about 69% to about 99.9%; from about 70% to about 99.9%; from about 71% to about 99.9%; from about 72% to about 99.9%; from about 73% to about 99.9%; from about 74% to about 99.9%; from about 75% to about 99.9%; from about 76% to about 99.9%; from about 77% to about 99.9%; from about 78% to about 99.9%; from about 79% to about 99.9%; from about 80% to about 99.9%; from about 81% to about 99.9%; from about 82% to about 99.9%; from about 83% to about 99.9%; from about 84% to about 99.9%; from about 85% to about 99.9%; from about 86% to about 99.9%; from about 87% to about 99.9%; from about 88% to about 99.9%; from about 89% to about 99.9%; from about 90% to about 99.9%; from about 91% to about 99.9%; from about 92% to about 99.9%; from about 93% to about 99.9%; from about 94% to about 99.9%; from about 95% to about 99.9%; from about 96% to about 99.9%; from about 97% to about 99.9%; from about 98% to about 99.9%; or from about 99% to about 99.9%, w/w of the total composition.

[0206] In some embodiments, an antimicrobial composition of the present disclosure can comprise: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; wherein the excipient ranges from about 0.01% to about 99.9%; from about 0.02% to about 99.9%; from about 0.03% to about 99.9%; from about 0.04% to about 99.9%; from about 0.05% to about 99.9%; from about 0.06% to about 99.9%; from about 0.07% to about 99.9%; from about 0.08% to about 99.9%; from about 0.09% to about 99.9%; from about 0.1% to about 99.9%; from about 0.2% to about 99.9%; from about 0.3% to about 99.9%; from about 0.4% to about 99.9%; from about 0.5% to about 99.9%; from about 0.6% to about 99.9%; from about 0.7% to about 99.9%; from about 0.8% to about 99.9%; from about 0.9% to about 99.9%; from about 1% to about 99.9%; from about 2% to about 99.9%; from about 3% to about 99.9%; from about 4% to about 99.9%; from about 5% to about 99.9%; from about 6% to about 99.9%; from about 7% to about 99.9%; from about 8% to about 99.9%; from about 9% to about 99.9%; from about 10% to about 99.9%; from about 11% to about 99.9%; from about 12% to about 99.9%; from about 13% to about 99.9%; from about 14% to about 99.9%; from about 15% to about 99.9%; from about 16% to about 99.9%; from about 17% to about 99.9%; from about 18% to about 99.9%; from about 19% to about 99.9%; from about 20% to about 99.9%; from about 21% to about 99.9%; from about 22% to about 99.9%; from about 23% to about 99.9%; from about 24% to about 99.9%; from about 25% to about 99.9%; from about 26% to about 99.9%; from about 27% to about 99.9%; from about 28% to about 99.9%; from about 29% to about 99.9%; from about 30% to about 99.9%; from about 31% to about 99.9%; from about 32% to about 99.9%; from about 33% to about 99.9%; from about 34% to about 99.9%; from about 35% to about 99.9%; from about 36% to about 99.9%; from about 37% to about 99.9%; from about 38% to about 99.9%; from about 39% to about 99.9%; from about 40% to about 99.9%; from about 41% to about 99.9%; from about 42% to about 99.9%; from about 43% to about 99.9%; from about 44% to about 99.9%; from about 45% to about 99.9%; from about 46% to about 99.9%; from about 47% to about 99.9%; from about 48% to about 99.9%; from about 49% to about 99.9%; from about 50% to about 99.9%; from about 51% to about 99.9%; from about 52% to about 99.9%; from about 53% to about 99.9%; from about 54% to about 99.9%; from about 55% to about 99.9%; from about 56% to about 99.9%; from about 57% to about 99.9%; from about 58% to about 99.9%; from about 59% to about 99.9%; from about 60% to about 99.9%; from about 61% to about 99.9%; from about 62% to about 99.9%; from about 63% to about 99.9%; from about 64% to about 99.9%; from about 65% to about 99.9%; from about 66% to about 99.9%; from about 67% to about 99.9%; from about 68% to about 99.9%; from about 69% to about 99.9%; from about 70% to about 99.9%; from about 71% to about 99.9%; from about 72% to about 99.9%; from about 73% to about 99.9%; from about 74% to about 99.9%; from about 75% to about 99.9%; from about 76% to about 99.9%; from about 77% to about 99.9%; from about 78% to about 99.9%; from about 79% to about 99.9%; from about 80% to about 99.9%; from about 81% to about 99.9%; from about 82% to about 99.9%; from about 83% to about 99.9%; from about 84% to about 99.9%; from about 85% to about 99.9%; from about 86% to about 99.9%; from about 87% to about 99.9%; from about 88% to about 99.9%; from about 89% to about 99.9%; from about 90% to about 99.9%; from about 91% to about 99.9%; from about 92% to about 99.9%; from about 93% to about 99.9%; from about 94% to about 99.9%; from about 95% to about 99.9%; from about 96% to about 99.9%; from about 97% to about 99.9%; from about 98% to about 99.9%; or from about 99% to about 99.9%, w/w of the total composition.

