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WO2024106740A1 - Composition pour lutter contre les champignons phytopathogènes ou réduire les mycotoxines, comprenant une solution de culture de bacillus velezensis jck-7158 ou son extrait, procédé de préparation, et procédé pour lutter contre les champignons phytopathogènes ou réduire les mycotoxines - Google Patents

Composition pour lutter contre les champignons phytopathogènes ou réduire les mycotoxines, comprenant une solution de culture de bacillus velezensis jck-7158 ou son extrait, procédé de préparation, et procédé pour lutter contre les champignons phytopathogènes ou réduire les mycotoxines Download PDF

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Publication number
WO2024106740A1
WO2024106740A1 PCT/KR2023/014822 KR2023014822W WO2024106740A1 WO 2024106740 A1 WO2024106740 A1 WO 2024106740A1 KR 2023014822 W KR2023014822 W KR 2023014822W WO 2024106740 A1 WO2024106740 A1 WO 2024106740A1
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WO
WIPO (PCT)
Prior art keywords
jck
fusarium
strain
phytopathogenic fungi
rhizoctonia solani
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Ceased
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PCT/KR2023/014822
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English (en)
Korean (ko)
Inventor
김진철
박애란
여유정
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Industry Foundation of Chonnam National University
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Industry Foundation of Chonnam National University
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Priority to JP2025528704A priority Critical patent/JP2025539800A/ja
Publication of WO2024106740A1 publication Critical patent/WO2024106740A1/fr
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    • 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
    • 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
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • 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
    • 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
    • C12R2001/07—Bacillus

Definitions

  • the present invention was made under the support of the Ministry of Agriculture, Food and rural Affairs under project identification number 1545025743 and detailed task number 320036-5.
  • the research management agency for the project was the Agriculture, Food and rural Development Technology Planning and Evaluation Institute, and the research project name was "Industrialization Technology for Response to Crop Viruses and Pests.”
  • Development Project the research project name is "Development of an eco-friendly biological control agent for rice red mold disease"
  • the research institution is Chonnam National University Industry-Academic Cooperation Foundation
  • the research period is 2022.01.01 to 2022.12.31.
  • the present invention relates to a composition for controlling phytopathogenic fungi containing Bacillus velezensis JCK-7158 strain, its culture medium or extract thereof, a method for producing the same, a method for controlling phytopathogenic fungi, and a method for reducing fungal toxins.
  • Fusarium head blight caused by various Fusarium species including Fusarium graminearum and F. asiaticum, occurs periodically in wheat, barley, corn, rice, etc. It occurred on a large scale and caused a lot of damage around the world. In particular, major outbreaks have occurred in the United States and Canada over the past few years, resulting in a sharp rise in global grain prices.
  • Red mold disease not only causes a decrease in grain production, but also deteriorates the quality of agricultural products by leaving mycotoxins such as trichothecene and zearalenone, which are harmful to livestock, in the infected area.
  • mycotoxins such as trichothecene and zearalenone, which are harmful to livestock, in the infected area.
  • trichothecene Types of nivalenol (NIV) and deoxynivalenol (DON) cause serious poisoning, including lowered immunity and digestive tract disorders.
  • the present inventors found that when a composition for controlling phytopathogenic fungi containing Bacillus velezensis JCK-7158 strain, its culture medium, or its extract was treated with phytopathogenic fungi, the control activity was significantly superior and the composition produced by the fungus was significantly superior. It was confirmed that the production of fungal toxins was also effectively reduced.
  • the purpose of the present invention is to provide a Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, which has antifungal activity.
  • Another object of the present invention is to provide a composition for controlling phytopathogenic fungi containing the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.
  • Another object of the present invention is iturin A, surfactin, 2,3-butanediol, 5-Methylhexan-2-one ), heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl -2-Heptanone (5-methyl-2-heptanone) to provide a composition for controlling plant pathogenic fungi containing at least one selected from the group consisting of.
  • Another object of the present invention is to provide a method for producing a composition for controlling phytopathogenic fungi, which includes a cultivation step of preparing a culture medium by culturing the Bacillus bellegensis JCK-7158 strain deposited under the accession number KCTC15169BP.
  • Another object of the present invention is iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone.
  • Another object of the present invention is to provide a composition for reducing fungal toxins containing the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.
  • Another object of the present invention is to provide a method for reducing fungal toxins, including a treatment step of treating the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.
  • culture medium in this specification refers to containing microorganisms after culturing them.
  • extract in this specification refers to separation from the culture medium of the strain, and the extract includes one or more selected from the group consisting of culture supernatant, culture filtrate, cell suspension, and fractions.
  • the extract includes iturin A, surfactin, 2,3-butanediol, 5-Methylhexan-2-one ), heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl It may contain one or more compounds selected from the group consisting of -2-heptanone (5-methyl-2-heptanone).
  • culture supernatant in this specification refers to the upper liquid obtained after removing most microorganisms from the culture medium through centrifugation.
  • culture filtrate in this specification refers to the liquid remaining after filtering and removing bacterial cells from the culture medium by performing centrifugation and filtration. Culture filtrate contains substances formed and excreted during the growth of microorganisms, so the substances can be purified or extracted.
  • culture filtrate means removing fungi from the culture solution by centrifugation and filtration.
  • the plant pathogenic fungus is Clarireedia jacksonii ( Clariredia jacksonii , (formerly called Sclerotinia homoeocarpa ), Rhizoctonia solani AG-4, Rhizoctonia solani AG-2-2 (IV) Large patch, Rhizoctonia solani Aju 2-2 (IV) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum ( Fusarium oxysporum f. sp. cucumerinum ), Fusarium oxysporum f. Esp. Lycopersici ( Fusarium oxysporum f. sp.
