WO2020262910A1 - Composition pour prévenir une maladie chez les plantes en utilisant une souche de brevibacillus brevis hk544 - Google Patents

Composition pour prévenir une maladie chez les plantes en utilisant une souche de brevibacillus brevis hk544 Download PDF

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WO2020262910A1
WO2020262910A1 PCT/KR2020/008127 KR2020008127W WO2020262910A1 WO 2020262910 A1 WO2020262910 A1 WO 2020262910A1 KR 2020008127 W KR2020008127 W KR 2020008127W WO 2020262910 A1 WO2020262910 A1 WO 2020262910A1
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cause
disease
strain
composition
plant diseases
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Korean (ko)
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김헌
최경자
한재우
최용호
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한국화학연구원
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention comprises any one or more selected from the group consisting of Brevibacillus brevis HK544 strain, the culture medium of the strain, the culture filtrate of the strain, the fraction of the culture filtrate, or edeine B1 It is to provide a composition for controlling plant diseases and a method for controlling plant diseases using the same.
  • Brevibacillus ( Brevibacillus spp.) is a rod-shaped Gram-positive bacteria that was reclassified into the genus Brevibacillus from Bacillus brevis cluster in 1996 through phylogenetic analysis based on 16S rRNA gene sequence. .
  • antimicrobial peptides eg, tostadin, edeine, spergualin, tauramamide, brevistin, Bac-GM100, laterosporulin, etc.
  • clinical antibiotics linear peptide gramicidin A and cyclic peptide gramicidin S. laterosporulin 10
  • Brevibacillus spp. is being used as a major material in research to search for new antibiotics.
  • tomato plants were treated with Fusarium oxysporum f. sp. lycopersici and Brevibacillus brevis strains simultaneously on tomato plants, the development of tomato wilt disease was greatly reduced, and the effect of promoting plant growth was found. Yes (J Phytopathol 2010, 158: 470-478).
  • Brevibacillus brevis strain and the antibiotic gramicidin S produced by this strain showed excellent activity in inhibiting spore germination and mycelial growth of Botrytis cinerea , but plants No disease control effect was suggested (Journal of Applied Microbiology 2001, 91, 652-659).
  • Brevibacillus brevis FJAT-0809-GLX strain is a strain that produces ethylparaben, an antibacterial active substance, and the culture filtrate containing ethylparaben was treated with plants like Lasiodiplodia theobromae . It has been reported that the incidence of plant diseases in apple trees was greatly reduced. The above-mentioned antimicrobial activity effects do not appear in all Brevibacillus spp., but exhibit different antimicrobial activity effects according to the characteristics of the Brevibacillus spp. strain.
  • Brevibacillus produces various kinds of peptide antibiotics with antimicrobial activity, so it can be actively used to develop new crop protection agents.
  • the present inventors discovered and identified a strain of Brevibacillus brevis HK544 exhibiting a control effect against major plant diseases, and completed the present invention by identifying a substance exhibiting antibacterial activity against plant pathogens from the culture medium.
  • An object of the present invention is to provide a Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain that exhibits plant disease control activity deposited with accession number KACC 81093BP.
  • Another object of the present invention is to provide a composition for controlling plant diseases containing as an active ingredient any one or more selected from the group consisting of the culture medium of the strain and the culture filtrate of the strain.
  • Another object of the present invention is to provide a composition for controlling plant diseases containing strign B1 as an active ingredient.
  • Another object of the present invention is a composition for controlling plant diseases containing as an active ingredient any one or more selected from the group consisting of the Brevibacillus brevis HK544 strain, the culture medium of the strain, and the culture filtrate of the strain. It is to provide a plant disease control method comprising the step of treating a plant disease control composition containing phosphorus B1 as an active ingredient on plants or soil in which plants are grown.
  • the object of the present invention is to provide a Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain that exhibits plant disease control activity deposited with the accession number KACC 81093BP.
  • the present invention provides a composition for controlling plant diseases containing as an active ingredient any one or more selected from the group consisting of the culture medium of the strain and the culture filtrate of the strain.
  • the present invention provides a plant disease control agent composition containing strign B1 as an active ingredient.
  • the present invention is to control plant diseases containing one or more selected from the group consisting of Brevibacillus brevis HK544 strain, the culture medium of the strain, and the culture filtrate of the strain as an active ingredient. It provides a plant disease control method comprising the step of treating a plant or plant disease control composition containing sacredn B1 as an active ingredient in a plant or soil for cultivating plants.
  • composition for controlling plant diseases using the Brevibacillus brevis HK544 strain according to the present invention exhibits excellent control effects against various plant diseases such as tomato gray mold disease, tomato blight, wheat red rust, pepper anthrax, etc. It can be usefully used for disease control purposes.
  • FIG. 1 is a diagram showing the colonies of the Brevibacillus brevis HK544 strain cultured in a tryptic soy agar medium.
  • Figure 2 is a diagram showing a phylogenetic tree created by comparing the Brevibacillus brevis HK544 strain with a related species based on the 16S rRNA gene sequence.