[0207] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient, wherein the concentration of the engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof ranges from about 0.000001% to about 99.9% w/w of the total composition, for example, 0.00001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 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%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% by weight of the total combination or composition. [0208] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and an excipient, wherein the concentration of the engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof ranges from about 0.1% to about 99.9%; from about 1% to about 99.9%; from about 2% to about 99.9%; from about 3% to about 99.9%; from about 4% to about 99.9%; from about 5% to about 99.9%; from about 6% to about 99.9%; from about 7% to about 99.9%; from about 8% to about 99.9%; from about 9% to about 99.9%; from about 10% to about 99.9%; from about 11% to about 99.9%; from about 12% to about 99.9%; from about 13% to about 99.9%; from about 14% to about 99.9%; from about 15% to about 99.9%; from about 16% to about 99.9%; from about 17% to about 99.9%; from about 18% to about 99.9%; from about 19% to about 99.9%; from about 20% to about 99.9%; from about 21% to about 99.9%; from about 22% to about 99.9%; from about 23% to about 99.9%; from about 24% to about 99.9%; from about 25% to about 99.9%; from about 26% to about 99.9%; from about 27% to about 99.9%; from about 28% to about 99.9%; from about 29% to about 99.9%; from about 30% to about 99.9%; from about 31% to about 99.9%; from about 32% to about 99.9%; from about 33% to about 99.9%; from about 34% to about 99.9%; from about 35% to about 99.9%; from about 36% to about 99.9%; from about 37% to about 99.9%; from about 38% to about 99.9%; from about 39% to about 99.9%; from about 40% to about 99.9%; from about 41% to about 99.9%; from about 42% to about 99.9%; from about 43% to about 99.9%; from about 44% to about 99.9%; from about 45% to about 99.9%; from about 46% to about 99.9%; from about 47% to about 99.9%; from about 48% to about 99.9%; from about 49% to about 99.9%; from about 50% to about 99.9%; from about 51% to about 99.9%; from about 52% to about 99.9%; from about 53% to about 99.9%; from about 54% to about 99.9%; from about 55% to about 99.9%; from about 56% to about 99.9%; from about 57% to about 99.9%; from about 58% to about 99.9%; from about 59% to about 99.9%; from about 60% to about 99.9%; from about 61% to about 99.9%; from about 62% to about 99.9%; from about 63% to about 99.9%; from about 64% to about 99.9%; from about 65% to about 99.9%; from about 66% to about 99.9%; from about 67% to about 99.9%; from about 68% to about 99.9%; from about 69% to about 99.9%; from about 70% to about 99.9%; from about 71% to about 99.9%; from about 72% to about 99.9%; from about 73% to about 99.9%; from about 74% to about 99.9%; from about 75% to about 99.9%; from about 76% to about 99.9%; from about 77% to about 99.9%; from about 78% to about 99.9%; from about 79% to about 99.9%; from about 80% to about 99.9%; from about 81% to about 99.9%; from about 82% to about 99.9%; from about 83% to about 99.9%; from about 84% to about 99.9%; from about 85% to about 99.9%; from about 86% to about 99.9%; from about 87% to about 99.9%; from about 88% to about 99.9%; from about 89% to about 99.9%; from about 90% to about 99.9%; from about 91% to about 99.9%; from about 92% to about 99.9%; from about 93% to about 99.9%; from about 94% to about 99.9%; from about 95% to about 99.9%; from about 96% to about 99.9%; from about 97% to about 99.9%; from about 98% to about 99.9%; or from about 99% to about 99.9%, wt/wt of the total combination or composition.

[0209] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient, wherein the concentration of the engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, ranges from about 0.1% to about 99%; from about 0.1% to about 98%; from about 0.1% to about 97%; from about 0.1% to about 96%; from about 0.1% to about 95%; from about 0.1% to about 94%; from about 0.1% to about 93%; from about 0.1% to about 92%; from about 0.1% to about 91%; from about 0.1% to about 90%; from about 0.1% to about 89%; from about 0.1% to about 88%; from about 0.1% to about 87%; from about 0.1% to about 86%; from about 0.1% to about 85%; from about 0.1% to about 84%; from about 0.1% to about 83%; from about 0.1% to about 82%; from about 0.1% to about 81%; from about 0.1% to about 80%; from about 0.1% to about 79%; from about 0.1% to about 78%; from about 0.1% to about 77%; from about 0.1% to about 76%; from about 0.1% to about 75%; from about 0.1% to about 74%; from about 0.1% to about 73%; from about 0.1% to about 72%; from about 0.1% to about 71%; from about 0.1% to about 70%; from about 0.1% to about 69%; from about 0.1% to about 68%; from about 0.1% to about 67%; from about 0.1% to about 66%; from about 0.1% to about 65%; from about 0.1% to about 64%; from about 0.1% to about 63%; from about 0.1% to about 62%; from about 0.1% to about 61%; from about 0.1% to about 60%; from about 0.1% to about 59%; from about 0.1% to about 58%; from about 0.1% to about 57%; from about 0.1% to about 56%; from about 0.1% to about 55%; from about 0.1% to about 54%; from about 0.1% to about 53%; from about 0.1% to about 52%; from about 0.1% to about 51%; from about 0.1% to about 50%; from about 0.1% to about 49%; from about 0.1% to about 48%; from about 0.1% to about 47%; from about 0.1% to about 46%; from about 0.1% to about 45%; from about 0.1% to about 44%; from about 0.1% to about 43%; from about 0.1% to about 42%; from about 0.1% to about 41%; from about 0.1% to about 40%; from about 0.1% to about 39%; from about 0.1% to about 38%; from about 0.1% to about 37%; from about 0.1% to about 36%; from about 0.1% to about 35%; from about 0.1% to about 34%; from about 0.1% to about 33%; from about 0.1% to about 32%; from about 0.1% to about 31%; from about 0.1% to about 30%; from about 0.1% to about 29%; from about 0.1% to about 28%; from about 0.1% to about 27%; from about 0.1% to about 26%; from about 0.1% to about 25%; from about 0.1% to about 24%; from about 0.1% to about 23%; from about 0.1% to about 22%; from about 0.1% to about 21%; from about 0.1% to about 20%; from about 0.1% to about 19%; from about 0.1% to about 18%; from about 0.1% to about 17%; from about 0.1% to about 16%; from about 0.1% to about 15%; from about 0.1% to about 14%; from about 0.1% to about 13%; from about 0.1% to about 12%; from about 0.1% to about 11%; from about 0.1% to about 10%; from about 0.1% to about 9%; from about 0.1% to about 8%; from about 0.1% to about 7%; from about 0.1% to about 6%; from about 0.1% to about 5%; from about 0.1% to about 4%; from about 0.1% to about 3%; from about 0.1% to about 2%; from about 0.1% to about 1%; or from about 0.1% to about 0.5%, wt/wt of the total composition.

[0210] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient, wherein the concentration of the excipient ranges from about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 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%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% by weight of the total composition.