  • Fusarium graminearum Fusarium asiaticum , Fusarium verticillioides , Pythium ultimum , Gaeumannomyces graminis , Phytophthora infestans , Botrytis Cinerea, and Colletotrichum coccodes.
  • rice red mold disease a plant pathogenic fungal disease
  • a composition for controlling phytopathogenic fungi containing the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract. Control activity against Fusarium graminearum or Fusarium asiaticum can be obtained.
  • Another aspect of the present invention is iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2- It is a composition for controlling phytopathogenic fungi containing at least one compound selected from the group consisting of 5-methyl-2-heptanone and 5-methyl-2-heptanone.
  • iturin A may be a compound of the following formula (1), its molecular weight is 1,043, and its molecular formula is C 48 H 74 O 14 N 12 .
  • the surfactin may be a compound of the following formula (2), the molecular weight is 1,036, and the molecular formula is C 53 H 93 O 13 N 7 .
  • the 2,3-butanediol may be a compound of the following formula (3), the molecular weight is 90, and the molecular formula is C 4 H 10 O 2 .
  • the 5-methylhexan-2-one may be a compound of the following formula (4), the molecular weight is 114, and the molecular formula is C 7 H 14 O.
  • the heptan-2-one may be a compound of the following formula 5, the molecular weight is 114, and the molecular formula is C 7 H 14 O.
  • the 2,5-dimethylpyrazine may be a compound of the following formula (6), the molecular weight is 108, and the molecular formula is C 6 H 8 N 2 .
  • the 6-methylheptan-2-one may be a compound of the following formula (7), the molecular weight is 128, and the molecular formula is C 8 H 16 O.
  • the 5-methyl-2-heptanone may be a compound of the following formula (8), the molecular weight is 128, and the molecular formula is C 8 H 16 O.
  • the phytopathogenic fungi include Claryredia jacksoni, Rhizoctonia solani AGE-4, Rhizoctonia solani AGE 2-2 (IV) Large Patch, Rhizoctonia solani AGE 2- 2 (IV) Brown Patch, Fusarium Oxiform F. Esp. Cucumerinum, Fusarium oxyformum f. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes.
  • composition for controlling plant pathogenic fungi may further include additives such as additives, extenders, nutrients, or sealing agents, but is not limited thereto.
  • the additives are polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene.
  • the extender may include one or more selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate. It is not limited.
  • the nutritional agent may include one or more selected from the group consisting of skim milk, soybean flour, rice, wheat, red clay, diatomaceous earth, bentonite, dextrin, glucose, and starch. , but is not limited to this.
  • Another aspect of the present invention relates to a method for producing a composition for controlling phytopathogenic fungi, which includes a culturing step of preparing a culture solution by culturing the Bacillus bellegensis JCK-7158 strain deposited under accession number KCTC15169BP.
  • the phytopathogenic fungi include Claryredia jacksoni, Rhizoctonia solani AGE-4, Rhizoctonia solani AGE 2-2 (IV) Large Patch, Rhizoctonia solani AGE 2- 2 (IV) Brown Patch, Fusarium Oxiform F. Esp. Cucumerinum, Fusarium oxyformum f. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, but is not limited thereto.
  • the culturing step may include preparing a culture filtrate from the culture solution by centrifuging and filtering the culture solution, but is not limited thereto.
  • the culture medium may include one or more selected from the group consisting of milk proteins such as skim milk, whey, and casein, sugars, homologs, and extracts, but is not limited thereto.
  • the method for producing a composition for controlling plant pathogenic fungi may further include a concentration step of concentrating the culture medium.
  • the method for producing a composition for controlling plant pathogenic fungi may further include a dilution step of diluting the culture medium.
  • the method for producing a composition for controlling phytopathogenic fungi may further include an extraction step of extracting components within the fungal cells.
  • the method for producing the composition for controlling fungi may include a fractionation step of obtaining an active fraction from the culture medium, but is not limited thereto.
  • the method for producing the composition for controlling fungi may include the following steps:
  • the step of obtaining the culture filtrate is performed at 2,000 to 5,000 rpm, 2,500 to 5,000 rpm, 3,000 to 5,000 rpm, 3,500 to 5,000 rpm, 4,000 to 5,000 rpm, 4,500 to 5,000 rpm, for example, 4,500 rpm. centrifugation It may include the step of doing so, but is not limited thereto.
  • the fractionation step may include fractionation with one or more solvents selected from the group consisting of butanol, ethyl acetate, chloroform, methanol, ethanol, and hexane, but is not limited thereto.
  • the fractionation step may consist of the following steps:
  • a third fractionation step in which the fractions obtained in the second fractionation step are eluted with a mixed solution of chloroform: methanol: water, and at least one type of fraction selected from the group consisting of the first to third fractions among the four fractions is obtained.
  • the mixing volume ratio of chloroform:methanol:water may be 50 to 60:30 to 40:5 to 10, preferably 52 to 58:35 to 40:7 to 9. It may be, for example, 55:36:8, but it is not limited to this.
  • the concentration step may include concentrating the fraction using a reduced pressure concentrator, but is not limited thereto.
  • the purification step may include purification using preparative high-performance liquid chromatography (HPLC), but is not limited thereto.
  • HPLC preparative high-performance liquid chromatography
  • the active fraction is Iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone, but is not limited thereto.
  • Another aspect of the present invention relates to a method for controlling phytopathogenic fungi, which includes a treatment step of treating the plant or soil with the Bacillus bellegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.
  • the treatment step may be performed by one or more methods selected from the group consisting of spraying, soil irrigation, surface spraying, root zone treatment, seed treatment, soaking, poisoning, and smoking, for example, soil It may be performed through irrigation, but is not limited to this.
  • “spraying” may be performed by one or more methods selected from the group consisting of spraying, misting, atomizing, powder spraying, granule spraying, lifetime use, and permanent use.