  • Figure 3 is a photograph confirming the control effect of red pepper anthrax disease of Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain culture medium and culture filtrate.
  • Figure 4 is a diagram showing the process of separating the antimicrobial active substance ideine B1 (edeine B1) from the Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain.
  • 5 is a diagram showing the results of ESI-MS cation analysis of edeine B1.
  • FIG. 6 is a diagram showing the chemical structure of edeine B1.
  • control includes not only the prevention and avoidance of diseases or pests, but also removal and death.
  • the present invention provides a Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain that exhibits plant disease control activity deposited with the accession number KACC 81093BP.
  • the strain may have the nucleotide sequence of SEQ ID NO: 1.
  • the strain may be isolated from the soil, and specifically, may be isolated from the soil of the Korea Research Institute of Chemical Technology.
  • the strain may have plant disease control activity.
  • the plant diseases include rice blast disease (cause: Magnaporthe oryzae ), tomato blight (cause: Phytophthora infestans ), wheat red rust (cause: Puccinia triticina ), barley powdery mildew (cause: Blumeria graminis f. sp. hordei ), pepper anthrax (cause bacterium) : Colletotrichum coccodes ), tomato gray mold disease (cause : Botrytis cinerea ), cabbage black blotches (cause : Alternaria brassicicola ), phalaenopsis bacterial brown spot blotch disease (cause: Acidovorax avenae subsp.
  • cattleyae peach bacterial pore disease (cause: Xanthomonas arboricola) .
  • pv pruni peach bacterial pore disease
  • kiwi gweyangbyeong pathogen:. Pseudomonas syringae pv actinidiae
  • fruit trees fire blight fruit trees fire blight (causative organisms: be Erwinia amylovora), but not limited to these.
  • the present invention provides a composition for controlling plant diseases containing as an active ingredient any one or more selected from the group consisting of the culture solution of the strain and the culture filtrate of the strain.
  • the strain is Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain deposited under the accession number KACC 81093BP and may have the characteristics as described above.
  • the culture solution may be obtained by culturing the strain, and the culture filtrate may be obtained by physically filtering the culture solution obtained by culturing the strain.
  • the plant diseases include rice blast disease (cause: Magnaporthe oryzae ), tomato blight (cause: Phytophthora infestans ), wheat red rust (cause: Puccinia triticina ), barley powdery mildew (cause: Blumeria graminis f. sp. hordei ), pepper anthrax (cause bacterium) : Colletotrichum coccodes ), tomato gray mold disease (cause : Botrytis cinerea ), cabbage black blotches (cause : Alternaria brassicicola ), phalaenopsis bacterial brown spot blotch disease (cause: Acidovorax avenae subsp.
  • cattleyae peach bacterial pore disease (cause: Xanthomonas arboricola) .
  • pv pruni peach bacterial pore disease
  • kiwi gweyangbyeong pathogen:. Pseudomonas syringae pv actinidiae
  • fruit trees fire blight fruit trees fire blight (causative organisms: be Erwinia amylovora), but not limited to these.
  • the plant may be any one or more selected from the group consisting of rice, tomato, barley, wheat, pepper, phalaenopsis, peach, kiwi, Chinese cabbage, apple tree, and pear tree.
  • the composition for controlling plant diseases according to the present invention may be a simple mixture of an active substance isolated from a Brevibacillus brevis HK544 strain, a culture solution thereof, or a culture filtrate thereof.
  • the composition for controlling plant diseases is a mixture of the culture medium or the active substance separated from the culture filtrate and an inert carrier, and the mixture is an emulsion, an emulsion, a fluidizing agent, a wettable powder, a granulated wettable powder, a powder, It is prepared by adding a surfactant and other necessary auxiliary agents to the mixture so that it can be formulated into granules or the like.
  • the above-mentioned composition for controlling plant diseases may be used as a seed treatment agent by itself or by adding other inactive ingredients.
  • liquid carriers examples include water; Alcohols such as methanol and ethanol; Ketones such as acetone and methyl ethyl ketone; Aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene and methyltaphthalene; Aliphatic hydrocarbons such as hexane, cyclohexane, kerosene and light oil; Esters such as ethyl acetate and butyl acetate; Nitriles such as acetonitrile and isobutyrnitrile; Ethers such as diisopropylether and dioxane; Acid amides such as N,N-dimethyl form amide and N,N-dimethylacetamide; Halogenated hydrocarbons such as dichloromethane, trichloroethane and carbon tetrachloride; Dimethyl sulfoxide; And vegetable oils such as soybean oil and cottonseed
  • solid carriers examples include fine powders or granules such as minerals such as kaolin clay, attapulgite clay, bentonite, montmorillonite, acid white clay, pilophyllite, talc, diatomaceous earth and talcite; Natural organic substances such as corn leaf stalk powder and walnut husk powder; Synthetic organic materials such as urea; Salts such as calcium carbonate and ammonium sulfate; Synthetic inorganic materials such as synthetic hydrated silicon oxide; As the liquid carrier, aromatic hydrocarbons such as xylene, alkylbenzene and methylnaphthalene; Alcohols such as 2-propanol, ethylene glycol, propylene glycol and ethylene glycol monoethyl ether; Ketones such as acetone, cyclohexanone and isophorone; Vegetable oils such as soybean oil and cottonseed oil; Petroleum aliphatic hydrocarbons, esters, dimethylsulfoxide, acetonit
  • surfactants include anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalene sulfonate form. Aldehyde polycondensate; And nonionic surfactants such as polyoxyethylene alkyl aryl ethers, polyoxyethylene alkylpolyoxypropylene block copolymers and sorbitan fatty acid esters and cationic surfactants such as alkyltrimethylammonium salts.
  • anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalene s
  • formulation aids examples include water-soluble polymers such as polyvinyl alcohol and polyvinylpyrrolidone, polysaccharides such as gum arabic, algylic acid and salts thereof, CMC (carboxymethyl-cellulose), xanthan rubber, inorganic substances such as aluminum magnesium silicate and alumina sol. (alumina sol), preservatives, colorants and stabilizers such as PAP (acid phosphate isopropyl) and BHT (butylhydroxytoluene).
  • water-soluble polymers such as polyvinyl alcohol and polyvinylpyrrolidone
  • polysaccharides such as gum arabic, algylic acid and salts thereof
  • CMC carboxymethyl-cellulose
  • xanthan rubber examples include aluminum magnesium silicate and alumina sol. (alumina sol), preservatives, colorants and stabilizers such as PAP (acid phosphate isopropyl) and BHT (butylhydroxytoluene).
  • PAP acid phosphat
  • the present invention provides a step of treating a plant, a seed thereof, or a habitat thereof with any one or more selected from the group consisting of the Brevibacillus brevis HK544 strain deposited with the accession number KACC 81093BP, a culture solution thereof, and a culture filtrate thereof. It provides a method for controlling plant diseases, characterized in that it comprises.
  • the treatment may be an indirect spraying of spraying the composition directly on the plant, spraying it on the soil in which the plant is growing, or spraying the cultivation medium of the plant.
  • the control method of the present invention includes treatment of stems and leaves of plants, treatment of places where plants grow (eg soil), treatment of seeds such as seed sterilization/seed coating, and treatment of roots.
  • Treatment of the stems and leaves in the control method of the present invention may include, in particular, application on the plant surface, such as spraying the stems and leaves.
  • the treatment of the soil includes, for example, spraying on the soil, mixing with the soil, and spraying the liquid treatment system into the soil (irrigation of the liquid treatment system, injection into the soil, dripping of the liquid treatment agent).
  • places to be treated are planting holes, furrows, around planting furrows, around planting furrows, the entire surface of the growth site, between soil and plants, between roots, and under the stems of plants. It includes the part, main furrow, growing soil, pond seat, seedling box, seedling tray, and seedling.
  • the treatment may be performed before spraying, during spraying, immediately after spraying, during a simulated cultivation period, before cultivation settling, cultivation settling, and at a growing period after cultivation settling.
  • the active ingredient may be applied to the plants at the same time, or a solid fertilizer such as a paste fertilizer containing the active ingredient may be applied to the soil.
  • the active ingredient can be mixed in an irrigation liquid, for example injected into an irrigation system (irrigation tube, irrigation pipe, sprinkler, etc.), mixed in a liquid overflowing between furrows, or mixed in a water culture medium. I can.
  • the irrigation liquid and active ingredients can be premixed and used for treatment by suitable irrigation methods, including, for example, irrigation methods mentioned above and other methods such as sprinkling and flooding.
  • the volatilization treatment method according to the control method of the present invention for example, the volatilization of the sprayed composition by spraying treatment on media such as soil for cultivating plants with the composition for controlling plant diseases of the present invention, hydroponic medium for culturing plants, and bed It is a method of protecting the plant from pests and diseases, and in addition, the composition may be placed around the plant to expose the plant to the volatilized gaseous composition.
  • the seed treatment method in the control method of the present invention is, for example, a method of treating seeds so as to be protected from pests with the composition for controlling plant diseases of the present invention, and a specific example thereof is a suspension of the composition for controlling plant diseases of the present invention in advance.
  • a spray treatment method of securing and spraying on the seed surface Spray treatment method of applying the wettable powder, emulsion, fluidizing agent, etc. of the composition for controlling plant diseases of the present invention on the seed surface by itself or by adding a small amount of water;
  • the amount of treatment may vary depending on the type of plant to be treated, the type and frequency of occurrence of pests to be controlled, the form of the formulation, the treatment period, and climatic conditions.
  • Emulsions, wettable powders, fluidizing agents, etc. are usually diluted with water and then sprayed for treatment.
  • the concentration of the active ingredient is usually in the range of 0.0001 to 3% by weight, preferably 0.0005 to 1% by weight.
  • Powders, granules, etc. are usually used for treatment without dilution.
