KR20240081510A - Composition for controlling plant disease or reducing mycotoxins comprising culture solution of Bacillus velezensis JCK-7158 Strain or extract thereof, manufacturing methods thereof and controlling method for phytopathogenic fungi or reducing method for mycotoxins - Google Patents

Abstract

The present invention relates to a composition for controlling phytopathogenic fungi containing the Bacillus velezensis JCK-7158 strain, its culture medium or extract thereof, a method for producing the same, and a method for controlling phytopathogenic fungi, which include the culture medium of the strain or its extract. The extract has excellent antifungal activity and induced resistance and has an excellent ability to reduce toxins produced by fungi, so it can be used to control plant pathogenic fungi.

Description

Composition for controlling phytopathogenic fungi or reducing mycotoxins comprising an extract of the culture medium or strain culture medium of Bacillus velegensis JCK-7158, a method for producing the same, and a method for controlling plant pathogenic fungi or reducing mycotoxins {Composition for controlling plant disease or reducing mycotoxins comprising culture solution of Bacillus velezensis JCK-7158 Strain or extract thereof, manufacturing methods thereof and controlling method for phytopathogenic fungi or reducing method for mycotoxins}

The present invention relates to a composition for controlling phytopathogenic fungi containing Bacillus velezensis JCK-7158 strain, its culture medium or extract thereof, a method for producing the same, a method for controlling phytopathogenic fungi, and a method for reducing fungal toxins.

Fusarium head blight, caused by various Fusarium species including Fusarium graminearum and F. asiaticum, occurs periodically in wheat, barley, corn, rice, etc. It occurred on a large scale and caused a lot of damage around the world. In particular, major outbreaks have occurred in the United States and Canada over the past few years, resulting in a sharp rise in global grain prices.

Red mold disease not only causes a decrease in grain production, but also deteriorates the quality of agricultural products by leaving mycotoxins such as trichothecene and zearalenone, which are harmful to livestock, in the infected area. In particular, trichothecene Types of nivalenol (NIV) and deoxynivalenol (DON) cause serious poisoning, including lowered immunity and digestive tract disorders.

In addition, as there is a significant correlation between the rate of fusarium contamination and the detection level of trichothecenes in domestic wheat cultivation complexes, contamination of mycotoxins caused by red mold disease poses a significant threat to public health. It is becoming.

To prevent red mold toxin contamination by chemical control, the timing of fungicide treatment is very important. In the case of wheat, spraying should be done when anthers first emerge from the ears, and in the case of barley, spraying should be made when ears emerge.

DMI (Demethylation Inhibitor) class disinfectants such as metconazole, propiconazole, tebuconazole+prothionazole, and tebuconazole are the most effective. Due to the local penetration ability and the residual problem of some drugs, they can only be used up to 30 days before harvest, so the control effect in the field is less than 50%, which has the disadvantage of not being effective.

In addition, the misuse of synthetic pesticides, which account for the majority of pesticides, is raising various problems such as soil, water and agricultural product contamination, toxicity, ecosystem disturbance, and the emergence of resistant strains.

Accordingly, recently, OECD countries are trying to reduce the use of synthetic pesticides by more than 40%, and as interest in well-being is increasing and demand for organic products is rapidly increasing, especially in developed countries, there is a need to control red mold disease. There is an urgent need to develop environmentally friendly biologics.

Accordingly, the present inventors found that when a composition for controlling phytopathogenic fungi containing Bacillus velezensis JCK-7158 strain, its culture medium, or its extract was treated with phytopathogenic fungi, the control activity was significantly superior and the composition produced by the fungus was significantly superior. It was confirmed that the production of fungal toxins was also effectively reduced.

Accordingly, the purpose of the present invention is to provide a Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, which has antifungal activity.

Another object of the present invention is to provide a composition for controlling phytopathogenic fungi containing the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.

Another object of the present invention is iturin A, sulpectin, 2,3-butanediol, 5-Methylhexan-2-one ), heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl -2-Heptanone (5-methyl-2-heptanone) to provide a composition for controlling plant pathogenic fungi containing at least one selected from the group consisting of.

Another object of the present invention is to provide a method for producing a composition for controlling phytopathogenic fungi, which includes a cultivation step of preparing a culture medium by culturing the Bacillus bellegensis JCK-7158 strain deposited under the accession number KCTC15169BP.