  • the term “drenchment” is a method of spraying a chemical that involves digging a hole in the soil or tree and injecting a chemical solution.
  • soil irrigation refers to a method of injecting or spraying a chemical solution into crop cultivation soil.
  • the phytopathogenic fungi include Claryredia jacksoni, Rhizoctonia solani AGE-4, Rhizoctonia solani AGE 2-2 (IV) Large Patch, Rhizoctonia solani AGE 2- 2 (IV) Brown Patch, Fusarium Oxiform F. Esp. Cucumerinum, Fusarium oxyformum f. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, but is not limited thereto.
  • Another aspect of the present invention is iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-
  • a method for controlling phytopathogenic fungi comprising at least one compound selected from the group consisting of 5-methyl-2-heptanone and 5-methyl-2-heptanone.
  • composition for reducing fungal toxins comprising the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.
  • the extract is iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone.
  • the fungal toxin may be produced by a phytopathogenic fungus, and the phytopathogenic fungus is Claryredia jacksoni, Rhizoctonia solani Aizu-4, Rhizoctonia solani Aiju 2-2 (IV) Raji. Patch, Rhizoctonia solani Aju 2-2 (IV) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum, Fusarium oxyformum f. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus.
  • Totrichum cocodes may be one or more species selected from the group consisting of Totrichum cocodes, for example, it may be produced by Fusarium asiaticum, and the fungal toxin may be Nivalenol (NIV). , but is not limited to this.
  • NAV Nivalenol
  • Another aspect of the present invention relates to a method for producing a composition for reducing fungal toxins, comprising a culturing step of preparing a culture medium by culturing the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP.
  • the extract is iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone.
  • the fungal toxin may be produced by a phytopathogenic fungus, and the phytopathogenic fungus is Claryredia jacksoni, Rhizoctonia solani Aizu-4, Rhizoctonia solani Aiju 2-2 (IV) Raji. Patch, Rhizoctonia solani Aju 2-2 (IV) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum, Fusarium oxyformum F. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, for example, it may be produced by Fusarium asiaticum, and the fungal toxin may be nivalenol, but is not limited thereto. no.
  • Another aspect of the present invention relates to a method for reducing fungal toxins, comprising a treatment step of treating the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.
  • the extract is iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone.
  • the fungal toxin may be produced by a phytopathogenic fungus, and the phytopathogenic fungus is Claryredia jacksoni, Rhizoctonia solani Aizu-4, Rhizoctonia solani Aiju 2-2 (IV) Raji. Patch, Rhizoctonia solani Aju 2-2 (IV) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum, Fusarium oxyformum f. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, for example, it may be produced by Fusarium asiaticum, and the fungal toxin may be nivalenol, but is not limited thereto. no.
  • the present invention relates to a composition for controlling phytopathogenic fungi or reducing fungal toxins containing Bacillus velezensis JCK-7158 strain, its culture medium or extracts thereof, a method for producing the same, and a method for controlling phytopathogenic fungi or reducing fungal toxins.
  • the culture medium of the above-mentioned strain or its extract has significantly excellent antifungal activity and induced resistance, it can be used for controlling plant pathogenic fungi or reducing fungal toxins.
  • Figure 1 is a family diagram analyzed by 16S rRNA gene base sequence of Bacillus velezensis JCK-7158 strain according to an embodiment of the present invention.
  • Figure 2a is a graph showing the extracellular protease activity of Bacillus velegensis JCK-7158 strain over time according to an embodiment of the present invention.
  • Figure 2b is a graph showing the extracellular chitinase activity of Bacillus velegensis JCK-7158 strain over time according to an embodiment of the present invention.
  • Figure 2c is a graph showing the extracellular cellulase activity of Bacillus velegensis JCK-7158 strain over time according to an embodiment of the present invention.
  • Figure 2d is a graph showing the extracellular gelatinase activity of Bacillus velegensis JCK-7158 strain over time according to an embodiment of the present invention.
  • Figure 3 is a photograph showing the extracellular enzyme activity of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figure 4 is a photograph showing the IAA production activity results of the Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figure 5 is a photograph showing the acetoin production activity of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figures 6a and 6b are photographs showing the mycelial growth inhibition activity of various plant pathogenic fungi by replacement culture of the Bacillus bellegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figure 7 is a diagram schematically illustrating the process of fractionation of antifungal active substances of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figure 8 shows the antifungal properties against Fusarium graminearum , a pathogenic strain, after developing the butanol fraction of the culture filtrate of Bacillus velegensis JCK-7158 strain by thin layer chromatography (TLC) according to an embodiment of the present invention. This is a photo analyzing activity.
  • Figure 9 is a thin layer chromatography photograph of four fractions isolated from the BF4 fraction of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figure 10 is a graph showing the results of positive ion mode LC/MS analysis of iturin A isolated from the BF4-1 fraction of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figure 11 is a graph showing the results of positive ion mode LC/MS analysis of surfactin isolated from the BF4-1 fraction of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figures 12a and 12b are photographs showing the mycelial growth inhibition activity of plant pathogenic fungi by the antifungal activity of volatile substances produced by the Bacillus belegensis JCK-7158 strain according to an embodiment of the present invention.
  • Figure 13 is a graph schematically illustrating the results of solid phase microextraction (SPME) GC-MS analysis of volatile organic compounds produced by the Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
  • SPME solid phase microextraction
  • Figure 14 is a photograph of testing the PR-1 gene expression activity according to the presence or absence of GUS expression of the Bacillus velegensis JCK-7158 strain in transgenic Arabidopsis thaliana according to an embodiment of the present invention.
  • Figure 15 is a graph showing the control effect on rice red mold disease (Fusarium head blight) caused by induced resistance of Bacillus belegensis JCK-7158 strain in greenhouse conditions according to an embodiment of the present invention.