  • control method of the present invention can be used in arable or uncultivated areas such as rice fields.
  • the plant diseases include rice blast disease (cause: Magnaporthe oryzae ), tomato blight (cause: Phytophthora infestans ), wheat red rust (cause: Puccinia triticina ), barley powdery mildew (cause: Blumeria graminis f. sp. hordei ), pepper anthrax (cause bacterium) : Colletotrichum coccodes ), tomato gray mold disease (cause : Botrytis cinerea ), cabbage black blotches (cause : Alternaria brassicicola ), phalaenopsis bacterial brown spot blotch disease (cause: Acidovorax avenae subsp.
  • cattleyae peach bacterial pore disease (cause: Xanthomonas arboricola) .
  • pv pruni peach bacterial pore disease
  • kiwi gweyangbyeong pathogen:. Pseudomonas syringae pv actinidiae
  • fruit trees fire blight fruit trees fire blight (causative organisms: be Erwinia amylovora), but not limited to these.
  • the plant may be any one or more selected from the group consisting of rice, tomato, barley, wheat, pepper, phalaenopsis, peach, kiwi, Chinese cabbage, apple tree, and pear tree.
  • the present inventors identified the HK544 strain isolated from the soil of the experimental pavement at the Korea Research Institute of Chemical Technology and deposited it as a patent strain (accession number: KACC81093BP) to the Agricultural Genetic Resource Center of the Rural Development Administration (see Fig. 1).
  • the present inventors treated the culture filtrate of the Brevibacillus brevis HK544 strain of the present invention on plants, and then examined the growth inhibitory activity against phytopathogenic fungi and bacteria, as a result of investigating the growth inhibitory activity against phytopathogenic fungi and bacteria, Chinese cabbage black blotch ( Alternaria brassicicola ). , Tomato gray mold ( Botrytis cinerea ), Capsicum anthrax ( Coletotrichum coccodes ), Phalaenopsis bacterial brown spot ( Acidovorax avenae subsp.
  • the present inventors treated the plant with the culture solution or culture filtrate of the strain, and then rice blast disease (pathogen: Magnaporthe oryzae ), tomato gray mold disease (pathogen: Botrytis cinerea ), tomato blight (pathogen: Phytophthora infestans ), wheat red rust (Pathogen: Puccinia triticina ), barley powdery mildew (pathogen: Blumeria graminis f. sp.
  • rice blast disease pathogen: Magnaporthe oryzae
  • tomato gray mold disease pathogen: Botrytis cinerea
  • tomato blight pathogen: Phytophthora infestans
  • wheat red rust Pathogen: Puccinia triticina
  • barley powdery mildew pathogen: Blumeria graminis f. sp.
  • the culture medium or culture filtrate of the strain of the present invention is tomato It was confirmed that it had a control effect on gray mold disease, tomato blight, wheat rust and pepper anthrax (see Table 2).
  • the present invention provides a composition for controlling plant diseases containing a compound represented by the following formula (1) and a stereoisomer thereof as an active ingredient.
  • the chemical is edeine B1.
  • the compound may be isolated from the Brevibacillus brevis HK544 strain, but is not limited thereto.
  • the plant diseases include rice blast disease (cause: Magnaporthe oryzae ), tomato blight (cause: Phytophthora infestans ), wheat red rust (cause: Puccinia triticina ), barley powdery mildew (cause: Blumeria graminis f. sp. hordei ), pepper anthrax (cause bacterium) : Colletotrichum coccodes ), tomato gray mold disease (cause : Botrytis cinerea ), cabbage black blotchy disease (cause : Alternaria brassicicola ), phalaenopsis bacterial brown spot disease (cause: Acidovorax avenae subsp.
  • cattleyae peach bacterial pore disease (cause: Xanthomonas arboricola) .
  • pv pruni peach bacterial pore disease
  • kiwi gweyangbyeong pathogen:. Pseudomonas syringae pv actinidiae
  • fruit trees fire blight fruit trees fire blight (causative organisms: be Erwinia amylovora), but not limited to these.
  • the plant may be any one or more selected from the group consisting of rice, tomato, barley, wheat, pepper, phalaenopsis, peach, kiwi, Chinese cabbage, apple tree, and pear tree.
  • the composition may have plant disease control activity.
  • the compound may be used in the form of an agrochemically acceptable salt, and may include all salts, hydrates, and solvates that may be prepared by conventional methods, as well as agrochemically acceptable salts.
  • the present invention provides a method for controlling plant diseases using a composition containing the compound as an active ingredient.
  • the treatment may be an indirect spraying of spraying the composition directly on the plant, spraying it on the soil in which the plant is growing, or spraying the cultivation medium of the plant.
  • the control method of the present invention includes treatment of stems and leaves of plants, treatment of places where plants grow (eg soil), treatment of seeds such as seed sterilization/seed coating, and treatment of roots.
  • Treatment of the stems and leaves in the control method of the present invention may include, in particular, application on the plant surface, such as spraying the stems and leaves.
