Antifungal peptides for controlling plant pathogens

Abstract

This disclosure relates to methods for increasing plant immunity or resistance to plant pathogens or pests, or inhibiting plant pathogens or pests, by supplying antimicrobial or antifungal oligopeptides. Antimicrobial or antifungal oligopeptides and nucleic acids encoding said oligopeptides are also provided herein. Compositions, kits, and cells comprising antimicrobial or antifungal oligopeptides are also provided.

Description

[Technical Field]

[0001] Reference to electronic sequence listings The contents of the electronic sequence listing (185952000940SEQLIST.xml; size: 19,752 bytes; and creation date: February 7, 2024) are incorporated herein by reference in their entirety.

[0002] This invention relates to oligopeptides for increasing plant immunity and inhibiting plant pathogens or pests, and to the use thereof.

[0003] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Applications No. 63 / 486,652, No. 63 / 486,653, and No. 63 / 486,654, all of which were filed on 23 February 2023, and the entire contents of each of them are incorporated herein by reference. [Background technology]

[0004] Fungal plant pathogens pose a significant threat to food security worldwide. For example, infections caused by the fungus Botrytis cinerea result in hundreds of millions of US dollars in crop losses annually worldwide (Bolton et al., 2006; Dean et al., 2012). Genetically modified organisms (GMOs) and other genetically modified approaches, as well as chemical pesticides, are widely used to control various plant pathogens, including fungal infections. However, these approaches have limited applicability and can have considerable environmental impacts. Therefore, there is a need for natural and sustainable solutions to control plant pathogens.

[0005] Therefore, there is a need for substances that regulate plant immunity and prevent infection by common plant pathogens such as fungi. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. 20020115849 [Patent Document 2] International Patent Application Publication No. 2020 / 047124 [Patent Document 3] U.S. Patent No. 8,581,036 [Patent Document 4] U.S. Patent No. 9,757,473 [Patent Document 5] WO / 2014 / 086835A1 [Patent Document 6] US2017 / 0057997 [Patent Document 7] US2020 / 0263189 [Patent Document 8] US20200399647 [Patent Document 9] WO / 2011 / 084061 [Patent Document 10] U.S. No. 6,586,211 Bl [Patent Document 11] PCT / EP2004 / 013131 [Patent Document 12] WO 00 / 13017 A2 [Patent Document 13] WO 90 / 00626 Al [Patent Document 14] EP 385 410 A2 [Patent Document 15] WO 94 / 12632 Al [Patent Document 16] WO 95 / 17413 Al [Patent Document 17] EP 316 018 A2 [Patent Document 18] EP 022 242 A2 [Patent Document 19] WO 99 / 14318 Al [Patent Document 20] EP-A-36776 [Non-patent literature]

[0007] [Non-licensed document 1] Robert W. Jackson, Plant Pathogenic Bacteria: Genomics and Molecular Biology, Horizon Scientific Press, 2009, ISBN 1904455379, 9781904455370 [Non-licensed document 2] Samuel S. Gnanamanickam, Plant-Associated Bacteria, Springer Publishing, 2007, ISBN 1402045379, 9781402045370 [Non-licensed document 3] Martin Dworkin, The Prokaryotes: a handbook on the biology of bacteria, Springer Publishing, 2006, ISBN 0387254927, 9780387254920

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0008] This disclosure is directed to a number of embodiments, not limited to the following embodiments: Embodiment A1: A method for increasing immunity in plants, comprising the step of supplying a plant with an oligopeptide containing the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof.

[0009] Embodiment A2: The method according to Embodiment A1, wherein the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0010] Embodiment A3: A method for increasing resistance in plants to plant pathogens or pests, comprising the step of supplying plants with an oligopeptide containing the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21 having one, two, three, or four amino acid substitutions, insertions, or deletions.

[0011] Embodiment A4: A method for inhibiting plant pathogens or pests in plants, comprising the step of supplying a plant with an oligopeptide containing the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21 having one, two, three, or four amino acid substitutions, insertions, or deletions.

[0012] Embodiment A5: The method according to Embodiment A3 or A4, wherein the plant pathogen or pest is selected from fungal pathogens or pests, bacterial pathogens or pests, and viral pathogens or pests.

[0013] Embodiment A6: The plant pathogen or pest is a fungus belonging to the genera Cercospora, Mycosphaerella, Glomerella, Cladosporium, Diplodia maydis, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme, Fusarium verticillioides, grass leaf spot fungus (Cochliobolus sativus), and Colletotrichum graminicola. Graminicola), Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, Phytophthora parasitica Parasitica, Phytophthora megasperma fsp. Glycinea, Macrohomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiolum, Fusarium oxysporumoxysporum), Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassicola cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae pv. Glycinea, Xanthomonas campestris pv. Phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, soybean rust fungus (Phakopsora pachyrigis) Pythium pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani; Canola: Albugo candidaCandida), Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solanii, Sclerotinia sclerotiolum, Mycosphaerella brassicola, Pythium urtimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganensis subsp. Insidiosum, Pythium urtimum, Pythium irregulare, Pythium splendens Pythium splendens), Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium arbo atrum albo-atrum), Xanthomonas campestris pv. Alfalfa, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfaalfalfae), Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Pseudomonas syringae pv. Atrofaciens, Urocystis agropyri, Xanthomonas campestris pv. translucence Translucens), Pseudomonas syringae pv. Syringae, Alternaria alternata, Cladosporium herbarum, Fusarium gramineum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Colletotrichum graminicola, Wheat powdery mildew fungus (Erysiphe graminis f.sp. tritici) Puccinia graminis f.sp. tritici, wheat black rust fungus (Puccinia recondite f.sp. tritici), wheat red rust fungus (Puccinia recondite f.sp. tritici)Pythium tritici), yellow rust fungus (Puccinia striiformis), wheat yellow spot fungus (Pyrenophora tritici repentis), Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium algenomanes Pythium arrhenomanes, Pythium urtimum, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solanii, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, Plasmopara halsteadii Sclerotinia halstedii, Sclerotinia sclerotiolum, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrohomina phaseolinaPhaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, sunflower rust fungus (Puccinia helianthi), Verticillium dahlia, Erwinia carotovorum pv. Carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Corn: Colletotrichum graminicola Pythium graminicola), Fusarium verticillioides var. subglutinans, Erwinia stewartia, F. verticillioides, Gibberella zeae (Fusarium graminealum), Stenocarpella maydis (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium urtimum, Pythium aphanidermatum (Pythium Aspergillus flavus, Bipolaris maydis, CochliobolusHelmintosporium carbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helmintosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polisola Polysora), Macrohomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. Nebraskense, Trichoderma viride, Claviceps sorghi, Pseudomonas avenae avenae), Erwinia chrysanthemi pv. Zea, Erwinia carotovora, Corn stunt spiroplasma, Diplodia macrosporamacrospora), Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium, Exserohilum turcicum, C. sabri Neorum (C. sublineolum), Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae pv. Syringae, Xanthomonas campestris pv. Holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrohomina phaseolina, Perconia circinate, Fusarium verticilioides verticillioides), Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana) reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solanii, Acremonium strictumThe method according to Embodiment A3 or A4, wherein the pathogen or pest is a fungal selected from Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminealum, Fusarium oxysporum, Pythium arrhenomanes, and Pythium graminicola.

[0014] Embodiment A7: The method according to Embodiment A3 or A4, wherein the plant pathogen or pest is a bacterial pathogen or pest selected from the genera Pseudomonas, Pantoea, and Erwinia.

[0015] Embodiment A8: Plant pathogens or pests include cucumber mosaic virus, tobacco mosaic virus, barley fusarium virus, alfalfa mosaic virus (Alfamovirus), apple chlorotic leaf spot virus (Trichovirus), apple rust viroid (Viroid), Arabis mosaic virus (Nepovirus), barley mild mosaic virus (Bymovirus), barley spotted leaf mosaic virus (Hordeivirus), barley yellow spotted mosaic virus (Faimovirus), and bean common mosaic virus (Bean common mosaic virus). Potyvirus (potyvirus genus), bean yellow mosaic virus (potyvirus genus), beet necrotic leaf vein yellow virus (Furovirus genus), cowpea mosaic virus (potyvirus genus), bean mosaic virus (potyvirus genus), broad bean virus (Fabavirus genus), butterbur mosaic virus (Carlavirus genus), carnation spotted virus (Carmovirus genus), carnation vein mottle virus (potyvirus genus), cauliflower mosaic virus (Caulimovirus genus), chrysanthemum spotted virus Rus (Cucumovirus), Tomato Aspermyvirus (Cucumovirus), Chrysanthemum Dwarf Viroid (Viroid), Citrus Mosaic Virus, Citrus Tristeza Virus (Closterovirus), Clover Vein Yellowing Virus (Potivirus), Cox's Foot Mottle Virus (Sobemovirus), Cucumber Green Spot Mosaic Virus (Tobamovirus), Cucumber Mosaic Virus (Cucumovirus), Cycad Necrotic Dwarf Virus (Nepovirus), Taro Mosaic Virus (Potivirus), Grapevine Algerian Latent VirusThe method according to Embodiment A3 or A4, wherein the pathogen or pest is a virus selected from among (Tombusvirus), konjac mosaic virus (Potivirus), melon necrotic spot virus (Calmovirus), mulberry ring spot virus (Nepovirus), and narcissus mosaic virus (Potexvirus). A plant virus is a virus that affects plants. Additional examples of plant viruses include Odontoglossum ring spot virus (Tobamovirus), papaya ring spot virus (Potivirus), peach latent mosaic viroid, peanut spot virus (Potivirus), peanut leaf spot virus (Potivirus), bean mosaic virus (Potivirus), peanut dwarf virus (Cucumovirus), potato virus A (Potivirus), potato virus M (Carlavirus), potato virus S (Carlavirus), potato virus X (Potexvirus), potato virus Y (Potivirus), and prune dwarf virus (Iralvirus). Ilarvirus, Prune necrotic ring spot virus (Ilarvirus genus), Radish leaf mosaic virus (Comovirus genus), Rice black streak dwarf virus (Fijivirus genus), Rice dwarf virus (Reovirus genus), Rice grassy stunt virus (Tenuivirus genus), Rice stripe leaf blight virus (Tenuivirus genus), Rice tunglobular spheroid virus (Sequivirus genus), Rice dwarf virus, Rice tunglobular spheroid virus (Sequivirus genus), Ryegrass spot virus, Satsuma dwarf virus (Nepovirus genus), Wheat mosaic virus (Soil-borne wheat mosaic virus)Viruses (Frovirus genus), Southern bean mosaic virus (Sobemovirus genus), Soybean mosaic virus (Potivirus genus), Soybean dwarf virus (Cukumovirus genus), Cucumber mosaic virus (Cukumovirus genus), Tobacco mosaic virus (Tobamovirus genus), Tobacco mosaic virus (Tobamovirus genus), Tobacco mosaic virus (Tobamovirus genus), Tobacco necrosis virus (Necrovirus genus), Tobacco stem necrosis virus (Tobravirus genus), Tobacco ring spot virus (Nepovirus genus), Tomato aspermivirus (Cukumovirus genus), Tomato black ring spot virus (Nepovirus genus), Tomato mosaic virus (Tobamovirus genus), Tomato ring spot virus (Nepovirus genus), Tomato yellow necrosis virus (Tospovirus genus), Turnip mosaic virus (Potivirus genus), Pumpkin mosaic virus 1 (Watermelon mosaic virus 1) Examples include 1) Potivirus (genus Potivirus), papaya ring spot virus (genus Potivirus), watermelon mosaic virus 2 (genus Potivirus), wheat yellow mosaic virus (genus Faimovirus), and zucchini yellow mosaic virus (genus Potivirus).

[0016] Embodiment A9: The method according to Embodiment A3 or A4, wherein the plant pathogen or pest is selected from the group consisting of Septoria tritisi, Botrytis cinerea, Alternaria solani, Rhizoctonia solani anastomosis, Phytophthora capsici, and Phytophthora infestans.

[0017] Embodiment A10: The method according to any one of Embodiments A1 to A9, wherein the oligopeptide is supplied by expressing a nucleic acid encoding the oligopeptide.

[0018] Embodiment A11: The method according to Embodiment A10, wherein a nucleic acid is operably linked to a heterogeneous promoter.

[0019] Embodiment A12: The method according to Embodiment A10 or A11, wherein the heterogeneous promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

[0020] Embodiment A13: The method according to Embodiment A12, wherein the inducible promoter is induced by fungal infection and may optionally be a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., promoters for phenylalanine ammonia lyase and chalcone synthase), a promoter associated with a gene that modifies the plant cell wall (e.g., promoters for hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase), a promoter associated with a gene encoding an enzyme that degrades the fungal cell wall (e.g., a promoter for chitinase or glucanase), or a promoter associated with a gene encoding a thaumatin-like protein, or optionally, the maize Mis1 promoter or the flax Fis1 promoter.

[0021] Embodiment A14: The method according to any one of Embodiments A1 to A9, wherein the oligopeptide is supplied by application to a plant or a part thereof.

[0022] Embodiment A15: The method according to Embodiment A14, wherein the plant part is a leaf, bud, root, shoot, flower part, or seed.

[0023] Embodiment A16: The method according to Embodiment A14 or A15, wherein the oligopeptide is applied as a coating to the seeds before planting.

[0024] Embodiment A17: The method according to any one of Embodiments A1 to A9, wherein the oligopeptide is supplied by application to soil in which plants are planted.

[0025] Embodiment A18: The method according to any one of Embodiments A1 to A9, wherein the oligopeptide is supplied by adding it to the water supplied to the plant.

[0026] Embodiment A19: The method according to any one of Embodiments A1 to A9, wherein the oligopeptide is supplied to seeds, fruits, and / or plant parts after harvest.

[0027] Embodiment A20: The method according to any one of Embodiments A14 to A19, wherein the application is by a spreader, power duster, boom sprayer, hand sprayer, spray duster, or granular spreader.

[0028] Embodiment A21: The method according to any one of Embodiments A1 to A20, wherein miPEP includes a secretion tag, a tag that facilitates entry into plant cells, or a nuclear localization tag.

[0029] Embodiment A22: The method according to Embodiment A21, wherein the tag that promotes entry into plant cells comprises a cell-permeable peptide linked to the amino acid sequence.

[0030] Embodiment A23: The cell-permeable peptide is a protein transduction domain, an amphiphilic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimer-type polycationic molecule, a vascular endothelial-cadherin peptide cell-permeable peptide, a transportan cell-permeable peptide, a monomer or dimer cell-permeable peptide of the HIV-1 TAT basic domain, a penetratin cell-permeable peptide, a synthetic cationic homoarginine oligopeptide cell-permeable peptide, a gamma-zein cell-permeable peptide, a corn (Zea mays) knotted 1 cell-permeable peptide, a Saccharomyces pombe TP10 cell-permeable peptide, a Candida albicans Zebra cell-permeable peptide, an Antennapedia sequence, a TAT sequence, an Antp-3A (Antp variant) sequence, or Buforin The method according to Embodiment A22, comprising a II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynB1 sequence, a Pep-7 sequence, an HN-1 sequence, a BGSC (Bis-Guanidinium-Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.

[0031] Embodiment B1: An isolated oligopeptide comprising the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, including one, two, three, or four amino acid substitutions, insertions, or deletions.

[0032] Embodiment B2: The oligopeptide according to Embodiment B1, which is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0033] Embodiment B3: The oligopeptide according to Embodiment B1 or B2, comprising one, two, three, or four amino acid substitutions, insertions, or deletions.

[0034] Embodiment B4: An oligopeptide according to any one of Embodiments B1 to B3, comprising proline at the N-terminus, aspartic acid at the C-terminus, or both.

[0035] Embodiment B5: An oligopeptide according to any one of Embodiments B1 to B4, comprising one or more D-amino acids.

[0036] Embodiment B6: The oligopeptide according to Embodiment B5, wherein one or more D-amino acids enhance the stability of the oligopeptide compared to an oligopeptide with the same amino acid sequence containing all L-amino acids.

[0037] Embodiment B7: The oligopeptide according to any one of Embodiments B1 to B6, comprising a secretion tag, a tag that promotes entry into plant cells, or a nuclear localization tag.

[0038] Embodiment B8: The oligopeptide according to Embodiment B7, wherein the tag that promotes entry into plant cells comprises a cell-permeable peptide linked to the amino acid sequence.