[0211] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient, wherein the concentration of the excipient ranges from about 0.1% to about 99.9%; from about 1% to about 99.9%; from about 2% to about 99.9%; from about 3% to about 99.9%; from about 4% to about 99.9%; from about 5% to about 99.9%; from about 6% to about 99.9%; from about 7% to about 99.9%; from about 8% to about 99.9%; from about 9% to about 99.9%; from about 10% to about 99.9%; from about 11% to about 99.9%; from about 12% to about 99.9%; from about 13% to about 99.9%; from about 14% to about 99.9%; from about 15% to about 99.9%; from about 16% to about 99.9%; from about 17% to about 99.9%; from about 18% to about 99.9%; from about 19% to about 99.9%; from about 20% to about 99.9%; from about 21% to about 99.9%; from about 22% to about 99.9%; from about 23% to about 99.9%; from about 24% to about 99.9%; from about 25% to about 99.9%; from about 26% to about 99.9%; from about 27% to about 99.9%; from about 28% to about 99.9%; from about 29% to about 99.9%; from about 30% to about 99.9%; from about 31% to about 99.9%; from about 32% to about 99.9%; from about 33% to about 99.9%; from about 34% to about 99.9%; from about 35% to about 99.9%; from about 36% to about 99.9%; from about 37% to about 99.9%; from about 38% to about 99.9%; from about 39% to about 99.9%; from about 40% to about 99.9%; from about 41% to about 99.9%; from about 42% to about 99.9%; from about 43% to about 99.9%; from about 44% to about 99.9%; from about 45% to about 99.9%; from about 46% to about 99.9%; from about 47% to about 99.9%; from about 48% to about 99.9%; from about 49% to about 99.9%; from about 50% to about 99.9%; from about 51% to about 99.9%; from about 52% to about 99.9%; from about 53% to about 99.9%; from about 54% to about 99.9%; from about 55% to about 99.9%; from about 56% to about 99.9%; from about 57% to about 99.9%; from about 58% to about 99.9%; from about 59% to about 99.9%; from about 60% to about 99.9%; from about 61% to about 99.9%; from about 62% to about 99.9%; from about 63% to about 99.9%; from about 64% to about 99.9%; from about 65% to about 99.9%; from about 66% to about 99.9%; from about 67% to about 99.9%; from about 68% to about 99.9%; from about 69% to about 99.9%; from about 70% to about 99.9%; from about 71% to about 99.9%; from about 72% to about 99.9%; from about 73% to about 99.9%; from about 74% to about 99.9%; from about 75% to about 99.9%; from about 76% to about 99.9%; from about 77% to about 99.9%; from about 78% to about 99.9%; from about 79% to about 99.9%; from about 80% to about 99.9%; from about 81% to about 99.9%; from about 82% to about 99.9%; from about 83% to about 99.9%; from about 84% to about 99.9%; from about 85% to about 99.9%; from about 86% to about 99.9%; from about 87% to about 99.9%; from about 88% to about 99.9%; from about 89% to about 99.9%; from about 90% to about 99.9%; from about 91% to about 99.9%; from about 92% to about 99.9%; from about 93% to about 99.9%; from about 94% to about 99.9%; from about 95% to about 99.9%; from about 96% to about 99.9%; from about 97% to about 99.9%; from about 98% to about 99.9%; or from about 99% to about 99.9%, wt/wt of the total composition.

[0212] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient, wherein the concentration of the excipient ranges from about 0.1% to about 99%; from about 0.1% to about 98%; from about 0.1% to about 97%; from about 0.1% to about 96%; from about 0.1% to about 95%; from about 0.1% to about 94%; from about 0.1% to about 93%; from about 0.1% to about 92%; from about 0.1% to about 91%; from about 0.1% to about 90%; from about 0.1% to about 89%; from about 0.1% to about 88%; from about 0.1% to about 87%; from about 0.1% to about 86%; from about 0.1% to about 85%; from about 0.1% to about 84%; from about 0.1% to about 83%; from about 0.1% to about 82%; from about 0.1% to about 81%; from about 0.1% to about 80%; from about 0.1% to about 79%; from about 0.1% to about 78%; from about 0.1% to about 77%; from about 0.1% to about 76%; from about 0.1% to about 75%; from about 0.1% to about 74%; from about 0.1% to about 73%; from about 0.1% to about 72%; from about 0.1% to about 71%; from about 0.1% to about 70%; from about 0.1% to about 69%; from about 0.1% to about 68%; from about 0.1% to about 67%; from about 0.1% to about 66%; from about 0.1% to about 65%; from about 0.1% to about 64%; from about 0.1% to about 63%; from about 0.1% to about 62%; from about 0.1% to about 61%; from about 0.1% to about 60%; from about 0.1% to about 59%; from about 0.1% to about 58%; from about 0.1% to about 57%; from about 0.1% to about 56%; from about 0.1% to about 55%; from about 0.1% to about 54%; from about 0.1% to about 53%; from about 0.1% to about 52%; from about 0.1% to about 51%; from about 0.1% to about 50%; from about 0.1% to about 49%; from about 0.1% to about 48%; from about 0.1% to about 47%; from about 0.1% to about 46%; from about 0.1% to about 45%; from about 0.1% to about 44%; from about 0.1% to about 43%; from about 0.1% to about 42%; from about 0.1% to about 41%; from about 0.1% to about 40%; from about 0.1% to about 39%; from about 0.1% to about 38%; from about 0.1% to about 37%; from about 0.1% to about 36%; from about 0.1% to about 35%; from about 0.1% to about 34%; from about 0.1% to about 33%; from about 0.1% to about 32%; from about 0.1% to about 31%; from about 0.1% to about 30%; from about 0.1% to about 29%; from about 0.1% to about 28%; from about 0.1% to about 27%; from about 0.1% to about 26%; from about 0.1% to about 25%; from about 0.1% to about 24%; from about 0.1% to about 23%; from about 0.1% to about 22%; from about 0.1% to about 21%; from about 0.1% to about 20%; from about 0.1% to about 19%; from about 0.1% to about 18%; from about 0.1% to about 17%; from about 0.1% to about 16%; from about 0.1% to about 15%; from about 0.1% to about 14%; from about 0.1% to about 13%; from about 0.1% to about 12%; from about 0.1% to about 11%; from about 0.1% to about 10%; from about 0.1% to about 9%; from about 0.1% to about 8%; from about 0.1% to about 7%; from about 0.1% to about 6%; from about 0.1% to about 5%; from about 0.1% to about 4%; from about 0.1% to about 3%; from about 0.1% to about 2%; from about 0.1% to about 1%; or from about 0.1% to about 0.5%, wt/wt of the total composition.