  • the treatment of the soil includes, for example, spraying on the soil, mixing with the soil, and spraying the liquid treatment system into the soil (irrigation of the liquid treatment system, injection into the soil, dripping of the liquid treatment agent).
  • places to be treated are planting holes, furrows, around planting furrows, around planting furrows, the entire surface of the growth site, between soil and plants, between roots, and under the stems of plants. It includes the part, main furrow, growing soil, pond seat, seedling box, seedling tray, and seedling.
  • the treatment may be performed before spraying, during spraying, immediately after spraying, during a simulated cultivation period, before cultivation settling, cultivation settling, and at a growing period after cultivation settling.
  • the active ingredient may be applied to the plants at the same time, or a solid fertilizer such as a paste fertilizer containing the active ingredient may be applied to the soil.
  • the active ingredient can be mixed in an irrigation liquid, for example injected into an irrigation system (irrigation tube, irrigation pipe, sprinkler, etc.), mixed in a liquid overflowing between furrows, or mixed in a water culture medium. I can.
  • the irrigation liquid and active ingredients can be premixed and used for treatment by suitable irrigation methods, including, for example, irrigation methods mentioned above and other methods such as sprinkling and flooding.
  • the volatilization treatment method according to the control method of the present invention for example, the volatilization of the sprayed composition by spraying treatment on media such as soil for cultivating plants with the composition for controlling plant diseases of the present invention, hydroponic medium for culturing plants, and bed It is a method of protecting the plant from pests and diseases, and in addition, the composition may be placed around the plant to expose the plant to the volatilized gaseous composition.
  • the seed treatment method in the control method of the present invention is, for example, a method of treating seeds so as to be protected from diseases and pests with the composition for controlling plant diseases of the present invention, and a specific example thereof is a suspension of the composition for controlling plant diseases of the present invention Spray treatment method of spraying on the surface; Spray treatment method of applying the wettable powder, emulsion, fluidizing agent, etc. of the composition for controlling plant diseases of the present invention on the seed surface by itself or by adding a small amount of water; An impregnation treatment method of impregnating seeds into a solution of the composition for controlling plant diseases of the present invention for a specific period of time; It includes a film coating treatment method and a pellet coating treatment method.
  • the amount of treatment may vary depending on the type of plant to be treated, the type and frequency of occurrence of pests to be controlled, the form of the formulation, the treatment period, and climatic conditions.
  • Emulsions, wettable powders, fluidizing agents, etc. are usually diluted with water and then sprayed for treatment.
  • the concentration of the active ingredient is usually in the range of 0.0001 to 3% by weight, preferably 0.0005 to 1% by weight.
  • Powders, granules, etc. are usually used for treatment without dilution.
  • control method of the present invention can be used in arable or uncultivated areas such as rice fields.
  • the present inventors isolated ideine B1 represented by Formula 1 from a fraction obtained by sequentially fractionating the culture filtrate of Brevibacillus brevis HK544 strain with butanol and acetone. (See Figs. 4 and 5).
  • the isolated edeine B1 is a phytopathogenic fungus cabbage black pattern pathogen ( Alternaria brassicicola ), tomato gray mold pathogen ( Botrytis cinerea ), pepper anthrax bacteria ( Coletotrichum coccodes ), rice blast fungus ( Magnaporthe oryzae ), tomato blight ( Phytophthora infestans ), and plant pathogenic bacteria, Erwinia amylovora , were confirmed to have excellent bactericidal activity (see Table 4).
  • edeine B1 is a phytopathogenic fungus cabbage black pattern pathogen ( Alternaria brassicicola ), tomato gray mold pathogen ( Botrytis cinerea ), pepper anthrax bacteria ( Coletotrichum coccodes ), rice blast fungus ( Magnaporthe oryzae ), tomato blight ( Phytophthora infestans ), and plant pathogenic bacteria, Erwinia amylovora , were confirmed to have excellent bactericidal activity (see Table 4).
  • HK544 a soil sample was collected from the soil of the experimental pavement at the Korea Research Institute of Chemical Technology and mixed with water. After filtering the debris using a 25 ⁇ m sieve, take 0.1 ml of the suspension and take 1/10 strength tryptic soy agar; 0.17% tryptone, 0.03% soytone, 0.025% dextrose, 0.05% NaCl, 0.25% K 2 HPO 4 , 1.5% agar) and then incubated for 3 days in a 25 °C incubator. The isolated bacteria obtained a single colony through passage 3 times, and was named as HK544 strain. Thereafter, until use in the experiment, the HK544 strain was immersed in a sterile 20% glycerol aqueous solution and stored at -80°.
  • HK544 strain used in the present invention is an ivory slime form when cultured in TSA (tryptic soy agar; 1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.5% NaCl, 2.5% K 2 HPO 4 , 1.5% agar) medium To form a colony of (Fig. 1).