[0039] Embodiment B9: The cell-permeable peptide is a protein transduction domain, an amphiphilic peptide, a synthetic cationic polypeptide, optionally polylysine, polyhistidine, or polyarginine, a dendrimer-type polycationic molecule, a vascular endothelial-cadherin peptide cell-permeable peptide, a transportan cell-permeable peptide, a monomer or dimer cell-permeable peptide of the HIV-1 TAT basic domain, a penetratin cell-permeable peptide, a synthetic cationic homoarginine oligopeptide cell-permeable peptide, a gamma-zein cell-permeable peptide, a maize knotted 1 cell-permeable peptide, a Saccharomyces pombe TP10 cell-permeable peptide, a Candida albicans zebra cell-permeable peptide, an Antennapedia sequence, a TAT sequence, an Antp-3A (Antp variant) sequence, or Buforin The oligopeptide according to Embodiment B8, comprising a II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynB1 sequence, a Pep-7 sequence, an HN-1 sequence, a BGSC (bis-guanidinium-spermidine-cholesterol) sequence, or a BGTC (bis-guanidinium-tren-cholesterol) sequence.

[0040] Embodiment C1: A composition comprising an oligopeptide according to any one of Embodiments B1 to B9, together with an agriculturally acceptable formulation agent which may include water, an organic solvent, a paraffinic oil, a vegetable oil, a dispersant, an emulsifier, a wetting agent, a buffer, a hydrotrope, a rheology modifier, an antifoaming agent and an antifoaming agent, an antifreeze, a biocide, a dye, a polymer wall, a catalyst, a thermosetting material, a crosslinking polymerizer, a UV protective agent, an antioxidant, and a chelating agent.

[0041] Embodiment C2: A composition comprising the oligopeptide described in any one of Embodiments B1 to B9 together with an agriculturally acceptable carrier, diluent, or excipient.

[0042] Embodiment C3: The composition according to Embodiment C2, wherein the agriculturally acceptable carrier, diluent, or excipient comprises a buffer and has a pH in the range of about 3.0 to about 9.0, or about 4.5 to about 8.0.

[0043] Embodiment C4: The composition according to any one of Embodiments C1 to C3, wherein the oligopeptide is in a concentration range of about 0.1 μg / ml to about 100 mg / ml, or about 5 μg / ml to about 5 mg / ml.

[0044] Embodiment C5: A composition according to any one of Embodiments C1 to C4, in the form of a finely-divided particulate solid, granules, pellets, wettable powder, dust, aqueous suspension, dispersion, gel, or emulsion.

[0045] Embodiment C6: The composition according to any one of Embodiments C1 to C5, further comprising one or more other active agents selected from the group consisting of pesticides, fertilizers, insecticides, attractants, sterilizers, mite control agents, anthelmintic agents, herbicides, biostimulants, biological preparations, and growth regulators.

[0046] Embodiment D1: A nucleic acid encoding an oligopeptide as described in any one of Embodiments B1 to B9.

[0047] Embodiment D2: A nucleic acid construct comprising the nucleic acid described in Embodiment D1, operably coupled to a promoter.

[0048] Embodiment D3: The nucleic acid construct according to Embodiment D2, wherein the promoter is a heterogeneous promoter.

[0049] Embodiment D4: The nucleic acid construct according to Embodiment D2 or D3, wherein the promoter is a constitutive promoter, a tissue-specific promoter, a developmental stage-specific promoter, or an inducible promoter.

[0050] Embodiment D5: The nucleic acid construct according to Embodiment D4, wherein the inducible promoter is induced by fungal infection and may optionally be a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., promoters for phenylalanine ammonia lyase and chalcone synthase), a promoter associated with a gene that modifies the plant cell wall (e.g., promoters for hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase), a promoter associated with a gene encoding an enzyme that degrades the fungal cell wall (e.g., a promoter for chitinase or glucanase), or a promoter associated with a gene encoding a thaumatin-like protein, or a maize Mis1 promoter or flax Fis1 promoter.

[0051] Embodiment E: A cell comprising the nucleic acid described in Embodiment D1 or the nucleic acid construct described in any one of Embodiments D2 to D5.

[0052] Embodiment F: The method according to any one of Embodiments A1 to A23, the oligopeptide according to any one of Embodiments B1 to B9, the composition according to any one of Embodiments C1 to C6, the nucleic acid construct according to any one of Embodiments D1 to D4, or the cell according to Embodiment E, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 16, or the oligopeptide comprises the amino acid sequence shown in one of SEQ ID NO: 19 or SEQ ID NO: 16 having one, two, three, or four amino acid substitutions, insertions, or deletions.

[0053] Embodiment A24: The method according to any one of Embodiments A2 to A23 and F, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 19, or the amino acid sequence shown in SEQ ID NO: 19 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and the plant pathogen or pest is selected from Pythium urtimum, Rhizoctonia solanii, Zymoseptoria tritici, Phytophthora cactorum, Botrytis cinerea, Microdochium nivale, Alternaria alternata, Pyrenophora tritici repentis (wheat yellow spot fungus), and Fusarium gramineum.

[0054] Embodiment A25: The method according to any one of Embodiments A2 to A23 and F, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 16, or the amino acid sequence shown in SEQ ID NO: 16 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and the plant pathogen or pest is selected from Zymoseptria tritisi, Phytophthora cactrum, Botrytis cinerea, Microdochium nivale, and Alternaria alternata.

[0055] Embodiment A26: The method according to any one of Embodiments A2 to A23 and F, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 19, or the amino acid sequence shown in SEQ ID NO: 19 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and the plant pathogen or pest is selected from Pythium urtimum, Rhizoctonia solanii, Zymoseptria tritisi, Botrytis cinerea, Alternaria alternata, Pyrenophora tritisi repentis, and Fusarium gramineum.

[0056] Embodiment A27: The method according to any one of Embodiments A2 to A23 and F, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 16, or the amino acid sequence shown in SEQ ID NO: 16 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and the plant pathogen or pest is selected from Zymoseptria tritisi, Botrytis cinerea, and Alternaria alternata. [Brief explanation of the drawing]

[0057] [Figure 1] This figure shows the results of applying oligopeptide MP18279 (right) to tomato leaves or fruits infected with Botrytis cinerea, compared to a control (left). The images shown are from 4 days after inoculation ("dpi") for tomato leaves and 7 days after inoculation for fruits. Black circles indicate typical B. cinerea symptoms, including brown lesions and gray hyphae. Leaves or fruits were treated with either MP18279 or water (solvent for MP18279) at the site of infection. Arrows indicate sites of infection without visible symptoms in MP18279-treated leaves or fruits. [Figure 2] This figure shows the results of a germination assay of B. cinerea spores treated with MP18279 (right) compared to an untreated control (left). Images were taken at either 0 hours (top), 24 hours (center), or 6 days (bottom) after peptide administration, as shown on the left. [Figure 3] This figure shows the results of a filamentous pathogen spore germination assay treated with MP18279 (gray bars) compared to a control peptide (black bars). The x-axis, from left to right, shows the filamentous pathogens tested, including Septoria tritisi, B. cinerea, Alternaria solanii, Rhizoctonia species, and Phytophthora capsici. The y-axis shows the EC50 at which spore germination is prevented at mg / L. The results are normalized to the germination level in samples treated with the control peptide, as indicated by the black dotted line. [Figure 4]This figure shows the results of a germination assay of filamentous pathogen spores treated with MP18279 compared to a scrambled control peptide ("sc control"). B. cinerea (left plot), S. tritisi (center plot), and Phytophthora capsici (right plot) were treated with MP18279 (three bars on the left of each plot) or the scrambled control peptide (three bars on the right of each plot). The y-axis shows the EC50 at which spore germination is prevented at mg / L. Three replicates are shown for each peptide and species combination, as indicated by the bar density. [Figure 5AB](Figure 5A) This figure shows the results of a germination assay of filamentous pathogen spores treated with MP18279, a truncated version of MP18279, a variant of MP18279, or a scrambled control peptide ("sc"). The truncated version of MP18279 was prepared by removing an amino acid from the C-terminus of the peptide. B. cinerea (left plot), S. tritisi (center plot), and P. capsici (right plot) were treated with full-length, 10-amino acid MP18279, a truncated version of MP18279 with lengths of 5-9 amino acids, or a scrambled control peptide. The x-axis of each plot shows the peptides used, from left to right, including the scrambled control, full-length, 10-amino acid MP18279, and truncated versions with lengths of 9, 8, 7, 6, and 5 amino acids. The y-axis of each plot shows the EC50 at which spore germination is prevented at mg / L. The truncated forms that were active in preventing spore germination are shown by bars on the checkerboard, and the truncated forms that were inactive are shown by light gray bars. The dotted line indicates the level of germination in samples treated with full-length MP18279. (Figure 5B) This figure shows the results of a germination assay of filamentous pathogen spores treated with MP18279, truncated forms of MP18279, variants of MP18279, or scrambled control peptide ("sc"). The truncated forms of MP18279 were prepared by removing amino acids from the N-terminus of the peptide. B. cinerea (left plot), S. tritisi (center plot), and P. capsici (right plot) were treated with full-length, 10-amino acid MP18279, 5-9 amino acid truncated forms of MP18279, or scrambled control peptide. The x-axis of each plot shows the peptides used, from left to right: scrambled control, full-length, 10-amino acid MP18279, and truncated forms of 9, 8, 7, 6, and 5 amino acids. The y-axis of each plot shows the EC50 at which spore germination is prevented at mg / L. Tronched forms that were active in preventing spore germination are shown with checkerboard bars, and truncated forms that were inactive are shown with light gray bars. The dotted line indicates the level of germination in samples treated with full-length MP18279. [Figure 5C] This figure shows the results of germination assays of filamentous pathogen spores treated with MP18279, a truncated form of MP18279, a variant of MP18279, or a scrambled control peptide ("sc"). S. tritisi spores were treated with a variant of MP18279 or a scrambled control peptide having amino acid additions and / or alterations. The y-axis of each plot represents the EC50 at which spore germination is prevented at mg / L. [Figure 6] This figure shows the level of B. cinerea control achieved in tomato leaves by various concentrations of MP18279. As shown on the x-axis, 2.5 × 10⁵ spores per 1 ml were incubated with 1 μM, 25 μM, 50 μM, 75 μM, or 100 μM MP18279 compared to a control sample ("Ctrl"). Spores and peptides were added as droplets to tomato leaves, and lesion size was measured after 2 days. The y-axis shows the percentage of B. cinerea control. Below the histogram, representative images showing the phenotype of B. cinerea lesions 2 days after peptide application are shown. The circled areas in the images are B. cinerea lesions. "ns" indicates not statistically significant. [Figure 7] (Figure 7A) This figure shows the results of spraying MP18279 onto tomato plants infected with B. cinerea (tomato gray mold). Representative images of tomato plants infected with B. cinerea (left) and tomato plants infected with B. cinerea and sprayed with 200 g / ha MP18279 one hour after infection (right) are shown. (Figure 7B) This figure shows the results of spraying MP18279 onto tomato plants infected with B. cinerea (tomato gray mold). It shows the percentage of disease control in tomato plants infected with B. cinerea and sprayed with 200 to 0.2 grams (g / ha) of MP18279 per hectare one hour after infection. The level of disease control shown is 4 days after infection ("dai"). The x-axis, from left to right, shows samples including the infected control (0% disease control) and samples treated with MP18279 at 200 g / ha (40% disease control), 20 g / ha (39% disease control), 2 g / ha (31% disease control), or 0.2 g / ha (13% disease control). The y-axis shows the percentage of disease control. [Figure 8] (Figure 8A) This figure shows the results of spray application of MP18279 to tomato plants followed by inoculation with the pathogen. It shows the percentage of disease control in tomato plants that were sprayed with 3 grams per hectare ("g / ha") of MP18279 and inoculated with Phytophthora infestance (Tomato Late Blight) 2 hours after MP18279 application. The x-axis shows samples from left to right, including infected control (0% disease control) and treatment with 3 g / ha MP18279 (32% disease control). The y-axis shows the percentage of disease control. (Figure 8B) This figure shows the results of spray application of MP18279 to wheat plants followed by inoculation with the pathogen. This graph shows the percentage of disease control in wheat plants that were sprayed with 300 grams ("g / ha") of MP18279 per hectare and inoculated with S. trichisi (wheat leaf blotch) 2 hours after MP18279 application. The x-axis shows samples from left to right, including infected control (0% disease control) and treatment with 300 g / ha MP18279 (38% disease control). The y-axis shows the percentage of disease control. [Figure 9](Figure 9A) This figure shows the results of an experiment analyzing the stability of MP18279 compared to a variant of MP18279 ("MP18279D1D10") that has D-amino acid residues at the N-terminus and C-terminus. It shows the level of peptide stability over time in a tomato leaf wash solution. The x-axis represents the storage time in units of time, and the y-axis represents the percentage of peptide purity. The results are shown for MP18279 (light gray line) and MP18279D1D10 (black line), showing a 20% difference in stability between the two peptides. (Figure 9B) This figure shows the results of an experiment analyzing the stability of MP18279 compared to a variant of MP18279 ("MP18279D1D10") that has D-amino acid residues at the N-terminus and C-terminus. It shows the level of B. cinerea control achieved by MP18279 and MP18279D1D10 in tomato leaves. The x-axis shows samples from left to right, including an infected control, a scrambled peptide control, MP18279, and MP18279D1D10. The y-axis shows the percentage of B. cinerea control. (Figure 9C) This figure shows the results of an experiment analyzing the stability of MP18279 compared to a variant of MP18279 ("MP18279D1D10") that has D-amino acid residues at the N-terminus and C-terminus. It shows the percentage of disease control in tomato plants sprayed with 30 g / ha MP18279 or MP18279D1D10 and subsequently infected with P. infestance (tomato blight). The x-axis shows samples from left to right, including an infected control, MP18279 (24% disease control), and MP18279D1D10 (22% disease control). The y-axis shows the percentage of P. infestation control. [Figure 10]This figure shows the amount of MP18279 present over time in a tomato leaf wash solution, compared to a 7-amino acid variant of MP18279 ("MP18865"). The x-axis, from left to right, shows samples containing MP18279 (91% peptide abundance) stored in the solution for 17-20 hours, MP18865 (97% peptide abundance) stored in the solution for 17-20 hours, MP18279 (0% peptide abundance) stored in the solution for 1 week, and MP18865 (12% peptide abundance) stored in the solution for 1 week. The y-axis shows the relative levels of the peptides. [Figure 11] This figure shows bar plot results of testing the antimicrobial activity of peptides MP19919, MP19594, and a negative control peptide against seven pathogens (plotted along the horizontal axis). The maximum half-volume effective concentration (EC50) in mg / L is plotted along the left vertical axis. The dotted horizontal lines correspond to the activity divisions plotted along the right vertical axis. Striped bars represent the activity of the negative control peptide, black bars represent the activity of MP19594, and bars with faint dots represent the activity of MP19919. The measured values ​​(mg / L) for each MP19594 and MP19919 activity are listed above each bar. [Figure 12] This figure shows bar plot results of radial growth measurements of three pathogen strains against peptides MP19919, MP19594, and a negative control peptide. The percentage control of the pathogen relative to the colony growth (mm) of the negative control (agar without additives) is plotted along the vertical axis. The tested peptides MP19919, MP19594, the negative control peptide, and the positive control Amistar® (a commercially available fungicide) are plotted along the horizontal axis. The percentage control is also shown above each bar. The pathogen strains involved are shown above each panel (Pythium ultimum for the left panel, Rhizoctonia solanii for the center panel, and Sclerotinia sclerotiolum for the right panel). [Figure 13]This figure shows bar plot results of the colony diameter (mm, y axis) of three pathogen strains when tested against peptides MP19919, MP19594, a negative control peptide, a positive control Amistar® (a commercially available fungicide), and a negative control of untreated agar (all plotted along the x-axis). Each measured colony diameter (mm) is also listed above each bar. The pathogen strains involved are shown above each panel (Pythium urtimum for the left panel, Rhizoctonia solanii for the center panel, and Sclerotinia sclerotiolum for the right panel). [Figure 14] This figure shows bar plot results of radial growth of three pathogen strains against peptides MP19919 and MP19594, a negative control peptide, a negative control of untreated agar, and a positive control of Amistar® (a commercially available fungicide), plotted along the horizontal axis. % colony growth compared to untreated agar is plotted along the vertical axis. % control is also shown above each bar. The pathogen strains involved are shown above each panel (Pythium urtimum for the left panel, Rhizoctonia solanii for the center panel, and Sclerotinia sclerotiolum for the right panel). [Modes for carrying out the invention]

[0058] Methods for increasing plant immunity, methods for increasing resistance in plants to plant pathogens or pests, and methods for inhibiting plant pathogens or pests are provided herein. Antimicrobial oligopeptides, nucleic acids encoding the oligopeptides, and compositions comprising the oligopeptides are also provided herein.