[0213] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient, wherein the concentration of the excipient ranges from about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 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%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% by weight of the total combination or composition.

[0214] In some embodiments, an antimicrobial composition of the present disclosure comprises: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient, wherein the concentration of the excipient ranges from about 0.1% to about 99.9%; from about 1% to about 99.9%; from about 2% to about 99.9%; from about 3% to about 99.9%; from about 4% to about 99.9%; from about 5% to about 99.9%; from about 6% to about 99.9%; from about 7% to about 99.9%; from about 8% to about 99.9%; from about 9% to about 99.9%; from about 10% to about 99.9%; from about 11% to about 99.9%; from about 12% to about 99.9%; from about 13% to about 99.9%; from about 14% to about 99.9%; from about 15% to about 99.9%; from about 16% to about 99.9%; from about 17% to about 99.9%; from about 18% to about 99.9%; from about 19% to about 99.9%; from about 20% to about 99.9%; from about 21% to about 99.9%; from about 22% to about 99.9%; from about 23% to about 99.9%; from about 24% to about 99.9%; from about 25% to about 99.9%; from about 26% to about 99.9%; from about 27% to about 99.9%; from about 28% to about 99.9%; from about 29% to about 99.9%; from about 30% to about 99.9%; from about 31% to about 99.9%; from about 32% to about 99.9%; from about 33% to about 99.9%; from about 34% to about 99.9%; from about 35% to about 99.9%; from about 36% to about 99.9%; from about 37% to about 99.9%; from about 38% to about 99.9%; from about 39% to about 99.9%; from about 40% to about 99.9%; from about 41% to about 99.9%; from about 42% to about 99.9%; from about 43% to about 99.9%; from about 44% to about 99.9%; from about 45% to about 99.9%; from about 46% to about 99.9%; from about 47% to about 99.9%; from about 48% to about 99.9%; from about 49% to about 99.9%; from about 50% to about 99.9%; from about 51% to about 99.9%; from about 52% to about 99.9%; from about 53% to about 99.9%; from about 54% to about 99.9%; from about 55% to about 99.9%; from about 56% to about 99.9%; from about 57% to about 99.9%; from about 58% to about 99.9%; from about 59% to about 99.9%; from about 60% to about 99.9%; from about 61% to about 99.9%; from about 62% to about 99.9%; from about 63% to about 99.9%; from about 64% to about 99.9%; from about 65% to about 99.9%; from about 66% to about 99.9%; from about 67% to about 99.9%; from about 68% to about 99.9%; from about 69% to about 99.9%; from about 70% to about 99.9%; from about 71% to about 99.9%; from about 72% to about 99.9%; from about 73% to about 99.9%; from about 74% to about 99.9%; from about 75% to about 99.9%; from about 76% to about 99.9%; from about 77% to about 99.9%; from about 78% to about 99.9%; from about 79% to about 99.9%; from about 80% to about 99.9%; from about 81% to about 99.9%; from about 82% to about 99.9%; from about 83% to about 99.9%; from about 84% to about 99.9%; from about 85% to about 99.9%; from about 86% to about 99.9%; from about 87% to about 99.9%; from about 88% to about 99.9%; from about 89% to about 99.9%; from about 90% to about 99.9%; from about 91% to about 99.9%; from about 92% to about 99.9%; from about 93% to about 99.9%; from about 94% to about 99.9%; from about 95% to about 99.9%; from about 96% to about 99.9%; from about 97% to about 99.9%; from about 98% to about 99.9%; or from about 99% to about 99.9%, wt/wt of the total composition. [0215] Agriculturally acceptable salts

[0216] As used herein, the term agriculturally acceptable salt, hydrates, solvates, crystal forms and individual isomers, enantiomers, tautomers, diastereomers and prodrugs of the an engineered, non-naturally occurring antimicrobial peptide described herein can be utilized.

[0217] In some embodiments, an agriculturally acceptable salt of the present disclosure possesses the desired pharmacological activity of the parent compound. Such salts include: acid addition salts, formed with inorganic acids; acid addition salts formed with organic acids; or salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion, aluminum ion; or coordinates with an organic base such as ethanolamine, and the like.

[0218] In some embodiments, agriculturally acceptable salts include conventional toxic or non-toxic salts. For example, in some embodiments, convention non-toxic salts include those such as fumarate, phosphate, citrate, chlorydrate, and the like. In some embodiments, the agriculturally acceptable salts of the present disclosure can be synthesized from a parent compound by conventional chemical methods. In some embodiments, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. In some embodiments, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is incorporated herein by reference in its entirety.

[0219] In some embodiments, an agriculturally acceptable salt can be one of the following: hydrochloride; sodium; sulfate; acetate; phosphate or diphosphate; chloride; potassium; maleate; calcium; citrate; mesylate; nitrate; tartrate; aluminum; or gluconate.

[0220] In some embodiments, a list of agriculturally acceptable acids that can be used to form salts can be: glycolic acid; hippuric acid; hydrobromic acid; hydrochloric acid; isobutyric acid; lactic acid (DL); lactobionic acid; lauric acid; maleic acid; malic acid (- L); malonic acid; mandelic acid (DL); methanesulfonic acid ; naphthalene-l,5-disulfonic acid; naphthalene-2- sulfonic acid; nicotinic acid; nitric acid; oleic acid; oxalic acid; palmitic acid; parnoic acid; phosphoric acid; proprionic acid; pyroglutamic acid (- L); salicylic acid; sebacic acid; stearic acid; succinic acid; sulfuric acid; tartaric acid (+ L); thiocyanic acid; toluenesulfonic acid (p); undecylenic acid; a l-hydroxy-2-naphthoic acid; 2,2-dichloroacetic acid; 2- hydroxyethanesulfonic acid; 2-oxoglutaric acid; 4-acetamidobenzoic acid; 4-aminosalicylic acid; acetic acid; adipic acid; ascorbic acid (L); aspartic acid (L); benzenesulfonic acid; benzoic acid; camphoric acid (+); camphor- 10-sulfonic acid (+); capric acid (decanoic acid); caproic acid (hexanoic acid); caprylic acid (octanoic acid); carbonic acid; cinnamic acid; citric acid; cyclamic acid; dodecylsulfuric acid; ethane- 1,2-disulfonic acid; ethanesulfonic acid; formic acid; fumaric acid; galactaric acid; gentisic acid; glucoheptonic acid (D); gluconic acid (D); glucuronic acid (D); glutamic acid; glutaric acid; or glycerophosphoric acid.

