WO2024111857A1 - Composition pour lutter contre les maladies des plantes, comprenant un bouillon de culture de la souche streptomyces sp. jck-8055 ou son extrait, son procédé de préparation et le procédé pour lutter contre les maladies les utilisant - Google Patents

Composition pour lutter contre les maladies des plantes, comprenant un bouillon de culture de la souche streptomyces sp. jck-8055 ou son extrait, son procédé de préparation et le procédé pour lutter contre les maladies les utilisant Download PDF

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WO2024111857A1
WO2024111857A1 PCT/KR2023/014816 KR2023014816W WO2024111857A1 WO 2024111857 A1 WO2024111857 A1 WO 2024111857A1 KR 2023014816 W KR2023014816 W KR 2023014816W WO 2024111857 A1 WO2024111857 A1 WO 2024111857A1
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jck
strain
streptomyces
disease
subsp
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PCT/KR2023/014816
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English (en)
Korean (ko)
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김진철
박애란
누엔티탄로안
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전남대학교산학협력단
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Priority claimed from KR1020220160448A external-priority patent/KR20240094098A/ko
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Publication of WO2024111857A1 publication Critical patent/WO2024111857A1/fr

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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
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • 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
    • A01N63/28—Streptomyces
    • 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
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • 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
    • C12R2001/465—Streptomyces

Definitions

  • This invention was made under the support of the Rural Development Administration under project identification number 1395077354 and project number RS-2020-RD008636.
  • the research management agency for the project is the Rural Development Administration, and the research project name is "Pest and pest damage reduction technology for pending issues such as fruit tree burn disease.”
  • Development the research project name is "Development of biological control technology for fruit tree burn disease (4 joint)"
  • the research institution is Chonnam National University Industry-Academic Cooperation Foundation, and the research period is 2023.01.01 to 2023.12.31.
  • This invention was made under the support of the Rural Development Administration under project identification number 1395072972 and detailed task number PJ0152962022.
  • the research management agency for the project was the Rural Development Administration, and the research project name was "Development of pest damage reduction technology for pending issues such as fruit tree burn disease",
  • the research project name is "Development of biological control technology for fruit tree burn disease (4 joint)"
  • the research institution is Chonnam National University Industry-Academic Cooperation Foundation, and the research period is 2022.01.01 to 2022.12.31.
  • the present invention relates to a composition for controlling plant diseases containing a culture medium of the Streptomyces sp. JCK-8055 strain or an extract thereof, a method for producing the same, and a method for controlling plant diseases using the same. More specifically, it relates to a composition for controlling plant diseases containing a culture medium of the Streptomyces sp. JCK-8055 strain or an extract thereof, a method for producing the same, and a method for controlling plant diseases using the same.
  • This relates to plant disease control technology using aureothricin and thiolutin, which are antibacterial active ingredients isolated from the JCK-8055 strain and its culture medium.
  • Plant pathogenic bacteria are mainly transmitted by agricultural tools and insects such as aphids and bees. When a bacterial disease occurs in one plant or tree, it is so contagious that the entire agricultural orchard is infected, and control is difficult.
  • Current methods for controlling bacterial diseases include using single or mixed agents of antibiotics and copper synthetic compounds, or cultivating resistant varieties. However, these methods are also ineffective under environmental conditions that are favorable to the occurrence and spread of plant pathogenic bacteria, and have several problems, such as contamination of the agricultural ecosystem due to the use of chemical disinfectants and the emergence of antibiotic-resistant strains.
  • Streptomyces is the largest genus of actinomycetes, and like other actinomycetes, it is a streptococcus and is known to have a gram-positive genome with a high GC content. Most Streptomyces, found primarily in soil and necrotic plants, produce spores and are characterized by a complex secondary metabolism that produces clinically useful naturally occurring antibiotics: neomycin, cyphamycin, grimemycin, botoromycin, and chloramphenicol. It is known to produce etc.
  • the present inventors confirmed that plant disease control activity was significantly superior when treated with Streptomyces sp. JCK-8055 strain, its culture medium, its extract, or isolated antibacterial active material.
  • the purpose of the present invention is to provide a strain of Streptomyces sp. JCK-8055 deposited under the accession number KCTC15168BP, which has plant disease control activity.
  • Another object of the present invention is to provide a composition for controlling plant diseases containing the Streptomyces sp JCK-8055 strain deposited under the accession number KCTC15168BP, its culture medium, or its extract.
  • Another object of the present invention is to provide a composition for controlling plant diseases containing at least one compound selected from the group consisting of aureothricin and thiolutin.
  • Another object of the present invention is to provide a method for producing a composition for controlling plant diseases, comprising a culturing step of preparing a culture medium by culturing the Streptomyces sp. JCK-8055 strain deposited under accession number KCTC15168BP.
  • Another object of the present invention is to provide a plant disease control method comprising a treatment step of treating the Streptomyces sp. JCK-8055 strain deposited under accession number KCTC15168BP, its culture medium, or its extract.
  • Another object of the present invention is to provide a method for controlling plant diseases comprising a treatment step of treating at least one compound selected from the group consisting of aureothricin and thiorutin.
  • Another object of the present invention relates to the use of the Streptomyces sp JCK-8055 strain deposited under accession number KCTC15168BP, its culture or extract thereof, and aureothricin or thiorutin, an antibacterial active substance isolated therefrom, for controlling plant diseases. will be.
  • the present invention relates to a composition for controlling plant diseases comprising the Streptomyces sp. JCK-8055 strain, its culture medium or extract thereof, a method for producing the same, and a control method using the same.
  • One aspect of the present invention is the Streptomyces sp. JCK-8055 strain deposited under the accession number KCTC15168BP, which has plant disease control activity.
  • the Streptomyces sp. JCK-8055 strain may contain 16S rRNA containing the base sequence of SEQ ID NO: 5.
  • the Streptomyces sp JCK-8055 strain may contain a gyrB gene containing the base sequence of SEQ ID NO: 6.
  • One aspect of the present invention is a composition for controlling plant diseases comprising the Streptomyces sp. JCK-8055 strain deposited under the accession number KCTC15168BP, its culture medium, or its extract.
  • 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.
  • 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 refers to the removal of bacterial cells from the culture solution by centrifugation and filtration.
  • the fraction may be fractionated with ethyl acetate from the culture medium or culture filtrate of the strain, and preferably contains at least one compound selected from the group consisting of aureothricin and thiorutin isolated from the fraction. It may be done, but it is not limited to this.
  • the plant disease may be caused by phytopathogenic microorganisms consisting of phytopathogenic fungi and phytopathogenic bacteria.
  • the phytopathogenic fungi include Rhizoctonia solani AG-4, Rhizoctonia solani AG-4, Rhizoctonia solani AG-2-2 (IV), Sclerotinia homoeocarpa , and Puja. It may be one or more species selected from the group consisting of Fusarium graminearum , Fusarium asiaticum, Phytophthora infestans, and Pythium ultimum. , for example, may be Fusarium asiaticum, but is not limited thereto.