[0059] The methods and oligopeptides of this disclosure are at least in part based on the applicant's remarkable discovery that oligopeptides can be used to modulate immunity and resistance to pathogens in plants. In particular, the applicant found that application of oligopeptides to plants eliminated infections by a wide range of fungal pathogens. Furthermore, the oligopeptides were effective in promoting immunity in both monocots and dicots. Moreover, the oligopeptides had the ability to inhibit fungal pathogens in vitro, and variants of the oligopeptides with higher stability also exhibited antifungal activity.

[0060] As used herein, “amino acid” or “amino acid residue” refers to any naturally occurring amino acid, any non-natural amino acid, any modified amino acid including derivatized amino acids, or any amino acid mimetic known in the art. In some embodiments, the amino acid is a D-amino acid. In some embodiments, the amino acid is an L-amino acid. Amino acids may be referred to by both common three-letter abbreviations and one-letter abbreviations.

[0061] As used herein, the terms “peptide” or “oligopeptide” refer to any peptide structure comprising or consisting of two or more amino acids, including chemical modifications and derivatives of amino acids. In some embodiments, the oligopeptide comprises an amino acid sequence that, in plants exposed to the sequence, is capable of inducing increased immunity or increased resistance to plant pathogens or pests. In some embodiments, the oligopeptide comprises an amino acid sequence that is capable of inhibiting plant pathogens or pests. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide. In some embodiments, the oligopeptide is 4 to 50 or 6 to 30 amino acids long.

[0062] As used herein, the term “purified” molecule refers to a biological or synthetic molecule that has been taken out of its natural environment, isolated or separated, and that does not contain any other naturally associated components.

[0063] The term "isolated" in relation to molecules including nucleic acids, constructs, vectors, etc., may refer to molecules that are not found in nature and / or exist in a situation where they would not be found in nature. The term "isolated" may also refer to molecules that have been isolated from a more complex solution or source, or that have undergone at least one process toward concentration or enrichment. However, the term "isolated" is never intended to limit the molecule to a specific location or state. For example, isolated nucleic acid molecules include nucleic acid molecules that have been introduced into the genome of a cell at a location where they are not found in nature, or nucleic acid molecules that are commensal to the offspring of a cell into which they have been introduced into the genome at a location where they are not found in nature.

[0064] The term "sequence identity" refers to the degree of similarity between two nucleic acid sequences or two amino acid sequences, and is expressed as sequence similarity, or otherwise called sequence identity. Sequence identity is often measured as percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are.

[0065] I. Method Methods for increasing immunity in plants are provided herein. Methods for increasing resistance in plants to plant pathogens or pests, and methods for inhibiting plant pathogens or pests are also provided herein.

[0066] peptide In some embodiments, the method includes the step of supplying oligopeptides to plants.

[0067] In some embodiments, the method includes the step of supplying a plant with an oligopeptide containing the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide contains an amino acid sequence having one, two, three, or four amino acid substitutions, insertions, or deletions compared to any of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acid long. In some embodiments, the oligopeptide contains proline at the N-terminus, aspartic acid at the C-terminus, or both. In some embodiments, the oligopeptide contains one or more D-amino acids. In some embodiments, one or more D-amino acids enhance the stability of the oligopeptide compared to an oligopeptide with the same amino acid sequence containing only L-amino acids. In some embodiments, the oligopeptide is an antimicrobial or antifungal oligopeptide.

[0068] Oligopeptide application In some embodiments, the method includes the step of supplying an oligopeptide to a plant. In some embodiments, the oligopeptide includes an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or an amino acid sequence having one, two, three, or four amino acid substitutions, insertions, or deletions thereof. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0069] Oligopeptides can be supplied to plants by any suitable method known in the art. In some embodiments, the method includes supplying oligopeptides to plants by foliar spraying, foliar immersion, drip irrigation, coating, mixing, pouring, powdering, spraying, soil irrigation, fumigation, soil injection, infiltration irrigation, sprinklers, or manual irrigation. In some embodiments, the method includes supplying oligopeptides to plants using a spreader, power duster, boom sprayer, hand sprayer, spray duster, or granular spreader. In some embodiments, the method includes supplying oligopeptides to plants by foliar spraying. In some embodiments, the method includes supplying oligopeptides to plants at a concentration of 2 g / ha to 300 g / ha. In some embodiments, the oligopeptide is a, 40g / ha, 45g / ha, 50g / ha, 55g / ha, 60g / ha, 65g / ha, 70g / ha, 75g / ha, 80g / ha, 85g / ha, 90g / ha, 95g / ha, 100g / ha, 110g / ha, 120g / ha, 130g / ha, 140g / ha, 150g / ha, 160g / ha, 170g / ha, Supplied in concentrations of 180g / ha, 190g / ha, 200g / ha, 220g / ha, 240g / ha, 260g / ha, 280g / ha or 300g / ha. In some embodiments, the oligopeptide is supplied to one or more leaves, buds, roots, shoots, flower parts, or seeds. In some embodiments, the oligopeptide is supplied to the plant or a part thereof after harvest. In some embodiments, the oligopeptide is supplied to the plant or a part thereof before harvest. In some embodiments, the oligopeptide is supplied to the plant before, during, or after infection with a pathogen or pest. In some embodiments, the oligopeptide is supplied to the plant by application to the soil in which the plant is planted. In some embodiments, the oligopeptide is supplied to the plant by addition to the water supplied to the plant.In some embodiments, the oligopeptide comprises an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or an amino acid sequence having one, two, three, or four amino acid substitutions, insertions, or deletions thereof. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0070] In some embodiments, the method includes the step of preparing a composition comprising an oligopeptide. In some embodiments, the composition is an agrochemically acceptable composition. In some embodiments, the composition comprises an oligopeptide and an agrochemically acceptable formulation agent. In some embodiments, the agrochemically acceptable formulation agent comprises one or more of the following: water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffers, hydrotropes, rheology modifiers, antifoaming and defoaming agents, antifreezes, biocides, dyes, polymer walls, catalysts, thermosetting materials, crosslinking polymerizers, UV protectants, antioxidants, and chelating agents. In some embodiments, the composition contains the oligopeptide in a concentration range from about 0.1 μg / ml to about 100 mg / ml, or from about 5 μg / ml to about 5 mg / ml. In some embodiments, the composition further comprises one or more other active agents selected from the group consisting of pesticides, fertilizers, insecticides, attractants, sterilizers, mite control agents, anthelmintic agents, herbicides, biostimulants, biological formulations, and growth regulators. In some embodiments, the composition is formulated as a liquid, gel, emulsion, suspension, capsule encapsulation, solid, powder, aerosol, paste, coating agent, spray, soil drenching agent, microcapsule, emulsion, or granule. In some embodiments, the composition takes the form of fine particulate solid, granule, pellet, hydrated powder, fine powder, aqueous suspension, dispersion, gel, or emulsion. In some embodiments, the agrochemically acceptable composition is formulated as a seed treatment, foliar spray, foliar dipping, ready-to-use (RTU) formulation, crop coating, flowable formulation, tank mix, aerosol, root dipping, soil treatment, dipping formulation, drenching formulation, or sprinkler formulation. In some embodiments, the agrochemically acceptable composition containing oligopeptides is a foliar spray composition. In some embodiments, the composition comprises an oligopeptide containing an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or an amino acid sequence having one, two, three, or four amino acid substitutions, insertions, or deletions thereof. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0071] In some embodiments, the method includes the step of preparing a composition comprising an oligopeptide and an agriculturally acceptable carrier, diluent, or excipient. In some embodiments, the composition comprises an oligopeptide and an agriculturally acceptable carrier, diluent, or excipient. In some embodiments, the agriculturally acceptable carrier, diluent, or excipient comprises a buffer with a pH ranging from about 3.0 to about 9.0, or from about 4.5 to about 8.0. In some embodiments, the agriculturally acceptable carrier comprises a solid carrier, a liquid carrier, a gel carrier, a suspension, or an emulsion. In some embodiments, the agriculturally acceptable carrier comprises an auxiliary agent, an inert component, a dispersant, a surfactant, a water-retaining agent, an emulsifier, a thickener, a wetting agent, a fertilizer, a mineral, a solvent, a tackifier, a binder, or a stabilizer. In some embodiments, the agriculturally acceptable composition comprises an oligopeptide and a surfactant or water-retaining agent. In some embodiments, the composition comprises an oligopeptide containing an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or an amino acid sequence having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and an agriculturally acceptable carrier, diluent, or excipient. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0072] In some embodiments, the oligopeptide is supplied by application to a plant or part thereof. In some embodiments, the plant part is a fruit, leaf, bud, root, shoot, flower part, or seed. In some embodiments, the oligopeptide is supplied to the plant or part thereof after harvest. In some embodiments, the oligopeptide is supplied to the plant or part thereof before harvest. In some embodiments, the oligopeptide is supplied to the plant by application to the soil in which the plant is planted. In some embodiments, the oligopeptide is supplied to the plant by addition to the water supplied to the plant. In some embodiments, the oligopeptide is applied to the plant by a spreader, power duster, boom sprayer, hand sprayer, spray duster, or granular spreader. In some embodiments, the oligopeptide contains an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0073] In some embodiments, the method includes the step of supplying oligopeptides to plants by expressing nucleic acids encoding oligopeptides. In some embodiments, the nucleic acids are operably linked to a heterogeneous promoter. In some embodiments, the heterogeneous promoter is a constitutive promoter, a tissue-specific promoter, a developmental stage-specific promoter, or an inducible promoter. In some embodiments, the nucleic acids are operably linked to an inducible promoter. In some embodiments, the inducible promoter is induced by fungal infection and may optionally be a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., promoters for phenylalanine ammonia lyase, chalcone synthase), a promoter associated with a gene that modifies the plant cell wall (e.g., promoters for hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase), a promoter associated with a gene that encodes an enzyme that degrades the fungal cell wall (e.g., promoters for chitinase or glucanase), a promoter associated with a gene that encodes a thaumatin-like protein, or a maize or flax promoter. For example, maize or flax promoters such as the Mis1 and Fis1 promoters, respectively, are promoters induced by fungal infection in plants and may be used in conjunction with the methods of this disclosure (U.S. Patent Application Publication No. 20020115849). In some embodiments, the nucleic acid expresses an oligopeptide comprising the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0074] In some embodiments, the method includes the step of supplying a nucleic acid encoding an oligopeptide to a plant. In some embodiments, the nucleic acid encoding the oligopeptide is supplied to the plant by transformation. In some embodiments, the transformation includes Agrobacterium-mediated transformation, microprojectile-mediated transformation, sonication, electroporation, or liposome or spheroplast-mediated vector delivery. In some embodiments, the nucleic acid encoding the peptide is added to the plant by genome editing. Gene editing may be carried out by any method known in the art, including, but not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), oligonucleotide-directed mutagenesis (ODM), clustered regularly interspaced short palindromic repeats (CRISPR) / Cas, or by gene writing (e.g., International Patent Application Publication 2020 / 047124). In some embodiments, the nucleic acid encoding the oligopeptide is added to a plant by crossing a first plant containing the nucleic acid encoding the oligopeptide with a second plant. In some embodiments, the plant containing the nucleic acid expresses the oligopeptide encoded by the nucleic acid. In some embodiments, the nucleic acid encodes an oligopeptide having an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0075] plant immunity In some embodiments, the method includes increasing immunity in plants. In some embodiments, the method includes supplying plants with an oligopeptide comprising an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions.

[0076] As used herein, “plant immunity” and “plant immunity” refer to the ability of a plant to recognize triggers, signatures, and / or patterns associated with exposure to a pathogen or pest, and to respond accordingly (e.g., through gene induction) to fight off and / or recover from the pathogen.

[0077] Any suitable method known in the art for determining plant immunity may be used. For example, plant immunity may be measured as a reduction in the number of pests and diseases in a plant compared to an untreated plant, or as a reduction in physical damage to the plant compared to an untreated plant. Physical damage includes herbivory and boring damage and may manifest as a variety of plant phenotypes, including, but not limited to, chewed or tattered leaves, broken leaves, tunnels in leaves, holes in stems, leaf deformation, leaf discoloration, leaf spots, wilting, stunting, rotten or dead stems, yellowing, breakage, or root damage. Convex hull analysis may be used to examine physical damage to infected plants.

[0078] In some embodiments, increasing plant immunity includes increasing disease control in plants by supplying them with antimicrobial oligopeptides. As used herein, the term “disease control” means the killing, reduction in the number, and / or reduction in growth, chewing, or normal physiological development of plant pathogens or pests at any or all life stages, and / or reduction in the effects of plant pest infection and / or parasitism. In some embodiments, increasing immunity in plants includes increasing disease control in plants by 10% to 90% compared to untreated infected plants. In some embodiments, increasing immunity in plants includes increasing disease control in plants by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% compared to untreated infected plants. In some embodiments, increasing immunity in plants includes increasing disease control in plants by 2 to 20 times compared to untreated infected plants. In some embodiments, increasing immunity in plants involves increasing disease control in plants by 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times compared to untreated infected plants. In some embodiments, the antimicrobial oligopeptide is an oligopeptide comprising the amino acid sequence shown in SEQ ID NOs. 1-5 and 14-21, or an amino acid sequence having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions thereof.

[0079] plant In some embodiments, the method includes the step of supplying an antimicrobial oligopeptide to a plant. In some embodiments, the antimicrobial oligopeptide is an oligopeptide comprising the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions.

[0080] In some embodiments, the plant is a dicotyledonous or monocotyledonous plant. In some embodiments, the plant is a crop. In some embodiments, the plant is a row crop, a fruiting plant, a tree, a vine, a vegetable, or an ornamental plant (e.g., ornamental flowers, trees, shrubs, ground cover plants, and turfgrass). In some embodiments, the plants are of the genus Solanum (solanum spp.) or Triticum (wheat spp.). Other exemplary plant types include, non-limitingly, Medicago sativa, almond (Prunus dulcis), Arabidopsis spp., Phaseolus spp., Fragaria spp., apple (Malus domestica), cherry (Prunus spp.), asparagus (Asparagus officinalis), Arabidopsis spp., banana (Musa spp.), barley (Hordeum vulgare), Phaseolus spp., Vaccinium spp., and cocoa (Theobroma). Cacao, Brassica spp., Carnation (Dianthus caryophyllus), Carrot (Daucus carota sativus), Cassava (Manihot esculentum), European cherry (Prunus avium), Chickpea (Cicer arietinum), Chicory (Cichorium intybus), Capsicum spp., Chrysanthemum spp., Coconut palm (Cocos nucifera), Coffee genus (Coffea spp.), Cotton (Gossypium hirsutum L), Vigna spp., Broad bean (Vicia faba), Cucumis (Cucumis sativus), Ribes spp., Date palm (Phoenix) (dactylifera), Solanum spp., Eucalyptus spp., Linum usitatissimum L.)), Pelargonium spp., Vitis spp., Guava (Psidium guajava), Hops (Humulus lupulus), Cannabis sativa and Cannabis spp., Iris spp.), Lettuce (Lactuca sativa), Citrus spp., Corn (Zea mays L), Mango (Mangifera indie), Mangosteen (Garcinia mangostan), Melon (Cucumis mel), Setaria spp., Echinochloa spp., Eleusine spp., Panicum spp., Pennisetum (spp.), oats (Avena sativ), Guinea oil palm (Elaeis guineensis), olive (Olea europaea), leeks (Allium spp.), papaya (Carica papaya), peaches (Prunus persica), pears (Pyrus spp.), peas (Pisum sativum), peanuts (Arachis hypogaea), peony (Paeonia spp.), petunias (Petunia spp.), pineapples (Ananas comosus), bananas (Musa spp.), European plums (Prunus domestica), poinsettias (Euphorbia pulcherrima), aspens (Populus spp.), pumpkins (Cucurbita spp.), rice (Oryza sativa) L), Rosa spp., Hevea brasiliensis, rye (Secale cereale), safflower (Carthamus tinctorius L), sesame (Sesame indium), sorghum bicolor, soybean (Glycine max L), Fragaria spp., beet (Beta vulgaris), sugarcane (Saccharum spp.)Examples include sunflower (Helianthus annuus), sweet potato (Ipomoea batatas), tea plant (Camellia sinensis), tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum), tulip (Tulipa spp.), Japanese walnut (Juglans spp. L), watermelon (Citrullus lanatus), bread wheat (Triticum aestivum), and yam (Dioscorea spp.). In some embodiments, the plants are wild plant varieties. In some embodiments, the plants are native plant varieties. In some embodiments, the plants are hybrid plant varieties. In some embodiments, the plants are genetically modified plants and / or gene-edited plants.

[0081] Plant pathogens and pests Methods for increasing resistance in plants to plant pathogens or pests are provided herein. As used herein, “resistance” or “increased resistance” in plants to pests or pathogens means that a plant is more capable of reducing the effects of pests or pathogens than a plant that is not resistant or has less resistance.