  • TSA tryptic soy agar
  • tryptone 0.3% soytone
  • dextrose 0.5% NaCl
  • K 2 HPO 4 1.5% agar
  • Fig. 1 Molecular phylogenetic analysis was performed based on the 16S rRNA gene sequence of the HK544 strain.
  • the HK544 strain was inoculated into TSA medium and cultured with shaking at 150 rpm for 2 days at 25°, using CTAB buffer, and then using a DNA extraction method to obtain DNA (100 ng/ ⁇ l) of the KH544 strain.
  • the 16S rRNA gene sequence of SEQ ID NO: 1 was obtained (SEQ ID NO: 1), and the 16S rRNA gene sequence of other strains registered in the NCBI BLASTn (http://www.ncbi. nlm.nih.gov) database was obtained. And comparative analysis. After sorting the nucleotide sequence with Clustal W using the MEGA 4.0 program, the similarity between genes was created as a neighbor-joining tree (Saitou and Nei, 1987). Molecular tree diagram was constructed using the neighbor-joining method, one of the distance matrix methods, and Bootstrap analysis (1,000 times) was performed using the algorithm of the MEGA program.
  • the HK544 strain formed the same group as the Brevibacillus brevis DSM 5760 strain (FIG. 2 ), and showed high homology (99.9% or more). Based on these results, the HK544 strain was identified as Brevibacillus brevis , and deposited as a patent strain (accession number: KACC81093BP) at the Agricultural Genetic Resource Center of the Rural Development Administration.
  • Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain was cultured in TSA medium (1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.5% NaCl, 2.5% K 2 HPO 4 , 1.5% agar), and then on TSA agar medium.
  • TSB medium sterilized TSB medium (tryptic soy broth; tryptic soy agar; 1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.5% NaCl, 2.5% K 2 HPO 4 ) by sealing the formed colonies in the face -Inoculation into a triangular flask and shaking culture (150 rpm) at 25 ⁇ C for 2 days to prepare a pre-culture solution.
  • the pre-culture was inoculated with 1% in a 2 L-triangle flask containing 400 ml of TSB medium, and cultured with shaking (150 rpm) at 25°C for 3 days after inoculation to prepare a HK544 culture solution.
  • Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain culture solution was centrifuged (10,000 ⁇ g, 20 min) and filtered through a filter paper (Whatman No. 1) to prepare a culture filtrate.
  • Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain culture solution or culture filtrate diluted in distilled water at 10%, 20%, 30% concentration was used for the plant disease control test.
  • Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain culture filtrate prepared according to Example 3 was used as a phytopathogenic fungus, such as cabbage black blotches ( Alternaria brassicicola ), tomato gray mold ( Botrytis cinerea ), capsicum anthrax ( Coletotrichum coccodes ). , Magnaporthe oryzae , Phytophthora infestans , and Phytophthora infestans and Phytophthora infestans , which are plant pathogenic bacteria, Acidovorax avenae subsp.
  • MIC minimum inhibitory concentration
  • MIC minimum inhibitory concentration
  • the culture filtrate of the HK544 strain in a complete inhibition of the 1.3% black pattern germs cabbage at a concentration (Alternaria brassicicola), tomato gray mold pathogen (Botrytis cinerea), pepper anthracnose fungus (Colletotrichum coccodes) It showed in vitro bactericidal activity.
  • the culture filtrate of the HK544 strain at a concentration of 10-20% showed the activity of completely inhibiting the growth of three kinds of phytopathogenic bacteria ( A. avenae subsp. cattleyae , X. arboricola pv. pruni , P. syringae pv. actinidiae ).
  • A. avenae subsp. cattleyae X. arboricola pv. pruni
  • P. syringae pv. actinidiae Done.
  • Rice blast disease (pathogen: Magnaporthe oryzae ), tomato gray mold disease (pathogen:), which are six major plant diseases, targeting the culture medium, culture filtrate, and dilutions thereof of the Brevibacillus brevis HK544 strain prepared in Example 3 above.
  • Botrytis cinerea tomato blight (pathogen: Phytophthora infestans ), wheat red rust (pathogen: Puccinia triticina ), barley powdery mildew (pathogen: Blumeria graminis f. sp. hordei ), capsicum anthrax (pathogen: Colletotrichum coccodes ). The effect was confirmed in greenhouse conditions.
  • Tween 20 0.025% (w/v) Tween 20 was added to the sample as an electrodepositing agent, and at this time, 0.025% (w/v) Tween 20 aqueous solution was used as an untreated group. Samples were spray-treated on rice, tomato, wheat, barley and pepper seedlings, and each pathogen was inoculated after 1 day to investigate the plant disease control activity of the culture solution or culture filtrate.
  • Tomato gray mold disease and tomato blight were examined 3 days after inoculation
  • rice blast disease 5 days after inoculation wheat red rust and barley powdery mildew were examined 7 days after inoculation.
  • the disease was onset in a humid room for 2 days, and then left in a constant temperature room at 25°C for 1 day, and 3 days after inoculation, the lesion area ratio (%) was investigated, and the following formula was substituted for control ( %) was obtained.