  • Figure 16 is a photograph showing the control effect on rice red mold disease caused by induced resistance of the Bacillus belegensis JCK-7158 strain under greenhouse conditions according to an embodiment of the present invention 7 days after inoculation.
  • Figure 17 is a graph showing the control effect on rice red mold disease by induced resistance of two formulations using Bacillus belegensis JCK-7158 strain in greenhouse conditions according to an embodiment of the present invention.
  • Figure 18 is a photograph showing the control effect on rice red mold disease by induced resistance of two formulations using the Bacillus belegensis JCK-7158 strain under greenhouse conditions according to an embodiment of the present invention, 7 days after inoculation.
  • Figure 19 is a graph showing the control effect on rice red mold disease by induced resistance of the Bacillus belegensis JCK-7158 strain preparation under field conditions according to an embodiment of the present invention.
  • Figure 20 is a photograph showing the control effect on rice red mold disease due to induced resistance of the Bacillus belegensis JCK-7158 strain preparation under field conditions according to an embodiment of the present invention two weeks after inoculation.
  • Figure 21 is a photograph showing the control effect on rice red mold disease due to induced resistance of the Bacillus belegensis JCK-7158 strain preparation under field conditions 4 weeks after inoculation according to an embodiment of the present invention.
  • the present invention relates to the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, which has antifungal activity.
  • % used to indicate the concentration of a specific substance is (weight/weight)% for solid/liquid, (weight/volume)% for solid/liquid, unless otherwise specified. /Liquid is (volume/volume)%.
  • a strain with antifungal activity against Fusarium graminearum ( F. graminearum ), which causes red mold disease in crops, was isolated from the stems of Shindongjin rice grown in Gokseong, Jeollanam-do.
  • the collected rice stems (10 g) were ground by adding 100 mL of sterilized water, and the ground stem samples were diluted 40 times with sterilized water and then plated on TSA (Tryptic Soy Agar) containing 1% of F. graminearum mycelium suspension. was plated on, cultured at 30°C for more than 3 to 7 days, and the JCK-7158 strain producing a clear zone was isolated.
  • Strain JCK-7158 isolated from rice stems, was identified molecularly through nucleotide sequence analysis of the 16S rRNA gene. The strain was inoculated into TSB medium and cultured with shaking at 1,580 rpm for 3 days at 30°C. Then, gDNA (genomic DNA) of the harvested strain was extracted using the I-genomic BYF DNA Extraction Mini kit (iNtRON, Korea) according to the protocol suggested by the manufacturer.
  • PCR-premix polymerase chain reaction-premix
  • primer set that can amplify the 16S rRNA of the strain
  • the PCR product of the amplified 16S rRNA gene was requested to Genotech (Daejeon, Korea) for base sequence analysis, and a total base sequence of 1352 bp (SEQ ID NO. 3) was obtained as the 16S rRNA coding base sequence of the isolated strain, JCK-7158 strain. got it
  • protease medium 1% skim milk + 1.5% agar, Difco
  • chitinase medium 1% colloidal chitin + 1.5% agar, Difco
  • gelatinase medium 10% gelatin + 1.5% agar, Duksan
  • cellulase medium (0.4% carboxymethyl cellulose sodium + 1.5% agar, Sigma) -Aldrich
  • HCl hydrochloric acid
  • 1 L of cold ethanol 99.9% was added, thoroughly mixed using a stirrer, and centrifuged at 4°C and 4500 rpm for 20 minutes. Afterwards, the supernatant was removed, and the chitin precipitate was washed 3-4 times with 0.1 M potassium phosphate buffer (pH 7.0) and used as colloidal chitin.
  • the prepared colloidal chitin was sterilized by high-pressure steam and stored at 4°C until use.
  • Gelatinase medium was solidified by adding 10% gelatin to LB solid medium, and other media were solidified by adding 1.5% agar.
  • a sterilized paper disc (0.8 cm, Advantec, Japan) was placed on top of each prepared medium, and 5 ⁇ L, 10 ⁇ L, and 15 ⁇ L of JCK-7158 strain culture filtrate was added to protease medium, chitinase medium, and cellulase medium. , 20 ⁇ L, 40 ⁇ L, and 60 ⁇ L were dispensed into the gelatinase medium, respectively.
  • As a negative control equal amounts of sterilized LB and medium were dispensed onto paper disks. The experiment was repeated three times, and the plate was maintained at 30°C to observe the clear zone according to extracellular enzyme activity.
  • the cellulase medium and chitinase medium were stained by adding 5 mL of Lugol's solution (2.5 g/L iodine and 5 g/L potassium iodide) to the plate and maintained for 10 minutes under dark conditions, and then the clear zone was observed.
  • Lugol's solution 2.5 g/L iodine and 5 g/L potassium iodide
  • the diameter of the clear zone for strain JCK-7158 was 34.87 mm after 9 days of treatment with 15 ⁇ L of the sample in the protease medium.
  • a clear zone of 35.72 mm after 5 days of treatment with 15 ⁇ L of sample in cellulose medium, 38.26 mm after 5 days of treatment of 15 ⁇ L of sample in chitinase medium, and 19.29 mm after 5 days of treatment of 60 ⁇ L of sample in gelatinase medium. could be observed.
  • the Bacillus velegensis JCK-7158 strain produces protease, chitinase, gelatinase, and cellulase.
  • IAA Indole-3-acetic acid
  • TSB medium supplemented with L-tryptophan (150 mg/L).
  • 1% of the -7158 strain culture was inoculated and cultured with shaking at 30°C and 150 rpm for 24 hours. Afterwards, the culture was centrifuged at 4°C and 10,000 rpm for 5 minutes and then filtered through a 0.2 ⁇ m sterile filter.
  • Example 6 Growth inhibitory activity through MIC test of plant pathogen mycelium according to treatment of Bacillus belegensis JCK-7158 strain
  • the growth inhibitory activity was measured for eight types of plant pathogens.