Another object of the present invention is to provide a method for controlling phytopathogenic fungi, which includes a treatment step of treating the Bacillus belegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.

Another object of the present invention is iturin A, sulpectin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone.

Another object of the present invention is to provide a composition for reducing fungal toxins containing the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.

Another object of the present invention is to provide a method for producing a composition for reducing fungal toxins, which includes a culturing step of preparing a culture medium by culturing the Bacillus velegensis JCK-7158 strain deposited under the accession number KCTC15169BP.

Another object of the present invention is to provide a method for reducing fungal toxins, including a treatment step of treating the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.

Another object of the present invention is to provide a use for controlling phytopathogenic fungi or reducing fungal toxins of Bacillus belegensis JCK-7158 strain, its culture medium, or extract thereof, deposited under accession number KCTC15169BP.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention relates to the Bacillus velezensis JCK-7158 strain deposited under the accession number KCTC15169BP, which has antifungal activity.

In the present invention, the Bacillus belegensis JCK-7158 strain may contain 16S rRNA containing the base sequence of SEQ ID NO: 3.

Another aspect of the present invention relates to a composition for controlling phytopathogenic fungi comprising the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.

The term “culture medium” in this specification refers to containing microorganisms after culturing them.

The term “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.

In the present invention, the extract is iturin A, sulpectin, 2,3-butanediol, 5-Methylhexan-2-one ), heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl It may contain one or more compounds selected from the group consisting of -2-heptanone (5-methyl-2-heptanone).

The term “culture supernatant” in this specification refers to the upper liquid obtained after removing most microorganisms from the culture medium through centrifugation.

The term “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.

In the present invention, culture filtrate means removing fungi from the culture solution by centrifugation and filtration.

In the present invention, the plant pathogenic fungus is Clarireedia jacksonii ( Clariredia jacksonii , (formerly called Sclerotinia homoeocarpa ), Rhizoctonia solani AG-4, Rhizoctonia solani AG-2-2 (IV) Large patch, Rhizoctonia solani Aju 2-2 (Ⅳ) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum ( Fusarium oxysporum f. sp. cucumerinum ), Fusarium oxysporum f. Esp. Lycopersici ( Fusarium oxysporum f. sp. lycopersici ), Fusarium graminearum , Fusarium asiaticum , Fusarium verticillioides , Pythium ultimum , Gaeumannomyces graminis , Phytophthora infestans , Botrytis Cinerea, and Colletotrichum coccodes. There may be one or more types selected, but it is not limited thereto.

In one embodiment of the present invention, rice red mold disease, a plant pathogenic fungal disease, is caused by using a composition for controlling phytopathogenic fungi containing the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract. Control activity against Fusarium graminearum or Fusarium asiaticum can be obtained.

Another aspect of the present invention is iturin A, sulpectin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2- It is a composition for controlling phytopathogenic fungi containing at least one compound selected from the group consisting of 5-methyl-2-heptanone and 5-methyl-2-heptanone.

In the present invention, iturin A may be a compound of the following formula (1), its molecular weight is 1,043, and its molecular formula is C ₄₈ H ₇₄ O ₁₄ N ₁₂ .

<Formula 1>

In the present invention, the sulpectin may be a compound of the following formula (2), the molecular weight is 1,036, and the molecular formula is C ₅₃ H ₉₃ O ₁₃ N ₇ .

<Formula 2>

In the present invention, the 2,3-butanediol may be a compound of the following formula (3), the molecular weight is 90, and the molecular formula is C ₄ H ₁₀ O ₂ .

<Formula 3>

In the present invention, the 5-methylhexan-2-one may be a compound of the following formula (4), the molecular weight is 114, and the molecular formula is C ₇ H ₁₄ O.

<Formula 4>

In the present invention, the heptan-2-one may be a compound of the following formula 5, the molecular weight is 114, and the molecular formula is C ₇ H ₁₄ O.

<Formula 5>

In the present invention, the 2,5-dimethylpyrazine may be a compound of the following formula (6), the molecular weight is 108, and the molecular formula is C ₆ H ₈ N ₂ .

<Formula 6>

In the present invention, the 6-methylheptan-2-one may be a compound of the following formula (7), the molecular weight is 128, and the molecular formula is C ₈ H ₁₆ O.