  • Control (%) [1-(Round area ratio of treated area / Area ratio of untreated area)] ⁇ 100
  • the culture medium or culture filtrate of the HK544 strain showed excellent control activity in controlling more than 90% of tomato gray mold and tomato blight even at a concentration of 10%, and against wheat red rust and pepper anthrax. It showed control activity.
  • the culture medium or culture filtrate of the HK544 strain exhibited an effect of controlling more than 80% of pepper anthrax at a concentration of 30%, which was comparable to the effect of treating the synthetic pesticide dithianon at a concentration of 50 ⁇ g/ml (Fig. 3 ).
  • the culture medium and the culture filtrate of the HK544 strain showed the effect of controlling 80% and 53% of wheat red rust at 30% concentration, respectively.
  • Brevibacillus brevis Brevibacillus brevis HK544 strain was cultured in TSA medium (1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.5% NaCl, 2.5% K 2 HPO 4 , 1.5% agar), and then formed in TSA medium.
  • TSA medium 1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.5% NaCl, 2.5% K 2 HPO 4 , 1.5% agar
  • 1 L triangle containing 200 ml of TSB medium (tryptic soy broth; tryptic soy agar; 1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.5% NaCl, 2.5% K 2 HPO 4 )
  • the flask was inoculated and shaken at 25°C for 2 days (150 rpm) to prepare a preculture.
  • the pre-culture was inoculated 1% into 10 2 L Erlenmeyer flasks containing 400 ml of TSB medium, followed by shaking culture (150 rpm) at 25° C. for 3 days after inoculation to prepare a HK544 culture solution (4 L). Thereafter, Brevibacillus brevis ( Brevibacillus brevis ) HK544 strain culture solution was centrifuged (10,000 ⁇ g, 20 min) and filtered through a filter paper (Whatman No. 1) to prepare a culture filtrate.
  • Brevibacillus brevis Brevibacillus brevis
  • HK544 strain culture solution was centrifuged (10,000 ⁇ g, 20 min) and filtered through a filter paper (Whatman No. 1) to prepare a culture filtrate.
  • HK544 culture filtrate (4 L) was loaded into a column ( ⁇ 8 cm ⁇ 40 cm) filled with polystyrene resin (Diaion HP-20, Mitsubishi Chemicals), and 20%, 40%, 60%, 80%, 100 % Methanol aqueous solution was sequentially eluted at 1 L, and finally, 6 fractions of F1-F6 were prepared by eluting with 100% acetone (1 l), and each fraction was dissolved in 5% concentration in Colletotrichum coccodes . When treated, it was confirmed that the F4 fraction exhibited strong bactericidal activity.
  • the F4 fraction (2 g) was loaded onto a column ( ⁇ 4 cm ⁇ 20 cm) filled with C18 resin (LiChroprep RP-18, Merck), and 20%, 40%, 50%, 60%, 70 using a vacuum pump. %, 90%, 100% methanol aqueous solution (containing 0.1% formic acid) to prepare 7 fractions of F41-F47, and when each fraction was treated with red pepper anthrax at a concentration of 200 ⁇ g/ml, F41 fraction It was confirmed that strong bactericidal activity appeared
  • the position of the hydrogen signal was determined through 1H-1H COSY, HSQC, and HMBC analysis, and the 1 H- and 13 C-NMR spectra in Table 3 and literature data were synthesized to determine the chemical structure of compound 1 as a linear peptide antibiotic ideine. It was determined as B1 (edeine B1) (Fig. 6).
  • B1 plant pathogenic fungus cabbage black pattern germ edeine B1 (Alternaria brassicicola) , tomato gray mold pathogen (Botrytis cinerea), red pepper anthracnose fungus (Colletotrichum coccodes), rice blast fungus (Magnaporthe oryzae), tomatoes Station pathogens (Phytophthora infestans ) and phytopathogenic bacteria ( Erwinia amylovora ) for growth inhibitory activity was determined by the microdilution method of liquid medium using a 96-well plate to determine the minimum inhibitory concentration (MIC).
  • MIC minimum inhibitory concentration
  • 100 ⁇ l wells were cultured to contain spores at a concentration of 1 ⁇ 10 4 spores/ml for fungi or 1 ⁇ 10 3 bacterial cells/ml for bacteria, and edeine B1 was 0.1, 0.2, 0.4 and 0.8, respectively.
  • edeine B1 was 0.1, 0.2, 0.4 and 0.8, respectively.
  • 1.6, 3.1, 6.3, 12.5, 25, 50, 100, 200 ⁇ g / ml were prepared so that the concentration.
  • the methanol content was less than 1%, and only 1% methanol was used as an untreated section, and the antibiotics cycloheximide and oxytetracycline were used as positive controls for phytopathogenic fungi and bacteria.
  • the concentration at which the growth of fungi and bacteria was completely inhibited was determined as the minimum inhibitory concentration (MIC) when incubated for 2-3 days at the optimum temperature for microbial growth and then observed with the naked eye.