[0082] Methods for inhibiting plant pathogens or pests in plants are also provided herein. As used herein, “inhibiting plant pathogens or pests” means killing, incapacitating, immobilizing, reducing in number, or otherwise making it substantially incapable of causing harm to plants.

[0083] In some embodiments, the method comprises the step of supplying a plant with an oligopeptide comprising an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide. In some embodiments, the plant pathogen or pest is a fungus, bacterium, virus, or eukaryote.

[0084] In some embodiments, the plant pathogen or pest is a fungus. Examples of fungal plant pathogens or pests that can be targeted by this method include, but are not limited to, species of Cercospora, Mycosferella, Glomerella, Cladosporium, Diplodia meidis, Fusarium oxysporum, Fusarium gramineum, Fusarium moniliforme, Fusarium verticilioides, grass leaf spot fungus (Cochliobolus satybus), Coletotrichum graminecola, Stagonospora nodorum, Stagonospora avenae, Stenocarpera meidis, Sclerotinia minor, Sclerotinia sclerotiolum, Sclerotinia species, Alternaria species, Phytophthora species, Botrytis species, wheat yellow spot fungus (Pyrenophora tritisi repentis), and Phytophthora parasitica. The following are some of the specific pathogens affecting major crops: Soybeans: Phytophthora megasperma fsp. glycinea, Macrohomina phaseolina, Rhizoctonia solanii, Sclerotinia sclerotiolum, Fusarium oxysporum, Diaporte phaseorum var. sodjae (Phomopsis sodjae), Diaporte phaseorum var. cauribola, S Clerodium rolfsii, Cercospora kikuchi, Cercospora sodina, Peronospora manshurica, Coletotrichum dematium (Coletotrichum trancatam), Corinespora cassicola, Septoria glycinea, Philosticta sodicola, Alternaria alternata, Pseudomonas syringae pv. glycinea, Xanthomonas campestris pv.Phaseoli, Microsphaera diffusa, Fusarium semitectum, Fialophora gregata, Gromerella glycineus, soybean rust fungus (Phacopsola pachyridi), Pythium aphanidermatum, Pythium ultimum, Pythium devarianum, Fusarium solani; Canola: Albugo candida, Alternaria brascae, Leptosphaeria macrans, Rhizoctonia solani, Sclerotinia sclerotiolum, Mycosphaerella brascicicola, Pythium ultimum, Peronospora parasitii Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter misiganensis subspecies insidiosum, Pythium ultimum, Pythium irregularle, Pythium splendens, Pythium devarianum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifolium, Forma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrotilla medicaginis, Fusarium oxysporum, Verticillium • Arbo atrum, Xanthomonas campestris pv. alfalfa, Aphanomyces euteices, Stemphylium herbarum, Stemphylium alfalfa, Coletotrichum trifoli, Leptosphaerina briociana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora melilotii, Stemphylium botryousum, Leptotrichilla medicaginis; Wheat: Pseudomonas syringae pv. atrofaciens, Urocystis agropyri, Xanthomonas campes Tris pv. translucence, Pseudomonas syringae pv. syringae, Alternaria alternata, Cladosporium herbarum, Fusarium gramineum, Fusarium avenaceum, Fusarium curmorum, Ustyrago tritisi, Ascochita tritisi, Cephalosporium gramineum, Coletotrichum graminicola, Wheat powdery mildew fungus (Ericife graminis f.sp. tritisi), Wheat black rust fungus (Psinia graminis f.sp. tritisi), Wheat red rust fungus (Psinia recondite f.sp.Pythium aphaniderma Pythium tum, Pythium argenomanes, Pythium urtimum, Vipolaris sorokiniana, Ergot fungus (Claviceps purpurea), Tiretia tritisi, Tiretia laevis, Ustirago tritisi, Tiretia indica, Rhizoctonia solanii, Pythium argenomanes, Pythium graminicola, Pythium afanidermatum; Sunflower: Plasmopara Halsteady, Sclerotinia sclerotiolum, Septoria helianthi, Homopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Forma macdonaldii, Macrohomina phaseolina, Elysife ticolacearum, Rhizopus oryzae, Rhizopus aritus, Rhizopus stronifer, Sunflower rust fungus (Pscinia helianthi), Verticillium daerie, Erwinia carotoborum pv. carotobora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Maize: Coletotrichum graminicola, Fusarium verticillioides var. subglutinans, Erwinia stewaltii, F.Bertisilioides, Gibberella zeae (Fusarium gramineum), Stenocarpera meidis (Diplodia meidis), Pythium irregularle, Pythium devarianum, Pythium graminicola, Pythium splendens, Pythium urtimum, Pythium aphanidermatum, Aspergillus flava, Vipolaris meidis O, T (Cochliobolus heterostrophus), Helmintosporium carbonum I, II & III (Cochliobolus carbonum), Exerohilum tulsicum I, II & III, Helmintosporium pediceratum, Physoderma meidis, Phyllosticta meidis, Cabatiera meidis, Cercospora sorgii, Ustylago meidis, Psinia sorgii, Psinia polisola, Macrohomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curbularia lunata, Curbularia inaequalis, Curbularia parescens, Clavibacter michiganense subspecies nebulaskense, Trichoderma viride, Claviceps sorgii, Pseudomonas avenae, Erwinia chrysanthemi pv. zea, Erwinia • Carotovora, Maize dwarf spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorgii, Peronosclerospora philippinensis, Peronosclerospora meidis, Peronosclerospora sakari, Sphaserotheca laeliana, Physopera zeae, Cephalosporium meidis, Cephalosporium acremonium; Sorghum: Exerohilum tulsicum, C. sublineorum, Cercospora sorgii, Gloeocercospora sorgii, Ascochita sorgina, Pseudomonas syringae pv. syringae, Xanthomonas campestris pv.Forsicola, Pseudomonas andropogonis, Psinia purpurea, Macrohomina phaseolina, Perconia circinata, Fusarium verticilioides, Alternaria alternata, Vipolaris sorgicola, Helmintosporium sorgicola, Curbularia lunata, Forma insidiosa, Pseudomonas avenae (Pseudomonas arboprecipitans), Ramlispora sorgii, Ramlispora sorgicola, Phylacara sakari, S Polysorum lerianum (Sfacerotheca leriana), Sfacerotheca cruenta, Sporisorum sorgii, Claviceps sorgii, Rhizoctonia solanii, Acremonium strictum, Sclerophthora macrospora, Pernosclerospora sorgii, Pernosclerospora philippinesis, Sclerospora graminicola, Fusarium gramineum, Fusarium oxysporum, Pythium argenomanes, and Pythium graminicola, etc.

[0085] In some embodiments, the plant pathogen or pest is a bacterium. Examples of plant pathogenic bacteria include those belonging to the genera Pseudomonas, Pantoea, and Erwinia. Additional plant pathogenic bacteria are listed in Robert W. Jackson, Plant Pathogenic Bacteria: Genomics and Molecular Biology, Horizon Scientific Press, 2009, ISBN 1904455379, 9781904455370; Samuel S. Gnanamanickam, Plant-Associated Bacteria, Springer Press, 2007, ISBN 1402045379, 9781402045370; Martin Dworkin et al., The Prokaryotes: a handbook on the biology of bacteria, Springer Press, 2006, ISBN 0387254927, 9780387254920; George N. Agrios, Plant pathology, Academic Press, 2005, ISBN It is listed in 0120445654, 9780120445653; and David W. Parry, Plant pathology in agriculture, CUP Archive Publishing, 1990, ISBN 0521368901, 9780521368902.

[0086] In some embodiments, the plant pathogen or pest is a virus. Examples of viruses that can cause infection in plants include, but are not limited to, cucumber mosaic virus, tobacco mosaic virus, barley fusarium virus, alfalfa mosaic virus (Alphamovirus), apple chlorotic leaf spot virus (Trichovirus), apple rust viroid (Viroid), Arabis mosaic virus (Nepovirus), barley mild mosaic virus (Faimovirus), barley spotted leaf mosaic virus (Hordeivirus), barley yellow spotted mosaic virus (Faimovirus), kidney bean mosaic virus (Potivirus), kidney bean yellow spotted mosaic virus (Potivirus), beet necrotic leaf vein yellowing virus (Frovirus), cowpea mosaic virus (Potivirus), kidney bean mosaic virus (Potivirus), broad bean wilt virus (Fabavirus), butterbur mosaic virus (Carlavirus), carnation spotted virus (Carlavirus) (Genus Potivirus), Carnation Vein Mottle Virus (Genus Potivirus), Cauliflower Mosaic Virus (Genus Caurimovirus), Chrysanthemum Micro-spot Virus (Genus Cucumovirus), Tomato Aspermy Virus (Genus Cucumovirus), Chrysanthemum Dwarf Viroid (Viroid), Citrus Mosaic Virus, Citrus Tristeza Virus (Genus Crosterovirus), Clover Leaf Vein Yellowing Virus (Genus Potivirus), Cox Foot Mottle Virus (Genus Sobemovirus), Cub Examples of plant viruses include cucumber green spot mosaic virus (Tobamovirus), cucumber mosaic virus (Cucumovirus), cycad necrotic dwarf virus (Nepovirus), taro mosaic virus (Potivirus), grapevine algeria latent virus (Thombusvirus), konjac mosaic virus (Potivirus), melon necrotic spot virus (Calmovirus), mulberry ring spot virus (Nepovirus), and narcissus mosaic virus (Potexvirus). Plant viruses are viruses that infect plants. Additional examples of plant viruses include odontoglossum ring spot virus (Tobamovirus), papaya ring spot virus (Potivirus), peach latent mosaic viroid,Peanut spot virus (Potivirus genus), peanut leaf spot virus (Potivirus genus), bean mosaic virus (Potivirus genus), peanut dwarf virus (Cucumovirus genus), potato virus A (Potivirus genus), potato virus M (Carlavirus genus), potato virus S (Carlavirus genus), potato virus X (Potexvirus genus), potato virus Y (Potivirus genus), prune dwarf virus (Iralvirus genus), prune necrotic ring spot virus ( Iralvirus genus), Radish leaf mosaic virus (Comovirus genus), Rice black streak dwarf virus (Fijivirus genus), Rice dwarf virus (Reovirus genus), Rice grassy stunt virus (Tenuivirus genus), Rice stripe leaf blight virus (Tenuivirus genus), Rice tunglobular virus (Cekivirus genus), Rice dwarf virus, Rice tunglobular virus (Cekivirus genus), Ryegrass spotted virus, Satsuma dwarf virus (Nepovirus genus), Wheat mosaic virus (Furovirus genus), Southern kidney bean Mosaic virus (Sobemovirus genus), Soybean mosaic virus (Potivirus genus), Soybean dwarf virus (Cukumovirus genus), Cucumber mosaic virus (Cukumovirus genus), Tobacco mosaic virus (Tobamovirus genus), Tobacco mosaic virus (Tobamovirus genus), Tomato mosaic virus (Tobamovirus genus), Tobacco necrosis virus (Necrovirus genus), Tobacco stem necrosis virus (Tobulavirus genus), Tobacco ring spot virus (Nepovirus genus), Tomato aspermivirus (Cukumovirus genus) Examples of plant viruses include *Rhus* (genus *Rhus*), tomato black ring spot virus (genus *Nepovirus*), tomato mosaic virus (genus *Tobamovirus*), tomato ring spot virus (genus *Nepovirus*), tomato yellow necrosis virus (genus *Tospovirus*), turnip mosaic virus (genus *Potivirus*), pumpkin mosaic virus 1 (genus *Potivirus*), papaya ring spot virus (genus *Potivirus*), pumpkin mosaic virus 2 (genus *Potivirus*), wheat stripe dwarf virus (genus *Baimovirus*), and zucchini yellow mosaic virus (genus *Potivirus*). For more plant viruses, see FC Bawden, *Plant Viruses and Virus Diseases*.It is listed in Biotech Books, 2002, ISBN 8176220647, 9788176220644.

[0087] In some embodiments, plant pathogens or pests are eukaryotes. In some embodiments, eukaryotes are insects or nematodes. Agricultural pests can be classified into chewing insects, sap-sucking insects, and soil insects. Common chewing insects include, for example, the beet armyworm (Spodoptera exigua), the diamondback moth (Plutella xylostella), the corn earworm (Heliothis zea, also known as ball worm and tomato fruit worm), blister beetles (Epicauta, etc.), the carrot flower weevil (Listronotus oregonensis, Hyperodes texana), and the cabbage looper (Trichopulsia ni). ni)), grasshoppers (several species), flea beetles (e.g., tobacco flea beetle (Epitrix hirtipennis), eggplant flea beetle (E. fuscula), potato flea beetle (E. cucumeri), and other species), fall armyworm (Spodoptera frugiperda), scale moths: Pyralidae (Lesser cornstalk borer) (Elasmopalpus lignosellus), Texas leafcutter ant (Atta texana), citrus leafminer (Phyllocnistis citrela) These include citrella, leafminer (Liriomyza spp.), and yellowstriped armyworm (Spodoptera ornithogalli).Common sap-sucking insects include, for example, brown marmorated bugs (e.g., Nezara viridula and other species), leafhoppers (Homalodisca spp. and Oncopmetopia spp.), whiteflies (e.g., silverleaf whitefly, greenhouse whitefly, cotton whitefly (tobacco whitefly (Bemisia tabaci), greenhouse whitefly (Trialeuroides vaporariorum)), psyllids (e.g., citrus psyllid), squash bugs (Anasa tristis), and leaffooted bugs (Leptoglossus genus). spp.)), leafhoppers (e.g., sugar beet leafhopper, Empoasca solana, aster leafhopper, Macrosteles fascifrons, Western potato leafhopper, Empoasca abrupta, grape leafhopper, variegated leafhopper (a species of leafhopper family), sugar beet leafhopper, Circulifer tenellus), aphids (Aphidoidea superfamily, e.g., peach aphid, false radish aphid, melon aphid, potato aphid, rosy apple aphid) These include aphids (Spirea aphids). Common licking insects include, but are not limited to, thrips (e.g., citrus thrips, western flower thrips (Frankliniella occidentalis), onion thrips (Thrips tabaci), melon thrips, and pepper thrips).Common soil insects include, for example, granulate cutworms (Feltia subterranea), mole crickets (e.g., northern mole cricket, Neocurtilla hexadactyla; southern mole cricket, Scapteriscus acletus), corn root worms (e.g., Diabrotica undecimpunctata howardi), pillbugs and woodlice (several species), ant weevils (Cylas formicarius elegantulus), ground insects (Phyllophaga spp.), and horsehair worms (several species). Exemplary nematodes that act as plant pathogens or pests can be classified into parasitic nematodes, such as root-knot nematodes including the genera Heterodera, Meloidogyne, and Globodera; cyst nematodes including the genera Heterodera, Meloidogyne (water cyst nematodes), and Globodera; and root-lesion nematodes including the genera Pratylenchus.

[0088] In some embodiments, the pathogen is a pathogen of the Solanum or Wheat genus. In some embodiments, the plant pathogen or pest is a fungus. In some embodiments, the fungus is Septoria tritisi, Botrytis cinerea, Alternaria solanii, Rhizoctonia species, Phytophthora capsici, Phytophthora capsicum, or Phytophthora infestans.

[0089] II. Oligo-Papilloma Antimicrobial oligopeptides and antifungal oligopeptides are also provided herein.

[0090] In some embodiments, the method includes the step of supplying a plant with an oligopeptide containing an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide contains an amino acid sequence having one, two, three, or four amino acid substitutions, insertions, or deletions compared to any of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide contains an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acid long. In some embodiments, the oligopeptide contains proline at the N-terminus, aspartic acid at the C-terminus, or both. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0091] In some embodiments, the oligopeptide contains a protease-resistant amino acid sequence. In some embodiments, the oligopeptide contains one or more modifications compared to a natural oligopeptide. In some embodiments, the oligopeptide contains one or more modifications that increase stability and / or half-life. In some embodiments, the oligopeptide contains one or more D-amino acids. In some embodiments, one or more D-amino acids enhance the stability of the oligopeptide in cells. In some embodiments, one or more D-amino acids enhance the stability of the oligopeptide compared to an oligopeptide with the same amino acid sequence containing all L-amino acids. In some embodiments, the oligopeptide contains a sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having one or more D-amino acids. In some embodiments, the oligopeptide contains an amino acid sequence having one, two, three, or four amino acid substitutions, insertions, or deletions compared to any of SEQ ID NOs: 1-5 and 14-21, and having one or more D-amino acids. In some embodiments, an oligopeptide containing one or more D-amino acids is an antimicrobial oligopeptide or an antifungal oligopeptide. In some embodiments, the oligopeptide contains a D-amino acid at its N-terminus. In some embodiments, the oligopeptide contains a D-amino acid at its C-terminus. In some embodiments, the oligopeptide contains D-amino acids at both its N-terminus and C-terminus.

