WO2024106754A1 - Composition pour lutter contre les maladies fongiques des plantes, les maladies bactériennes ou les maladies provoquées par les nématodes, comprenant le fluide de culture de la souche lysobacter enzymogenes jck-1421 ou son extrait, procédé de préparation, et procédé pour lutter contre les maladies fongiques des plantes, les maladies bactériennes ou les maladies provoquées par les nématodes - Google Patents

Composition pour lutter contre les maladies fongiques des plantes, les maladies bactériennes ou les maladies provoquées par les nématodes, comprenant le fluide de culture de la souche lysobacter enzymogenes jck-1421 ou son extrait, procédé de préparation, et procédé pour lutter contre les maladies fongiques des plantes, les maladies bactériennes ou les maladies provoquées par les nématodes Download PDF

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WO2024106754A1
WO2024106754A1 PCT/KR2023/015634 KR2023015634W WO2024106754A1 WO 2024106754 A1 WO2024106754 A1 WO 2024106754A1 KR 2023015634 W KR2023015634 W KR 2023015634W WO 2024106754 A1 WO2024106754 A1 WO 2024106754A1
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jck
strain
disease
diseases
nematode
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English (en)
Korean (ko)
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김진철
박애란
손지연
유난희
하아름
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전남대학교산학협력단
주식회사 잰153바이오텍
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Publication of WO2024106754A1 publication Critical patent/WO2024106754A1/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
    • 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
    • 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
    • A01P5/00—Nematocides
    • 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

Definitions

  • the present invention relates to a Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases comprising the strain, a culture thereof, or an extract thereof, a method for producing the composition, and the above. It relates to a method for controlling plant fungal diseases, bacterial diseases, or nematode diseases using a composition.
  • Types of biological control against plant pathogens include direct antagonism, direct/indirect antagonism, and indirect antagonism.
  • Direct antagonism includes overlapping parasitism and predation mechanisms by various fungi and bacteria, and direct and indirect antagonism includes control by antibiotics and degradative enzymes.
  • Indirect antagonism includes competition and induced resistance mechanisms.
  • induced resistance is a phenomenon in which the host plant's resistance to pathogen invasion is induced by specific biotic or abiotic factors. It improves the plant's defense ability against microbial invasion or similar stress, and once expressed, is maintained for a long period of time. there is. Recently, interest in induced resistance mechanisms has increased in terms of improving agricultural ecosystems and biological control of various plant diseases.
  • Lysobacter enzymogenes is a Gram-negative bacterium that does not form spores, exists in the form of a rod-shaped bacillus, and moves through gliding motility. Colonies are usually cream, pink or tan in color and have a very mucilaginous character. Lyzobacter enzymogenes produces extracellular enzymes such as protease, chitinase, and ⁇ -1,3-glucanase, producing a wide range of fungi and nematodes. It has high growth inhibitory activity by decomposing the structural components of pathogenic microorganisms.
  • the present inventors found that when a composition containing Lysobacter enzymogenes JCK-1421 strain, its culture medium, or its extract was treated against phytopathogenic fungal diseases, bacterial diseases, and nematode diseases, the resistance-inducing activity in host plants was significantly higher. It was confirmed to be excellent.
  • the purpose of the present invention is to provide a Lyzobacter enzymogenes JCK-1421 strain having antifungal, antibacterial and antinematode activities.
  • Another object of the present invention is to provide a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases, comprising the Lyzobacter enzymogenes JCK-1421 strain, its culture medium, or its extract.
  • Another object of the present invention is to provide a method for producing a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases, including a culturing step of culturing the Lyzobacter enzymogenes JCK-1421 strain.
  • Another object of the present invention is to provide a method for controlling plant fungal diseases, bacterial diseases, or nematode diseases, including a treatment step of treating the Lyzobacter enzymogenes JCK-1421 strain, its culture solution, or its extract.
  • Another object of the present invention relates to the use of Lyzobacter enzymogenes JCK-1421 strain, its culture solution, or its extract for controlling plant fungal diseases, bacterial diseases, or nematode diseases.
  • the present invention relates to a Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases comprising the strain, a culture thereof, or an extract thereof, a method for producing the composition, and the above. It relates to a method for controlling plant fungal diseases, bacterial diseases, or nematode diseases using a composition.
  • Lysobacter enzymogenes JCK-1421 strain which has antifungal, antibacterial and antinematode activities.
  • the Lyzobacter enzymogenes JCK-1421 strain may be the Lyzobacter enzymogenes JCK-1421 strain deposited under the accession number KCTC 15126BP.
  • the Lyzobacter enzymogenes JCK-1421 strain of the present invention may contain 16S rRNA containing the base sequence of SEQ ID NO: 3.
  • the Lyzobacter enzymogenes JCK-1421 strain of the present invention was deposited with the Korea Research Institute of Bioscience and Biotechnology KCTC (Korean Collection for Type Cultures) under the accession number KCTC 15126BP on October 6, 2022.
  • Another aspect of the present invention is a plant fungal disease, bacterial disease, or nematode disease comprising a Lysobacter enzymogenes JCK-1421 strain, a culture thereof, or an extract thereof, which has antifungal, antibacterial, and antinematode activity. It relates to a composition for pest control.
  • the Lyzobacter enzymogenes JCK-1421 strain may be the Lyzobacter enzymogenes JCK-1421 strain deposited under the accession number KCTC 15126BP.
  • the Lyzobacter enzymogenes JCK-1421 strain of the present invention may contain 16S rRNA containing the base sequence of SEQ ID NO: 3.
  • the fungus is Fusarium graminearum , Fusarium oxyform F. Esp. Cucumerium ( Fusarium oxysporum f. sp. cucumerium ), Rhizoctonia solani AG2-2 Brown patch, Rhizoctonia solani AG2-2 Large patch (AG2-2 Large patch) , Rhizoctonia solani AG-4, Sclerotinia homoeocarpa , and Pythium aphanidermatum.
  • it is not limited to this.
  • the bacteria include Ralstonia solanacearum , Pectobacterium carotovorum subsp . carotovorum , Xanthomonas euvesicatoria , And it may be one or more species selected from the group consisting of Erwinia amylovora , but is not limited thereto.
  • the nematode may be one or more species selected from the group consisting of Meloidogyne incognita and Bursaphelenchus xylophilus , but is not limited thereto.
  • plant fungal diseases include cucumber pruning disease caused by Rhizoctonia solani, Fusarium oxysform F. Esp. Cucumber vine scorch caused by Fusarium oxysporum f. sp. cucumerinum , lawn blight caused by Sclerotinia homoeocarpa , and Pythium aphanidermatum. It may be, but is not limited to, one or more species selected from the group consisting of grass pythium blight caused by Fusarium graminearum and rice red mold disease caused by Fusarium graminearum .
  • plant bacterial diseases include cabbage soft rot caused by Pectobacterium carotovora subsp. carotovora , and tomato caused by Ralstonia solanacearum. It may be, but is limited to, one or more species selected from the group consisting of green blight, pepper bacterial spot caused by Xanthomonas euvesicatoria , and apple burn caused by Erwinia amylovora. It doesn't work.
  • the plant nematode disease is one selected from the group consisting of tomato root-knot nematode disease caused by Meloidogyne incognita , and pine wilt nematode disease caused by Bursaphelenchus xylophilus. It may be more than one species, but is not limited to this.
  • culture in this specification means containing microorganisms after culturing them.
  • culture supernatant in this specification refers to the upper liquid obtained by removing most microorganisms from the culture medium through centrifugation, and is also referred to as “supernatant liquid.”
  • 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.