[0221] In some embodiments, agriculturally acceptable salt can be any organic or inorganic addition salt.

[0222] In some embodiments, the agriculturally acceptable salts may use an inorganic acid and an organic acid as a free acid. The inorganic acid may be hydrochloric acid, bromic acid, nitric acid, sulfuric acid, perchloric acid, phosphoric acid, etc. The organic acid may be citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methane sulfonic acid, gluconic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methane sulfonic acid, ethane sulfonic acid, 4-toluene sulfonic acid, salicylic acid, citric acid, benzoic acid, malonic acid, etc. [0223] In some embodiments, the agriculturally acceptable salts include alkali metal salts (sodium salts, potassium salts, etc.) and alkaline earth metal salts (calcium salts, magnesium salts, etc.). For example, the acid addition salt may include acetate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisilate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthalate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharate, stearate, succinate, tartrate, tosylate, trifluoroacetate, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, zinc salt, etc., and among them, hydrochloride or trifluoroacetate may be used.

[0224] In yet other embodiments, the agriculturally acceptable salt can be a salt with an acid such as acetic acid, propionic acid, butyric acid, formic acid, trifluoroacetic acid, maleic acid, tartaric acid, citric acid, stearic acid, succinic acid, ethylsuccinic acid, lactobionic acid, gluconic acid, glucoheptonic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, laurylsulfuric acid, malic acid, aspartic acid, glutaminic acid, adipic acid, cysteine, N-acetylcysteine, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, hydroiodic acid, nicotinic acid, oxalic acid, picric acid, thiocyanic acid, undecanoic acid, polyacrylate or carboxyvinyl polymer.

[0225] In some embodiments, the agriculturally acceptable salt can be prepared from either inorganic or organic bases. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, ferrous, zinc, copper, manganous, aluminum, ferric, manganic salts, and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally-occurring substituted amines, and cyclic amines, including isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2- dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and the like. Preferred organic bases are isopropylamine, diethylamine, ethanolamine, piperidine, tromethamine, and choline.

[0226] In some embodiments, agriculturally acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977), the disclosure of which is incorporated herein by reference in its entirety.

[0227] In some embodiments, the salts of the present disclosure can be prepared in situ during the final isolation and purification of the compounds of the disclosure, or separately by reacting the free base function with a suitable organic acid. Examples of agriculturally acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other agriculturally acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further agriculturally acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

[0228] Exemplary descriptions of pharmaceutically acceptable salts is provided in P. H. Stahl and C. G. Wermuth, (editors), Handbook of Pharmaceutical Salts: Properties, Selection and Use, John Wiley & Sons, Aug 23, (2002), the disclosure of which is incorporated herein by reference in its entirety.

[0229] METHODS OF USING THE ANTIMICROBIAL PEPTIDE

[0230] Methods for protecting plants, plant parts, and seeds

[0231] The present disclosure provides methods of combating, controlling, or inhibiting a microbe comprising: applying an antimicrobially-effective amount of: (1) an engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and/or (2) an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient; to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe.

[0232] In some embodiments, the present disclosure provides a method for controlling pathogenic microbes, comprising contacting the pathogenic microbes with an antimicrobially- effective amount of the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and/or antimicrobial compositions of the present disclosure. [0233] In some embodiments, the present disclosure provides a method for controlling a pathogenic microbe in agronomic and/or nonagronomic applications, comprising contacting the pathogenic microbe or its environment, a solid surface, including a plant surface or part thereof, with an antimicrobially effective amount of antimicrobial combinations and/or antimicrobial compositions of the present disclosure.

[0234] In some embodiments, to achieve contact with the antimicrobial combinations and/or antimicrobial compositions of the present disclosure, to protect a field crop from microbes, the antimicrobial combinations and/or antimicrobial compositions of the present disclosure is typically applied to the seed of the crop before planting, to the foliage (e.g., leaves, stems, flowers, fruits) of crop plants, or to the soil or other growth medium before or after the crop is planted.

[0235] One embodiment of a method of the present disclosure is to contact microbes with antimicrobial combinations and/or antimicrobial compositions of the present disclosure via spraying. Alternatively, a granular formulation comprising the antimicrobial combinations and/or antimicrobial compositions of the present disclosure, can be applied to the plant foliage or the soil. Antimicrobial combinations and/or antimicrobial compositions of the present disclosure can also be effectively delivered through plant uptake by contacting the plant with the antimicrobial combinations and/or antimicrobial compositions of the present disclosure, applied as a soil drench of a liquid formulation, a granular formulation to the soil, a nursery box treatment or a dip of transplants. Of note is an antimicrobial composition of the present disclosure in the form of a soil drench liquid formulation. Also of note is a method for inhibiting or killing a microbe comprising contacting the microbe or its environment with an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof.

[0236] In some illustrative embodiments, the illustrative method contemplates a soil environment, wherein the antimicrobial combinations and/or antimicrobial compositions of the present disclosure is applied to the soil as a soil drench formulation. Of further note is that an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof is also effective by localized application to the locus of microbe infection. Other methods of contact with a microbe that is sensitized to the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, include application of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and/or antimicrobial composition of the present disclosure, by direct and residual sprays, aerial sprays, gels, seed coatings, microencapsulations, systemic uptake, baits, ear tags, boluses, foggers, fumigants, aerosols, dusts and many others. One embodiment of a method of contact is a dimensionally stable fertilizer granule, stick or tablet comprising a combination or composition of the present disclosure.

[0237] In some embodiments, an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and/or antimicrobial compositions of the present disclosure are also useful in seed treatments for protecting seeds from pathogenic microbes. In the context of the present disclosure and claims, treating a seed means contacting the seed with an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and/or antimicrobial composition of the present disclosure. This seed treatment protects the seed from pathogenic microbes and generally can also protect roots and other plant parts in contact with the soil of the seedling developing from the germinating seed. The seed treatment may also provide protection of foliage by translocation of the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof within the developing plant. Seed treatments can be applied to all types of seeds, including those from which plants genetically transformed to express specialized traits will germinate.