[0092] In some embodiments, the oligopeptide includes a tag. In some embodiments, the tag is a secretory tag, a tag that facilitates entry into plant cells, or a nuclear localization tag. In some embodiments, the oligopeptide includes a tag at the N-terminus, C-terminus, or both. In some embodiments, the oligopeptide includes an amino acid sequence shown in one of SEQ ID NOs. 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, and a tag.

[0093] In some embodiments, the oligopeptide further comprises a cell-permeable peptide linked to the amino acid sequence of the oligopeptide. In some embodiments, the cell-permeable peptide increases intracellular delivery and activity in cells. Useful cell-permeable peptides in this method are a type of short peptide that translocates across the cell membrane and acts as a nanocarrier for the delivery of proteins to plant cells. Exemplary cell-permeable peptide families include, but are not limited to, cell-permeable peptides derived from protein transduction domains, amphiphilic peptides, and synthetic cationic polypeptides such as polylysine, polyhistidine, and polyarginine, or dendrimer-type polycationic molecules. Cell-permeable peptides that may be used in conjunction with the oligopeptides of this disclosure include, but are not limited to, vascular endothelial-cadherin peptide cell-permeable peptides, transportan cell-permeable peptides, monomers and dimers of the HIV-1 TAT basic domain cell-permeable peptides, penetratin cell-permeable peptides, synthetic cationic homoarginine oligopeptide cell-permeable peptides (see Eudes and Chugh, (2008) Plant Signal Behav. 3: pp. 549-550), and gamma-zein cell-permeable peptides (see U.S. Patent No. 8,581,036 incorporated herein by reference). Additional non-limiting examples of cell-permeable peptides and other internally distributed molecules include maize knotted 1 cell-permeable peptide, Saccharomyces pombe TP10 cell-permeable peptide, Candida albicans zebra cell-permeable peptide, Antennapedia sequence, TAT sequence, Antp-3A (Antp variant), Buforin II, MAP (model amphiphilic peptide), K-FGF, Ku70, prions, pVEC, SynB1, Pep-7, HN-1, BGSC (bis-guanidinium-spermidine-cholesterol), and BGTC (bis-guanidinium-tren-cholesterol).Additional cell-permeable peptides are described in U.S. Patent No. 9,757,473, WO / 2014 / 086835A1, U.S.2017 / 0057997, U.S.2020 / 0263189, U.S.20200399647, and WO / 2011 / 084061 (the contents of each are incorporated by reference for these peptides). In some embodiments, the cell-permeable peptide includes C-terminal modification, N-terminal modification, or both. In some embodiments, the oligopeptide includes a cell-permeable peptide linked to the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions.

[0094] In some embodiments, the oligopeptide comprises an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, and an N-terminal and / or C-terminal residue. In some embodiments, the oligopeptide comprises one or more proline residues at the N-terminus or C-terminus. In some embodiments, the N-terminal and / or C-terminal residues correspond to cleavage tags. For example, the residues may be part of a peptide bond that is cleaved by a chemical (e.g., acidic cleavage) or enzymatic reaction. In some embodiments, the oligopeptide comprises an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, and an N-terminal proline, methionine, tryptophan, glycine, or cysteine, and / or a C-terminal methionine, aspartic acid, cysteine, asparagine, or tryptophan. In some embodiments, the oligopeptide comprises an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, and an N-terminal proline and / or C-terminal aspartate. In some embodiments, the oligopeptide is 4-50 or 6-30 amino acid long. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0095] In some embodiments, the oligopeptides provided herein are produced by cleavage of concatemers of multiple oligopeptides that are linked together and expressed as a single polypeptide chain. In some embodiments, the cleavage results in short (one or two) amino acid tags at the N-terminus and / or C-terminus of the oligopeptides. In some embodiments, the oligopeptides are produced by a method comprising expressing a fusion polypeptide comprising a polypeptide that forms an inclusion body in a cell, operably linked to two or more oligopeptides by peptide bonds. In some embodiments, the oligopeptides are produced by a method comprising expressing a fusion polypeptide comprising a carrier polypeptide operably linked to two or more oligopeptides by peptide bonds, and releasing the two or more oligopeptides from the carrier polypeptide. In some embodiments, the two or more oligopeptides in the fusion polypeptide are released from and / or from each other by sequence-specific chemical cleavage of the peptide bond. In some embodiments, the peptide bond is an Asp-Pro bond, and the sequence-specific cleavage is carried out using acetic acid. In some embodiments, the release of two or more oligopeptides from the carrier polypeptide is carried out in the absence of a chaotropic agent. In some embodiments, the release of two or more oligopeptides from the carrier polypeptide is carried out without a column-based purification step. In some embodiments, the two or more oligopeptides are 4 to 50 amino acid long. In some embodiments, the two or more oligopeptides contain the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the released oligopeptides contain N-terminal proline and / or C-terminal aspartic acid.In some embodiments, the released oligopeptides contain an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, as well as an N-terminal proline residue and a C-terminal aspartic acid. In some embodiments, two or more oligopeptides are antimicrobial oligopeptides or antifungal oligopeptides.

[0096] Oligopeptide synthesis In some embodiments, the method includes a step of synthesizing oligopeptides by chemical synthesis. Various approaches have been developed for the chemical synthesis of peptides, such as solid-phase approaches. Solid-phase peptide synthesis is a method for chemically synthesizing peptides on a solid support. In solid-phase peptide synthesis, amino acids or peptides are typically attached to the solid support via their C-terminus. New amino acids are added to the bound amino acids or peptides by a coupling reaction. Translation-based approaches for peptide synthesis have also been developed, in which peptides are produced from an encoding transcript by in vitro translation.

[0097] In some embodiments, the method includes a step of synthesizing the oligopeptide by biosynthesis. Any suitable method of biosynthesis of the peptide may be used. The biosynthesis may include expressing the peptide in cells and purifying the peptide from the cells.

[0098] In some embodiments, the synthesis of oligopeptides involves expressing a fusion polypeptide comprising a polypeptide that forms an inclusion body in cells, operably linked to two or more oligopeptides by peptide bonds. In some embodiments, the synthesis of oligopeptides involves expressing a fusion polypeptide comprising a carrier polypeptide operably linked to two or more oligopeptides by peptide bonds, and releasing the two or more oligopeptides from the carrier polypeptide. In some embodiments, the synthesis of oligopeptides involves expressing a fusion polypeptide comprising a carrier polypeptide operably linked to two or more oligopeptides by peptide bonds, and releasing the two or more oligopeptides from the carrier polypeptide. In some embodiments, the two or more oligopeptides in the fusion polypeptide are released from and / or from each other by sequence-specific chemical cleavage of the peptide bond. In some embodiments, the peptide bond is an Asp-Pro bond, and the sequence-specific cleavage is carried out using acetic acid. In some embodiments, the release of two or more oligopeptides from the carrier polypeptide is carried out in the absence of a chaotropic agent. In some embodiments, the release of two or more oligopeptides from a carrier polypeptide is carried out without a column-based purification step. In some embodiments, the two or more oligopeptides are 4 to 50 amino acid long. In some embodiments, the two or more oligopeptides contain an N-terminal proline, a C-terminal aspartic acid, or both. In some embodiments, the oligopeptides contain an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, and tags. In some embodiments, the two or more oligopeptides are antimicrobial oligopeptides or antifungal oligopeptides.

[0099] In some embodiments, the method includes a step of synthesizing nucleic acids encoding oligopeptides. Synthetic nucleic acids (DNA, RNA, or analogs thereof) may be prepared using a column-based synthesizer or produced from existing nucleic acids by PCR or in vitro transcription. Other methods of nucleic acid synthesis include, for example, U.S. No. 6,586,211 Bl, PCT / EP2004 / 013131, WO 00 / 13017 A2, S. Rayner et al., PCR Methods and Applications 8 (7), pp. 741-747, 1998; WO 90 / 00626 Al, EP 385 410 A2, WO 94 / 12632 Al, WO 95 / 17413 Al, EP 316 018 A2, EP 022 242 A2, LE Sindelar and JM Jaklevic, Nucl. Acids Res. 23 (6), pp. 982-987, 1995; DA Lashkari, Proc. Nat. Acad. Sci. USA 92 (17), pp. 7912-7915, 1995, and described in WO 99 / 14318 Al. Nucleic acids can also be synthesized by biological synthesis, for example, by introducing nucleic acids into cells (e.g., bacteria), allowing the nucleic acids to replicate, and then isolating the newly synthesized nucleic acids.

[0100] III. Composition Compositions containing oligopeptides are also provided. In some embodiments, the composition contains isolated oligopeptides. In some embodiments, the composition contains purified oligopeptides. In some embodiments, the oligopeptides contain the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21.

[0101] In some embodiments, the composition comprises an oligopeptide having an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the composition comprises an oligopeptide having an amino acid sequence having one, two, three, or four amino acid substitutions, insertions, or deletions compared to one of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide. In some embodiments, the oligopeptide contains proline at its N-terminus, aspartic acid at its C-terminus, or both. In some embodiments, the oligopeptide contains one or more D-amino acids. In some embodiments, one or more D-amino acids enhance the stability of the oligopeptide compared to an oligopeptide of the same amino acid sequence containing all L-amino acids. In some embodiments, the oligopeptide further comprises a cell-permeable peptide linked to the amino acid sequence of the oligopeptide.

[0102] In some embodiments, the composition comprises oligopeptides and agriculturally acceptable formulation agents. In some embodiments, the agriculturally acceptable formulation agents include one or more of the following: water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffers, hydrotropes, rheology modifiers, antifoaming and defoaming agents, antifreezes, biocides, dyes, polymer walls, catalysts, thermosetting materials, crosslinking polymerizers, UV protectants, antioxidants, and chelating agents.

[0103] In some embodiments, the composition contains oligopeptides in a concentration range from about 0.1 μg / ml to about 100 mg / ml, or from about 5 μg / ml to about 5 mg / ml. In some embodiments, the composition contains oligopeptides in concentrations of about 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 50 μg / ml, 60 μg / ml, 70 μg / ml, 80 μg / ml, 90 μg / ml, 1 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml.

[0104] In some embodiments, the composition further comprises one or more other active agents selected from the group consisting of pesticides, fertilizers, insecticides, attractants, sterilizers, mite control agents, anthelmintic agents, herbicides, biostimulants, biological formulations, and growth regulators. In some embodiments, the composition is formulated as a liquid, gel, emulsion, suspension, capsule encapsulation, solid, powder, aerosol, paste, coating agent, spray, soil drenching agent, microcapsule, emulsion, or granule. In some embodiments, the composition takes the form of fine particulate solid, granule, pellet, hydrated powder, fine powder, aqueous suspension, dispersion, gel, or emulsion. In some embodiments, agronomically acceptable compositions are formulated as seed treatment, foliar spray, foliar dipping, ready-to-use (RTU) formulations, crop coatings, flowable formulations, tank mixes, aerosols, root dipping, soil treatment, dipping formulations, drenching formulations, or sprinkler formulations. In some embodiments, the composition is formulated for application to post-harvest plants or parts thereof. In some embodiments, the composition is formulated for application to a plant or part thereof before harvest. In some embodiments, the plant part is a fruit, leaf, bud, root, shoot, flower part, or seed. In some embodiments, the composition is formulated for application to a plant before, during, or after infection with a pathogen or pest. In some embodiments, the composition is formulated for application to the soil in which the plant is planted. In some embodiments, the composition is formulated for application to a plant by addition to the water supplied to the plant.

[0105] In some embodiments, the composition comprises an oligopeptide and an agriculturally acceptable carrier, diluent, or excipient. In some embodiments, the agriculturally acceptable carrier, diluent, or excipient comprises a buffer with a pH ranging from about 3.0 to about 9.0. In some embodiments, the agriculturally acceptable carrier, diluent, or excipient comprises a buffer with a pH ranging from about 3.0 to about 4.0, about 4.0 to about 5.0, about 5.0 to about 6.0, about 6.0 to about 7.0, about 7.0 to about 8.0, or about 8.0 to about 9.0. In some embodiments, the agriculturally acceptable carrier, diluent, or excipient comprises a buffer with a pH ranging from about 4.5 to about 8.0. In some embodiments, the agriculturally acceptable carrier comprises a solid carrier, a liquid carrier, a gel carrier, a suspension, or an emulsion. In some embodiments, the agriculturally acceptable carrier comprises an auxiliary agent, an inert component, a dispersant, a surfactant, a water-retaining agent, an emulsifier, a thickener, a wetting agent, a fertilizer, a mineral, a solvent, a tackifier, a binder, or a stabilizer. In some embodiments, the agriculturally acceptable composition comprises an oligopeptide and a surfactant or water-retaining agent.

[0106] IV. Nucleic acids Nucleic acids encoding oligopeptides are also provided.

[0107] In some embodiments, the nucleic acid encodes an oligopeptide comprising an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide. In some embodiments, the oligopeptide comprises an amino acid sequence having 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions compared to any of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acid long. In some embodiments, the oligopeptide further comprises an N-terminal proline and a C-terminal aspartic acid. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0108] In some embodiments, nucleic acids encoding oligopeptides are operably linked to a promoter, for example, in a nucleic acid construct. In some embodiments, the promoter may be a heterogeneous promoter. In some embodiments, the promoter may be a constitutive promoter, a tissue-specific promoter, a developmental stage-specific promoter, or an inductive promoter. In some embodiments, the promoter is an inductive promoter. In some embodiments, the inductive promoter may be a promoter induced by fungal infection and optionally associated with genes involved in phenylpropanoid metabolism (e.g., promoters for phenylalanine ammonia lyase, chalcone synthase), a promoter associated with genes that modify plant cell walls (e.g., promoters for hydroxyproline-rich glycoproteins, glycine-rich proteins, and peroxidases), a promoter associated with genes encoding enzymes that degrade fungal cell walls (e.g., promoters for chitinase or glucanase), a promoter associated with genes encoding thaumatin-like proteins, or maize or flax promoters named Mis1 and Fis1.

[0109] In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a polypeptide that forms an inclusion body in cells, operably linked to two or more oligopeptides. In some embodiments, the nucleic acid encodes a fusion polypeptide comprising a carrier polypeptide operably linked to two or more oligopeptides. In some embodiments, the operable linkage comprises a peptide bond that is highly sensitive to sequence-specific chemical cleavage. In some embodiments, the operable linkage is an Asp-Pro bond, and the sequence-specific cleavage is cleavage by acetic acid. In some embodiments, the oligopeptides are 4 to 50 amino acid long. In some embodiments, the oligopeptides contain N-terminal proline and C-terminal aspartic acid. In some embodiments, the nucleic acid operably linked to the promoter encodes an oligopeptide comprising the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0110] In some embodiments, the nucleic acid encodes an oligopeptide further comprising a tag. In some embodiments, the tag is a secretory tag, a tag that facilitates entry into plant cells, or a nuclear localization tag.

[0111] The nucleic acids of this disclosure may be prepared using any techniques known in the art. These may include, but are not limited to, cloning, DNA isolation, amplification, and purification, enzymatic reactions including DNA ligases, DNA polymerases, restriction endonucleases, and others of the same kind, and various separation techniques such as gel electrophoresis and chromatography.Some standard techniques are found in Ausubel et al., (1992) Current Protocols in Molecular Biology, Green / Wiley, New York, NY; Sambrook et al., (1989) Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory, Plainview, NY; Maniatis et al., (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al., (ed.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982); Practical Methods in Molecular Biology; Glover (ed.) (1985); DNA Cloning, Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985); Nucleic Acid Hybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979); Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York; and Ausubel et al. (1992) Current Protocols in Molecular Biology, Greene / Wiley, New York, NY.The abbreviations and nomenclature used are considered standard in this field and are commonly used in professional journals such as those cited herein.

[0112] vector Vectors comprising nucleic acids encoding any of the fusion polypeptides, carrier polypeptides, or oligopeptides disclosed herein are also provided herein.

[0113] A "vector" is a nucleic acid capable of transporting another nucleic acid. A vector can be, for example, a plasmid, virus, cosmid, or phage. An "expression vector" is a vector capable of directing the expression of a protein encoded by one or more genes contained within it, when it is present in a suitable environment. An example of a vector is one that can autonomously replicate and express a structural gene product present in the DNA segment to which they are operably linked. Thus, a vector can contain the previously described replicons and selection markers. Examples of vectors include, but are not limited to, expression vectors.