  • the JCK-6019 strain was inoculated into TSB liquid medium and then cultured with shaking at 150 rpm for 3 days at 30°C.
  • the culture was centrifuged at 10,000 MIC (Minimum Inhibitory Concentration) test was performed by applying the 96-well microtiter plate method, and the plant pathogenic fungi were Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (IV) Large patch, Fusarium graminearum, and Fusarium asiaticum. , Gaeumannomyces graminis, Phytophthora infestans, Botrytis cinerea, and Colletotrichum coccodes were used.
  • the plant pathogenic fungi used in the experiment were inoculated into PDB (potato dextrose broth, Becton, Dickinson and Co., Sparks, MD, USA) medium and cultured in an incubator at 25°C for 7 days. After stationary culture, the weight of the mycelium was measured, sterilized distilled water was added to a concentration of 50 mg/mL, homogenized using a homogenizer at 10,000 rpm for 5 seconds, and then added to PDB medium to obtain a fungal suspension with a 1% concentration.
  • PDB potato dextrose broth, Becton, Dickinson and Co., Sparks, MD, USA
  • the phytopathogenic fungal suspension was treated with culture filtrate samples of the Bacillus velegensis JCK-7158 strain of the present invention to a concentration of 10%, 5%, 2.5%, 1.25%, 0.625%, 0.313%, 0.156%, and 0.078%, respectively. was performed three times.
  • the treated plate was sealed and cultured in an incubator at 25°C for 3-5 days to examine the minimum inhibitory concentration (MIC) of pathogens.
  • MIC Minimum growth inhibitory concentration
  • MIC Minimum growth inhibitory concentration for various plant pathogenic fungi in culture filtrate samples of Bacillus belegensis JCK-7158 strain No. plant pathogenic fungi MIC value (%)
  • the JCK-7158 strain had an antifungal activity of 1.25 against Clarireedia jacksonii and Fusarium graminearum . It showed the strongest antifungal activity with a MIC value of %, and was also effective against Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2(IV) Large patch, Gaeumannomyces graminis , Botrytis cinerea, Colletotrichum coccodes , and Phytophthora infestans with a MIC value of 5%. It showed strong activity. In addition, it was confirmed that Fusarium asiaticum had antifungal activity as it had an MIC value of 10%.
  • Bacillus belegensis JCK-7158 strain of the present invention has strong antifungal activity against various plant pathogenic fungi used in the MIC test results.
  • Example 7 Growth inhibition activity through replacement culture of plant pathogens according to treatment of Bacillus belegensis JCK-7158 strain
  • Rhizoctonia solani AG-4 Rhizoctonia solani AG 2-2
  • Large patch Rhizoctonia solani AG 2-2
  • Brown patch Fusarium oxysporum f. sp. cucumerinum , Fusarium oxysporum f.sp. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytophthora infestans, Botrytis cinerea, and Colletotrichum coccodes were used.
  • the culture medium of the Bacillus belegensis JCK-7158 strain of the present invention was streaked to a length of 5 cm at a point 2 cm away from the edge of the PDA plate, and the mycelial pieces of each pathogen were cut with a cork borer with a diameter of 6 mm to form JCK-7158. It was inoculated at a distance of 5 cm from the strain. The untreated group was inoculated only with mycelial fragments of the pathogen. After inoculation with fragments of pathogenic fungal hyphae and the culture medium of the Bacillus belegensis JCK-7158 strain of the present invention, the cells were cultured in an incubator at 25°C.
  • the mycelial growth radius of the pathogen was measured according to the growth rate of the pathogen, and the experiment was repeated three times. Antifungal activity was calculated by measuring the radius (R2) of pathogen hyphae in the direction of the JCK-7158 strain and measuring the radius (R1) of pathogen hyphae in control conditions. Both values were converted to percent mycelial growth inhibition using the following formula:
  • the Bacillus belegensis JCK-7158 strain of the present invention As a result of performing replacement culture of the Bacillus belegensis JCK-7158 strain of the present invention against various plant pathogenic fungi, as can be seen in Figure 6 and Table 4, the Bacillus belegensis JCK-7158 strain was effective against all pathogens used in the experiment. showed growth inhibitory activity. Among them, the growth of all molds used in the experiment except Pythium ultimum was inhibited by about 50%. Therefore, it was confirmed that the JCK-7158 strain had strong antifungal activity against various plant pathogenic fungi used in the experiment even through replacement culture.
  • the strain was inoculated into TSB liquid medium and then cultured with shaking at 150 rpm for 3 days at 30°C.
  • the isolation process of the antibacterial active substance produced by the Bacillus velegensis JCK-7158 strain was performed according to the process shown in FIG. 7.
  • the culture medium (total 3 L) was centrifuged at 4500 rpm for 20 minutes to obtain the supernatant of the culture medium.
  • the supernatant (290 mL) was fractionated twice with equal amounts of ethyl acetate (EtOAc) and butanol (BuOH) to obtain an organic solvent layer and an aqueous solution layer of the ethyl acetate layer and the butanol layer, then concentrated under reduced pressure, and each fraction layer was washed with acetone. , re-dissolved in methanol and water.
  • EtOAc ethyl acetate
  • BuOH butanol
  • each fraction layer was washed with acetone. , re-dissolved in methanol and water.
  • Fusarium graminearum was used as an antifungal activity test pathogen.
  • the selected active fraction the BF4 fraction
  • These four fractions and the standard substances iturin, fengycin, and surfactin purchased from Sigma-Aldrich were loaded into thin layer chromatography (TLC) and chloroform:methanol:water (55:36). :8, v/v/v) After development in solvent conditions, it was read by UV at 254 and 365 nm wavelengths and visualized by spraying p-anisaldehyde and water.