  • the composition of the present invention contains, in addition to the above-mentioned strain, its culture, a concentrate of the culture, a dried product of the culture, and/or a culture supernatant of the above-mentioned strain as an active ingredient, a culture containing the bacterial cells, an extract of the bacterial cells, a concentrate thereof, a concentrate, It may include dried matter, and, if necessary, diluted liquid, diluted matter, etc., and may include culture medium and any state obtained by processing the culture.
  • composition of the present invention can be formulated by conventional methods and can be prepared in the form of dry powder or liquid fertilizer.
  • the microbial preparation according to the present invention can be prepared in liquid form, and can be used in the form of powder by adding an extender, or can be formulated and granulated.
  • the formulation there is no particular limitation to the formulation.
  • the composition can be manufactured by adding additives such as additives, extenders, and nutrients.
  • the additives are polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene.
  • the bulking agent and nutritional agent may be one or more selected from the group consisting of skim milk (medium), soybean flour, rice, wheat, red clay, diatomaceous earth, bentonite, dextrin, glucose, and starch, but are limited thereto. no.
  • the disintegrant may be one or more selected from the group consisting of bentonite, talc, dialite, kaoline, and calcium carbonate, but is not limited thereto. no.
  • Another aspect of the present invention relates to a method for producing a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases, including a culturing step of culturing the Lysobacter enzymogenes JCK-1421 strain.
  • the Lyzobacter enzymogenes JCK-1421 strain may be the Lyzobacter enzymogenes JCK-1421 strain deposited under the accession number KCTC 15126BP.
  • the Lyzobacter enzymogenes JCK-1421 strain of the present invention may contain 16S rRNA containing the base sequence of SEQ ID NO: 3.
  • the fungus is Fusarium graminearum , Fusarium oxyform F. Esp. Cucumerium ( Fusarium oxysporum f. sp. cucumerium ), Rhizoctonia solani AG2-2 Brown patch, Rhizoctonia solani AG2-2 Large patch (AG2-2 Large patch) , Rhizoctonia solani AG-4, Sclerotinia homoeocarpa , and Pythium aphanidermatum.
  • it is not limited to this.
  • the bacteria include Ralstonia solanacearum , Pectobacterium carotovorum subsp . carotovorum , Xanthomonas euvesicatoria , And it may be one or more species selected from the group consisting of Erwinia amylovora , but is not limited thereto.
  • the nematode may be one or more species selected from the group consisting of Meloidogyne incognita and Bursaphelenchus xylophilus , but is not limited thereto.
  • plant fungal diseases include cucumber pruning disease caused by Rhizoctonia solani, Fusarium oxysform F. Esp. Cucumber vine scorch caused by Fusarium oxysporum f. sp. cucumerinum , lawn blight caused by Sclerotinia homoeocarpa , and Pythium aphanidermatum. It may be, but is not limited to, one or more species selected from the group consisting of grass pythium blight caused by Fusarium graminearum and rice red mold disease caused by Fusarium graminearum .
  • plant bacterial diseases include cabbage soft rot caused by Pectobacterium carotovora subsp. carotovora , and tomato caused by Ralstonia solanacearum. It may be, but is limited to, one or more species selected from the group consisting of green blight, pepper bacterial spot caused by Xanthomonas euvesicatoria , and apple burn caused by Erwinia amylovora. It doesn't work.
  • the plant nematode disease is one selected from the group consisting of tomato root-knot nematode disease caused by Meloidogyne incognita , and pine wilt nematode disease caused by Bursaphelenchus xylophilus. It may be more than one species, but is not limited to this.
  • 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 fungal diseases, bacterial diseases, or nematode diseases may further include a concentration step of concentrating the culture solution.
  • the method for producing a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases may further include a dilution step of diluting the culture solution.
  • the method for producing a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases may further include an extraction step of extracting components within the bacterial cells.
  • the production method may include a fractionation step of obtaining an active fraction from the culture medium, but is not limited thereto.
  • the fractionation step may include the following steps:
  • the step of obtaining the culture filtrate involves centrifuging the culture at 2000 to 5000 rpm, 2500 to 5000 rpm, 3000 to 5000 rpm, 3500 to 5000 rpm, 4000 to 5000 rpm, 4500 to 5000 rpm, for example, 4500 rpm. It may include the step of doing so, but is not limited thereto.
  • the fractionation step may include fractionating with one or more solvents selected from the group consisting of butanol, ethyl acetate, methanol, ethanol, and hexane, but is not limited thereto.
  • the concentration step may include concentrating the fraction using a reduced pressure concentrator, but is not limited thereto.
  • the purification step may include purification by preparative HPLC (high-performance liquid chromatography), but is not limited thereto.
  • Another aspect of the present invention relates to a method for controlling plant fungal diseases, bacterial diseases, or nematode diseases, comprising the step of treating a culture of Lysobacter enzymogenes JCK-1421 strain or an extract thereof with a composition.
  • composition treatment steps include spraying (e.g., spraying, misting, atomizing, powder spraying, granule spraying, surface application, permanent use, etc.), soil irrigation (e.g., mixing, irrigation, etc.), and surface. It may be performed by one or more methods selected from the group consisting of use (e.g., application, smearing, coating, etc.), immersion, poisoning, smoke application, and seed treatment, but is not limited thereto.
  • “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 amount of composition used can be appropriately determined depending on its formulation, damage situation, application method, application location, etc.
  • the content that overlaps with the composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases is included in this document. Considering the complexity of the specification, it is omitted.
  • the present invention relates to a Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases comprising the strain, a culture thereof, or an extract thereof, a method for producing the composition, and the above.
  • This relates to a method for controlling plant fungal diseases, bacterial diseases, or nematode diseases using a composition, which can effectively control various plant diseases and nematode diseases at the same time by promoting plant growth and inducing resistance in host plants when applied.
  • Figure 1 shows the results of testing the presence or absence of resistance-inducing activity through GUS gene expression analysis by treatment with JCK-1421 strain in Arabidopsis seedlings transformed with a GUS-labeled vector in the PR-1 promoter according to an embodiment of the present invention. It represents.
  • Figure 2 shows the results of phylogenetic analysis based on the 16S rRNA gene sequence of the Lysobacter enzymogenes JCK-1421 strain according to an embodiment of the present invention.
  • Figure 3 shows the extracellular enzyme activity of Lyzobacter enzymogenes JCK-1421 strain according to an embodiment of the present invention.
  • Figure 4 shows the plant growth hormone IAA production activity of the Lyzobacter enzymogenes JCK-1421 strain according to an embodiment of the present invention.
  • Figure 5 shows the plant growth promoting activity in grass two weeks after treatment with the Lyzobacter enzymogenes JCK-1421 strain culture medium according to an embodiment of the present invention.
  • Figure 6 shows the in vitro antifungal activity by replacement culture of the Lyzobacter enzymogenes JCK-1421 strain according to an embodiment of the present invention.
  • Figure 7a is a graph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on cucumber mottled disease according to an embodiment of the present invention.
  • Figure 7b is a photograph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on cucumber mottled disease according to an embodiment of the present invention.
  • Figure 8a is a graph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on cucumber vine scorch disease according to an embodiment of the present invention.
  • Figure 8b is a photograph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on cucumber vine scorch disease according to an embodiment of the present invention.
  • Figure 9a is a graph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on lawn coin blight according to an embodiment of the present invention.
  • Figure 9b is a photograph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on lawn coin blight according to an embodiment of the present invention.
  • Figure 10a is a graph showing the control effect of Lyzobacter enzymogenes JCK-1421 strain on lawn pythium blight according to an embodiment of the present invention.
  • Figure 10b is a photograph showing the control effect of Lyzobacter enzymogenes JCK-1421 strain on lawn pythium blight according to an embodiment of the present invention.