[0238] One method of seed treatment is by spraying or dusting the seed with an antimicrobial combination and/or antimicrobial composition of the present disclosure before sowing the seeds. Compositions formulated for seed treatment generally have an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and a film former or adhesive agent. Therefore, typically, a seed coating composition of the present disclosure consists of an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and a film former or adhesive agent. Seeds can be coated by spraying a flowable suspension concentrate directly into a tumbling bed of seeds and then drying the seeds. Alternatively, other composition types such as wetted powders, solutions, suspoemulsions, emulsifiable concentrates and emulsions in water can be sprayed on the seed. This process is particularly useful for applying film coatings on seeds. Various coating machines and processes are available to one skilled in the art. Suitable processes include those listed in P. Kosters et al., Seed Treatment: Progress and Prospects, 1994 BCPC Monograph No. 57, and references listed therein, the disclosures of which are incorporated herein by reference in their entireties.

[0239] The treated seed typically comprises an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof in an amount ranging from about 0.01 g to 1 kg per 100 kg of seed (i.e. from about 0.00001 to 1% by weight of the seed before treatment). A flowable suspension formulated for seed treatment typically comprises from about 0.5 to about 70% of the active ingredient, from about 0.5 to about 30% of a film-forming adhesive, from about 0.5 to about 20% of a dispersing agent, from 0 to about 5% of a thickener, from 0 to about 5% of a pigment and/or dye, from 0 to about 2% of an antifoaming agent, from 0 to about 1% of a preservative, and from 0 to about 75% of a volatile liquid diluent.

[0240] Methods of using the antimicrobial compositions and combinations

[0241] In some embodiments, the present disclosure provides a method of using an antimicrobially-effective amount of: (1) an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and/or (2) an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient; to inhibit growth of a microbe; wherein said method comprises, preparing the combination and then applying said combination to the microbe or its environment.

[0242] In some embodiments, the present disclosure provides a method of using an antimicrobially-effective amount of: (1) an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, and/or (2) an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; to combat, control, inhibit, or kill pathogenic microbes (e.g., plant pathogenic microbes).

[0243] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a microbe comprising: applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe. [0244] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a microbe comprising: applying an antimicrobially-effective amount of an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; and an excipient; the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe.

[0245] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a pathogenic microbe comprising: applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof to the locus of the microbe, or to a plant or animal susceptible to an attack by the pathogenic microbe.

[0246] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a pathogenic microbe comprising: applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; the locus of the microbe, or to a plant or animal susceptible to an attack by the pathogenic microbe.

[0247] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a microbe comprising: (1) applying an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof; the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; or (2) applying an antimicrobially-effective amount of an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; wherein the an engineered, non-naturally occurring antimicrobial peptide of SEQ ID NO: 1, or an agriculturally acceptable salt thereof.

[0248] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a microbe comprising: (1) applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; or (2) applying an antimicrobially-effective amount of an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; wherein the engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof, comprises, consists essentially of, or consists of an amino acid sequence as set forth in SEQ ID NO: 1.

[0249] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a microbe comprising: (1) applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; or (2) applying an antimicrobially-effective amount of an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; wherein the microbe is any deleterious microbe as described herein.

[0250] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a microbe comprising: (1) applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; or (2) applying an antimicrobially-effective amount of an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; wherein the microbe is a microbe belonging to the genera: Monilinia, Botrytis, Fusarium, Alternaria, Verticillium, Phytophthora, Colletotrichum, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, Pythium, Phoma, Gaeumannomces, Leptoshaeria, or Puccinia.

[0251] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a microbe comprising: (1) applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; or (2) applying an antimicrobially-effective amount of an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; wherein the microbe is a microbe belonging to the genera: Monilinia or Botrytis.

[0252] In some embodiments, the present disclosure provides a method of combating, controlling, or inhibiting a microbe comprising: (1) applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; or (2) applying an antimicrobially-effective amount of an antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof and an excipient; the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe; wherein the microbe is a Monilinia fructicola or a Botrytis cinerea.

[0253] Any of the methods described herein can be practiced using an engineered, non- naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof described herein (e.g. an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1), and any of the excipients described herein.

[0254] PATHOGENIC MICROBES

[0255] “Pathogenic microbe” refers to any microbe that is deleterious or pathogenic to an organism; e.g., any microbe that causes or exacerbates an infection or disease in a living organism. For example, in some embodiments, a pathogenic microbe of the present disclosure can be a pathogen to plants, e.g., a phytopathogen, such as a bacterium, a protozoan, or a fungus; in other embodiments, a pathogenic microbe can be a pathogen to animals. As used herein, a “plant pathogenic microbe” or “plant pathogen” can refer to a microbe that can cause disease into whole plants, plant tissues, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0256] In some embodiments, the present disclosure provides a method for combating, controlling, or inhibiting microbes, e.g., pathogenic microbes.

[0257] In some embodiments, the present disclosure provides a method for combating, controlling, or inhibiting microbes, e.g., pathogenic microbes, wherein the pathogenic microbe is a. Fusarium sp., Altemaria sp., Verticillium sp., Phytophthora sp., Colletotrichum sp., Botrytis sp., Cercospora sp., Phakopsora sp. Rhizoctonia sp., Sclerotinia sp., Pythium sp., Phoma sp., Gaeumannomces sp. Leptoshaeria sp., Puccinia sp., Ascochyta sp., Diplodia sp., Erysiphe sp., Gaeumanomyces sp., Helminthosporium sp., Macrophomina sp., Nectria sp., Peronospora sp., Phymatotrichum sp., Plasmopara sp., Podosphaera sp., Pyrenophora sp., Pyricularia sp., Scerotium sp., Septoria sp., Thielaviopsis sp., Uncinula sp., or Venturia sp.

[0258] In some embodiments, the pathogenic microbe is a pathogenic fungus. In some embodiments, the pathogenic fungus is a dermatophyte. In some embodiments, the dermatophyte is selected from the group consisting of Trichophyton ruhrum, Trichophyton inter digitale, Trichophyton violaceum, Trichophyton tonsurans, Trichophyton soudanense, Trichophyton mentagrophytes, Microsporum flavum, Epidermophyton floccosum, and Microsporum gypseum.