[0114] In some embodiments, the vector is a bacterial vector. In some embodiments, the vector is a yeast vector. Examples of expression vectors that can be used in prokaryotic host cells include the cloning vector pET plasmid (Novagen, Madison, Wis., USA) or vectors derived from commercially available plasmids such as pBR322 (ATCC 37017). The pBR322 vector contains genes for ampicillin and tetracycline resistance and therefore provides a simple means for identifying transformed cells. To construct an expression vector using pBR322, a suitable promoter and a DNA sequence encoding one or more polypeptides of the present invention are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM-1 (Promega Biotec, Madison, Wis., USA). Other commercially available vectors include those specifically designed for protein expression; these include the pMAL-p2 and pMAL-c2 vectors (New England Biolabs, Beverly, Mass., USA) used for the expression of proteins fused with maltose-binding proteins.

[0115] In bacterial systems, several expression vectors may be advantageously selected depending on the intended use of the variant squalene synthase enzyme, whether isolation of the enzyme is desired, and the desired purity level. For example, if large quantities are to be produced, a vector directing the expression of a high-level fusion protein product that can be easily purified may be desirable.

[0116] In yeast, several vectors containing constitutive or inducible promoters can be used. For an overview, see Current Protocols in Molecular Biology, Vol. 2, edited by Ausubel et al., Greene Publish. Assoc. & Wiley Interscience, Chapter 13 (1988); Bitter et al., Expression and Secretion Vectors for Yeast, Methods in Enzymology, edited by Wu & Grossman, 31987, Acad. Press, NY, Vol. 153, pp. 516-544 (1987); Glover, DNA Cloning, Vol. II, IRL Press, Wash., DC, Chapter 3 (1986); Bitter, Heterologous Gene Expression in Yeast, Methods in Enzymology, edited by Berger & Kimmel, Acad. Press, NY, Vol. 152, pp. 673-684 (1987); and The Molecular Biology of the Yeast Saccharomyces, edited by Strathem et al., Cold Spring Harbor Press, Vols. I and II (1982). Constitutive yeast promoters such as ADH1 or LEU2, or inducible promoters such as GAL4, may be used (Cloning in Yeast, Chapter 3, R. Rothstein: DNA Cloning Vol. 11, A Practical Approach, DM Glover (ed.), IRL Press, Wash., DC (1986)). Alternatively, vectors that promote the integration of foreign DNA sequences into yeast or bacterial chromosomes may be used.

[0117] Commonly used promoter sequences for recombinant prokaryotic host cell expression vectors include the bacteriophage T7 promoter (Studier and Moffatt, J. Mol. Biol. 189: 113 (1986)), β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; Goeddel et al., Nature 281:544 (1979)), tryptophan (tap) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); EP-A-36776), and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; p. 412 (1982)). Particularly useful prokaryotic host cell expression systems utilize the phage λ PL promoter and the c1857ts thermally stable repressor sequence. Plasmid vectors available from the American Type Culture Collection (ATCC) that incorporate derivatives of the PL promoter include plasmid pHUB2 (commonly present in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (commonly present in E. coli RR1 (ATCC 53082)).

[0118] V. Cell Cells containing antimicrobial or antifungal oligopeptides are also provided. In some embodiments, the cells contain oligopeptides comprising the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the cells are host cells.

[0119] In some embodiments, the cells are plant cells. In some embodiments, the cells are plant cells. In some embodiments, the plant cells are dicotyledonous or monocotyledonous plant cells. In some embodiments, the plant cells are crop-derived cells. In some embodiments, the plant cells are cells derived from Solanum and Wheat plants. In some embodiments, the cells are transgenic cells. In some embodiments, the cells express antimicrobial or antifungal oligopeptides.

[0120] In some embodiments, the cells are bacterial cells. In some embodiments, the bacteria are Escherichia coli strains. In some embodiments, the cells are cells capable of forming inclusion bodies. In some embodiments, the cells are prokaryotic cells. In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are fungal cells. In some embodiments, the cells are algal cells. In some embodiments, the cells are animal cells. In some embodiments, the animal cells are mammalian cells or insect cells.

[0121] Useful microbial host cells in the present invention include, for example, intestinal bacteria (e.g., Escherichia and Salmonella), as well as bacteria such as Bacillus, Acinetobacter, Streptomyces, Methylobacterium, Rhodococcus, and Pseudomonas; and cyanobacteria such as Rhodobacter and Synechocystis. a); Yeasts such as those of the genera Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia, Yarrowia, and Torulopsis; as well as filamentous fungi such as Aspergillus and Arthrobotrys, and algae, but not limited to these.

[0122] In some embodiments, the host cell contains at least one copy of a nucleic acid sequence encoding an oligopeptide. At least one copy of the nucleic acid sequence encoding the oligopeptide may be present on a chromosome of a prokaryotic (bacterial) cell or on one chromosome of a eukaryotic cell. Alternatively, at least one copy of the nucleic acid sequence encoding the oligopeptide may be present on a vector or plasmid present in the cell. Such a host cell can be a prokaryotic or a eukaryotic cell. If it is a prokaryotic cell, it can be a bacterial cell. If it is a eukaryotic cell, it can be a yeast cell, plant cell, or animal cell. Suitable host cells are described herein.

[0123] In some embodiments, the cell contains an oligopeptide, or a nucleic acid encoding the oligopeptide, comprising the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the oligopeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 1-5 and 14-21. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acid long. In some embodiments, the oligopeptide further comprises N-terminal proline and C-terminal aspartic acid. In some embodiments, the oligopeptide comprises a secretory tag, a tag that facilitates entry into plant cells, or a nuclear localization tag. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0124] VI. Kit Some aspects of this disclosure provide a kit for supplying oligopeptides to plants, including an attached document containing oligopeptides and instructions for use. In some embodiments, the oligopeptides are antimicrobial oligopeptides or antifungal oligopeptides.

[0125] In some embodiments, the kit includes an oligopeptide comprising an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21 having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, and a package insert containing instructions for use regarding the application of the oligopeptide to plants. In some embodiments, the oligopeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of SEQ ID NOs: 1-5 and 14-21, and the package insert contains instructions for use regarding the application of the oligopeptide to plants. In some embodiments, the oligopeptide is 4-50 or 5-30 amino acid long. In some embodiments, the oligopeptide further comprises N-terminal proline and C-terminal aspartic acid. In some embodiments, the oligopeptide comprises a secretion tag, a tag that facilitates entry into plant cells, or a nuclear localization tag. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0126] In some embodiments, the kit includes a composition comprising an oligopeptide and accompanying documentation including instructions for use regarding the application of the composition. In some embodiments, the composition comprises an oligopeptide and an agrochemically acceptable formulation agent. In some embodiments, the agrochemically acceptable formulation agent comprises one or more of the following: water, organic solvents, paraffinic oils, vegetable oils, dispersants, emulsifiers, wetting agents, buffers, hydrotropes, rheological modifiers, antifoaming and defoaming agents, antifreezes, biocides, dyes, polymer walls, catalysts, thermosetting materials, crosslinking polymerizers, UV protectants, antioxidants, and chelating agents. In some embodiments, the composition takes the form of a fine particulate solid, granules, pellets, hydrated powder, fine powder, aqueous suspension, dispersion, gel, or emulsion. In some embodiments, the composition further comprises one or more other activators selected from the group consisting of pesticides, fertilizers, insecticides, attractants, sterilizers, mite control agents, anthelmintic agents, herbicides, biostimulants, biological formulations, and growth regulators. In some embodiments, the kit includes an oligopeptide comprising an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

[0127] In some embodiments, the kit comprises a composition comprising an oligopeptide and an agrochemically acceptable carrier, diluent, or excipient. In some embodiments, the kit comprises a composition comprising the oligopeptide and a package insert containing instructions for use on applying the oligopeptide to a plant or a part thereof. In some embodiments, the part of the plant is a leaf, bud, root, shoot, flower part, or seed. In some embodiments, the package insert contains instructions for supplying the composition to the plant by application to the soil in which the plant is planted. In some embodiments, the package insert contains instructions for supplying the composition by addition to the water supplied to the plant. In some embodiments, the package insert contains instructions for applying the composition by a spreader, power duster, boom sprayer, hand sprayer, spray duster, or granular spreader. In some embodiments, the kit comprises an oligopeptide comprising an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, having 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, and a package insert containing instructions for use on applying the oligopeptide to a plant.

[0128] Kits are also provided herein that include cells containing an oligopeptide or a nucleic acid encoding the oligopeptide, and an accompanying document containing instructions for use regarding the application of the cells to plants. In some embodiments, the cells contain an oligopeptide having an amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, with 0, 1, 2, 3, or 4 amino acid substitutions, insertions, or deletions, and the accompanying document contains instructions for use regarding the application of the cells to plants. In some embodiments, the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide. [Examples]

[0129] This disclosure is described in further detail in the following embodiments, which are not intended in any way to limit the scope of the disclosure as claimed. The accompanying drawings are intended to be an integral part of the specification and description of this disclosure. The following embodiments are provided for illustrative purposes only and are not intended to limit the claimed disclosure.

[0130] (Example 1) Screening of antimicrobial oligopeptides targeting members of the Slmir169 family; identification of antimicrobial oligopeptides that eliminate Botrytis infections. The following examples describe the screening process leading to the identification of MP18279, an oligopeptide with antifungal activity.

[0131] Materials and methods screening Botrytis cinerea was grown on potato dextrose agar (PDA) in darkness at 25°C. Actively growing hyphae were collected from the plates, dissolved in water, and filtered through Medifab cloth. Spores present in the filtrate were counted, and 2.5 × 10⁶ spores were found in potato dextrose broth (PDB). 5 The final concentration was adjusted to the number of spores / ml.

[0132] Tomato leaves, three weeks after germination, were inoculated with 5 μl of spore solution at six sites on mature leaves. Twenty-two peptides were chemically synthesized (SB-Peptide, Grenoble, France) and tested for activity against Botrytis cinerea. The peptides were diluted to a concentration of 100 μM in Milli-Q water. Each peptide was added daily to the sites of future Botrytis inoculation (three prophylactic treatments). Spores were placed on the leaves as droplets as described above, and the plants were kept in a moist environment to allow for the initiation of infection. At 24 and 48 hours post-infection, 5 μl of 100 μM peptide was added to the infection sites. After 72 hours, the lesion area was measured and compared to a control treatment in which Milli-Q water was applied at the same time as the peptide.

[0133] Application of MP18279 to tomatoes infected with Botrytis cinerea MP18279 (SEQ ID NO: 1) was applied to Botrytis cinerea infections on tomato leaves and fruits. 100 μM of MP18279 was applied directly to the infection site twice daily (for leaves) or five times daily (for fruits) (see Figure 1).

[0134] result MP18279 completely eliminates Botrytis infection in tomato leaves and fruits. A total of 22 candidate antimicrobial oligopeptides were analyzed. Peptide activity was evaluated by applying the peptides to lesions induced by B. cinerea infection in tomato plants. Typically, infected tomato plants showed brownish symptoms at the site of infection, caused by the spread of the pathogen in the tissue.

[0135] Application of the 10-amino acid peptide MP18279 (SEQ ID NO: 1) resulted in the complete elimination of symptoms. MP18279 was identified in the main transcripts of the tomato (Solanum lycopersicum) SlymIR169b, potato (Solanum tuberosum) StumIR169c, and chili pepper (Capsicum annuum) mIR169 gene families. Plant tissues treated with MP18279 remained healthy and green compared to heavily infected control plants (Figure 1). Infected control tomato fruits exhibited typical B. cinerea symptoms, such as the development of gray mycelium at the infection site, while tomato plants treated with the peptide showed no visible symptoms during the observed period (Figure 1). Therefore, MP18279 completely eliminated B. cinerea infection in tomato leaves and fruits.

[0136] (Example 2) MP18279 prevents the germination of Botrytis cinerea. The following examples describe experiments investigating the effect of MP18279 on the germination of B. cinerea.

[0137] Materials and methods Germination assay 400 spores of Botrytis cinerea were incubated in either water (control) or 100 μM MP18279 (SEQ ID NO: 1) in darkness for 24 hours and analyzed by microscopic observation. The spores were then deposited on potato glucose agar (PGA) medium, and their development (spore germination and mycelial growth) was analyzed after 6 days (see Figure 2).

[0138] result MP18279 prevents the germination of Botrytis cinerea. To understand the mechanism of action of MP18279's antifungal effect, B. cinerea spores were incubated with MP18279, and spore germination and hyphal growth were monitored. Under control conditions, spores germinated after 24 hours. In contrast, no germination was observed in MP18279-treated spores. After 6 days, gray mycelium grew on the control plate, while no growth was observed on the MP18279-treated plate (Figure 2). Therefore, MP18279 directly inhibited the germination of B. cinerea spores.

[0139] (Example 3) MP18279 inhibits the germination of various fungal and oomycete pathogens. This example describes an experiment to test the ability of MP18279 to prevent the germination of the fungal pathogens B. cinerea, Septoria tritisi, Alternaria solani, and Rhizoctonia solani anastomosis, as well as the oomycete Phytophthora capsisi.

[0140] Materials and methods Germination assay For the initial germination assay, B. cinerea, S. tritisi, A. solani, Rhizoctonia solani anastomosis, and P. capsici were grown on PDA agar plates, spores were collected, and diluted in 1% PDA. 96-well plates were prepared, each containing 90 μL of the same number of spores of each pathogen. Peptides were prepared by resuspending them in 100 mM 2-(N-morpholino)ethanesulfonate buffer (MES), and 10 μL of the peptide solution was added to each well to reach final concentrations of 300 mg / L, 100 mg / L, 33 mg / L, or 10 mg / L. Peptide MP18021 (SEQ ID NO: 3), which has no known antifungal activity, was used as a control peptide. Blank (MES) and Proline standards were also used as negative and positive controls, respectively. Final evaluations were performed 2–7 days later, depending on pathogen growth. The amount of fungal growth is compared to a control treatment and scored against a predefined key, and the resulting decrease in growth per million (ppm) is expressed as the EC (Energy Convergence). 50 The concentration was determined to be (mg / L).

[0141] Subsequently, B. cinerea, S. tritisi, and P. capsici spores were incubated with MP18279 or a scrambled control peptide at 300 mg / L, 100 mg / L, 30 mg / L, or 10 mg / L. The scrambled control peptide MP18753 (SEQ ID NO: 13) consisted of all the amino acids of the initial MP18279 sequence, but in a random order. Spore germination was visually detected, and EC (Emission Control) was performed. 50 This was calculated for the control peptide and MP18279 (see Figure 4).

[0142] result In vitro activity of MP18279 on spore germination in five different pathogens. The ability of MP18279 to inhibit spore germination was tested on the species of B. cinerea (fungus, gray mold), S. tritisi (fungus, wheat leaf blight), A. solani (fungus, summer blight), Rhizoctonia (fungus, root rot), and P. capsisi (oomycete, pepper blight). MP18279 reduced the germination of S. tritisi, P. capsisi, B. cinerea, and Rhizoctonia spores (Figure 3). MP18279 showed the greatest effect in preventing spore germination of S. tritisi and P. capsisi. B. cinerea and Rhizoctonia were similarly affected after peptide incubation. Finally, MP18279 showed no effect whatsoever on A. solani growth, which suggests selectivity for a specific pathogen (Figure 3). As a control, spores were incubated with an unrelated peptide sequence at the same concentration. The control peptide did not affect spore germination under in vitro conditions.

[0143] In summary, MP18279 showed inhibitory activity against four of the five filamentous pathogens tested, including both fungi and oomycetes.

[0144] Confirmation of in vitro activity of MP18279 against three different pathogens. S. tritisi, P. capsici, and B. cinerea were further investigated because spore germination in these species was significantly affected by the presence of MP18279. For all pathogens tested, the EC of MP18279 was examined. 50 This is the EC of the scrambled control peptide. 50 The levels were significantly lower (Figure 4). Scrambled peptides showed no effect on germination in either B. cinerea or S. tritisi, and only a slight effect on the growth of Phytophthora species.

[0145] In conclusion, the ability of MP18279 to inhibit spore formation by three filamentous pathogens (including both fungi and oomycetes) was shown to depend on the specific amino acid sequence of MP18279, as the scrambled peptide control did not produce any effect on spore germination.

[0146] (Example 4) Identification of the 7-amino acid residue region of MP18279 necessary for antipathogen activity. The following examples describe experiments to limit the region of MP18279 necessary for antifungal activity. Specifically, various truncated forms of MP18279 were prepared and tested for their ability to inhibit fungal or oomycete germination. The seven amino acid residue sequence of MP18279 necessary for its activity was identified.

[0147] Materials and methods Production of a shortened version of MP18279

[0148] [Table 1]

[0149] Germination assay Germination assays were performed using B. cinerea, S. tritisi, and P. capsici spores as described in Example 3 above (see Figures 5A, 5B, and 5C).

[0150] result For all tested pathogens, the 9-amino acid, 8-amino acid, and 7-amino acid truncated (truncate from the C-terminus) versions of MP18279 were more active than the original 10-amino acid sequence (see Figure 5A, black line). Reducing the peptide to 6-amino acid and 5-amino acid lengths resulted in a loss of peptide activity.