  • the active fraction BF4-1 was observed to contain iturin as a result of thin layer chromatography, but as a result of LC/MS analysis of the BF4-1 fraction, positive ion mode peaks were found at molecular weights of 1043.55 and 1036.69, respectively, as shown in Figures 10 and 11 below. It was found that these substances were iturin A (C 48 H 74 O 14 N 12 , molecular weight 1,043) and surfactin (C 53 H 93 O 13 N 7 , molecular weight 1,036).
  • Example 9 Antifungal activity assay using volatile organic compounds produced by Bacillus velegensis JCK-7158 strain
  • Rhizoctonia solani AG 2-2 Large patch, Rhizoctonia solani AG 2-2 (IV) of volatile organic compounds produced by the Bellezensis JCK-7158 strain of the present invention Brown patch, Fusarium oxysporum f. sp. cucumerinum , Fusarium oxysporum f. sp.
  • Antifungal activity was tested against lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytophthora infestans, Botrytis cinerea, and Colletotrichum coccodes .
  • a Bi-Petri Dish (SPL Life Sciences Co., Ltd., Pocheon, Gyeonggi-do, Korea) with a separated center was used. TSA was dispensed on one side of the plate, and PDA was dispensed on the other side. After dispensing, the culture medium of the JCK-7158 strain was smeared and cultured, and a piece of pathogen mycelium with a diameter of 6 mm was inoculated into the PDA on the other side and cultured at 25°C. In the untreated group, only the pathogen was inoculated into the PDA without smearing the culture medium of the JCK-7158 strain.
  • the experiment was performed in three repetitions, and all plates were sealed twice with parafilm, cultured at 25°C, and the mycelial growth diameter of the pathogen was measured.
  • untreated group only pathogens were inoculated without smearing the culture medium of the strain.
  • Antifungal activity was calculated by measuring the length and width (R1, R2) of pathogen hyphae and measuring the length and width (R3, R4) of pathogen hyphae in control conditions. Both values were converted to percent mycelial growth inhibition using the following formula: The results are shown in the table below.
  • the antifungal activity of volatile organic compounds produced by the JCK-7158 strain against plant pathogenic fungi was confirmed, and the volatile substances produced by the JCK-7158 strain inhibited the growth of various plant pathogenic fungi.
  • the volatile substances produced by the JCK-7158 strain inhibited the growth of various plant pathogenic fungi.
  • Botrytis cinerea which causes gray mold disease on fruits and crops
  • Fusarium graminearum which causes red mold disease on grains. They showed excellent pathogenic mycelia inhibition effects of 60.50% and 48.66%, respectively. Therefore, it was confirmed that the JCK-7158 strain of the present invention produces volatile substances showing antifungal activity against various plant pathogenic fungi.
  • SPME solid phase microextraction
  • GC-MS Gas chromatography-mass spectrometry
  • the material adsorbed on the SPME fiber was injected and exposed for 1 minute to be desorbed and then analyzed using a GC-MS (Shimadzu GC-MS QP2010, Shimadzu) equipped with a DB-5MS capillary column (30 m co., Kyoto, Japan). He was used as the mobile phase, and the flow rate was maintained at 1.0 mL/min.
  • the inlet temperature was set at 250°C, and the column temperature was maintained at 60°C for 2 minutes, then increased at a rate of 10°C/min to 250°C and maintained at 250°C for 20 minutes.
  • the ionization voltage of the mass spectrometer was 70 eV, and the analysis was performed in positive ion mode with a scan of 50 to 400 m/z at 200°C.
  • the obtained mass spectrum was accurately identified by comparison with data from the WILEY8 Library, and the content of each substance was expressed as the area ratio of the TIC (total ion chromatogram) peak.
  • the volatile organic compounds produced by the JCK-7158 strain of the present invention were detected as 5 substances within 7 minutes.
  • the chemical structures of the five substances were confirmed by comparing their mass spectrum with those in the library, and as a result of GC-MS analysis and mass spectrum analysis, the five volatile compounds were 5-methylhexan-2-one, heptan- They were identified as 2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanon. It was thought that these substances produced by the JCK-7158 strain of the present invention inhibit the growth of the plant pathogenic fungi used in the experiment.
  • Example 11 Preparation of formulation using culture medium of Bacillus velegensis JCK-7158 strain
  • the Bacillus belegensis JCK-7158 strain was inoculated into 20 mL of TSB medium, and then cultured with shaking at 30°C and 150 rpm for 3 days. did. 20% of Flocet Lite (oxidized starch) was added to the culture solution harvested after culturing for 3 days and spray-dried at 140°C while stirring. The spray-dried samples obtained as a result of spray-drying were prepared as described in Tables 9 and 10 below to prepare JCK-7158 strain suspension concentrate (SC) and wettable powder (WP).
  • SC JCK-7158 strain suspension concentrate
  • WP wettable powder
  • Example 12 Resistance induction activity assay of JCK-7158 strain in Arabidopsis thaliana
  • the PR -1 gene is used as a marker gene to test whether resistance is induced in plants using a series of signaling systems that result in the expression of PR-1 protein. Therefore, in order to test the resistance-inducing activity of Bacillus belegensis JCK-7158 strain culture using the PR-1 gene system, Arabidopsis thaliana transformed with a GUS-labeled vector in the PR-1 promoter was used. .
  • the surface of Arabidopsis seeds was sterilized using 95% ethanol, and secondary surface sterilization was performed using a bleach solution prepared with 2% NaOCl and 0.05% Tween-20. The bleaching solution remaining on the seeds was washed with sterile distilled water and soaked at 4°C for 48 hours.