  • the plant pathogenic bacteria include Acidovorax avenae subsp. cattleyae , Acidovorax kojiaci, Agrobacterium tumerfaciens , and Burkorderia glume. ( Burkholderia glumae ), Clavibacter michiganensis subsp. michiganensi ), Erwinia amylovora , Erwinia pyrifoliae , Pectobacterium Pectobacterium carotovora subsp. carotovora , Pectobacterium chrysanthemi , Pseudomonas syringae pv.
  • Ralstonia solana It may be one or more species selected from the group consisting of Ralstonia solanacearum , Xanthomonas arboricola pv . pruni , and Xanthomonas euvesicatoria , for example. , Erwinia amylovora, Pectobacterium carotovora subspicis carotovora, and Ralstonia solanacearum, but are not limited thereto.
  • the plant disease may be selected from the group consisting of red mold disease, green blight, burn disease, and soft rot, for example, rice red mold disease caused by Fusarium asiaticum, Ralstonia sola It may be, but is limited to, tomato green blight caused by Nacearum, apple fire blight caused by Erwinia amylobora, and cabbage soft rot caused by Pectobacterium carotobora subspicis carotobora. It doesn't work.
  • One aspect of the present invention is a composition for controlling plant diseases comprising at least one compound selected from the group consisting of aureothricin and thiolutin.
  • the aureothricin may be a compound of the following formula (1), the molecular weight is 242.3, and the molecular formula is C 9 H 10 N 2 O 2 S 2 .
  • the thiorutin may be a compound of the following formula (2), the molecular weight is 228.3, and the molecular formula is C 8 H 8 N 2 O 2 S 2 .
  • the plant disease may be caused by phytopathogenic microorganisms consisting of phytopathogenic fungi and phytopathogenic bacteria.
  • the phytopathogenic fungi include Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (IV), Skrelotinia homoeocarpa, Fusarium graminearum, Fusarium asiaticum, and Pi. It may be one or more species selected from the group consisting of Thophthora infestans and Pycium ultimum, for example, Fusarium asiaticum, but is not limited thereto.
  • the phytopathogenic bacteria include Acidoborax avenae subspecis Cattleya, Acidoborax kojiyasi, Agrobacterium tuberfaciens, Burkolderia glume, Clavibacter misiganensis subspecis misiganensis, Erwinia amylovora, Erwinia pirofoliae, Pectobacterium carotovora subspicis carotovora, Pectobacterium chrysantemi, Pseudomonas syringae pasova latrimans, Ralstonia solanacearum It may be one or more species selected from the group consisting of Xanthomonas arboricola Passoba prunii, and Xanthomonas uvejicatoria, for example, Erwinia amylovora, Pectobacterium carotovora subspicis caro It may be Tobora, and Ralstonia solanacearum, but
  • the plant disease may be selected from the group consisting of red mold disease, green blight, burn disease, and soft rot, for example, rice red mold disease caused by Fusarium asiaticum, Ralstonia sola It is selected from the group consisting of tomato green blight caused by Nacearum, apple burn blight caused by Erwinia amylobora, and cabbage soft rot caused by Pectobacterium carotovora subspicis carotobora. However, it is not limited to this.
  • composition for controlling plant diseases 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.
  • One aspect of the present invention is a method for producing a composition for controlling plant diseases, comprising a culturing step of preparing a culture solution by culturing the Streptomyces sp. JCK-8055 strain deposited under the accession number KCTC15168BP.
  • the plant disease may be caused by phytopathogenic microorganisms consisting of phytopathogenic fungi and phytopathogenic bacteria.
  • the phytopathogenic fungi include Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (IV), Skrelotinia homoeocarpa, Fusarium graminearum, Fusarium asiaticum, and Pi. It may be one or more species selected from the group consisting of Thophthora infestans and Pycium ultimum, for example, Fusarium asiaticum, but is not limited thereto.
  • the phytopathogenic bacteria include Acidoborax avenae subspecis Cattleya, Acidoborax kojiyasi, Agrobacterium tuberfaciens, Burkolderia glume, Clavibacter misiganensis subspecis misiganensis, Erwinia amylovora, Erwinia pirofoliae, Pectobacterium carotovora subspicis carotovora, Pectobacterium chrysantemi, Pseudomonas syringae pasova latrimans, Ralstonia solanacearum It may be one or more species selected from the group consisting of Xanthomonas arboricola Passoba prunii, and Xanthomonas uvejicatoria, for example, Erwinia amylovora, Pectobacterium carotovora subspicis caro It may be Tobora, and Ralstonia solanacearum, but
  • the plant disease may be selected from the group consisting of red mold disease, green blight, burn disease, and soft rot, for example, rice red mold disease caused by Fusarium asiaticum, Ralstonia sola It is selected from the group consisting of tomato green blight caused by Nacearum, apple burn blight caused by Erwinia amylobora, and cabbage soft rot caused by Pectobacterium carotovora subspicis carotobora. However, it is not limited to this.
  • the culturing step may be performed under temperature conditions of 10 to 40°C, preferably 10 to 35°C, 10 to 30°C, 15 to 40°C, 15 to 35°C, 15 to 30°C, It may be carried out under temperature conditions of 20 to 40°C, 20 to 35°C, 20 to 30°C, 25 to 40°C, or 25 to 35°C, for example, carried out under temperature conditions of 25 to 30°C. However, it is not limited to this.
  • the culturing step may be performed under conditions of pH 5 to 11, and preferably may be performed under conditions of pH 6 to 10, but is not limited thereto.
  • the culturing step may be performed under medium conditions where NaCl is present at a concentration of 0 to 4%, and preferably may be performed under medium conditions where NaCl is present at a concentration of 0 to 3%. It is not limited.
  • 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 diseases may additionally include a concentration step of concentrating the culture solution, and the concentration step may be performed using a reduced pressure concentrator, but is not limited thereto.
  • the method for producing a composition for controlling plant diseases may additionally include a dilution step of diluting the culture medium.
  • the method for producing a composition for controlling plant diseases may additionally include an extraction step of extracting components within the bacterial cells.
  • the production method additionally includes a fractionation step of obtaining an antibacterial active material by fractionating the culture medium or culture filtrate with one or more solvents selected from the group consisting of butanol, ethyl acetate, chloroform, methanol, ethanol, and hexane. It may be.
  • the fractionation step may include the following steps:
  • the ethyl acetate layer was eluted with a mixed solution of chloroform:methanol with a mixing volume ratio of 80:20 to 100:0 using high performance liquid chromatography (HPLC), and among 131 fractions, 5 showing antibacterial activity was obtained.
  • HPLC high performance liquid chromatography
  • the culture filtrate is adjusted to 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 be obtained by doing so, but is not limited to this.
  • the second fractionation step may be performed by selecting the mixing volume ratio of chloroform:methanol to be 85:15 to 100:0, preferably 90:10 to 100:0, for example For example, it may be performed by selecting 95:5 to 100:0, but is not limited to this.