  • Figure 11a is a graph showing the control effect of Lyzobacter enzymogenes JCK-1421 strain on rice red mold disease according to an embodiment of the present invention.
  • Figure 11b is a photograph showing the control effect of Lyzobacter enzymogenes JCK-1421 strain against rice red mold disease according to an embodiment of the present invention.
  • Figure 12a is a graph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on cabbage soft rot according to an embodiment of the present invention.
  • Figure 12b is a photograph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on cabbage soft rot according to an embodiment of the present invention.
  • Figure 13a is a graph showing the control effect of Lyzobacter enzymogenes JCK-1421 strain on tomato green blight according to an embodiment of the present invention.
  • Figure 13b is a photograph showing the control effect of Lyzobacter enzymogenes JCK-1421 strain on tomato green blight according to an embodiment of the present invention.
  • Figure 14a is a graph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on pepper bacterial spot disease according to an embodiment of the present invention.
  • Figure 14b is a photograph showing the control effect of the Lyzobacter enzymogenes JCK-1421 strain on pepper bacterial spot disease according to an embodiment of the present invention.
  • Figure 15 shows the control effect of Lyzobacter enzymogenes JCK-1421 strain on apple burn blight 10 days after inoculation with the pathogen in Chinese pearleaf crabapple seedlings according to an embodiment of the present invention.
  • Figure 16a is a graph showing the control effect on apple burn blight 7 and 10 days after inoculation with the pathogen in M9 seedlings of the Lyzobacter enzymogenes JCK-1421 strain according to an embodiment of the present invention.
  • Figure 16b is a photograph showing the control effect on apple burn blight 7 and 10 days after inoculation with the pathogen in M9 seedlings of the Lyzobacter enzymogenes JCK-1421 strain according to an embodiment of the present invention.
  • Figure 17a shows the control effect of Lyzobacter enzymogenes JCK-1421 strain against tomato root-knot nematode disease according to an embodiment of the present invention, 6 weeks after inoculation with the pathogen, a) galling index inhibition effect, b) egg cyst This is expressed as a suppression effect on egg masses.
  • Figure 17b shows the control effect of the Lyzobacter enzymogenes JCK-1421 strain against tomato root-knot nematode disease in accordance with an embodiment of the present invention as a photograph of the roots 6 weeks after inoculation with the pathogen.
  • Figure 18 shows the effect of controlling pine wilt disease after 4 weeks when the Lyzobacter enzymogenes JCK-1421 strain was treated on pine seedlings according to an embodiment of the present invention.
  • Figure 19 shows the effect of controlling pine wilt disease after 6 weeks when pine seedlings are treated with Lyzobacter enzymogenes JCK-1421 strain according to an embodiment of the present invention.
  • the present invention relates to the Lysobacter enzymogenes JCK-1421 strain deposited under the accession number KCTC 15126BP, which has antifungal, antibacterial and antinematode activities.
  • % 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)%.
  • Arabidopsis thaliana was transformed with a GUS-labeled vector in the PR-1 promoter, and a gene related to induced resistance was used. PR-1 gene expression was examined. Resistance is induced in plants through the salicylic acid signaling system, and as a result, 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.
  • Transformed Arabidopsis seeds were first surface sterilized with 70% ethanol for 30 seconds and then secondarily surface sterilized with bleach solution (2% NaOCl + 0.05% tween-20) for 5 minutes. Afterwards, the bleach solution remaining on the surface of the seeds was washed 3-4 times with sterilized water and soaked at 4°C for 2 days. The soaked seeds were placed on 1/2 Murashige - Skoog solid medium (MS, 2.2 g MS salts, 10 g sucrose, 8 g phyto agar/L, Duchefa) supplemented with 50 ⁇ g/mL Kanamycin, and sealed with 3M tape. Then, they were cultured in a plant growth incubator at 25°C (16-hour photoperiod, 80% relative humidity).
  • bleach solution 2% NaOCl + 0.05% tween-20
  • strain JCK-1421 which shows GUS activity through expression of the PR-1 gene, was selected.
  • the JCK-1421 strain was blue in all treatments of culture broth, culture filtrate, and cell suspension, of which the culture broth had a wider and darker color. showed. In contrast, the medium used as a negative control did not show GUS activity. Through this, it was confirmed that the JCK-1421 strain has resistance-inducing activity by expressing the PR-1 gene, a resistance marker gene.
  • Strain JCK-1421 which was selected as a strain with resistance-inducing activity in plants, was identified molecularly through 16S rRNA gene sequence analysis.
  • the strain was inoculated into LB liquid medium (Luria-Bertani, Becton, Dickinson and Co., Sparks, MD, USA) and then cultured with shaking at 150 rpm at 30°C for 24 hours.
  • the harvested strain was incubated with iNtRON's I-genomic BYF DNA. Genomic DNA (gDNA) of the strain was extracted according to the protocol using the Extraction Mini Kit.
  • 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 gene PCR product was submitted to Genotech (Daejeon, Korea) for base sequence analysis, and a total base sequence of 1388 bp (SEQ ID NO. 3) was obtained as the 16S rRNA coding base sequence of the isolated strain, JCK-1421 strain. got it As can be seen in Figure 2, as a result of comparing the base sequence of the GenBank database using NCBI's BlastN search, the JCK-1421 strain was identified as Lysobacter enzymogenes .
  • the 16S rRNA base sequence of the strain was uploaded to the GenBank database as OP420512, and the selected strain was named Lysobacter enzymogenes JCK-1421 strain and was registered at the Korea Research Institute of Bioscience and Biotechnology KCTC (Korean Collection) on October 6, 2022. for Type Cultures and was given the accession number KCTC 15126BP.
  • protease chitinase
  • gelatinase 1% colloidal chitin + 1.5% agar, Difco
  • gelatinase medium % gelatin + 1.5% agar, Duksan
  • cellulase medium (0.4% carboxymethyl cellulose sodium + 1.5% agar, Sigma-Aldrich) was prepared, and chitinase medium was XL1 medium (5 g/L peptone (Difco), 5 g/L yeast extract (Difco), 5 g/L NaCl) was prepared by adding colloidal chitin, and this medium was designated XL1+C.
  • Colloidal chitin added to the medium was first added to crab shell powder with hydrochloric acid (HCl) (10 g crab shell powder/150 mL HCl) and stirred for 6 hours. After 6 hours, 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 using 0.1 M potassium phosphate buffer (pH 7.0). The prepared colloidal chitin was sterilized by high-pressure steam and stored at 4°C until use. For the gelatin medium, 10% gelatin was added to LB solid medium, and other media were solidified by adding 1.5% agar.
  • HCl hydrochloric acid
  • a sterilized paper disc (0.8 cm, Advantec, Japan) was placed on top of each prepared medium, and 2.5 ⁇ L, 5 ⁇ L, and 10 ⁇ L of JCK-1421 strain culture filtrate were added for protease and 30 ⁇ L for chitinase and cellulase, respectively.
  • Gelatinase was dispensed in amounts of 10 ⁇ L, 60 ⁇ L, and 90 ⁇ L, respectively, and 10 ⁇ L, 20 ⁇ L, and 30 ⁇ L of gelatinase.
  • As a negative control equal amounts of sterilized LB and medium were dispensed onto paper discs. The experiment was repeated three times, and the plate was maintained at 30°C to observe clear zones according to extracellular enzyme activity.
  • the cellulase medium was stained by adding 5 mL of Lugol's solution (2.5 g/L iodine and 5 g/L potassium iodide) to the plate, and the plate was 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 JCK-1421 strain had a clear zone diameter of 24.61 mm after 9 days of treatment with 10 ⁇ L of protease and chitinase. was 17.25 mm after 7 days of 90 ⁇ L treatment, gelatinase was 29.42 mm after 4 days of 30 ⁇ L treatment, and cellulase was 28.67 mm after 5 days of 90 ⁇ L treatment.