  • the soaked seeds were placed on Murashige-Skoog Agar (MS agar) supplemented with 50 ppm kanamycin using a sterilized toothpick and cultured in a plant growth incubator at 25°C. After 12 days of culture, the culture medium, culture filtrate, cell suspension, liquid hydrate (SC), and powder hydrate (WP) of the Bacillus belegensis JCK-7158 strain of the present invention were added to each well of a 24-well plate in sterile water at 500, 1,000, It was diluted 2,000- and 4,000-fold and treated at 2.5 mL each. Two Arabidopsis plants were submerged in each well and maintained at room temperature for 48 hours in an orbital shaker.
  • MS agar Murashige-Skoog Agar
  • SC liquid hydrate
  • WP powder hydrate
  • the staining solution was removed, immersed in 70% ethanol for 1 hour, and then immersed in 90% ethanol several times at hourly intervals to remove unnecessary pigments such as chlorophyll. After immersing several times and all the pigment was removed, the expression of the PR-1 gene was confirmed by checking whether it was blue using a microscope.
  • the expression of the PR-1 gene was confirmed by the presence or absence of GUS expression, and the induction resistance and induction activity were tested.
  • the culture broth (CB), culture filtrate (CF), and cell suspension (Culture Broth (CB)) of the Bacillus belegensis JCK-7158 strain were the same as the salicylic acid (SA) treatment group, which is a positive control.
  • SA salicylic acid
  • Cell and the two types of preparations prepared in Example 11, liquid hydrate (SC) and powder hydrate (WP), were confirmed to induce expression of the PR-1 gene by expressing GUS, and through this, Bacillus belegensis JCK It was confirmed that strain -7158 can control pathogenic fungi by inducing resistance in plants through a mechanism similar to salicylic acid.
  • Example 13 Control effect on rice red mold disease by induced resistance of Bacillus belegensis JCK-7158 strain in greenhouse conditions
  • Samkwang rice seeds were used to investigate the in vivo control activity of rice red mold disease by induced resistance of the JCK-7158 strain of the present invention under greenhouse conditions.
  • compound fertilizer for base fertilizer (Soil Love 21, Namhae Chemical) was dissolved in water and applied evenly to the irrigated rice paddy soil. Afterwards, the young seedlings grown in a constant temperature and humidity room were transplanted into the center of the Wagner pot filled with 70% paddy soil. Before transplanting the pot, the drainage hole at the bottom of the pot was blocked with a silicone stopper, and then the pot was prepared by laying Styrofoam balls (4 cm in diameter, 10 pieces/pot) and potting mats (14 cm in diameter).
  • the Wagner pots were filled with water, and they were grown in a glass greenhouse (minimum temperature of 20-25°C, maximum temperature of 30-35°C) and used in experiments. 20 days after pot transplantation, the first fertilization was done with SuperAli (14 kg/10 a, Namhae Chemical), and 2 months after pot transplantation, the second fertilization was done with NK 24 (N:P:K, 24-0-12, FarmHannong). Additional fertilization was carried out.
  • Fusarium asiaticum strain one of the causative bacteria of rice red mold disease, was inoculated into PDA medium and cultured for 5 days at 25°C.
  • Five agar plugs (0.1 cm ⁇ 7H 2 O, 1 g NH 4 NO 3 , and 1 g KH 2 PO 4 , 1 L distilled water) and cultured with shaking at 25°C and 150 rpm for 4 days.
  • the pathogen culture was filtered through 4 layers of gauze to remove hyphae, and the filtered spore suspension was adjusted to a concentration of 2 did.
  • the adjusted spore suspension was centrifuged at 4°C and 4,000 rpm for 10 minutes, the supernatant was discarded to remove the medium components, and then suspended in the same amount of 0.05% Tween-80 and used as an inoculum in this experiment.
  • JCK-7158 induced resistance against rice red mold disease caused by F. asiaticum .
  • Treatment of the JCK-7158 strain was performed on 11-week-old rice plants after pot transplantation, using JCK-7158 strain culture medium, culture filtrate, cell suspension, liquid hydrate (SC), and powder hydrate (WP) 1,000 times 2 weeks and 1 week before pathogen inoculation. , was diluted 2,000 times and applied as a foliar spray to saturate the rice ears and stems.
  • Play Emulsion (Peulrei, ai 13% Difenoconazole + 13% Propiconazole EC, Syngenta Korea), a publicly available drug for rice red mold disease, was diluted 2,000 times 1 day before pathogen inoculation.
  • Pathogen inoculation was carried out 7 days after the second drug treatment, by spraying the same amount of F. asiaticum spore suspension (2 When manufacturing the drug, Tween-20, an electrodeposition agent, was added to the JCK-7158 strain culture medium, culture filtrate, and cell suspension at a level of 250 ⁇ g/mL. Immediately after inoculation, it was treated in a wet room using a plastic bag for 3 days, and the experiment was repeated 5 times per treatment group. Seven days after inoculation, the severity of rice red mold disease according to the affected area and the control value compared to the untreated group were calculated.
  • the 1,000-fold dilution of the JCK-7158 strain culture showed a control activity of 49.88% and 34.71% in the 2,000-fold dilution compared to the untreated group, and the 1,000-fold dilution of the JCK-7158 strain culture filtrate showed 43.88% and 38.82% in the 2,000-fold dilution. It showed control activity of 47.88% at 1,000-fold dilution and 42.59% at 2,000-fold dilution of JCK-7158 strain cell suspension. It was observed that the control agent was effective in a concentration-dependent manner in all treatments.
  • Play which was used as a control agent, showed a control value of 34.41% at a 2,000-fold dilution, and the control activity of the treatment was superior to that of the control agent, confirming that the JCK-7158 strain effectively controls rice red mold disease through induced resistance. there was.
  • the 1,000-fold dilution of JCK-7158 liquid wetting agent (SC) showed a control activity of 63.87% and 35.43% at a 2,000-fold dilution, and 34.40% and a 2,000-fold dilution of JCK-7158 strain powder wetting agent (WP).