  • the antibacterial active material may be one or more compounds selected from the group consisting of aureothricin and thiorutin, but is not limited thereto.
  • One aspect of the present invention is a plant disease control method comprising a treatment step of treating the Streptomyces sp. JCK-8055 strain deposited under accession number KCTC15168BP, its culture medium, or its extract.
  • the extract may be one or more selected from the group consisting of culture supernatant, culture filtrate, cell suspension, and fractions from the Streptomyces sp. JCK-8055 strain culture medium.
  • 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.
  • “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 plant disease may be caused by phytopathogenic microorganisms consisting of phytopathogenic fungi and phytopathogenic bacteria.
  • the phytopathogenic fungi include Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (IV), Skrelotinia homoeocarpa, Fusarium graminearum, Fusarium asiaticum, and Pi. It may be one or more species selected from the group consisting of Thophthora infestans and Pycium ultimum, for example, Fusarium asiaticum, but is not limited thereto.
  • the phytopathogenic bacteria include Acidoborax avenae subspecis Cattleya, Acidoborax kojiyasi, Agrobacterium tuberfaciens, Burkolderia glume, Clavibacter misiganensis subspecis misiganensis, Erwinia amylovora, Erwinia pirofoliae, Pectobacterium carotovora subspicis carotovora, Pectobacterium chrysantemi, Pseudomonas syringae pasova latrimans, Ralstonia solanacearum It may be one or more species selected from the group consisting of Xanthomonas arboricola Passoba pruni, and Xanthomonas euvejicatoria, for example, Erwinia amylovora, Pectobacterium carotovora subspicis caro It may be Tobora, and Ralstonia solanacearum, but
  • the plant disease may be selected from the group consisting of red mold disease, green blight, burn disease, and soft rot, for example, rice red mold disease caused by Fusarium asiaticum, Ralstonia sola It is selected from the group consisting of tomato green blight caused by Nacearum, apple burn blight caused by Erwinia amylobora, and cabbage soft rot caused by Pectobacterium carotovora subspicis carotobora. However, it is not limited to this.
  • One aspect of the present invention is a method for controlling plant diseases comprising a treatment step of treating at least one compound selected from the group consisting of aureothricin and thiorutin.
  • 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.
  • the plant disease may be caused by phytopathogenic microorganisms consisting of phytopathogenic fungi and phytopathogenic bacteria.
  • the phytopathogenic fungi include Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (IV), Skrelotinia homoeocarpa, Fusarium graminearum, Fusarium asiaticum, and Pi. It may be one or more species selected from the group consisting of Thophthora infestans and Pycium ultimum, for example, Fusarium asiaticum, but is not limited thereto.
  • the phytopathogenic bacteria include Acidoborax avenae subspecis Cattleya, Acidoborax kojiyasi, Agrobacterium tuberfaciens, Burkolderia glume, Clavibacter misiganensis subspecis misiganensis, Erwinia amylovora, Erwinia pirofoliae, Pectobacterium carotovora subspicis carotovora, Pectobacterium chrysantemi, Pseudomonas syringae pasova latrimans, Ralstonia solanacearum It may be one or more species selected from the group consisting of Xanthomonas arboricola Passoba prunii, and Xanthomonas uvejicatoria, for example, Erwinia amylovora, Pectobacterium carotovora subspicis caro It may be Tobora, and Ralstonia solanacearum, but
  • the plant disease may be selected from the group consisting of red mold disease, green blight, burn disease, and soft rot, for example, rice red mold disease caused by Fusarium asiaticum, Ralstonia sola It is selected from the group consisting of tomato green blight caused by Nacearum, apple burn blight caused by Erwinia amylobora, and cabbage soft rot caused by Pectobacterium carotovora subspicis carotobora. However, it is not limited to this.
  • the present invention relates to a composition for controlling plant diseases comprising the Streptomyces sp. JCK-8055 strain, its culture medium or extract thereof, a method for producing the same, and a control method using the same.
  • Figure 1 is a schematic diagram analyzed by 16S rRNA and gyrB gene nucleotide sequences of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figure 2 shows various media (1: ISP1; 2: ISP2; 3: ISP3; 4: ISP4; 5: ISP5; 6: ISP1; 7: Marine agar; 8: Bennet; 9: PDA) according to an embodiment of the present invention.
  • 10 This is a photograph showing the color of the colony of Streptomyces sp. JCK-8055 strain cultured in TSA).
  • Figure 3 is a flowchart showing the process of fractionation of antibacterial active substances of Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figures 4a and 4b are graphs showing the results of high performance liquid chromatography (HPLC) and UV spectrum analysis of the antibacterial active substance SYF1 of Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figures 5a and 5b are graphs showing the results of HPLC and UV spectrum analysis of the antibacterial active substance SYF2 of Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figures 6a and 6b show the results of LC-MS analysis of the antibacterial active substance SYF1 of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figures 7a and 7b show the results of LC-MS analysis of the antibacterial active substance SYF2 of Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figure 8 shows the treatment of Streptomyces sp. JCK-8055 strain and antibacterial active substances 1 day before inoculation (DBI) and 1 day after inoculation (DAI) with pathogens according to an embodiment of the present invention. This is a graph showing the effect of controlling tomato green blight.
  • Figure 9 is a photograph showing the effect of controlling tomato green blight according to treatment with the Streptomyces sp JCK-8055 strain and antibacterial active material 1 day before and 1 day after pathogen inoculation according to an embodiment of the present invention, 7 days after pathogen inoculation.
  • Figure 10 shows the effect of controlling apple burn disease according to the treatment of powdered wettable powder (JCK-8055-EtOAc WP 10%) prepared using the Streptomyces sp JCK-8055 strain 1 day before pathogen inoculation according to an embodiment of the present invention. This is a graph showing .
  • Figure 11 is a photograph confirming the effect of controlling apple burn disease 14 days after pathogen inoculation according to treatment of powdered moisturizer prepared using Streptomyces sp JCK-8055 strain 1 day before pathogen inoculation according to an embodiment of the present invention.
  • Figure 12 is a graph showing the effect of controlling tomato green blight according to the spraying method of the Streptomyces sp. JCK-8055 strain culture filtrate according to an embodiment of the present invention.
  • Figure 13 is a photograph showing the effect of controlling tomato green blight according to the spraying method of the culture filtrate of Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention 7 days after inoculation with the pathogen.
  • Figure 14 is a graph showing the effect of controlling apple burn disease according to the spraying method of the Streptomyces sp. JCK-8055 strain culture filtrate according to an embodiment of the present invention.
  • Figure 15 is a photograph showing the apple burn disease control effect according to the spraying method of the Streptomyces sp JCK-8055 strain culture filtrate according to an embodiment of the present invention 14 days after pathogen inoculation.