[0259] In some embodiments, the pathogenic microbe is derived from organisms belonging to the Aspergillus, Cryptococcus, Penicillium, Rhizopus, Apophysomyces, Cunninghamella, Saksenaea, Rhizomucor, Syncephalostrum, Cokeromyces, Actinomucor, Pythium, Fusarium, Histoplasmosis, or Blastomyces genus.

[0260] In some embodiments, the pathogenic microbe is derived from organisms belonging to Candida genus. In some embodiments, the pathogenic microbes derived from the Candida genus are from the species Candida albicans, C. glabrata, C parasilosis, C. tropicalis, or C. krusei.

[0261] Any of the pathogenic microbes described herein can combatted, controlled, or inhibited using an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1, and any of the excipients described herein.

EXAMPLES

[0262] Example 1. Recombinant Expression of Antimicrobial Peptide NCR13a

[0263] A polynucleotide operable to encode the engineered non-natural NCR13a antimicrobial peptide having the amino acid sequence of SEQ ID NO: 1, was cloned into a yeast expression vector, pKLACl, resulting in an expression vector pLB602 as shown in FIG. 1.

Transcription of the NCR13a peptide expression cassette is controlled by a yeast promoter, pLAC4, and expresses a fusion protein, a yeast a-mating factor prepro peptide fusion to the N- terminus of the NCR13a peptide, which directs the final mature NCR13a peptide secretion out of the cells. The wild-type mature peptide WT-NCR13 was cloned in the same way resulting an expression vector called pLB603. WT-NCR13 is used as a comparator peptide. The sequence of WT-NCR13 from Cicer arietinum was disclosed and published in Montiel, J. et al. Terminal Bacteroid Differentiation Is Associated With Variable Morphological Changes in Legume Species Belonging to the Inverted Repeat-Lacking Clade, Molecular Plant-Microbe Interactions; Vol. 29, No.3:210-219 (2016) (Table 1), the disclosure of which is incorporated herein by reference in its entirety. The antimicrobial peptide of the present disclosure consisting of the amino acid sequence of SEQ ID NO: 1 is referred to in FIG. 1 as NCR13a and is used synonymously throughout the present disclosure.

[0264] The pLB602 (encoding NCR13a) and pLB603 (encoding wild-type (WT)-NCR13 - SEQ ID NO: 3) vectors were separately transformed into K. lactis strains and integrated into the K. lactis genome, resulting in expression strains which stably express and secrete the desired peptides. To compare the yields of the NCR13a and WT-NCR13 peptides from the expression strains, 45 transformants from each transformation were picked and cultured in 48-well deepwell plates at 23.5°C for 6 days for the peptide yield evaluation. The peptide yield evaluation was performed by reverse phase HPLC runs with the spent medium which contained the secreted native WT-NCR13 or NCR13a peptides. Surprisingly the NCR13a strains (pLB602-YCT) had much higher peptide yield than the native WT-NCR13 strains (pLB603-YCT) as shown in FIG.

2. In average, pLB602-YCT strain showed >10 folds higher yield than that of pLB603-YCT strains when expressed in K. lactis.

[0265] However, the pLB602 and pLB603 vectors are designed to multiply integrate into the K. lactis genome. The higher yield from the pLB602 expression cassette containing strains may result from the more integrations. Therefore, in another set of experiments, 21 pLB602- YCT strains (NCR13a peptide) and 21 pLB603-YCT strains (Native WT-NCR13 peptide) from the Deep- well cultures were picked for gDNA extraction and Yield-Integration relation study. [0266] The genome DNAs (gDNA) from the pLB602-YCT and pLB603-YCT strains was extracted using Chelex 100 glass beads. The qPCR reactions were set up for all gDNAs extracted from pLB602 and pLB603 strains to identify integrated target gene copies in the expression strain genomes using Luna universal qPCR master mix reagent (New England Biolab) and QuanStudio 3 Real-time PCR system (Thermo-Fisher). The deep-well culture peptide yields of the pLB602-YCT and pLB603-YCT strains were plotted to the corresponding qPCR resulted integrated-target-gene copies, as shown in FIG. 3. The slopes of the plots indicate peptide yield from strain per integrated target gene copy in the strain genome (gene productivity). As shown in FIG. 3, NCR13a gene productivity was 10 mg/mL per gene copy, significantly higher than that of WT-NCR13, which was 0.2 mg/mL per gene copy. The unexpected result indicated that the engineered non-natural NCR13a peptide can be expressed significantly more efficiently in the K. lactis expression system than the WT-NCR13 peptide.

[0267] Example 2. Anti-fungal activity spectrum study of NCR13a

[0268] The NCR13a peptide (SEQ ID NO:1) was purified from the expression strain spent medium using cation exchange chromatography using Nuvia HR-S resin (Bio-Rad Laboratories). The anti-fungal activity of the purified NCR13a was evaluated in the anti-fungal bioassays against Aspergillus fumigatus, Botrytis cinerea, Monilinia fructicola, Wilsonomyces carpophilus, Collectotrichum gloeospiorides and Collectotrichum acutatum. In brief, the fungal strains were cultured in potato dextrose agar plate. The fungal spores were collected from the agar plates and suspended into the PBS buffer and enumerated by plating. The enumerated spores were inoculate into the 100 pL SFM medium (K2HPO4 2.5mM, MgSCL 50pM, CaCh 50pM, FeSCL 5pM, C0CI2 O.lpM, CuSCL O.lpM, Na2MoO4 2pM, H3BO3 0.5pM, KI O.lpM, ZnSO4 0.5pM, MnSCL 0.1 pM, glucose 10 g/L, asparagine 1 g/L, methionine 20 mg/L, myoinositol 2 mg/L, biotin 0.2 mg/L, thiamine-HCl Img/L, pyridoxine-HCl 0.2 mg/L) in sterile Coming 96-well tissue culture plates, which contains 2-fold series diluted purified NCR13a peptide, to achieve the final desired cell density of approximately 2.5xl0⁴ CFU/mL. The antifungal bioassay plates were incubated at 25°C for 48-72 hours. The Minimum Inhibition Concentration (MIC) values were recorded as the lowest concentration of the NCR13a peptide that inhibited 100% growth compared to the growth control without the NCR13a peptide.