[0151] The truncation of amino acids at the N-terminus resulted in an immediate loss of peptide activity, demonstrating that amino acids at positions 1-7 of the N-terminus are essential and necessary for peptide activity (Figure 5B).

[0152] Regarding S. tritisi, the MP18279 variant (with amino acid addition and / or modification) was still active (Figure 5C). These modifications did not interfere with peptide activity.

[0153] (Example 5) Concentration-dependent antifungal activity of MP18279 in plants The following example describes an experiment to investigate the minimum concentration of peptide required to affect the growth of Botrytis cinerea in tomato leaves.

[0154] Materials and methods Botrytis cinerea - Tomato pathogen response system Using a B. cinerea and tomato pathogen response system, we investigated the minimum peptide concentration required to affect B. cinerea growth in tomato leaves. To do this, 2.5 × 10⁻⁶ 5 Spores were incubated with peptides or water (control) at concentrations of 100 μM, 75 μM, 50 μM, 25 μM, and 1 μM for 1 hour, and then the spore solution was applied as droplets to tomato leaves. For each treatment, 6 droplets were applied to 6 leaves, resulting in n=36 per treatment. Lesion size was measured after 2 days and compared to control infection (see Figure 6).

[0155] result The effects of MP18279 activity at different concentrations were tested in tomato leaves in plants. Incubation with 100 μM or 75 μM MP18279 resulted in almost 100% control of B. cinerea, with no lesions or very small lesions (Figure 6). 50 μM of the peptide resulted in 80% control of B. cinerea, but reducing the peptide concentration to below 25 μM did not affect B. cinerea growth, producing lesions of the same size as the control lesions, and showing only 0-20% control of B. cinerea (Figure 6).

[0156] Therefore, MP18279 concentrations in the range of 50–100 μM were effective in controlling Botrytis infection in tomato leaves.

[0157] (Example 6) Spray application of MP18279 provides control of crop diseases. The following examples describe the testing of MP18279 applied as a spray to different plant / pathogen systems to test its ability to control pathogens in crops.

[0158] Materials and methods Application of MP18279 spray to tomato plants infected with tomato gray mold. Tomato plants three weeks after germination were spray-infected with Botrytis cinerea (tomato gray mold). One hour after infection, the plants were sprayed with MP18279 (SEQ ID NO: 1) at varying doses ranging from 200 to 0.2 grams per hectare (g / ha). Bulbous hulls were analyzed daily, and the severity of disease symptoms was visually inspected four days after infection. Using the hullous hull measurements, disease control between infected and peptide-treated plants was calculated (Figures 7A and 7B). The peptides were diluted to their respective final concentrations in TankMix-006 (pH 7.0; 1.44 g / L alkyl polyglucoside, 0.5 g / L polysorbate 20, 0.1 g / L polydimethylsiloxane oil, and 1.96 g / L 2-ethanesulfonic acid).

[0159] Spray application of MP18279 to tomato plants that will later be inoculated with tomato blight fungus and wheat plants that will later be inoculated with wheat rust fungus. Tomato plants were sprayed with 3 grams of MP18279 per hectare, and wheat plants were sprayed with 300 grams of MP18279 per hectare. Two hours after peptide application, tomato plants were inoculated with Phytophthora infestans (tomato blight fungus), and wheat plants were inoculated with Septoria tritisi (wheat leaf blight) (see Figures 8A and 8B). As standard fungicidal controls, inoculated tomato plants were sprayed with Revus SC, and inoculated wheat plants were sprayed with fluxapyroxad.

[0160] result Spray application of MP18279 provides 40% disease control against tomato gray mold. To further test MP18279's ability to control pathogens in crops, MP18279 was applied as a spray to different plant / pathogen systems. When tomato plants were treated with 200 grams and 20 grams of the peptide per hectare, 40% disease control against B. cinerea was observed (Figure 7A). Furthermore, there was a concentration-dependent effect of the applied peptide, with a loss of activity of MP18279 at 0.2 grams per hectare (Figure 7B).

[0161] In summary, the application of MP18279 by spraying significantly improved disease control against tomato gray mold.

[0162] Broad-spectrum activity of MP18279: Spray application of MP18279 provides disease control against tomato blight and wheat rust. Further tests were conducted to determine whether MP18279 provides disease control against other filamentous pathogens such as rust fungi or oomycetes. Specifically, the spray application of MP18279 was tested on tomato plants that would later be inoculated with tomato blight (Figure 8A) and wheat plants infected with wheat rust (Figure 8B).

[0163] Based on the percentage of infected leaf area compared to untreated inoculated plants, the fungicide used as a control provided 100% control, while the application of MP18279 resulted in disease control of 30% or more (Figures 8A and 8B). Therefore, spray application of MP18279 can be used against multiple filamentous pathogens.

[0164] (Example 7) Stability analysis of MP18279 and its variants The following examples describe experiments analyzing the stability of MP18279, as well as the synthesis of stable variants of MP18279 having D amino acids at the N-terminus and C-terminus.

[0165] Materials and methods Synthesis of MP18279D1D10, a variant of MP18279 having D amino acid residues at the N-terminus and C-terminus. Peptide MP182879D1D10 was chemically synthesized by SB-peptide (Grenoble, France).

[0166] Tomato leaf wash-off solution degradation assay For the preparation of the tomato leaf washing solution, leaves from plants grown in a growth chamber for two weeks after germination were cut and placed in a 50 mL Falcon tube. Then, 2 mL of purified water was added to the tube and vortexed. The solution was then collected in a 1.5 mL tube and centrifuged at 500 x g, 20°C, for 4 minutes. The supernatant was then added to the peptide stock solution.

[0167] For the preparation of the analytical sample, the peptide was diluted directly into an analytical vial with a 200 μL insert to reach a final concentration of 0.5 g / L. The 200 μL sample volume was obtained from either 20 μL of peptide stock solution and 180 μL of tomato leaf wash solution or MiliQ water.

[0168] To compare the biological stability of MP18279 and MP18279D1D10 in a tomato leaf wash solution, samples were stored at 20°C for 6, 9, or 48 hours. Subsequently, 5–10 μl were used for high-performance liquid chromatography (HPLC) analysis. The resulting chromatograms were then compared and plotted on the same curve graph, and the percentage relative amount of the peptide over time was calculated.

[0169] Application of Botrytis cinerea spores and peptides to tomato leaves MP18279 and MP18279D1D10 at 100 μM, 2.5 × 10 5 Isolated B. cinerea spores were incubated with the spores at a concentration of 1 / mL for 30 minutes and applied to leaves as droplets (5 μl) (see Figure 9B). As an infection control, spores were applied without further treatment. As a peptide control, spores were incubated with 100 μM MP19075 (SEQ ID NO: 4) (which contains the same amino acids as MP18279 but has a scrambled sequence).

[0170] Application of MP18279 and MP18279D1D10 spray to tomato plants infected with tomato blight. Tomato plants were sprayed with MP18279 (SEQ ID NO: 1) or MP18279D1D10 at a dose of 30 grams per hectare. Two hours after spraying, the plants were inoculated with P. infestans (see Figure 9C).

[0171] result The MP18279 variant exhibits higher stability along with similar antifungal activity. Experiments were conducted to test whether the stability of MP18279 could be improved. The stability of MP18279 was tested in a solution washed off tomato leaves, representing microorganisms present on the leaf surface. MP18279 rapidly degraded after 40 hours of storage in this solution (Figure 9A).

[0172] To overcome this biological degradation, a variant of MP18279 was chemically synthesized in which the first and last amino acids in the sequence were replaced with the D-form of each amino acid. While not intending to be bound by theory, since D-amino acids are not naturally occurring, it was predicted that the MP18279 variant with D-amino acids at the N-terminus and C-terminus would be degraded by microorganisms to a lower degree than the peptide which consists entirely of L-amino acids. Indeed, the modified peptide ("MP18279D1D10") showed a 20% improvement in stability compared to MP18279 when stored in leaf wash solution for the same amount of time (Figure 9A).

[0173] Two independent studies were conducted to further analyze the effect of the D-amino acid MP18279 on the biological activity. In the first experiment, the peptide was incubated with isolated B. cinerea spores and applied as droplets to leaves. Necrotic lesions were observed two days after spore infection under control conditions containing either buffer or the scrambled sequence of MP18279. In contrast, both peptides MP18279 and MP18279D1D10, when used at 100 μM, were able to completely eliminate disease symptoms (Figure 9B). Furthermore, the activity of MP18279D1D10 as a spray application was later validated in tomato plants infected with Phytophthora infestans, and both peptides showed similar levels of disease control between 22–24% (Figure 9C). Thus, MP18279D1D10 retained its full biological activity, although it was less prone to degradation.

[0174] Finally, the stability of MP18279 and its shorter 7-amino acid variant ("MP18865") was compared in a tomato leaf wash solution. After one week, MP18279 was almost completely degraded, while MP18865 was still present at 12% (Figure 10). Furthermore, the disease control capabilities of MP18279 and MP18865 were tested using tomato gray mold or tomato late blight assays. No significant difference was observed between the two peptides (Table 2). Therefore, the shorter variant was more stable and exhibited the same biological activity as the original peptide under the tested conditions.

[0175] [Table 2]

[0176] (Example 8) Genotoxicity testing of MP18279 The following examples describe the results of genotoxicity screening conducted to test whether MP18279 induces genetic damage.

[0177] result Genotoxicity screening was performed on MP18279. Bacterial reverse mutation testing (AMES; Table 3) and in vitro micronucleus testing (MNT; Table 4) using mammalian cells showed no signs of any observed genotoxicity for MP18279.

[0178] [Table 3]

[0179] [Table 4]

[0180] (Example 9) Antimicrobial selectivity of MP19919 and MP19594 The following examples describe the results of antimicrobial screening conducted to test whether MP19919 (SEQ ID NO: 19) and / or MP19594 (SEQ ID NO: 16) exhibit antimicrobial activity against Pythium urtimum, Rhizoctonia solanii, Sclerotinia sclerotiolum, Zymoseptria tritisi, Phytophthora cactrum, Botrytis cinerea, Microdochium nivale, Alternaria alternata, Pyrenophora tritisi repentis, and / or Fusarium graminealum, compared to a positive control commercially available antifungal agent and / or negative control peptide. Pathogen classification and importance (relevance) are listed in Table 5. MP19919 (SEQ ID NO: 19) showed selective antimicrobial activity against all tested pathogens except Sclerotinia sclerotiolum. MP19594 (SEQ ID NO: 16) showed selective antimicrobial activity only against Zymoseptria tritisi, Phytophthora cactrum, Botrytis cinerea, Microdochium nivale, and Alternaria alternata. The negative control peptide showed no antimicrobial activity against any of the tested pathogens.

[0181] Method Measurement of the maximum semi-effective concentration (EC 50 ) The peptide antimicrobial activities of MP19919 and MP19594 were tested against seven microbial strains: Zymoseptoria tritici, Phytophthora cactorum, Botrytis cinerea, Microdochium nivale, Alternaria alternata, Fusarium graminearum, and the wheat yellow spot pathogen (Pyrenophora tritici-repentis). The negative control peptide was 11 amino acids long with an N-terminal P residue and a C-terminal D residue and had no sequence identity to either MP19919 or MP19594. The peptides were tested at amounts of 300 ppm, 100 ppm, 30 ppm, and 10 ppm, respectively, each being poured directly into the growth medium in 96-well plates. A negative control without peptide (no addition of compound / peptide) and two positive controls containing the chemical fungicide Proline 275 (one at 10 mg / L and one at 50 mg / L). Two replicates were performed for each dose of each peptide and control, and one measurement per well and two wells per concentration were measured. EC 50 was calculated as the amount of peptide required to induce 50% growth control against each tested pathogen, and the calculations were provided by Fera Science Ltd. (York, United Kingdom).

[0182] Measurement of growth control The ability to control pathogen colony growth was tested for MP19919, MP19594, and the negative control peptide along with a blank negative control of agar without addition and a positive control of the commercially available fungicide Amistar®. The pathogens tested were Pythium ultimum, Rhizoctonia solani, and the sclerotinia pathogen (Sclerotinia sclerotiorum). 100 ppm (or 100 mg / L) of each tested peptide was applied to the supplemented agar, and the subsequent growth of each respective pathogen colony was measured as the diameter of the colony in millimeters. The positive control was applied at 0.5 ppm (or 0.5 mg / L), and the negative control was left untreated.

[0183] Two replicates were performed for each peptide and control, resulting in four measurements per sample.

[0184] Growth was calculated as the corresponding colony growth for each peptide relative to the colony growth of a negative control (untreated agar). This can be calculated by dividing the radial growth of the relevant colonies of the peptide by the radial growth of the relevant colonies of the negative control.

[0185] Growth control was calculated as the corresponding colony growth (mm) for each peptide relative to the colony growth (mm) for the negative control (untreated agar). This can be calculated by subtracting the radial growth (mm) of the corresponding colony for the peptide from the radial growth (mm) of the corresponding colony for the negative control, and then dividing the difference by the radial growth (mm) of the corresponding colony for the negative control. This can also be calculated by subtracting the % growth from 100% (equivalent to the % growth of the negative control).

[0186] [Table 5A]

[0187] [Table 5B]

[0188] result Peptide MP19919 is EC 50 When measured, it showed antimicrobial activity against all tested pathogens. It exhibited EC levels of less than 50 mg / L against Z. tritisi, P. cactrum, B. cinerea, and M. nivare. 50 This indicates that the EC2 levels are less than 100 mg / L for A. alternata, F. graminealum, and P. tritisi repentis. 50 This was shown (Figure 11, bars with faint dots). Peptide MP19594 exhibited selective antimicrobial activity against Z. tritisi and P. cactrum at EC levels of less than 50 mg / L. 50, as well as EC levels less than 100 mg / L for A. alternata, F. graminealum, P. tritisi repentis, B. cinerea, and M. nivare. 50 It had (Figure 11, black bar). The negative control peptide was EC 50 When measured, it did not show significant antimicrobial activity against any of the tested pathogens.

[0189] In testing for % control, peptide MP19919, calculated from the % growth shown in Figure 14 (calculated from radial growth shown in Figure 13), provided 28% and 36% control of P. urtimum and R. solani, respectively, in vitro at 100 ppm (Figure 12). MP19594 provided 13% control of P. urtimum. Neither peptide provided significant control against S. sclerothiorum, and neither the negative control peptide nor MP19594 provided significant control against any of the three tested pathogens.

[0190] Table 5 shows the pathogens against which peptide MP19919 and / or MP19594 showed antimicrobial activity (marked "active").

Claims

1. A method for increasing immunity in plants, comprising the step of supplying plants with an oligopeptide containing the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof.

2. The method according to claim 1, wherein the oligopeptide is an antimicrobial oligopeptide or an antifungal oligopeptide.

3. A method for increasing resistance in plants to plant pathogens or pests, comprising the step of supplying plants with an oligopeptide containing the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21 having one, two, three, or four amino acid substitutions, insertions, or deletions.

4. A method for inhibiting plant pathogens or pests in plants, comprising the step of supplying a plant with an oligopeptide containing the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21 having one, two, three, or four amino acid substitutions, insertions, or deletions.

5. The method according to claim 3 or 4, wherein the plant pathogen or pest is selected from fungal pathogens or pests, bacterial pathogens or pests, and viral pathogens or pests.