  • Figure 16 is a photograph showing the PR-1 gene expression inducing activity of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figure 17 shows the effect of controlling apple burn disease due to induced resistance by treating the powdered wettable powder prepared using the Streptomyces sp JCK-8055 strain 10 days and 3 days before pathogen inoculation twice according to an embodiment of the present invention. This is the graph shown.
  • Figure 18 shows the effect of controlling apple burn disease by induced resistance following two treatments of powdered wettable powder prepared using the Streptomyces sp JCK-8055 strain 10 days and 3 days before pathogen inoculation according to an embodiment of the present invention. This photo was taken 14 days after inoculation with the pathogen.
  • Figure 19 is a graph showing the effect of controlling rice red mold disease by induced resistance of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figure 20 is a photograph showing the effect of controlling rice red mold disease by induced resistance of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention two weeks after inoculation with the pathogen.
  • Figure 21 is a graph showing the effect of controlling tomato green blight by seed coating of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figure 22 is a photograph showing the effect of controlling tomato green blight by seed coating of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention 7 days after inoculation with the pathogen.
  • Figure 23 is a graph showing the effect of controlling cabbage soft rot by seed coating of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention.
  • Figure 24 is a photograph showing the effect of controlling cabbage soft rot by seed coating of the Streptomyces sp. JCK-8055 strain according to an embodiment of the present invention 8 days after inoculation with the pathogen.
  • the present invention relates to the Streptomyces sp. JCK-8055 strain deposited under accession number KCTC15168BP, which has plant disease control activity.
  • % used to indicate the concentration of a specific substance means (weight/volume)% for solid/solid, (weight/volume)% for solid/liquid, and Liquid/liquid is (volume/volume)%.
  • a strain with control activity against plant pests was isolated from healthy pepper roots adjacent to a frequent pepper blight area in Daejeon. To remove rhizosphere soil from the roots of collected plants, they were washed with water and then surface sterilized with 70% ethanol (EtOH) for 5 minutes and 1.4% bleach solution (NaOCl) for 20 minutes. To neutralize the bleach solution, the surface was sterilized with 2% Na 2 S. 2 O 3 was treated.
  • the neutralized root sample was washed several times with sterilized water and triturated in Phosphate Buffered Saline (pH 7.0).
  • the root samples of the ground plants were diluted 10-fold and then mixed with Tryptic Soy Agar (TSA, pancreatic digest of casein 2 g, papaic digest of soybean 5 g, sodium chlroride 5 g, agar 15 g, pH 7.3). -7.5)
  • TSA Tryptic Soy Agar
  • the strain was isolated by smearing on a plate and culturing at 30°C for more than 3 to 7 days.
  • Strain JCK-8055 isolated from plant roots through the present invention was identified molecularly through nucleotide sequence analysis of 16S rRNA and gyrB gene.
  • the strain was plated on yeast extract malt extract agar (IPS2) and cultured with shaking at 180 rpm for 7 days at 28°C. Then, the genomic DNA (gDNA) of the harvested strain was extracted according to the protocol using the I-genomic BYF DNA Extraction Mini kit (iNtRON, Korea). did.
  • gDNA of the strain and PCR-premix (Polymerase chain reaction-premix) from iNtRON Biotechnology, and 16S rRNA (SEQ ID NO: 1/SEQ ID NO: 2) and gyrB (SEQ ID NO: 3/SEQ ID NO: 4) of the strain. After mixing primer sets that can amplify, the gene was amplified through PCR.
  • PCR-premix Polymerase chain reaction-premix
  • PCR started at 95°C for 5 minutes, repeated 30 times at 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 90 seconds, and then amplified at 72°C for 10 minutes and ended at 4°C.
  • the amplified 16S rRNA and gyrB gene PCR products were submitted to Genotech (Daejeon, Korea) for base sequence analysis, and the 16S rRNA and gyrB coding base sequences of the isolated strain, JCK-8055, were obtained with a total of 1,392 bp (SEQ ID NO. 5).
  • a nucleotide sequence of 1,224 bp was obtained.
  • strain Streptomyces sp. JCK-8055 and deposited in KCTC (Korean Collection for Type Cultures) on November 2, 2022, and given accession number KCTC15168BP.
  • the species specificity of bacteria in the genus Streptomyces is determined by the pigment produced, and the biosynthetic process of this pigment is known to be correlated with antibiotic biosynthesis.
  • ISP1 (3 g yeast extract, 5 g tryptone, 20 g agar, pH 7.0-7.2 per 1 L),
  • ISP2 (4 g yeast extract, 10 g malt extract, 4 g dextrose, 20 g agar, pH 7.0-7.2 per 1 L),
  • ISP3 (oatmeal 20 g, ferric sulphate heptahydrate 0.001 g, manganses chloride tetrahydrate 0.001 g, zinc sulphate heptahydrate 0.001 g, agar 20 g, pH 7.3-7.5 per liter),
  • ISP4 (10 g of soluble starch per 1 L, 1 g of K 2 HPO 4 , 1 g of MgSO 4 . 7H 2 O, 1 g of NaCl, 2 g of (NH 4 ) 2 SO 4 , 2 g of CaCO 3 , 1 mL of trace salt solution , agar 20 g, pH 7.2-7.4),
  • ISP5 (1 g of L-asparagine, 1 g of K 2 HPO 4 , 0.001 g of ferric sulphate heptahydrate, 0.001 g of manganses chloride tetrahydrate, 0.001 g of zinc sulphate heptahydrate, 20 g of agar, pH 7.4-7.6 per L),
  • ISP6 per 1 L, peptone 15 g, proteose peptone 5 g, yeast extract 1 g, ferric ammonium citrate 0.5 g, K 2 HPO 4 1 g, Na 2 S 2 O 3 0.08 g, agar 20 g, pH 7.2-7.4
  • marine agar peptone 5 g, yeast extract 1 g, ferric citrate 0.1 g, sodium chlroride 19.45 g, magnesium chloride 5.9 g, magnesium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium bicarbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 34 mg, boric acid 22 mg, sodium silicate 4 mg, sodium fluoride 2.4 mg, ammonium nitrate 1.6 mg, disodium phosphate 8 mg, agar 20 g, pH 7.6-7.8),
  • Bennet (beef extract 1 g, glucose 10 g, enzyme digest of casein 2 g, yeast extract 1 g, pH 7.3-7.5),
  • PDA potato starch 4 g, dextrose 20 g, agar 15 g, pH 5.6-5.8
  • TSA pancreatic digest of casein 2 g, papaic digest of soybean 5 g, sodium chlroride 5 g, agar 15 g, pH 7.3-7.5
  • TSB medium pancreatic digest of casein 17 g, papaic digest of soybean 3 g, sodium chlroride 5 g, dextrose 2.5 g, dipotassium phosphate 2.5
  • base medium g, pH 7.3-7.5
  • protease, amylase, gelatinase, and cellulase of the Streptomyces sp. JCK-8055 strain were measured.
  • the activity of the proteolytic enzyme protease was determined by inoculating the Streptomyces sp. JCK-8055 strain in a medium supplemented with 1% skim milk and 1.5% agar and then culturing to determine the presence or absence of a clear zone. Measured.