[0269] The antifungal activity results are summarized in the table 2. The NCR13a peptide had potent anti-fungal activity against Botrytis cinerea, Monilinia fructicola and Wilsonomyces carpophilus, mild activity against Collectotrichum gloeospiorides and Aspergillus fumigatus, and no detectable activity up to 128 ppm against Colletotrichum acutatum.

[0270] Table 2. Minimal inhibitory concentrations (MICs) of the antimicrobial peptide NCR13a against various pathogenic microorganisms are shown.

[0271] Example 3. Antifungal activity comparison between NCR13a and WT- NCR13

[0272] The antifungal activities of the engineered, non-natural antimicrobial peptide NCR13a (SEQ ID NO: 1) and the Wild-type NCR13 (WT-NCR13) (SEQ ID NO: 3) were tested and compared against the following fungal organisms: K. lactis, Botrytis cinerea and Monilinia fructicola.

[0273] In brief, the K. lactis strain was cultured in LSM medium (K2HPO4 5mM, (NH₄)₂SO₄ 7.5mM, MgSO₄ lOOpM, FeCl₃ lOpM, C0CI2 0.2pM, CuSO₄0.2pM, Na₂MoO₄4pM, H3BO3 1 pM, KI 0.2 pM, ZnSO₄ IpM, MnSO₄ 0.2pM, glucose 20g/L, asparagine 2g/L, methionine 40mg/L, myo-inositol 4 mg/L, biotin 0.4 mg/L, thiamine-HCl 2mg/L, pyridoxine- HC10.4mg/L, yeast extract lOOmg/L) for 24 hour and then resuspended into LSM medium to final OD600 of 0.02 before anti-fungal bioassay setup. The filament fungal strains (Botrytis cinerea and Monilinia fructicola) were cultured in potato dextrose agar plate for 4-6 days at room temperature. The fungal spores were collected from the agar plates and suspended into the sterile water and enumerated using a Petroff-Hausser cell counting chamber. Next 5 pL of the final fungal cell suspensions were then inoculate into the lOOpL LSM (for K. lactis strain) or SFM medium (for filament fungal strains) in sterile Coming 96-well tissue culture plates, which contains 2-fold series diluted purified NCR13a or WT-NCR13 peptide, to achieve the final desired cell density of OD600nm of 0.001 for K. lactis and approximately 3xl0⁴ CFU/mL for the filament fungi.

[0274] The anti-fungal bioassay plates were incubated at 30°C for K. lactis and at 25°C for the filament fungi with the control fungi growth without the peptides. The growth of the fungal species, Kluyveromyces lactis (K. lactis strain YCT306), Monilinia fructicola (M. fructicola and Botrytis cinerea, (B. cinerea) in the anti-fungal bioassay was assessed via OD600 measurement after incubation with NCR13a, WT-NCR13 at varying concentrations, and at 30°C for 48 hours (K. lactis) or at 25°C for 72 hours (M. fructicola, and B. cinerea). The OD600 values of the fungal cell treated by different concentrations of NCR13a or WT-NCR13 peptides were normalized to the control OD600 without NCR13a or WT-NCR13 treatment to determine the growth inhibition percentage, and then plotted to the corresponding treated peptide concentrations. The resulting dose-response curves were fitted with the Hill equation. The halfinhibition peptide concentration (IC50) was then determined from the fitting curve, which represents potency of the peptide anti-fungal activities.

[0275] Materials were as follows: Pure WT-NCR13 was chemically synthesized by Bachem (Bachem Americas, Inc.; 3132 Kashiwa Street, Torrance, CA 90505, USA); recombinant, non-naturally produced NCR13a as produced and described in Example 1. The amino acid sequence of WT-NCR13 is as follows:

TKPCQSDKDCKKFACRKPKVPKCINGFCKCVR (SEQ ID NO: 3).

[0276] As shown in Table 3 and FIGs. 4-6, NCT13a and WT-NCR13 had almost indistinguishable anti-fungal activities against K. lactis, Botrytis cinerea and Monilinia fructicola. Overall both NCR13a and WT-NCR13 had IC50 between 2 - 3 ppm, nearly 100 fold more potent than its activity against K. lactis, which had IC50S between 200 - 300ppm.

[0277] Table 3. Summary of the anti-fungal activities (IC50) of NCR13a and WT-NCR13 against K. lactis strain YCT306, M. fructicola, or B. cinerea.

Claims

1. An engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1.

2. An antimicrobial composition comprising: an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1, and an excipient.

3. A method of combating, controlling, or inhibiting a microbe comprising: applying an antimicrobially-effective amount of an engineered, non-naturally occurring antimicrobial peptide, or an agriculturally acceptable salt thereof consisting of an amino acid sequence set forth in SEQ ID NO: 1, to the locus of the microbe, or to a plant or animal susceptible to an attack by the microbe.

4. The method of claim 3, wherein the microbe is a pathogenic fungus.

5. The method of claim 4, wherein the pathogenic fungus is a plant-specific pathogenic fungus, or phytopathogenic fungus.

6. The method of claim 5, wherein the phytopathogenic fungus is a phytopathogenic fungus belonging to the genera: Monilinia, Botrytis, Fusarium, Venturia, Wilsonomyces, Botryosphaeria, Penicillium, Rhizopus, Aspergillus, Podosphaera, Erysiphe, Golovinomyces, Leveillula, Peronospora, Pseudoperonospora, Plasmopara, Bremia, Cladosporium, Neofabraea, Microdochium, Marssonina, Sclerotinia, Rhizopus, Didymella, Alternaria, Verticillium, Phytophthora, Colleto trichum, Cercospora, Phakopsora, Rhizoctonia, Sclerotinia, Pythium, Phoma, Gaeumannomces, Leptoshaeria, or Puccinia.

7. The method of claim 6, wherein the microbe is a microbe belonging to the genera: Monilinia or Botrytis.

8. The method of claim 7, wherein the microbe is a Monilinia fructicola or a Botrytis cinerea.