  • MIC minimum inhibitory
  • Phytopathogenic microorganisms Minimum inhibitory concentration ( ⁇ g/ml) Idine B1 Cyclohexamide Oxytetracycline mold Alternaria brassicicola 0.4 12.5 na Botrytis cinerea 1.6 100 na Colletotrichum coccodes 12.5 50 na Magnaporthe oryzae 12.5 1.6 na Phytophthora infestans 0.1 0.2 na Germ Erwinia amylovora 1.6 na 1.6 na, not analyzed
  • edeine B1 (edeine B1) exhibited a wide range of antibacterial activity against various phytopathogenic fungi.
  • edeine B1 is a cabbage black blotch ( Alternaria brassicicola ) at a concentration of 0.4 ⁇ g / ml, a tomato gray fungus ( Botrytis cinerea ) at a concentration of 1.6 ⁇ g / ml, pepper anthrax bacteria ( Colletotrichum coccodes ) at a concentration of 12.5 ⁇ g/ml, rice blast bacteria ( Magnaporthe oryzae ) at a concentration of 12.5 ⁇ g/ml, and tomato blight bacteria ( Phytophthora infestans ) at a concentration of 0.1 ⁇ g/ml.
  • edeine B1 showed a bactericidal activity that completely inhibited the phytopathogenic bacteria Erwinia amylovora at a concentration of 1.6 ⁇ g/ml, which is similar to the antibiotic oxytetracycline. Shows the effect.
  • Brevibacillus brevis ATCC 8186 For the comparison of bactericidal activity between the closely related strains of the HK544 strain, Brevibacillus brevis ATCC 8186, Brevibacillus brevis ATCC 8185 from the Korean Agricultural Culture Collection (KACC) , Brevibacillus brevis DSM 30, Brevibacillus reuszeri DSM 9887, Brevibacillus sp.
  • KACC Korean Agricultural Culture Collection
  • FMR09-05 Brevibacillus panachihumi DCY35, Brevibacillus borstelensis DSM 6347, Brevibacillus parabrevis ) NBRC 12334, Brevibacillus choshinensis DSM 8552, Brevibacillus agri DSM 6348, Brevibacillus formosus DSM 9885 was sold.
  • the pre-sold strain was cultured in TSA (tryptic soy agar; 1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.5% NaCl, 2.5% K 2 HPO 4 , 1.5% agar) medium, and then a single colony formed on agar medium 250 ml-triangle flask containing 50 ml of TSB medium (tryptic soy broth; tryptic soy agar; 1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.5% NaCl, 2.5% K 2 HPO 4 ) Was inoculated and cultured with shaking at 25°C for 3 days (150 rpm) to prepare a culture solution, centrifuged (10,000 ⁇ g, 20 min) and filtered through a filter paper (Whatman No.
  • Yeast bacteria Saccharomyces cerevisiae
  • YPG medium 1% yeast extract, 2% peptone, 2% glucose
  • the antibacterial activity of the Brevibacillus sp. strain against was evaluated. Sterilized distilled water was used in the untreated section.
  • yeast bacteria Saccharomyces cerevisiae
  • YPG medium 5 ml
  • the yeast bacteria cultured with shaking (150 rpm) at 30° for 1 day were used as the inoculum, as measured by UV/Vis spectroscopy, 600 nm It was diluted with sterilized distilled water so that the absorbance at was 0.3.
  • the yeast suspension was inoculated into a 96-well plate at a concentration of 1%. After inoculation of the yeast, the cells were cultured at 30° for 1 day, and the absorbance at 600 nm was measured to investigate the growth of the yeast. By substituting in the following equation, the growth inhibition rate (%) of yeast was investigated.
  • the bactericidal activity of the culture filtrate of the Brevibacillus brevis HK544 strain was the most excellent.
  • the culture filtrate of HK544 strain was treated at a concentration of 10%, the growth of yeast bacteria was inhibited by 93% compared to the untreated group, but other strains did not have the effect of inhibiting the growth of yeast bacteria.

Abstract

la présente invention a pour but de fournir une composition destinée à prévenir les maladies chez les plantes, contenant, en tant que principe actif, un ou plusieurs éléments choisis dans le groupe constitué par une souche Brevibacillus brevis HK544, un milieu de culture de la souche, un filtrat de culture de la souche, et une fraction du filtrat de culture ou de l'édéine B1, et de fournir un procédé de prévention des maladies chez les plantes à l'aide de la composition. La composition pour prévenir les maladies chez les plantes, de la présente invention, présente un excellent effet de prévention sur diverses maladies chez les plantes telles que la pourriture grise de la tomate, le mildiou de la tomate, l'anthracnose et celle due à Puccinia recondita, ce qui permet d'être efficacement utilisable dans la prévention de maladies chez les plantes.
PCT/KR2020/008127 2019-06-27 2020-06-24 Composition pour prévenir une maladie chez les plantes en utilisant une souche de brevibacillus brevis hk544 WO2020262910A1 (fr)

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