  • Lysobacter enzymogenes JCK-1421 strain produced protease, chitinase, gelatinase, and cellulase, and among these, gelatinase was produced the most.
  • JCK- 1% of strain 1421 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. 1 mL of the culture filtrate was mixed with 2 mL of Salkowski's reagent (150 mL H 2 SO 4 , 250 mL sterilized water, 7.5 mL 0.5 M FeCl 3 ⁇ 6H 2 O).
  • Lyzobacter enzymogenes JCK-1421 strain was inoculated into LB agar medium using a disposable loop and then cultured at 30°C for 2 days. A single colony of the cultured strain was inoculated into 5 mL of LB liquid medium and cultured with shaking at 30°C and 150 rpm for 24 hours. The optical density of the pre-culture solution was measured at 600 nm using a UV-VIS spectrophotometer, and the value of OD 600 was obtained. The strain suspension adjusted to 0.1 (1.0 x 10 8 CFU/mL) was inoculated at 1% in LB liquid medium. Afterwards, it was cultured with shaking at 30°C and 150 rpm for 24 hours and used as the main culture medium.
  • the grass was cut to a certain length, and the JCK-1421 strain culture was diluted 500-, 1,000-, and 2,000-fold and 20 mL per pot was applied to the soil.
  • Horicure a.i. 25% Tebuconazole EC, Farm Hannong
  • LB liquid medium without strain was diluted 500 times and treated.
  • the photoperiod was examined for 16 hours a day in a constant temperature and humidity room at 25°C, and the experiment was repeated 3 times with 2 plants per treatment group. After 2 weeks of treatment, the growth of the grass was visually inspected.
  • Example 6 Lyzobacter enzymogenes against phytopathogenic fungi ( Lysobacter enzymogenes ) of strain JCK-1421 in vitro antifungal activity
  • cucumerinum cucumber vine peck disease
  • Sclerotinia homoeocarpa grass blight
  • Rhizoctonia solani AG2-2 LP, lawn large patch disease
  • Rhizoctonia solani AG2-2 BP, grass brown patch disease
  • Pythium aphanidermatum grass pythium blight
  • Fusarium graminearum rice red mold disease
  • aphanidermatum was inoculated on corn meal agar (CMA, Sigma-Aldrich, India) medium, and the remaining strains were inoculated on potato dextrose agar (PDA, Becton, Dickinson and Co., Sparks, MD, USA) medium. , cultured statically in an incubator at 25°C.
  • agar plugs of plant pathogens grown at 25°C were separated using a cork bore (0.6 cm in diameter), and the separated agar plugs were inoculated at a point 2 cm away from the edge of the PDA.
  • Colonies of the JCK-1421 strain cultured on LB solid medium were inoculated against an agar plug (5 cm in length). Afterwards, they were cultured in an incubator at 25°C, and the degree of mycelial growth of the pathogen was observed after 2-7 days.
  • Example 7 Lyzobacter enzymogenes against plant pathogenic bacteria ( Lysobacter enzymogenes ) of strain JCK-1421 in vitro antibacterial activity
  • the adjusted bacterial suspension was injected into each well of a 96 well plate, and the final concentration of the sample was 10, 5, 2.5, 1.25, and 0.625%. Streptomycin sulfate was used as a positive control, and an untreated group treated with only bacterial suspension was used as a negative control. All plates were cultured in an incubator at 30°C to examine bacterial growth, and each experiment was repeated three times.
  • Example 8 Lyzobacter enzymogenes against plant pathogenic nematodes ( Lysobacter enzymogenes ) of strain JCK-1421 in vitro Nematicidal activity
  • the purpose of this study was to investigate the direct nematicidal activity of strain JCK-1421 by conducting in vitro tests on various plant pathogenic nematodes and examining the growth inhibitory activity of nematodes.
  • To investigate direct nematicidal activity two types of plant pathogenic nematodes, sweet potato root-knot nematode ( Meloidogyne incognita ) and pine tree nematode ( Bursaphelenchus xylophilus ), were used.
  • 1% NaOCl sodium hypochlorite, v/v
  • 1% NaOCl sodium hypochlorite, v/v
  • the collected nematode eggs were used for in vitro egg hatching inhibition activity tests.
  • the modified Baermann funnel method was used to hatch at 28°C for 3 days to separate second instar larvae.
  • the collected nematode egg suspension and larva suspension were adjusted to 150 nematode eggs and 50 second instar larvae per 100 ⁇ L, respectively, and were injected into each well of a 96 well plate.
  • the final concentration of the culture filtrate was treated to be 20% and 10%.
  • the experiment was repeated 3 times, and after treatment, the treatment was maintained for 3 days at 100% relative humidity under dark conditions at room temperature. Afterwards, it was observed using a microscope (LeicaDM IL LED; Leica Microsystems CMS GmbH, Wetzlar, Germany), and if the nematode's body was straight and motionless, it was considered dead. The experiment was repeated three times each, and the nematode mortality (%) and egg hatching inhibition rate of sweet potato root-knot nematode were calculated according to the formulas described below.
  • Nematode mortality (%) [(mortality rate of control group - mortality rate of treatment group)/(mortality rate of control group)] ⁇ 100
  • Egg hatch inhibition (%) [(Egg hatching rate of control group - Egg hatching rate of treatment group)/Egg hatching rate of control group] ⁇ 100
  • Cultured pine wilt nematodes were harvested using the Baermann funnel method, then counted under an optical microscope, adjusted to 50 nematodes per 100 ⁇ L, and injected into each well of a 96 well plate. The final concentration of the culture filtrate was treated to be 20% and 10%. The experiment was repeated 3 times, and after treatment, the treatment was maintained for 3 days at 100% relative humidity under dark conditions at room temperature. Afterwards, it was observed using a microscope (LeicaDM IL LED; Leica Microsystems CMS GmbH, Wetzlar, Germany), and if the nematode's body was straight and motionless, it was considered dead. The experiment was performed in three repetitions each, and the nematode mortality rate (%) of pine wilt nematode was calculated according to the formula described below.
  • Nematode mortality (%) [(mortality rate of control group - mortality rate of treatment group)/(mortality rate of control group)] ⁇ 100
  • Example 9 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Culture and preparation of preparations for controlling various plant diseases of JCK-1421 strain
  • JCK-1421 20 SC (Suspension concentrate, liquid) was prepared by adding 20% of the culture medium of the JCK-1421 strain and the spray dried product of the culture medium. Wetting agent) was used in this experiment.
  • strain culture the strain was inoculated into LB agar medium using a disposable loop and then cultured at 30°C for 2 days. A single colony of the cultured strain was inoculated into 5 mL of LB liquid medium and cultured with shaking at 30°C and 150 rpm for 24 hours.
  • the optical density of the pre-culture solution was measured at 600 nm using a UV-VIS spectrophotometer, and the value of OD 600 was obtained.
  • the strain suspension adjusted to 0.1 (1.0 x 10 8 CFU/mL) was inoculated at 1% into LB liquid medium. Afterwards, it was cultured with shaking at 30°C and 150 rpm for 24 hours and used as the main culture medium.
  • JCK-1421 For the formulation of JCK-1421, a total of 3 L of the main culture medium of the JCK-1421 strain cultured with shaking at 30°C and 150 rpm for 24 hours was spray dried.
  • the spray dryer was manufactured under the conditions of an injection temperature of 195°C and a discharge temperature of 93°C or higher. Afterwards, the spray-dried product was used to formulate JCK-1421 20 SC, which contains 20% of the spray-dried culture medium of the JCK-1421 strain.