  • Each pear diluted solution showed a control activity of 30.42%. It was observed that the control agent was effective in a concentration-dependent manner in all treatments. Play, which was used as a control agent, showed a control value of 44.87% at a 2,000-fold dilution, and the control value of JCK-7158 liquid hydrate (SC) at a 1,000-fold dilution (63.87%) was superior to the control agent, effectively controlling rice red mold disease. Control was confirmed. Therefore, it was confirmed that a highly effective rice red mold disease control agent could be developed in the future by using the resistance-inducing activity of the JCK-7158 strain.
  • Example 14 Control effect on rice red mold disease by induced resistance of Bacillus belegensis JCK-7158 strain preparation under field conditions
  • Bacillus belegensis JCK-7158 liquid moisturizer (SC) described in Example 11 was applied to Samkwang rice seeds. was processed. Bacillus belegensis JCK-7158 liquid moisturizer (SC) was diluted 1,000 times and 2,000 times and sprayed on the ears of Samkwang rice, the target plant, 2 weeks and 1 week before inoculation with the pathogen, and F. asiaticum was inoculated to inoculate Bacillus belegensis. The control effect of the JCK-7158 agent was investigated. The control agent was Play emulsion (13% propiconazole + 13% difenoconazole, Syngenta Korea Co., Ltd.) diluted 2,000 times and used 1 day before pathogen inoculation.
  • the inoculant to be inoculated into rice under field conditions was F. asiaticum, which causes rice red mold disease, by adjusting it to 2.0 It was prepared by adding.
  • the rice ears treated with the inoculum were sprayed with distilled water once in a zipper bag (8 cm was removed and water was sprayed twice daily. Treatment was repeated 3 times based on 30 rice heads per treatment group.
  • the 1,000-fold dilution of JCK-7158 liquid hydration agent (SC) showed 48.81% of the control activity compared to the untreated group, and 20.56% of the control activity at 2,000-fold dilution
  • the 1,000-fold dilution of JCK-7158 liquid hydration agent (SC) showed a control activity of 50.28% compared to the untreated group and 17.5% in a 2,000-fold dilution.
  • the control effect was concentration-dependent in the treatment group, and the results of the greenhouse experiment were similar.
  • Play which was used as a control agent, showed control values of 32.57% and 35.21% in a 2,000-fold dilution 2 and 4 weeks after inoculation with the pathogen, respectively, and JCK-7158 liquid moisturizer showed control values for rice red mold disease at both times. (SC) It was confirmed that the control value of the 1,000-fold diluted solution was superior to the control drug.
  • Example 15 Effect of reducing toxin production in rice red mold disease by induced resistance of Bacillus belegensis JCK-7158 strain preparation under field conditions
  • the effect of treatment with the liquid moisturizer for the JCK-7158 strain of the present invention is to reduce the production of nivalenol (NIV), a fungal toxin produced by F. asiaticum , a pathogen causing rice red mold disease, under packaging conditions.
  • NMV nivalenol
  • F. asiaticum a fungal toxin produced by F. asiaticum
  • SC Bacillus velegensis JCK-7158 liquid hydrator
  • Bacillus belegensis JCK-7158 liquid moisturizer was diluted 1,000 times and 2,000 times and sprayed on the ears of Samkwang rice, the target plant, 2 weeks and 1 week before inoculation with the pathogen, and Fusarium asiaticum was inoculated to inoculate Bacillus belle.
  • the control effect of Gensis JCK-7158 preparation was investigated.
  • the control agent was Play emulsion (13% propiconazole + 13% difenoconazole, Syngenta Korea Co., Ltd.) diluted 2,000 times and used 1 day before pathogen inoculation.
  • the inoculant to be inoculated into rice under field conditions is Fusarium asiaticum , which causes rice red mold disease, adjusted to 2.0 It was prepared by adding.
  • the rice ears treated with the inoculum were sprayed with distilled water once in a zipper bag (8 cm was removed and water was sprayed twice daily. Treatment was repeated 3 times based on 30 rice heads per treatment group.
  • treatment with JCK-7158 liquid moisturizer had the effect of reducing nivalenol by 40.62% compared to the untreated treatment, and reduced nivalenol, a fungal toxin, compared to Play, a chemical disinfectant (toxin reduction effect of 21.12%). It was confirmed that the effect was excellent.
  • the JCK-7158 strain is an eco-friendly strain with excellent control activity as an alternative to the use of chemical pesticides, which has the problem that it can only be used up to 30 days before harvest due to residual problems. It is expected to become a cornerstone in the development of control agents for various plant diseases.
  • the present invention relates to a composition for controlling phytopathogenic fungi containing Bacillus velezensis JCK-7158 strain, its culture medium or extract thereof, a method for producing the same, a method for controlling phytopathogenic fungi, and a method for reducing fungal toxins.

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Abstract

La présente invention concerne : une composition pour lutter contre les champignons phytopathogènes, comprenant une souche Bacillus velezensis, sa solution de culture ou son extrait ; un procédé de préparation ; et un procédé de lutte contre les champignons phytopathogènes. La solution de culture de la souche ou son extrait présente d'excellentes activités antifongiques et une résistance induite, ainsi qu'une excellente capacité à réduire les toxines produites par les champignons, et peut donc être utilisée pour lutter contre les champignons phytopathogènes.
PCT/KR2023/014822 2022-11-17 2023-09-26 Composition pour lutter contre les champignons phytopathogènes ou réduire les mycotoxines, comprenant une solution de culture de bacillus velezensis jck-7158 ou son extrait, procédé de préparation, et procédé pour lutter contre les champignons phytopathogènes ou réduire les mycotoxines Ceased WO2024106740A1 (fr)

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CN119613484A (zh) * 2024-12-06 2025-03-14 中国海洋大学 一种芽孢杆菌产生的抗真菌肽的分离纯化、鉴定及其应用

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