  • Amylase activity was measured in medium supplemented with 2% starch and 1.5% agar, gelatinase activity was measured in medium supplemented with 10% gelatin and 1.5% agar, and cellulase activity was measured in medium supplemented with 0.4% carboxymethylcellulose (CMC). and Streptomyces sp. JCK-8055 strains were inoculated and cultured in media supplemented with 1.5% agar, respectively, and the presence or absence of growth inhibitory rings was measured.
  • CMC carboxymethylcellulose
  • the Streptomyces sp. JCK-8055 strain was confirmed to possess protease protease, gelatinase, and cellulase activities excluding amylase.
  • Example 4 Isolation and structural identification of antibacterial active substances of Streptomyces sp. JCK-8055 strain
  • the strain was treated with ISP2 (4 g of yeast extract, 10 g of malt extract, 4 g of dextrose, 20 g of agar, pH 7.0-7.2 per 1 L). After inoculation into the liquid medium, culture was performed with shaking at 180 rpm for 7 days at 28°C. The isolation process of the antibacterial active substance produced by the Streptomyces sp. JCK-8055 strain was performed as shown in FIG. 3.
  • the culture solution (total of 2 L) was filtered through 4 layers of sterilized gauze to obtain a culture filtrate.
  • the culture filtrate (1.6 L) was fractionated twice with equal amounts of ethyl acetate (EtOAc) and butanol (BuOH) to obtain the organic solvent layer and aqueous solution layer of the ethyl acetate layer and the butanol layer, then concentrated under reduced pressure and each ethyl acetate layer.
  • the concentrate, butanol layer concentrate, and aqueous layer concentrate were re-dissolved in acetone, methanol, and water.
  • the separated materials were divided into groups with similar patterns, and the antibacterial activity fraction was retested using tested pathogens.
  • the yellow fraction that showed activity was separated using a Sephadex LH20 column using 100% methanol as a solvent, and among the separated fractions, the fraction with antibacterial activity was purified by prep-TLC.
  • antibacterial active substances SYF1 and SYF2 were selected.
  • the elution solvent at this time was distilled water to which 0.1% trifluoroacetic acid (TFA) was added and acetonitrile to which 0.1% trifluoroacetic acid was added, and 0.1% trifluoroacetic acid was used at a flow rate of 1 ml/min.
  • the content of acetonitrile to which fluoroacetic acid was added was given a gradient from 20% to 100% and developed for 30 minutes.
  • UV spectra were calibrated at 360 nm. As a result, it was confirmed that the antibacterial active substances SYF1 and SYF2 were pure.
  • SYF1 was detected at a retention time of 10.68 minutes
  • SYF2 was detected at a retention time of 9.88 minutes.
  • Example 5 Measurement of minimum inhibitory concentration of plant pathogenic fungi or bacteria according to treatment with antibacterial active material isolated from Streptomyces sp JCK-8055 strain culture filtrate
  • the minimum concentration of inhibition of mycelial growth of plant pathogens was measured using the culture filtrate of the Streptomyces sp. JCK-8055 strain of the present invention and the antibacterial active substance isolated from the JCK-8055 strain.
  • the JCK-8055 strain was inoculated into TSB liquid medium and then cultured at 28°C with shaking at 180 rpm for 7 days. The culture was centrifuged at 8,000 Samples were treated with plant pathogen mycelia at concentrations of 0.16, 0.32, 0.63, 1.25, 2.5, 5, and 10%.
  • the antibacterial active substances SYF1 and SYF2 isolated from strain JCK-8055 were treated with plant pathogen mycelium at a concentration of 0.2-200 ⁇ g/mL.
  • the plant pathogen mycelium used in this example was prepared by adjusting the mycelium cultured for 7 days at 25°C in PDB medium to a concentration of 500 ⁇ g/mL (w/v) and grinding using a blender (DAIHAN, HG-15D). did. 180 ⁇ L of ground mycelium was added to each well of a 96-well plate, and then 20 ⁇ L of sample was dispensed thereto to treat the sample at a concentration of 10%. Then, 100 ⁇ L was taken from the well treated at a concentration of 10%, dispensed into the next well, and mixed with 100 ⁇ L of ground mycelia to prepare a concentration of 5%. 2.5% to 0.16% was also diluted in the same manner as above. The degree of mycelial growth was measured after 24 hours at 25°C, and the experiment was repeated three times for each treatment group.
  • the culture filtrate of the Streptomyces sp. JCK-8055 strain was used in various plants such as Rhizoctonia solani AG 2-2, Rhizoctonia solani AG-4, Sclerotinia homoeocarpa , Fusarium graminearum , Phytophthora infestans , and Pythium ultimum .
  • the substances (SYF1-aureothricin, SYF2-thiorutin) showed excellent antifungal activity with MIC values ranging from 0.78 to 50 ⁇ g/mL against all plant pathogenic fungi used in the test.
  • the minimum growth inhibition concentration of plant pathogenic bacteria was measured using the culture filtrate of the Streptomyces sp. JCK-8055 strain of the present invention and the antibacterial active substance isolated from the JCK-8055 strain.
  • Acidovorax avenae subsp. cattleyae Acidovorax kojiaci , Agrobacterium tumerfaciens , Burkholderia glumae , Clavibacter michiganesis subsp. michiganensi , two species of Erwinia amylovora , Erwinia pyrifoliae , and Pectobacterium carotovorum subsp.
  • a single colony of the pathogen grown on tryptic soy agar (TSA, Becton, Dickinson and Co., Sparks, MD, USA) was inoculated on TSB (tryptic soy broth, Becton, Dickinson and Co., Sparks, MD, USA) medium. Cultured with shaking at 30°C and 150 rpm for 1-3 days. Afterwards, the optical density of the pathogen culture was measured at 600 nm using a UV-VIS spectrophotometer, and the OD 600 value was adjusted to 0.1 (1.0
  • the adjusted bacterial suspension was injected into each well of a 96 well plate, and the final concentration of the JCK-8055 strain culture filtrate was 10, 5, 2.5, 1.25, and 0.625%.
  • the antibacterial active substances SYF1 and SYF2 isolated from the JCK-8055 strain and streptomycin sulfate as a positive control were used at a concentration of 0.2-200 ⁇ g/mL, and an untreated group treated with only the bacterial suspension was used as a negative control. did. All plates were cultured in an incubator at 30°C to examine bacterial growth, and the experiment was repeated three times each.
  • Pectobacterium carotovorum subsp. carotovorum and Ralstonia solanacearum For all plant pathogenic bacteria used in the experiment except for the two antibacterial active substances (SYF1-aureothricin, SYF2-thiorutin) isolated from the JCK-8055 strain, MIC values were lower than that of streptomycin sulfate, a positive control. Therefore, it showed high antibacterial activity at low concentrations.
  • the antibacterial active substance isolated from the JCK-8055 strain can be effectively used in the development of a control agent for plant pathogenic bacteria.