  • Example 10 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of strain JCK-1421 on cucumber blight disease
  • Nebakja' seeds Cucumis sativus L. cv. Nebakja, Syngenta Korea
  • filter paper 9 cm in diameter Petri
  • cucumber seeds were sown in a plastic pot (6 cm in diameter, 6.5 cm in height) with horticultural medium, and irradiated with light for 16 hours a day in a constant temperature and humidity room at 25°C. It was grown for 6 days.
  • R. solani AG-4 the pathogen responsible for cucumber wilt disease, was inoculated into brown rice (Deungdae Agricultural Company, Jeollanam-do, Korea) soaked in distilled water for 24 hours and then sterilized under high pressure steam 3 times, and cultured for 3 weeks at 25°C. Pathogens cultured for 3 weeks were placed in an aluminum dish, covered with gauze, and dried at room temperature for 3 days. The dried material was ground with a blender, filtered through a 0.5 to 0.85 mm sieve, and used as an inoculum.
  • inoculum 0.75 g was evenly mixed in 1 L of horticultural medium 6 days after sowing, placed in a plastic pot (7 cm in diameter, 6 cm in height), and transplanted into cucumber seedlings grown for 6 days after sowing.
  • the culture medium and preparation (JCK-1421 20 SC) of the JCK-1421 strain were used as described in Example 9. prepared as follows.
  • the JCK-1421 strain culture medium was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea) to prepare the drug to be diluted 500-fold, 1,000-fold, and 2,000-fold, respectively.
  • the JCK-1421 20 SC preparation was prepared using the JCK-1421 strain culture medium. It was prepared by diluting at the same dilution ratio as that of .
  • Zallocen dispenser concentrate (DC): ai 30% Hymexazol + 5% Penthiopyrad, Korea Samgong) and eggplant eggs (WP: ai 10% Etidiazole + 55% Thiophanate-methyl, FarmHannong) were used at 1,000 doses. It was diluted 2,000 times and used as a soil irrigation treatment with 10 mL 1 hour after pathogen inoculation. The culture medium of the JCK-1421 strain and the JCK-1421 20 SC sample were treated with soil drench at 10 mL per pot 4 days before inoculation with the pathogen for cucumber wilt disease.
  • the pot After inoculation with pathogens and treatment with chemicals, the pot was placed in a constant temperature and humidity room at 25°C and irradiated with a light source for 12 hours a day. The experiment was repeated 3 times, 3 per treatment group, and the disease incidence (%) of cucumber prickly pear disease was investigated 4 days after inoculation, and the control value (%) compared to the untreated group was calculated according to the following formula.
  • Control value (%) 100 ⁇ [(Incidence rate in untreated group - Incidence rate in treated group) / Incidence rate in untreated group]
  • the JCK-1421 strain culture showed control values of 67%, 83%, and 79%, respectively, compared to the untreated group at 500-fold, 1,000-fold, and 2,000-fold dilutions. Additionally, JCK-1421 20 SC showed control values of 75%, 88%, and 75% at 500-, 1,000-, and 2,000-fold dilutions, respectively. Zallocen, used as a control agent, showed control values of 100% and 83% at 1,000- and 2,000-fold dilutions, and eggplant showed control values of 92% and 75% at 1,000- and 2,000-fold dilutions, respectively. JCK-1421 strain culture medium and JCK-1421 20 SC preparation showed the highest control effect at 1,000-fold dilution.
  • JCK-1421 20 SC 1,000-fold dilution had no statistically significant difference from the control agent, eggplant. Therefore, it can be said that strain JCK-1421, which has no direct antifungal activity against R. solani AG-4, the causative agent of cucumber hairy disease, induced resistance in cucumbers and controlled cucumber hairy disease. It is believed that it will be possible to develop a highly effective cucumber blight disease control agent using this in the future.
  • Example 11 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of strain JCK-1421 against cucumber vine scorch disease
  • the pathogen F. oxysporum f. sp.
  • the cultured mycelia of cucumerinum were cut using a cork bore (0.6 cm in diameter), and five agar plugs were inoculated into PDB medium and cultured with shaking at 25°C and 150 rpm for 7 days. Afterwards, the culture was harvested, filtered through 4 layers of gauze to remove mycelia, and a suspension of pathogen spores was prepared to a concentration of 2.5 ⁇ 10 6 spore/mL under a microscope (Axio Imager.A2, Carl Zeiss, Germany) using a hemocytometer. The prepared spore suspension was used as an inoculum to investigate the effect of controlling cucumber vine scorch disease. Pathogen inoculation was performed 7 days after sowing by inoculating the soil with 10 mLl of pathogen spore suspension inoculum per pot.
  • JCK-1421 20 SC The JCK-1421 strain culture medium was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea) to prepare the drug to be 500-fold, 1,000-fold, 2,000-fold, 4,000-fold, and 8,000-fold, respectively.
  • JCK-1421 20 SC preparation was prepared by diluting at the same ratio as the dilution ratio of the JCK-1421 strain culture medium.
  • Zallocen dispenser concentrate (DC): ai 30% Hymexazol + 5% Penthiopyrad, Korea Samgong) and Eggplant Egg (WP: ai 10% Etridazole + 55% Thiophanate-methyl, Farm Hannong) were used at 1,000 doses. It was diluted two-fold and used as a 10 mL soil drench 1 hour after pathogen inoculation.
  • the prepared JCK-1421 strain culture and preparations were treated with soil irrigation at 10 ml per pot 4 days before pathogen inoculation. After completing both inoculation and chemical treatment, the pot was irradiated with light for 12 hours a day in a constant temperature and humidity room at 25°C.
  • the experiment was repeated 3 times, 3 per treatment group, and was evaluated with a disease index ranging from 0 to 4 depending on the degree of disease 30 days after inoculation with the pathogen.
  • the JCK-1421 strain showed an excellent control effect against cucumber vine peck disease, and like cucumber vine peck disease, the causative agent of cucumber vine peck disease , F. oxysporum f. sp. It can be said that the JCK-1421 strain, which has no direct antifungal activity on cucumerinum , induced resistance in cucumbers and controlled cucumber vine scorch disease. In the future, it is believed that it will be possible to develop a highly effective cucumber vine scorch control agent using the induced resistance of the JCK-1421 strain.
  • Example 12 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of JCK-1421 strain on lawn blight
  • Sclerotinia homoeocarpa the pathogen of lawn blight
  • twice-high-pressure steam-sterilized wheat bran-rice husk medium (9 g wheat bran, 1.5 g rice husk, 10 mL distilled water; 250 mL Erlenmeyer flask) and cultured at 25°C for 7 days.
  • 110 mL of distilled water containing 200 ppm streptomycin sulfate was added and the cells were triturated.
  • the prepared bacterial suspension was used as an inoculum to investigate the effect of controlling lawn blight.
  • a 1 cm deep hole was made in the center of the pot, and 3.5 ml of S. homoeocarpa inoculum was inoculated per pot.
  • the JCK-1421 strain culture medium and JCK-1421 20 SC were prepared by the method described in Example 9.
  • the JCK-1421 strain culture medium was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea) to prepare the drug to be diluted 500-fold, 1,000-fold, and 2,000-fold, respectively.
  • the JCK-1421 20 SC preparation was prepared using the JCK-1421 strain culture medium. It was prepared by diluting at the same diluting ratio as that of .
  • Horikuo (EC: ai 25% Tebuconazole, FarmHannong) was diluted 2000 times and 20 mL per pot was irrigated into the soil 1 hour after pathogen inoculation.
  • the prepared JCK-1421 strain culture and preparations were treated with soil irrigation at a rate of 20 mL per pot 4 days before pathogen inoculation.