<Formula 7>

In the present invention, the 5-methyl-2-heptanone may be a compound of the following formula (8), the molecular weight is 128, and the molecular formula is C ₈ H ₁₆ O.

<Formula 8>

In the present invention, the phytopathogenic fungi include Claryredia jacksoni, Rhizoctonia solani AGE-4, Rhizoctonia solani AGE 2-2 (IV) Large Patch, Rhizoctonia solani AGE 2- 2 (Ⅳ) Brown Patch, Fusarium Oxiform F. Esp. Cucumerinum, Fusarium oxyformum f. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes.

In the present invention, the composition for controlling plant pathogenic fungi may further include additives such as additives, extenders, nutrients, or sealing agents, but is not limited thereto.

In the present invention, the additives are polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene. The group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80. It may include one or more types selected from, but is not limited to this.

In the present invention, the extender may include one or more selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate. It is not limited.

In the present invention, the nutritional agent may include one or more selected from the group consisting of skim milk, soybean flour, rice, wheat, red clay, diatomaceous earth, bentonite, dextrin, glucose, and starch. , but is not limited to this.

Another aspect of the present invention relates to a method for producing a composition for controlling phytopathogenic fungi, which includes a culturing step of preparing a culture solution by culturing the Bacillus bellegensis JCK-7158 strain deposited under accession number KCTC15169BP.

In the present invention, the phytopathogenic fungi include Claryredia jacksoni, Rhizoctonia solani AGE-4, Rhizoctonia solani AGE 2-2 (IV) Large Patch, Rhizoctonia solani AGE 2- 2 (Ⅳ) Brown Patch, Fusarium Oxiform F. Esp. Cucumerinum, Fusarium oxyformum F. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, but is not limited thereto.

In the present invention, 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.

In the present invention, 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.

In the present invention, the method for producing a composition for controlling plant pathogenic fungi may further include a concentration step of concentrating the culture medium.

In the present invention, the method for producing a composition for controlling plant pathogenic fungi may further include a dilution step of diluting the culture medium.

In the present invention, the method for producing a composition for controlling phytopathogenic fungi may further include an extraction step of extracting components within the fungal cells.

In the present invention, the method for producing the composition for controlling fungi may include a fractionation step of obtaining an active fraction from the culture medium, but is not limited thereto.

In the present invention, the method for producing the composition for controlling fungi may include the following steps:

A culture filtrate obtaining step of obtaining a culture filtrate from the culture medium;

A fractionation step of obtaining fractions by fractionating the culture filtrate with a solvent;

A concentration step of concentrating the fractions to obtain a concentrate; and

A purification step in which the concentrate is purified and the active fraction is selected.

In the present invention, the step of obtaining the culture filtrate is performed at 2,000 to 5,000 rpm, 2,500 to 5,000 rpm, 3,000 to 5,000 rpm, 3,500 to 5,000 rpm, 4,000 to 5,000 rpm, 4,500 to 5,000 rpm, for example, 4,500 rpm. centrifugation It may include the step of doing so, but is not limited to this.

In the present invention, the fractionation step may include fractionation with one or more solvents selected from the group consisting of butanol, ethyl acetate, chloroform, methanol, ethanol, and hexane, but is not limited thereto.

The fractionation step may consist of the following steps:

A first fractionation step of obtaining a butanol layer by fractionating the culture medium or the culture filtrate obtained therefrom with butanol;

A second fractionation step of eluting the butanol layer obtained in the first fractionation step with a mixed solution of chloroform: methanol: water, and obtaining the fourth or fifth fraction among the five fractions; and

A third fractionation step in which the fractions obtained in the second fractionation step are eluted with a mixed solution of chloroform: methanol: water, and at least one type of fraction selected from the group consisting of the first to third fractions among the four fractions is obtained.

Independently in the second or third fractionation step, the mixing volume ratio of chloroform:methanol:water may be 50 to 60:30 to 40:5 to 10, preferably 52 to 58:35 to 40:7 to 9. It may be, for example, 55:36:8, but it is not limited to this.

In the present invention, the concentration step may include concentrating the fraction using a reduced pressure concentrator, but is not limited thereto.

In the present invention, the purification step may include purification using preparative high-performance liquid chromatography (HPLC), but is not limited thereto.