  • Example 6 Control effect of Streptomyces sp JCK-8055 strain and active substance fraction
  • Tomato Samgwang Seed Lycopersicon esculentum Mill. cv. Seokwang, Farhannong Co., Seoul, Korea
  • the square connection pot was filled with topsoil, and tomato seeds were sown using tweezers and grown for 4 weeks in a constant temperature and humidity room at 25°C under a photoperiod of 16 hours per day.
  • the plants used for green blight were transplanted into pots (7 cm in diameter, 6 cm in height) using top soil.
  • the Ralstonia solanacearum strain the causative agent of tomato green blight
  • TSA medium the causative agent of tomato green blight
  • Colonies of cultured R. solanacearum were harvested in 50 mL conical tubes using distilled water and a cell scraper.
  • the optical density was measured at 600 nm using a UV-VIS spectrophotometer and the OD 600 value was adjusted to 0.1 (1.0
  • the JCK-8055 strain was inoculated into TSB liquid medium, and then Cultured at °C for 7 days with shaking at 180 rpm. The culture was centrifuged at 8,000
  • the JCK-8055 strain culture filtrate treatment group (CF) was treated with 5 and 10 times the JCK-8055 strain culture filtrate in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea) on Seogwang tomato seedlings 4 weeks after sowing. , was diluted 20 times and irrigated into the soil at 20 mL per pot one day before and one day after inoculation with the pathogen.
  • the active substance fraction treatment group (SYF) the active substance fraction containing aureothricin and thiorutin isolated from the JCK-8055 strain culture was diluted to concentrations of 20 ⁇ g/mL and 100 ⁇ g/mL in the same manner and administered per port. The soil was irrigated with 20 mL each.
  • Buranmycin (a.i. Streptomycin sulfate 20% WP, Farm Hannon) was diluted 1,000 times and used in the same manner.
  • Samples and control agents were used as soil irrigation at 20 mL per pot 1 day before inoculation (DBI) or 1 day after inoculation (DAI). The purpose was to confirm the preventive effect by treating the sample and control drug 1 day before pathogen inoculation, and to see the therapeutic effect by treating the sample and control drug 1 day after pathogen inoculation.
  • DBI 1 day before inoculation
  • DAI 1 day after inoculation
  • Disease index consists of 6 levels from 0 to 5 depending on the severity of symptoms (disease index, 0: no symptoms; 1: 1-2 leaves wilted; 2: 3-4 leaves wilted; 3: 5-6 leaves)
  • the control effect was calculated based on the evaluation (4: wilting of most leaves; 5: death).
  • the control value (%) compared to the untreated group was calculated according to the following equation.
  • control values were 27.27%, 41.41%, and 42.42%, respectively, compared to the untreated group.
  • active substance fraction (SYF) containing two types of active substances was diluted to concentrations of 20 ⁇ g/mL and 100 ⁇ g/mL and treated 1 day after pathogen inoculation, control values were 54.54% and 72.73%, respectively, compared to the untreated group. It showed a concentration-dependent tomato green blight control effect and showed a superior tomato green blight treatment effect than the positive control buramycin treatment (3.03% control), which had almost no treatment effect.
  • both the JCK-8055 strain culture filtrate and the active substance fraction (SYF) containing two active substances isolated from the JCK-8055 strain culture showed excellent preventive effects when treated 1 day before pathogen inoculation and therapeutic effects 1 day after pathogen inoculation. It was confirmed that buramycin, which was used as a reference drug, had a preventive effect, but had almost no therapeutic effect. Therefore, it was confirmed that it is possible to develop an effective tomato green blight control agent in the future by using the antibacterial activity of the JCK-8055 strain.
  • Apple seedlings (M9, height 15 ⁇ 5 cm) were used to investigate the in vivo control activity of the Streptomyces sp. JCK-8055 strain and active substance fraction of the present invention against apple burn disease. 5 mL of the sample was applied as a foliar spray to the leaves of apple seedlings, and then apple burn pathogen ( Erwinia amylovora TS3128, 5 mL) was inoculated into the seedlings with the sample pre-treated.
  • apple burn pathogen Erwinia amylovora TS3128, 5 mL
  • the chemical pesticide Agrepto (a.i.: Streptomycin 20% WP, Kyungnong) was diluted 2,000 times the commercial concentration and sprayed on the leaves at 5 mL per seedling 1 day before inoculation with the apple blight pathogen.
  • the inoculated plants were covered with a plastic cover to maintain 100% relative humidity, placed in a constant temperature and humidity room at 25°C, maintained in dark conditions for 2 days, and then examined for disease severity while maintaining a 12-hour photoperiod and 75% relative humidity. . There were 3 repetitions for each treatment, and each repetition consisted of 3 ports, and the same experiment was repeated 3 times to evaluate efficacy.
  • the disease index was divided and tested as follows (index 0: no symptoms; index 1: partial necrosis of the tip area, which is the stem growth meristem; index 2: complete necrosis of the tip area; index 5: petiole necrosis of terminal leaves; index 10: Petiole necrosis of leaves and stems).
  • Control value (%) (virulence rate in untreated group - virulence rate in treated group) / virulence rate in untreated group ⁇ 100%.
  • Example 7 Control effect of foliar spray and soil drench treatment of Streptomyces sp. JCK-8055 strain
  • the square connection port was filled with topsoil, and tomato seeds ( Lycopersicon esculentum Mill. cv. Seokwang, Farhannong Co. ., Seoul, Korea) were sown and grown for 4 weeks in a constant temperature and humidity room at 25°C under a photoperiod of 16 hours per day. Afterwards, the plants used for green blight were transplanted into pots (7 cm in diameter, 6 cm in height) using top soil.
  • Tomato green blight pathogen inoculum and culture filtrate of JCK-8055 strain were prepared in the same manner as in Example 6-1.
  • the culture filtrate (CF) of the JCK-8055 strain was diluted 250-, 500-, and 1,000-fold on Seogwang tomato seedlings 4 weeks after sowing, and in the case of soil irrigation (SD), 20 mL each per pot. , in the case of foliar spray (FS), 5 mL of diluted culture filtrate was treated per pot.
  • SD soil irrigation
  • FS foliar spray
  • the JCK-8055 strain culture filtrate showed the highest control effect in soil drench (SD) treatment at a 500-fold dilution, and based on this, it was confirmed that the JCK-8055 strain showed an excellent control effect against tomato green blight.
  • tomato green blight was controlled more effectively by inducing resistance in tomatoes through pretreatment of the samples. Therefore, it was confirmed that it would be possible to develop an effective tomato green blight control agent in the future by using the resistance-inducing activity of the JCK-8055 strain.
  • Apple seedlings (M9, height 15 ⁇ 5 cm) were used to investigate the in vivo control activity of apple burn disease by the treatment method of the Streptomyces sp. JCK-8055 strain of the present invention. 5 mL of the sample was applied to the leaves of apple seedlings as a foliar spray or 20 mL was injected into the soil, and then the apple blight pathogen ( Erwinia amylovora TS3128, 5 mL) was inoculated by foliar spray on the seedlings that had been pre-treated with the sample.