  • 20 ml of JCK-1421 strain culture and preparation chemicals per pot were applied to creeping bentgrass 3 weeks after sowing, and the S. homoeocarpa mycelial suspension prepared 4 days after treatment was applied to the soil. 3.5 mL was inoculated per pot.
  • the pot After completing pathogen inoculation and chemical treatment, the pot was filled with water in a tray and covered with a plastic chamber (width 35 cm, length 28 cm, height 16 cm) to maintain 100% relative humidity, and kept in a constant temperature and humidity room at 25°C for 12 hours a day. was investigated. The experiment was repeated twice, three times per treatment group. 13 days after inoculation, the disease area rate was investigated, and the control value (%) compared to the untreated group was calculated according to the following equation.
  • the JCK-1421 strain culture medium was 500-fold, 1000-fold, and 2000-fold diluted compared to the untreated group.
  • the control values were 86%, 92%, and 85%, respectively.
  • JCK-1421 20 SC showed control values of 66%, 83%, and 55% at 500-, 1,000-, and 2,000-fold dilutions, respectively.
  • Horikur, used as a control agent, showed a control value of 94% at a 2,000-fold dilution.
  • JCK-1421 strain culture and JCK-1421 20 SC each showed the highest control effect at 1,000-fold dilution.
  • all dilution treatments of JCK-1421 strain culture used in the experiment and JCK-1421 20 SC 1,000-fold dilution were grass coins. It showed excellent activity in controlling blight, and there was no statistically significant difference from the control drug Horikur. Therefore, it was confirmed that strain JCK-1421 showed an excellent control effect against lawn blight. Since it had no direct antifungal activity on S. homoeocarpa, the causative agent of lawn blight, it could be said that it controlled lawn blight by inducing resistance in the grass. . In the future, it is believed that it will be possible to develop a highly effective lawn blight control agent using the induced resistance of the JCK-1421 strain.
  • Pythium aphanidermatum strain a lawn pythium blight pathogen
  • corn meal agar medium CMA, Sigma-Aldrich, India
  • the agar plugs were separated from the cultured mycelia of P. aphanidermatum using a cork bore (0.5 cm in diameter), and the separated agar plugs (2 per pot) were used as an inoculum source.
  • the JCK-1421 strain culture medium and JCK-1421 20 SC were prepared by the method described in Example 9.
  • the JCK-1421 strain culture medium was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea) to make the drug diluted 500-fold, 1,000-fold, and 2,000-fold, respectively.
  • the JCK-1421 20 SC preparation was prepared using the JCK-1421 strain culture medium. It was prepared by diluting at the same diluting ratio as that of .
  • Heritiji a.i.
  • Azoxystrobin 50% WG was diluted 10,000 times and 10 ml per pot was injected into the soil 1 hour after pathogen inoculation.
  • 10 ml of JCK-1421 strain culture and preparation chemicals prepared on creeping bentgrass 3 weeks after sowing were applied to the soil for each pot.
  • 2 pathogen agar plugs were placed per pot. The inoculation was done by turning the grass surface so that the mycelia were in contact with each other.
  • the pot After completing pathogen inoculation and chemical treatment, the pot was filled with water in a tray and covered with a plastic chamber (width 35 cm, length 28 cm, height 16 cm) to maintain 100% relative humidity, and kept in a constant temperature and humidity room at 25°C for 12 hours a day. was investigated. The experiment was repeated twice, three times per treatment group. Seven days after inoculation, the disease area rate was investigated, and the control value (%) compared to the untreated group was calculated according to the following formula.
  • JCK-1421 strain culture medium and JCK-1421 20 SC each showed the highest control effect at 1,000-fold dilution.
  • all dilution treatments of JCK-1421 strain culture medium used in the experiment were compared with the 1,000-fold dilution treatment of JCK-1421 20 SC preparation. It showed an excellent control effect on lawn pythium blight with no statistically significant difference from the drug Heritage.
  • the JCK-1421 strain showed an excellent control effect against lawn Pyceum blight. Since it had no direct antifungal activity on P. aphanidermatum , the causative agent of lawn Pyceum blight, it induced resistance in grass and controlled lawn Pyceum blight. It can be said that it was done. Therefore, it is believed that it is possible to develop not only a lawn fungus blight control agent using the induced resistance of the JCK-1421 strain, but also a highly effective lawn Pythium blight control agent.
  • Example 14 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of strain JCK-1421 on rice red mold disease
  • Samkwang rice seeds were used to investigate the in vivo control activity of rice red mold disease by induced resistance of the JCK-1421 strain.
  • the pot was placed on a yellow tray with a hole at the bottom so that the bottom of the pot was submerged in water, and cultured in a 30°C constant temperature and humidity room covered with a non-woven fabric for the seed bed.
  • the non-woven fabric was removed, and the rice was grown for 4 weeks under a photoperiod of 16 hours per day, then transplanted into Wagner pots (top diameter 17.5 cm, bottom diameter 16 cm, height 19.8 cm, NF-5).
  • compound fertilizer for base fertilizer Soil Love 21, Namhae Chemical
  • 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.
  • 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.
  • 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-1421 induced resistance against rice red mold disease caused by F. graminearum .
  • Treatment of the JCK-1421 strain was performed on 11-week-old rice after pot transplantation, 2 weeks before and 1 week before pathogen inoculation, by diluting the JCK-1421 strain culture medium and JCK-1421 20 SC preparation 1,000-fold and 2,000-fold and drenching the rice ears and stems. Foliar spray treatment was applied to wet the area.
  • the JCK-1421 strain culture was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea).
  • the control agent was Nonbra (ai 30% Ferimzone + 10% Tricyclazole WP, Kyungnong), 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. graminearum spore suspension (5 ⁇ 10 5 spores/mL) on the leaves for each treatment group.
  • Tween-20 an electrodeposition agent
  • the JCK-1421 strain culture medium was 1,000-fold and 2,000-fold diluted compared to the untreated group, respectively. It showed a control value of 92% and 21%.
  • JCK-1421 20 SC formulation showed control values of 34% and 47% at 1,000- and 2,000-fold dilutions, respectively.
  • Nonbra used as a control agent showed a control value of 46% at a 1,000-fold dilution.
  • the JCK-1421 strain culture medium showed the highest control effect at 1,000-fold dilution and JCK-1421 20 SC at 2,000-fold dilution.
  • the 1,000-fold dilution of JCK-1421 strain culture showed higher control activity than the control drug, the synthetic pesticide Nonbra.
  • strain JCK-1421 showed an excellent control effect against rice red mold disease, and since it had no direct antifungal activity against F. graminearum , the causative agent of rice red mold disease, it induced resistance in rice and controlled rice red mold disease. It can be said that it was done. 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-1421 strain.
  • Example 15 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect on cabbage soft rot of strain JCK-1421
  • Chungwangbom cabbage seeds Brassica campestris L. ssp. pekinensis (Lour.) Rupr. cv. Chungwang, Sakata Korea
  • the black square connection pot square 4 Seeds were sown. After sowing, the plants were grown for 3 weeks in a constant temperature and humidity room at 25°C under a photoperiod of 16 hours per day. Then, 5- or 6-leaf cabbage plants were transplanted into larger pots (7 cm in diameter, 6 cm in height) 24 hours before sample treatment. used.
  • the culture medium of the JCK-1421 strain and JCK-1421 20 SC were prepared by the method described in Example 9.
  • the JCK-1421 strain culture medium was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea) to prepare the drug to be diluted 500-fold, 1,000-fold, 2,000-fold, and 4,000-fold, respectively.
  • the JCK-1421 20 SC preparation was prepared as JCK- It was prepared by diluting at the same dilution ratio as the 1421 strain culture medium.