In the present invention, the active fraction is Iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone, but is not limited thereto.

Another aspect of the present invention relates to a method for controlling phytopathogenic fungi, which includes a treatment step of treating the plant or soil with the Bacillus bellegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.

In the present invention, the treatment step may be performed by one or more methods selected from the group consisting of spraying, soil irrigation, surface spraying, root zone treatment, seed treatment, soaking, poisoning, and smoking, for example, soil It may be performed through irrigation, but is not limited to this.

In the present invention, “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.

As used herein, 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.

As used herein, the term “soil irrigation” refers to a method of injecting or spraying a chemical solution into crop cultivation soil.

In the present invention, the phytopathogenic fungi include Claryredia jacksoni, Rhizoctonia solani AGE-4, Rhizoctonia solani AGE 2-2 (IV) Large Patch, Rhizoctonia solani AGE 2- 2 (Ⅳ) Brown Patch, Fusarium Oxiform F. Esp. Cucumerinum, Fusarium oxyformum F. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, but is not limited thereto.

Another aspect of the present invention is iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2- A method for controlling phytopathogenic fungi comprising at least one compound selected from the group consisting of 5-methyl-2-heptanone and 5-methyl-2-heptanone.

Another aspect of the present invention relates to a composition for reducing fungal toxins comprising the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.

In the present invention, the extract is iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone.

The fungal toxin may be produced by a phytopathogenic fungus, and the phytopathogenic fungus is Claryredia jacksoni, Rhizoctonia solani Aizu-4, Rhizoctonia solani Aiju 2-2 (IV) Raji. Patch, Rhizoctonia solani Aju 2-2 (Ⅳ) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum, Fusarium oxyformum F. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, for example, it may be produced by Fusarium asiaticum, and the fungal toxin may be Nivalenol (NIV). , but is not limited to this.

Another aspect of the present invention relates to a method for producing a composition for reducing fungal toxins, comprising a culturing step of preparing a culture medium by culturing the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP.

In the present invention, the extract is iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone.

The fungal toxin may be produced by a phytopathogenic fungus, and the phytopathogenic fungus is Claryredia jacksoni, Rhizoctonia solani Aizu-4, Rhizoctonia solani Aiju 2-2 (IV) Raji. Patch, Rhizoctonia solani Aju 2-2 (Ⅳ) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum, Fusarium oxyformum f. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, for example, it may be produced by Fusarium asiaticum, and the fungal toxin may be nivalenol, but is not limited thereto. no.

Another aspect of the present invention relates to a method for reducing fungal toxins, comprising a treatment step of treating the Bacillus velegensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract.

In the present invention, the extract is iturin A, sulpactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone.

The fungal toxin may be produced by a phytopathogenic fungus, and the phytopathogenic fungus is Claryredia jacksoni, Rhizoctonia solani Aizu-4, Rhizoctonia solani Aiju 2-2 (IV) Raji. Patch, Rhizoctonia solani Aju 2-2 (Ⅳ) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum, Fusarium oxyformum f. Esp. Lycopassici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticilloides, Pythium ultimum, Gaumanomyces graminis, Phytophthora infestans, Botrytis cinerea, and Coleus. It may be one or more species selected from the group consisting of Totrichum cocodes, for example, it may be produced by Fusarium asiaticum, and the fungal toxin may be nivalenol, but is not limited thereto. no.

In the method for producing the composition for controlling plant pathogenic fungi and the method for controlling plant pathogenic fungi, content that overlaps with the composition for controlling plant pathogenic fungi is omitted in consideration of the complexity of the present specification.

The present invention relates to a composition for controlling phytopathogenic fungi or reducing fungal toxins containing Bacillus velezensis JCK-7158 strain, its culture medium or extracts thereof, a method for producing the same, and a method for controlling phytopathogenic fungi or reducing fungal toxins. As the culture medium of the above-mentioned strain or its extract has significantly excellent antifungal activity and induced resistance, it can be used for controlling plant pathogenic fungi or reducing fungal toxins.