  • the apple blight pathogen Erwinia amylovora TS3128, 5 mL
  • the JCK-8055 strain was inoculated into TSB liquid medium and shaken at 180 rpm for 7 days at 28°C. Cultured. The culture was centrifuged at 8,000 The JCK-8055 strain culture filtrate (CF) was diluted 250 times in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea) and applied at 20 mL per pot for soil drench (SD) per apple seedling and foliar spray ( In the case of FS), 5 mL of equally diluted culture filtrate was treated per pot.
  • Apple seedlings were treated with samples twice, and 3 days after the second sample treatment, 5 mL of E. amylovora TS3128 bacterial suspension (OD 600 0.3, 3.0 It was inoculated by spraying.
  • the inoculated plants were covered with a plastic cover to maintain 100% relative humidity, placed in a constant temperature and humidity room at 25°C, maintained in dark conditions for 2 days, and then examined for disease severity while maintaining a 12-hour photoperiod and 75% relative humidity. .
  • the co-occurrence index was divided by 10.
  • the disease prevention value was analyzed in the same manner as in Example 6-2.
  • the JCK-8055 strain showed an excellent control effect against apple burn disease.
  • apple burn disease was controlled more effectively by inducing resistance in apples through pretreatment of the samples. Therefore, it was confirmed that it would be possible to develop an apple burn disease control agent with excellent efficacy by using the resistance-inducing activity of the JCK-8055 strain in the future.
  • Example 8 Resistance induction activity assay of JCK-8055 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 the Streptomyces sp. JCK-8055 strain 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 a second surface sterilization was performed using a bleach solution prepared with 2% NaOCl and 0.05% Tween-20.
  • the bleach solution remaining on the seeds was washed using sterilized 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.
  • each well of a 24-well plate was treated with 2.5 mL of the culture medium of the Streptomyces sp. JCK-8055 strain of the present invention, diluted 100, 500, and 1,000 times in sterilized water, and 2 Arabidopsis plants were submerged in each well. and kept at room temperature for 48 hours in an orbital shaker. After 48 hours, to fix the reaction, it was immersed in 90% acetone at -20°C for 1 hour and washed twice with 0.1 M sodium phosphate buffer (pH 7.0).
  • a staining solution 100mM Sodium phosphate buffer, 0.1 % Triton X-100, 2mM It was kept in a water tank for 24 hours.
  • the dyeing 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 was tested through the Arabidopsis thaliana assay system.
  • SA salicylic acid
  • streptoma Expression of GUS in the culture medium of the Seth Esp JCK-8055 strain strongly induced the expression of the PR-1 gene.
  • Streptomyces sp. JCK-8055 strain can control pathogenic bacteria by inducing resistance in plants through a mechanism similar to salicylic acid.
  • JCK-8055 CB- a powdered moisturizer
  • JCK-8055 strain preparation Treatment of the JCK-8055 strain preparation was performed by foliar spraying apple seedlings about 15 cm in height with the JCK-8055 preparation diluted 1,000 times, 5 mL per seedling, twice, 10 days and 3 days before inoculation with the pathogen.
  • the biological pesticide Serifel AI: Bacillus amyloliquefaciens MBI600 (11%) WP, Farm Hannong
  • Foliar spray treatment was applied. Three days after the secondary treatment with the sample and control agent, 5 mL of E. amylovora TS3128 bacterial suspension (OD 600 0.3, 3.0
  • the inoculated plants were covered with a plastic cover to maintain 100% relative humidity, placed in a constant temperature and humidity room at 25°C, maintained in dark conditions for 2 days, and then examined for disease severity while maintaining a 12-hour photoperiod and 75% relative humidity. . There were 3 repetitions for each treatment, and each repetition consisted of 3 ports, and the same experiment was repeated 3 times to evaluate efficacy. The degree of co-occurrence was analyzed in the same manner as in Example 6-2.
  • Samkwang rice Oryza sativa cv. Samkwang seeds were used to investigate the in vivo control activity of rice red mold disease by induced resistance of the JCK-8055 strain of the present invention.
  • 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). After transplanting, 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.
  • the pathogen Fusarium asiaticum the causative agent of rice red mold disease
  • PDA medium was inoculated into PDA medium and cultured for 6 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 grown at a concentration of 5 Adjusted.
  • 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-8055 strain culture filtrate was diluted 1,000 times 2 weeks before and 1 week before pathogen inoculation on 11-week-old rice plants after pot transplantation, and foliar spray was applied to saturate the rice ears and stems.
  • the JCK-8055 strain culture filtrate was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea).
  • the control drug was Play (Peulrei, ai 13% Difenoconazole + 13% Propiconazole EC, Syngenta Korea), a publicly available drug for rice red mold disease, 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 (5 F. asiaticum spore suspension was inoculated by adding Tween-20, a spreader, at a level of 500 ⁇ 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. Two weeks 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.
  • strain JCK-8055 effectively controls rice red mold disease through induced resistance. 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-8055 strain.
  • Example 9 Control effect by seed coating of Streptomyces sp. JCK-8055 strain
  • the JCK-8055 strain was inoculated into TSB medium and cultured with shaking at 28°C and 180 rpm for 7 days, followed by 4,000 rpm.
  • Cell fraction was obtained from the sediment by centrifugation at rpm for 10 minutes. After suspending by adding sterilized water equal to the volume of the obtained cell fraction, a cell suspension to be used for seed coating was prepared.
  • Seed coating by the JCK-8055 strain was done by soaking tomato seeds ( Lycopersicon esculentum Mill. cv. Seokwang, Farhannong Co., Seoul, Korea) for 24 hours using the prepared cell suspension of the JCK-8055 strain.
  • Coated and untreated 'Seogwang' seeds were immersed in filter paper (9 cm diameter Petri dish) containing distilled water and germinated in an incubator at 28°C for 24 hours, then placed in a long horticultural medium in a plastic pot (6 cm diameter, Tomato seeds were sown in a container with a height of 6.5 cm) and grown for 4 weeks in a constant temperature and humidity room at 25°C under a photoperiod of 16 hours per day. Afterwards, the plants used for green blight were transplanted into pots (7 cm in diameter, 6 cm in height) using top soil.
  • the Ralstonia solanacearum strain the causative agent of tomato green blight
  • TSA medium the causative agent of tomato green blight
  • Colonies of cultured R. solanacearum were harvested in 50 mL conical tubes using distilled water and a cell scraper.
  • the optical density was measured at 600 nm using a UV-VIS spectrophotometer and the OD 600 value was adjusted to 0.1 (1.0 x 10 CFU/mL), and the prepared suspension was used as an inoculum.
  • the inoculated pot was placed in a yellow tray with a closed bottom, the tray was filled with water, and the photoperiod was irradiated for 12 hours a day in a constant temperature and humidity room at 30°C.