6. The plant pathogens or pests include Cercospora spp., Mycosphaerella spp., Glomerella spp., Cladosporium spp., Diplodia maydis, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme, Fusarium verticillioides, grass leaf spot fungus (Cochliobolus sativus), and Colletotrichum graminicola. Graminicola), Stagonospora nodorum, Stagonospora avenae, Stenocarpella maydis, Sclerotinia minor, Sclerotinia sclerotiorum, Sclerotinia sp., Alternaria spp., Phytophthora spp., Botrytis spp., Pyrenophora tritici-repentis, Phytophthora parasitica Parasitica, Phytophthora megasperma fsp. Glycinea, Macrohomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiolum, Fusarium oxysporumoxysporum), Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Peronospora manshurica, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassicola cassiicola), Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas syringae pv. Glycinea, Xanthomonas campestris pv. Phaseoli, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, soybean rust fungus (Phakopsora pachyrigis) Pythium pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Fusarium solani; Canola: Albugo candidaCandida), Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solanii, Sclerotinia sclerotiolum, Mycosphaerella brassicola, Pythium urtimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibacter michiganensis subsp. Insidiosum, Pythium urtimum, Pythium irregulare, Pythium splendens Pythium splendens), Pythium debaryanum, Pythium aphanidermatum, Phytophthora megasperma, Peronospora trifoliorum, Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Verticillium arbo atrum albo-atrum), Xanthomonas campestris pv. Alfalfa, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium alfalfaalfalfa), Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrichila medicaginis; Pseudomonas syringae pv. Atrofaciens, Urocystis agropyri, Xanthomonas campestris pv. translucence Translucens), Pseudomonas syringae pv. Syringae, Alternaria alternata, Cladosporium herbarum, Fusarium gramineum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Colletotrichum graminicola, Wheat powdery mildew fungus (Erysiphe graminis f.sp. tritici) Puccinia graminis f.sp. tritici, wheat black rust fungus (Puccinia recondite f.sp. tritici), wheat red rust fungus (Puccinia recondite f.sp. tritici)Pythium tritici), yellow rust fungus (Puccinia striiformis), wheat yellow spot fungus (Pyrenophora tritici repentis), Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Pythium aphanidermatum, Pythium algenomanes Pythium arrhenomanes, Pythium urtimum, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solanii, Pythium arrhenomannes, Pythium gramicola, Pythium aphanidermatum, Plasmopara halsteadii halstedii), Sclerotinia sclerotiolum, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldiimacdonaldii), Macrohomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, sunflower rust fungus (Puccinia helianthi), Verticillium dahlia, Erwinia carotovorum pv. Carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis tragopogonis; Maize: Colletotrichum graminicola, Fusarium verticillioides var. subglutinans, Erwinia stewartia, F. verticillioides, Gibberella zeae (Fusarium graminealum), Stenocarpella maydis (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens (Pythium Pythium splendens, Pythium urtimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T(Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III (Exserohilum turcicum I, II & III), Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polisola Polysora), Macrohomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Clavibacter michiganense subsp. Nebraskense, Trichoderma viride, Claviceps sorghi, Pseudomonas avenae avenae), Erwinia chrysanthemi pv. Zea, Erwinia carotovora, Corn stunt spiroplasmaspiroplasma), Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Peronosclerospora maydis, Peronosclerospora sacchari, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium acremonium), Exserohilum turcicum, C. C. sublineolum, Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae pv. Syringae, Xanthomonas campestris pv. Holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrohomina phaseolina, Perconia circinate, Fusarium verticilioides verticillioides), Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana) reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solanii, Acremonium strictumThe method according to claim 3 or 4, wherein the pathogen or pest is a fungal selected from Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, Fusarium graminealum, Fusarium oxysporum, Pythium arrhenomanes, and Pythium graminicola.

7. The method according to claim 3 or 4, wherein the plant pathogen or pest is a bacterial pathogen or pest selected from the genera Pseudomonas, Pantoea, and Erwinia.

8. Plant pathogens or pests include cucumber mosaic virus, tobacco mosaic virus, barley fusarium virus, alfalfa mosaic virus (Alfamovirus), apple chlorotic leaf spot virus (Trichovirus), apple rust viroid (Viroid), Arabis mosaic virus (Nepovirus), barley mild mosaic virus (Bymovirus), barley spotted leaf mosaic virus (Hordeivirus), barley yellow spotted mosaic virus (Faimovirus), and bean common mosaic virus (Bean common mosaic virus). Potyvirus (potyvirus genus), bean yellow mosaic virus (potyvirus genus), beet necrotic leaf vein yellow virus (Furovirus genus), cowpea mosaic virus (potyvirus genus), bean mosaic virus (potyvirus genus), broad bean virus (Fabavirus genus), butterbur mosaic virus (Carlavirus genus), carnation spotted virus (Carmovirus genus), carnation vein mottle virus (potyvirus genus), cauliflower mosaic virus (Caulimovirus genus), chrysanthemum spotted virus Rus (Cucumovirus), Tomato Aspermyvirus (Cucumovirus), Chrysanthemum Dwarf Viroid (Viroid), Citrus Mosaic Virus, Citrus Tristeza Virus (Closterovirus), Clover Vein Yellowing Virus (Potivirus), Cox's Foot Mottle Virus (Sobemovirus), Cucumber Green Spot Mosaic Virus (Tobamovirus), Cucumber Mosaic Virus (Cucumovirus), Cycad Necrotic Dwarf Virus (Nepovirus), Taro Mosaic Virus (Potivirus), Grapevine Algerian Latent VirusThe method according to claim 3 or 4, wherein the pathogen or pest is a virus selected from among (Tombusvirus), konjac mosaic virus (Potivirus), melon necrotic spot virus (Calmovirus), mulberry ring spot virus (Nepovirus), and narcissus mosaic virus (Potexvirus). Plant viruses are viruses that infect plants. Additional examples of plant viruses include Odontoglossum ring spot virus (Tobamovirus), papaya ring spot virus (Potivirus), peach latent mosaic viroid, peanut spot virus (Potivirus), peanut leaf spot virus (Potivirus), bean mosaic virus (Potivirus), peanut dwarf virus (Cucumovirus), potato virus A (Potivirus), potato virus M (Carlavirus), potato virus S (Carlavirus), potato virus X (Potexvirus), potato virus Y (Potivirus), and prune dwarf virus (Iralvirus). Ilarvirus, Prune necrotic ring spot virus (Ilarvirus genus), Radish leaf mosaic virus (Comovirus genus), Rice black streak dwarf virus (Fijivirus genus), Rice dwarf virus (Reovirus genus), Rice grassy stunt virus (Tenuivirus genus), Rice stripe leaf blight virus (Tenuivirus genus), Rice tunglobular spheroid virus (Sequivirus genus), Rice dwarf virus, Rice tunglobular spheroid virus (Sequivirus genus), Ryegrass spot virus, Satsuma dwarf virus (Nepovirus genus), Wheat mosaic virus (Soil-borne wheat mosaic virus)Viruses (Frovirus genus), Southern bean mosaic virus (Sobemovirus genus), Soybean mosaic virus (Potivirus genus), Soybean dwarf virus (Cukumovirus genus), Cucumber mosaic virus (Cukumovirus genus), Tobacco mosaic virus (Tobamovirus genus), Tobacco mosaic virus (Tobamovirus genus), Tobacco necrosis virus (Necrovirus genus), Tobacco stem necrosis virus (Tobravirus genus), Tobacco ring spot virus (Nepovirus genus), Tomato aspermivirus (Cukumovirus genus), Tomato black ring spot virus (Nepovirus genus), Tomato mosaic virus (Tobamovirus genus), Tomato ring spot virus (Nepovirus genus), Tomato yellow necrosis virus (Tospovirus genus), Turnip mosaic virus (Potivirus genus), Pumpkin mosaic virus 1 (Watermelon mosaic virus 1) Examples include 1) Potivirus (genus Potivirus), papaya ring spot virus (genus Potivirus), watermelon mosaic virus 2 (genus Potivirus), wheat yellow mosaic virus (genus Faimovirus), and zucchini yellow mosaic virus (genus Potivirus).

9. The method according to claim 3 or 4, wherein the plant pathogen or pest is selected from the group consisting of Septoria tritisi, Botrytis cinerea, Alternaria solani, Rhizoctonia solani anastomosis, Phytophthora capsici, and Phytophthora infestans.

10. The method according to any one of claims 1 to 9, wherein the oligopeptide is supplied by expressing a nucleic acid encoding the oligopeptide.

11. The method according to claim 10, wherein the nucleic acid is operably linked to a heterogeneous promoter.

12. The method according to claim 10 or 11, wherein the heterogeneous promoter is a constitutive promoter, a tissue-specific promoter, or an inducible promoter.

13. The method according to claim 12, wherein the inducible promoter is induced by fungal infection and is optionally a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., promoters for phenylalanine ammonia lyase and chalcone synthase), a promoter associated with a gene that modifies the plant cell wall (e.g., promoters for hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase), a promoter associated with a gene encoding an enzyme that degrades the fungal cell wall (e.g., promoters for chitinase or glucanase), or a promoter associated with a gene encoding a thaumatin-like protein, or optionally, the maize Mis1 promoter or the flax Fis1 promoter.

14. The method according to any one of claims 1 to 9, wherein the oligopeptide is supplied by application to a plant or a part thereof.

15. The method according to claim 14, wherein the part of the plant is a leaf, bud, root, shoot, flower part, or seed.

16. The method according to claim 14 or 15, wherein the oligopeptide is applied as a coating to the seeds before planting.

17. The method according to any one of claims 1 to 9, wherein the oligopeptide is supplied by application to soil in which plants are planted.

18. The method according to any one of claims 1 to 9, wherein the oligopeptide is supplied by adding it to water supplied to the plant.

19. The method according to any one of claims 1 to 9, wherein the oligopeptide is supplied to seeds, fruits and / or plant parts after harvest.

20. The method according to any one of claims 14 to 19, wherein the application is by a spreader, power duster, boom sprayer, hand sprayer, spray duster, or granular spreader.

21. The method according to any one of claims 1 to 20, wherein miPEP includes a secretion tag, a tag that promotes entry into plant cells, or a nuclear localization tag.

22. The method according to claim 21, wherein the tag that promotes entry into plant cells comprises a cell-permeable peptide linked to the amino acid sequence.

23. Cell-permeable peptides include protein transduction domains, amphiphilic peptides, synthetic cationic polypeptides, optionally polylysine, polyhistidine, or polyarginine, dendrimer-type polycationic molecules, vascular endothelial-cadherin peptide cell-permeable peptides, transportan cell-permeable peptides, monomers or dimers of the HIV-1 TAT basic domain cell-permeable peptide, penetratin cell-permeable peptide, synthetic cationic homoarginine oligopeptide cell-permeable peptide, gamma-zein cell-permeable peptide, maize (Zea mays) knotted 1 cell-permeable peptide, Saccharomyces pombe TP10 cell-permeable peptide, Candida albicans Zebra cell-permeable peptide, Antennapedia sequence, TAT sequence, Antp-3A (Antp variant) sequence, Buforin The method according to claim 22, comprising a II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynB1 sequence, a Pep-7 sequence, an HN-1 sequence, a BGSC (Bis-Guanidinium-Spermidine-Cholesterol) sequence, or a BGTC (Bis-Guanidinium-Tren-Cholesterol) sequence.

24. An isolated oligopeptide containing the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, or the amino acid sequence shown in one of SEQ ID NOs: 1-5 and 14-21, including one, two, three, or four amino acid substitutions, insertions, or deletions.

25. The oligopeptide according to claim 24, which is an antimicrobial oligopeptide or an antifungal oligopeptide.

26. The oligopeptide according to claim 24 or 25, comprising one, two, three, or four amino acid substitutions, insertions, or deletions.

27. The oligopeptide according to any one of claims 24 to 26, comprising proline at the N-terminus, aspartic acid at the C-terminus, or both.

28. An oligopeptide according to any one of claims 24 to 27, comprising one or more D-amino acids.

29. The oligopeptide according to claim 28, wherein one or more D-amino acids enhance the stability of the oligopeptide compared to an oligopeptide having the same amino acid sequence containing only L-amino acids.

30. The oligopeptide according to any one of claims 24 to 29, comprising a secretion tag, a tag that promotes entry into plant cells, or a nuclear localization tag.

31. The oligopeptide according to claim 30, wherein the tag that promotes entry into plant cells comprises a cell-permeable peptide linked to the amino acid sequence.

32. Cell-permeable peptides include protein transduction domains, amphiphilic peptides, synthetic cationic polypeptides, optionally polylysine, polyhistidine, or polyarginine, dendrimer-type polycationic molecules, vascular endothelial-cadherin peptide cell-permeable peptides, transportan cell-permeable peptides, monomers or dimers of the HIV-1 TAT basic domain cell-permeable peptide, penetratin cell-permeable peptide, synthetic cationic homoarginine oligopeptide cell-permeable peptide, gamma-zein cell-permeable peptide, maize knotted 1 cell-permeable peptide, Saccharomyces pombe TP10 cell-permeable peptide, Candida albicans zebra cell-permeable peptide, Antennapedia sequence, TAT sequence, Antp-3A (Antp variant) sequence, Buforin The oligopeptide according to claim 31, comprising a II sequence, a K-FGF sequence, a Ku70 sequence, a prion sequence, a pVEC sequence, a SynB1 sequence, a Pep-7 sequence, an HN-1 sequence, a BGSC (bis-guanidinium-spermidine-cholesterol) sequence, or a BGTC (bis-guanidinium-tren-cholesterol) sequence.

33. A composition comprising an oligopeptide according to any one of claims 24 to 32, together with an agriculturally acceptable formulation agent which may include water, an organic solvent, paraffinic oil, vegetable oil, a dispersant, an emulsifier, a wetting agent, a buffer, a hydrotrope, a rheology modifier, an antifoaming agent and an antifoaming agent, an antifreeze, a biocide, a dye, a polymer wall, a catalyst, a thermosetting material, a crosslinking polymerizer, a UV protective agent, an antioxidant, and a chelating agent.

34. A composition comprising the oligopeptide according to any one of claims 24 to 32, together with an agriculturally acceptable carrier, diluent, or excipient.

35. The composition according to claim 34, wherein the agriculturally acceptable carrier, diluent, or excipient comprises a buffer having a pH in the range of about 3.0 to about 9.0, or about 4.5 to about 8.

0.

36. The composition according to any one of claims 33 to 35, wherein the oligopeptide is in a concentration range of about 0.1 μg / ml to about 100 mg / ml, or about 5 μg / ml to about 5 mg / ml.

37. The composition according to any one of claims 33 to 36, taking the form of a fine particulate solid, granules, pellets, hydrated powder, fine powder, aqueous suspension, dispersion, gel, or emulsion.

38. The composition according to any one of claims 33 to 37, further comprising one or more other active agents selected from the group consisting of pesticides, fertilizers, insecticides, attractants, sterilizers, mite control agents, anthelmintic agents, herbicides, biostimulants, biological preparations, and growth regulators.

39. A nucleic acid encoding an oligopeptide according to any one of claims 24 to 32.

40. A nucleic acid construct comprising the nucleic acid according to claim 39, operably coupled to a promoter.

41. The nucleic acid construct according to claim 40, wherein the promoter is a heterogeneous promoter.

42. The nucleic acid construct according to claim 40 or 41, wherein the promoter is a constitutive promoter, a tissue-specific promoter, a developmental stage-specific promoter, or an inducible promoter.

43. The nucleic acid construct according to claim 42, wherein the inducible promoter is induced by fungal infection and optionally is a promoter associated with a gene involved in phenylpropanoid metabolism (e.g., promoters for phenylalanine ammonia lyase and chalcone synthase), a promoter associated with a gene that modifies the plant cell wall (e.g., promoters for hydroxyproline-rich glycoprotein, glycine-rich protein, and peroxidase), a promoter associated with a gene encoding an enzyme that degrades the fungal cell wall (e.g., promoters for chitinase or glucanase), or a promoter associated with a gene encoding a thaumatin-like protein, or a maize Mis1 promoter or flax Fis1 promoter.

44. A cell comprising the nucleic acid described in claim 39 or the nucleic acid construct described in any one of claims 40 to 43.

45. The method according to any one of claims 1 to 23, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 19 or SEQ ID NO: 16, or the oligopeptide comprises the amino acid sequence shown in one of SEQ ID NO: 19 or SEQ ID NO: 16 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof; the oligopeptide according to any one of claims 25 to 32; the composition according to any one of claims 33 to 38; the nucleic acid construct according to any one of claims 39 to 43; or the cell according to claim 44.

46. The method according to any one of claims 2 to 23 and 45, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 19, or the amino acid sequence shown in SEQ ID NO: 19 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and the plant pathogen or pest is selected from Pythium urtimum, Rhizoctonia solani, Zymoseptoria tritici, Phytophthora cactorum, Botrytis cinerea, Microdochium nivale, Alternaria alternata, Pyrenophora tritici repentis, and Fusarium gramineum.

47. The method according to any one of claims 2 to 23 and 45, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 16, or the amino acid sequence shown in SEQ ID NO: 16 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and the plant pathogen or pest is selected from Zymoseptria tritisi, Phytophthora cactrum, Botrytis cinerea, Microdokium nivale, and Alternaria alternata.

48. The method according to any one of claims 2 to 23 and 45, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 19, or the amino acid sequence shown in SEQ ID NO: 19 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and the plant pathogen or pest is selected from Pythium urtimum, Rhizoctonia solanii, Zymoseptria tritisi, Botrytis cinerea, Alternaria alternata, Pyrenophora tritisi repentis, and Fusarium graminealum.

49. The method according to any one of claims 2 to 23 and 45, wherein the oligopeptide comprises the amino acid sequence shown in SEQ ID NO: 16, or the amino acid sequence shown in SEQ ID NO: 16 having one, two, three, or four amino acid substitutions, insertions, or deletions thereof, and the plant pathogen or pest is selected from Zymoseptria tritisi, Botrytis cinerea, and Alternaria alternata.

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