  • Sungbocycline a.i.
  • Oxytetracycline 17% WP, Sungbo Chemical) pesticide was diluted 1,000 times 1 day before pathogen inoculation and then 10 ml per pot was irrigated into the soil.
  • Treatment of the JCK-1421 strain involves transplanting cabbage at the 5- or 6-leaf stage 3 weeks after sowing into a larger pot, and 24 hours later, irrigating the soil with the prepared JCK-1421 strain culture medium and preparation chemicals at a rate of 10 ml per pot. 4 days after drug treatment, 10 ml of pathogens were inoculated per pot.
  • the cabbage soft rot pathogen, Pectobacterium carotovorum subsp. carotovorum (Pcc) strain was inoculated into TSA medium and cultured at 30°C for 24 hours. Cultured Pcc colonies were harvested in 50 mL conical tubes using distilled water and a cell scraper. Afterwards, 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 Inoculation was done by soil drench at a rate of ml. 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 10 days later.
  • Pcc Pectobacterium carotovorum subsp. carotovorum
  • 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 (%) compared to the untreated group was calculated according to the following equation.
  • Control value (%) 100 ⁇ [(Incidence rate in untreated group - Incidence rate in treated group) / Incidence rate in untreated group]
  • the JCK-1421 strain culture was diluted 500 times, 1,000 times, 2,000 times, and 4,000 times. showed control values of 65%, 25%, 0%, and 38%, respectively, compared to the untreated group.
  • the JCK-1421 20 SC formulation showed control values of 70%, 95%, 93%, and 7% at 500-fold, 1,000-fold, 2,000-fold, and 4,000-fold dilutions, respectively.
  • Sungbocycline, used as a control agent showed a control value of 27% at a 1,000-fold dilution.
  • the JCK-1421 strain culture medium showed the highest control effect at 500-fold dilution and the JCK-1421 20 SC preparation at 1,000-fold dilution, respectively, and the JCK-1421 strain culture medium showed the highest control effect at 500-fold and 4,000-fold dilution. showed higher control activity than the reference drug, Sungbocycline, in all treatments except 4,000 times.
  • strain JCK-1421 showed an excellent control effect against soft rot of cabbage, and the causative agent of soft rot of cabbage , P. carotovorum subsp. Since there was no direct antibacterial activity on carotovorum , it can be said that cabbage soft rot was controlled by inducing resistance in cabbage. Therefore, it was confirmed that a highly effective cabbage soft rot control agent could be developed in the future by using the resistance-inducing activity of the JCK-1421 strain.
  • Example 15 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of JCK-1421 strain on tomato green blight
  • Tomato Samgwang seeds were used to investigate the in vivo control activity of tomato green blight caused by induced resistance of the JCK-1421 strain.
  • the square connection port was filled with topsoil, and tomato seeds ( Lycopersicon esculentum Mill. cv. Seokwang, Farhannong Co., Seoul, Korea) were sown using tweezers, and the photoperiod was irradiated for 16 hours a day in a constant temperature and humidity room at 25°C for 4 days. It was grown for a week. 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 8 CFU/mL), and the prepared suspension was used as an inoculum.
  • the culture medium and formulation (JCK-1421 20 SC) of the JCK-1421 strain were used as described in Example 9. Ready.
  • the JCK-1421 strain culture medium and JCK-1421 20 SC preparation were diluted 500-fold, 1,000-fold, 2,000-fold, and 4,000-fold on Seogwang tomato seedlings 4 weeks after sowing, and then injected into the soil at 20 mL each per pot. Drench treatment was performed.
  • the JCK-1421 strain culture was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea).
  • Sungbocycline (ai Oxytetracycline 17% WP, Sungbo Chemical) was diluted 2,000-fold and 4,000-fold and used as a soil drench at 10 ml per pot 1 day before pathogen inoculation.
  • 10 mL of R. solanacearum bacterial suspension (OD 600 0.1, 1.0 x 10 8 CFU/mL) per pot was inoculated as a pathogen inoculum 4 days after drug treatment.
  • 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 with a disease index ranging from 0 to 5 depending on the severity of symptoms.
  • 1 wilting of 1-2 leaves
  • 2 wilting of 3-4 leaves
  • 3 wilting of 5-6 leaves
  • 4 wilting of most leaves
  • 5 death, etc.
  • Six stages were investigated, and the control effect was calculated accordingly.
  • the control value (%) compared to the untreated group was calculated according to the following equation.
  • Control value (%) 100 ⁇ [(Incidence rate in untreated group - Incidence rate in treated group) / Incidence rate in untreated group]
  • the JCK-1421 strain culture medium was 500-fold, 1,000-fold, 2,000-fold, and 4,000-fold
  • the diluted solution showed control values of 15%, 76%, 59%, and 29%, respectively, compared to the untreated group.
  • the JCK-1421 20 SC formulation showed control values of 54%, 66%, 88%, and 78% at 500-, 1,000-, 2,000-, and 4,000-fold dilutions, respectively.
  • Sungbocycline used as a control agent, showed control values of 80% and 54% at 2,000- and 1,000-fold dilutions, respectively.
  • the JCK-1421 strain culture medium showed the highest control effect at a 1,000-fold dilution, and the JCK-1421 20 SC preparation showed the highest control effect at a 2,000-fold dilution. 20
  • the JCK-1421 strain showed an excellent control effect against tomato green blight, and since it had no direct antibacterial activity against R.
  • solanacearum the causative agent of tomato green blight
  • tomato green blight was controlled by inducing resistance in tomatoes. You can. Therefore, it was confirmed that it is possible to develop an effective tomato green blight control agent in the future by using the resistance-inducing activity of the JCK-1421 strain.
  • Example 16 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of strain JCK-1421 on pepper bacterial spot disease
  • the culture medium of the JCK-1421 strain and JCK-1421 20 SC were prepared by the method described in Example 9.
  • Treatment of the JCK-1421 strain is performed on pepper seedlings at the 5- or 6-leaf stage after sowing, 24 hours after transplanting, dilute the JCK-1421 strain culture medium and JCK-1421 20 SC preparation 2,000-fold and 4,000-fold and administer 5 per seedling, respectively.
  • Foliar spray treatment was performed mL at a time.
  • the JCK-1421 strain culture was diluted in distilled water containing 250 ppm of Tween 20 (Duksan science, Seoul, Korea).
  • Sungbocycline (ai Oxytetracycline 17% WP, Sungbo Chemical) was diluted 2,000 times and used as a foliar spray of 5 mL per seedling 1 day before pathogen inoculation.
  • 5 mL of X. euvesicatoria bacterial suspension (OD 600 0.1, 1.0 .
  • 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 24 hours, and then given a 12-hour photoperiod and examined for disease severity 11 days later.
  • Incidence was expressed as an index from 0 to 7. 0: no symptoms, 1: no symptoms, 2: necrotic lesions on some leaflets, 3: lesions combined on some leaves, 4: lesions combined on many leaves, 5: many leaflets Indicated that lesions had occurred, 6: Severe lesions and fallen leaves had occurred, 7: The plant had died.
  • the disease control value was calculated using the following formula.
  • Control value (%) 100 ⁇ [(Incidence rate in untreated group - Incidence rate in treated group) / Incidence rate in untreated group]
  • Example 17 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of JCK-1421 strain on apple burn disease
  • Apple seedlings (M9 family Chinese pearleaf crabapple, height 15 ⁇ 5 cm) were used to investigate the in vivo control activity of apple burn disease by induced resistance of the JCK-1421 strain.