  • the experiment was repeated 3 times, 3 per treatment group, and symptoms were confirmed 9 days after inoculation to investigate the degree of disease.
  • the severity of disease was evaluated in the same manner as in Example 6-1.
  • the JCK-8055 strain was treated with TSB. After inoculation into the medium, the cells were cultured with shaking at 28°C and 180 rpm for 7 days, and then centrifuged at 4,000 rpm for 10 minutes to obtain cell fractions from the sediment. After suspending by adding sterilized water equal to the volume of the obtained cell fraction, a cell suspension to be used for seed coating was prepared.
  • Seed coating by strain JCK-8055 was coated by soaking cabbage 'Chunkwang' seeds ( Brassica rapa subsp. pekinensis L. cv. Chunkwnag, Sacada Korea) for 24 hours using the prepared bacterial suspension of strain JCK-8055. Coated and untreated 'Chungwang' seeds were immersed in filter paper (9 cm diameter Petri dish) containing distilled water and germinated in an incubator at 28°C for 24 hours. Then, the long horticultural medium was placed in a plastic pot (6 cm diameter, Cabbage seeds were sown in a container with a height of 6.5 cm) and grown in a constant temperature and humidity room at 25°C with irradiated light for 16 hours a day.
  • Cabbages at the 5- or 6-leaf stage were transplanted into plastic cups with a diameter of 7.5 cm 24 hours before treatment.
  • cabbage grown by sowing untreated seeds was used and inoculated with pathogens
  • a premium pesticide Ilpum, ai. oxolinic acid 20% WP, Dongbang Agro
  • Soil irrigation was performed at 20 mL each. All treatments were inoculated with cabbage soft rot pathogen 1 day after the positive control treatment.
  • colonies of the pathogen Pectobacterium Cerotoborium subspecies Cerotoborium cultured for 3 days in TSA medium were harvested with sterilized water to prepare a bacterial suspension, and 600% using a spectrophotometer (Bio-Rad).
  • the OD value in nm was adjusted to 0.1 (107 CFU/ml), and then the bacterial suspension containing 10 mM magnesium chloride was inoculated into the soil at a rate of 20 mL per pot.
  • the inoculated plants were placed in a constant temperature and humidity room at 30°C and maintained in dark conditions for 24 hours. Afterwards, a 12-hour photoperiod and 100% relative humidity were maintained, and the degree of disease was examined 8 days later.
  • the severity of disease is an index from 0 to 5 (0: no symptoms, 1: one or two thin lesions, 2: two or more thin lesions, 3: bleaching of leaves) occurrence, 4: leaf necrosis occurred, 5: complete death), and the control value (%) was calculated compared to the untreated group.
  • the present invention relates to a composition for controlling plant diseases containing a culture medium of the Streptomyces sp. JCK-8055 strain or an extract thereof, a method for producing the same, and a method for controlling plant diseases using the same. More specifically, it relates to a composition for controlling plant diseases containing a culture medium of the Streptomyces sp. JCK-8055 strain or an extract thereof, a method for producing the same, and a method for controlling plant diseases using the same.
  • This relates to plant disease control technology using aureothricin and thiolutin, which are antibacterial active ingredients isolated from the JCK-8055 strain and its culture medium.

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Abstract

La présente invention concerne une souche Streptomyces sp. JCK-8055, une composition pour la lutte contre les maladies des plantes, contenant un bouillon de culture de la souche ou son extrait, un procédé de préparation correspondant et un procédé de lutte les utilisant. Il a été découvert que la composition contenant le bouillon de culture de la souche et la substance active antimicrobienne isolée à partir de celle-ci présente une excellente activité pour lutter contre les maladies et induire une résistance, et peut donc être utilisée comme bioagent respectueux de l'environnement pour le prétraitement des plantes ou comme agent d'enrobage des semences.
PCT/KR2023/014816 2022-11-25 2023-09-26 Composition pour lutter contre les maladies des plantes, comprenant un bouillon de culture de la souche streptomyces sp. jck-8055 ou son extrait, son procédé de préparation et le procédé pour lutter contre les maladies les utilisant WO2024111857A1 (fr)

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KR1020220160448A KR20240094098A (ko) 2022-11-25 스트렙토마이세스 에스피 jck-8055 균주의 배양액 또는 이의 추출물을 포함하는 식물병 방제용 조성물, 이의 제조 방법 및 이를 이용한 방제 방법
KR10-2022-0160448 2022-11-25

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100812649B1 (ko) * 2006-07-20 2008-03-13 한국생명공학연구원 식물 병원균에 대한 항균 활성을 갖는 스트렙토마이세스 속ｖｓｖ-12 ｋｃｔｃ１０９３６ｂｐ 및 이를 이용한 식물병원균 방제제
CN115261422A (zh) * 2022-06-30 2022-11-01 塔里木大学 一种新物种来源硫藤黄菌素的制备方法及应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100812649B1 (ko) * 2006-07-20 2008-03-13 한국생명공학연구원 식물 병원균에 대한 항균 활성을 갖는 스트렙토마이세스 속ｖｓｖ-12 ｋｃｔｃ１０９３６ｂｐ 및 이를 이용한 식물병원균 방제제
CN115261422A (zh) * 2022-06-30 2022-11-01 塔里木大学 一种新物种来源硫藤黄菌素的制备方法及应用

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* Cited by examiner, † Cited by third party
Title
KANG MIN-KYOUNG, KIM JONG-HOON; LIU MIN-JIAO; JIN CHUN-ZHI; PARK DONG-JIN; KIM JUNHEON; SUNG BONG-HYUN; KIM CHANG-JIN; SON KWANG-H: "New discovery on the nematode activity of aureothin and alloaureothin isolated from endophytic bacteria Streptomyces sp. AE170020", SCIENTIFIC REPORTS, NATURE PUBLISHING GROUP, US, vol. 12, no. 1, 1 January 2022 (2022-01-01), US , XP093174377, ISSN: 2045-2322, DOI: 10.1038/s41598-022-07879-w *
LE KHANH DUY, KIM JEUN, NGUYEN HOA THI, YU NAN HEE, PARK AE RAN, LEE CHUL WON, KIM JIN-CHEOL: "Streptomyces sp. JCK-6131 Protects Plants Against Bacterial and Fungal Diseases via Two Mechanisms", FRONTIERS IN PLANT SCIENCE, vol. 12, XP093042990, DOI: 10.3389/fpls.2021.726266 *
STURDIKOVA M, PROKSA B, UHRIN D, FUSKA J: "Regulation of biosynthesis of thiolutin and aureothricin in Streptomyces kasugaensis", FOLIA MICROBIOLOGICA., SPRINGER NETHERLANDS, NL, vol. 35, no. 4, 1 August 1990 (1990-08-01), NL , pages 278 - 283, XP009554877, ISSN: 0015-5632, DOI: 10.1007/BF02821279 *

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