  • streptomycin sulfate was diluted to 100 ⁇ g/ml, or Serifel biopesticide (active ingredient: Bacillus amyloliquefaciens MBI600 (11%) WP, FarmHannong, Korea) was diluted 2,000 times the commercial concentration and administered at 5 mL per seedling. Each was treated with foliar spray.
  • Serifel biopesticide active ingredient: Bacillus amyloliquefaciens MBI600 (11%) WP, FarmHannong, Korea
  • foliar spraying was performed twice, 10 days and 3 days before pathogen inoculation, and 3 days after the second treatment , E. amylovora TS3128 bacteria were applied to each apple seedling as an inoculum for causing apple burn disease.
  • Control price (%) (virulence rate in untreated group - virulence rate in treated group) / virulence rate in untreated group ⁇ 100%.
  • the control effect of the Lyzobacter enzymogenes JCK-1421 strain against apple burn disease was examined 7 and 10 days after inoculation with the pathogen in M9 seedlings, and the results showed that the JCK-1421 20 SC formulation was 1,000 times
  • the diluted solution treatment group showed a control value of 100% compared to the untreated group both 7 and 10 days after pathogen inoculation.
  • the 1,000-fold diluted solution treatment group of JCK-1421 20 SC formulation showed the control activity of Seripel in the biopesticide control treatment group (7 days after inoculation). It was confirmed that it had a much better disease control effect than (48%, 40% control 10 days after vaccination).
  • strain JCK-1421 showed excellent control effect against apple burn blight by inducing resistance through pre-treatment before inoculation with the pathogen. Therefore, it was confirmed that it is possible to develop a highly effective fruit tree burn disease control agent in the future by using the control activity of the JCK-1421 strain.
  • Example 18 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of strain JCK-1421 against tomato root-knot nematode disease
  • Tomato 'Samkwang' seeds were used to investigate the in vivo control activity of tomato root-knot nematode disease by induced resistance of strain JCK-1421.
  • the square connection port was filled with topsoil, and tomato seeds ( Lycopersicon esculentum Mill. cv. Seokwang, Farhannong Co., Seoul, Korea) were sown using tweezers, and the photoperiod was irradiated for 16 hours a day in a constant temperature and humidity room at 25°C for 4 days. It was grown for a week. Afterwards, the plants used for root-knot nematode disease were transplanted into pots (diameter 12 cm, height 11 cm) using mixed soil (sand: top soil, 1:1, v/v).
  • 1% NaOCl Sodium hypochlorite, v/v
  • 1% NaOCl Sodium hypochlorite, v/v
  • the collected root-knot nematode eggs were adjusted to 8,000 nematode eggs per 10 mL of nematode egg suspension and then used as an inoculum.
  • the culture medium and formulation (JCK-1421 20 SC) of the JCK-1421 strain were used as described in Example 9. prepared as follows.
  • the JCK-1421 strain culture medium and JCK-1421 20 SC were diluted 500-fold, 1,000-fold, and 2,000-fold on Seogwang tomato seedlings 4 weeks after sowing, and 20 mL of each pot was applied to the soil.
  • Terranova (ai 1.68% Abamectin SC, Syngenta Korea), a standard drug for root-knot nematode disease, was diluted 5,000 times the commercial concentration and used as a soil drench at 10 mL per pot 1 day before pathogen inoculation.
  • 10 mL per pot was inoculated with M. incognita nematode egg suspension as pathogenic nematodes 4 days after drug treatment.
  • the inoculated pots were subjected to a photoperiod of 12 hours per day in a constant temperature and humidity room at 25°C.
  • the experiment was repeated 3 times, 2 per treatment group, and 6 weeks after inoculation, the bone formation index (Galling index) was evaluated from 0 to 5 depending on the degree of bone formation in tomato roots.
  • Investigation was conducted in six stages, including -100% (Kim et al., 2018), and egg masses were measured after soaking and staining tomato roots in 0.0015% phloxine B (Daejeong Hwageum Co., Ltd.) for 1 hour, and control was carried out accordingly. The effect was calculated.
  • Control value (%) 100 ⁇ [(Incidence rate in untreated group - Incidence rate in treated group) / Incidence rate in untreated group]
  • the JCK-1421 20 SC preparation showed the highest egg hatching and osteogenesis inhibition effects in the 2,000-fold dilution, especially in the treatment group with the 500-fold dilution of JCK-1421 strain culture and all JCK-1421 20 SC preparation treatments, which is the control drug. It showed excellent tomato root-knot nematode disease control activity with no statistically significant difference from Terranova. Therefore, it can be said that the JCK-1421 strain controlled tomato root-knot nematode disease by inducing resistance in tomatoes because it did not have direct nematicidal activity against M.
  • Example 19 Lyzobacter enzymogenes ( Lysobacter enzymogenes ) Control effect of strain JCK-1421 against pine wilt disease
  • the culture medium of the JCK-1421 strain for sample processing was prepared by the method described in Example 9.
  • the prepared culture medium of the JCK-1421 strain was dissolved in an aqueous solution containing 250 ⁇ g/mL Tween 20 so that the optical density was at the level of 0.8, then placed in a micro-spray bottle and inoculated at 5 mL per pine tree 2 weeks before and 1 week before pine nematode inoculation.
  • the foliar spray was applied.
  • Emamectin benzoate a nematicidal substance
  • EB Emamectin benzoate
  • pine wilt nematodes were cultured as follows. First, Botrytis cinerea, which pine wilt nematodes feed on, was inoculated and cultured in an incubator at 25°C for 7 days. Bursaphelenchus xylophilus (National Institute of Forest Science) was inoculated onto the cultured Botrytis cinerea and then cultured in an incubator at 25°C for 7 days. Cultured pine wilt nematodes were harvested using the Baermann funnel method, and their numbers were examined under an optical microscope and adjusted to 20,000/ml.
  • the present invention relates to a Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases comprising the strain, a culture thereof, or an extract thereof, a method for producing the composition, and the above. It relates to a method for controlling plant fungal diseases, bacterial diseases, or nematode diseases using a composition.

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Abstract

La présente invention concerne : une souche Lysobacter enzymogenes JCK-1421 ; une composition pour lutter contre les maladies fongiques des plantes, les maladies bactériennes ou les maladies provoquées par les nématodes, comprenant la souche, son produit de culture ou son extrait ; un procédé de préparation de la composition ; et un procédé pour lutter contre les maladies fongiques des plantes, les maladies bactériennes ou les maladies provoquées par les nématodes au moyen de la composition.
PCT/KR2023/015634 2022-11-14 2023-10-11 Composition pour lutter contre les maladies fongiques des plantes, les maladies bactériennes ou les maladies provoquées par les nématodes, comprenant le fluide de culture de la souche lysobacter enzymogenes jck-1421 ou son extrait, procédé de préparation, et procédé pour lutter contre les maladies fongiques des plantes, les maladies bactériennes ou les maladies provoquées par les nématodes WO2024106754A1 (fr)

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KR20090116244A (ko) * 2008-05-06 2009-11-11 고려대학교 산학협력단 고추 역병에 대한 길항 효과를 나타내는 토양 근권세균라이조박터 엔지모제네스 균주 ｉｓｅ１３
CN112760267A (zh) * 2021-02-09 2021-05-07 中国农业科学院蔬菜花卉研究所 一株拮抗野油菜黄单胞菌的产酶溶杆菌cx06及其应用

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KR20090116244A (ko) * 2008-05-06 2009-11-11 고려대학교 산학협력단 고추 역병에 대한 길항 효과를 나타내는 토양 근권세균라이조박터 엔지모제네스 균주 ｉｓｅ１３
CN112760267A (zh) * 2021-02-09 2021-05-07 中国农业科学院蔬菜花卉研究所 一株拮抗野油菜黄单胞菌的产酶溶杆菌cx06及其应用

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