Figure 1 is a family diagram analyzed by 16S rRNA gene base sequence of Bacillus velezensis JCK-7158 strain according to an embodiment of the present invention.
Figure 2a is a graph showing the extracellular protease activity of Bacillus velegensis JCK-7158 strain over time according to an embodiment of the present invention.
Figure 2b is a graph showing the extracellular chitinase activity of Bacillus velegensis JCK-7158 strain over time according to an embodiment of the present invention.
Figure 2c is a graph showing the extracellular cellulase activity of Bacillus velegensis JCK-7158 strain over time according to an embodiment of the present invention.
Figure 2d is a graph showing the extracellular gelatinase activity of Bacillus velegensis JCK-7158 strain over time according to an embodiment of the present invention.
Figure 3 is a photograph showing the extracellular enzyme activity of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 4 is a photograph showing the IAA production activity results of the Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 5 is a photograph showing the acetoin production activity of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 6 is a photograph showing the mycelial growth inhibition activity of various plant pathogenic fungi by replacement culture of the Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 7 is a diagram schematically illustrating the process of fractionation of the antifungal active substance of the Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 8 shows the antifungal properties against Fusarium graminearum , a pathogenic strain, after developing the butanol fraction of the culture filtrate of Bacillus velegensis JCK-7158 strain by thin layer chromatography (TLC) according to an embodiment of the present invention. This is a photo analyzing activity.
Figure 9 is a thin layer chromatography photograph of four fractions isolated from the BF4 fraction of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 10 is a graph showing the results of positive ion mode LC/MS analysis of iturin A isolated from the BF4-1 fraction of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 11 is a graph showing the results of positive ion mode LC/MS analysis of sulfectin isolated from the BF4-1 fraction of Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 12 is a photograph showing the mycelial growth inhibition activity of plant pathogenic fungi by the antifungal activity of volatile substances produced by the Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 13 is a graph schematically illustrating the results of solid phase microextraction (SPME) GC-MS analysis of volatile organic compounds produced by the Bacillus velegensis JCK-7158 strain according to an embodiment of the present invention.
Figure 14 is a photograph of testing the PR-1 gene expression activity according to the presence or absence of GUS expression of the Bacillus velegensis JCK-7158 strain in transgenic Arabidopsis thaliana according to an embodiment of the present invention.
Figure 15 is a graph showing the control effect on rice red mold disease (Fusarium head blight) caused by induced resistance of Bacillus belegensis JCK-7158 strain in greenhouse conditions according to an embodiment of the present invention.
Figure 16 is a photograph showing the control effect on rice red mold disease caused by induced resistance of the Bacillus belegensis JCK-7158 strain in greenhouse conditions according to an embodiment of the present invention 7 days after inoculation.
Figure 17 is a graph showing the control effect on rice red mold disease by induced resistance of two formulations using Bacillus belegensis JCK-7158 strain in greenhouse conditions according to an embodiment of the present invention.
Figure 18 is a photograph showing the control effect on rice red mold disease by induced resistance of two formulations using the Bacillus belegensis JCK-7158 strain under greenhouse conditions according to an embodiment of the present invention, 7 days after inoculation.
Figure 19 is a graph showing the control effect on rice red mold disease by induced resistance of the Bacillus belegensis JCK-7158 strain preparation under field conditions according to an embodiment of the present invention.
Figure 20 is a photograph showing the control effect on rice red mold disease due to induced resistance of the Bacillus belegensis JCK-7158 strain preparation under field conditions according to an embodiment of the present invention two weeks after inoculation.
Figure 21 is a photograph showing the control effect on rice red mold disease due to induced resistance of the Bacillus belegensis JCK-7158 strain preparation under field conditions 4 weeks after inoculation according to an embodiment of the present invention.

Hereinafter, the present invention is explained in detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, “%” 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)%.

Example 1. Isolation of JCK-7158 strain with antibacterial activity against red mold

A strain with antifungal activity against Fusarium graminearum ( F. graminearum ), which causes red mold disease in crops, was isolated from the stems of Shindongjin rice grown in Gokseong, Jeollanam-do. The collected rice stems (10 g) were ground by adding 100 mL of sterilized water, and the ground stem samples were diluted 40 times with sterilized water and then plated on TSA (Tryptic Soy Agar) containing 1% of F. graminearum mycelium suspension. was plated on, cultured at 30°C for more than 3 to 7 days, and the JCK-7158 strain producing a clear zone was isolated.

Example 2. Molecular biological analysis and phylogenetic analysis of JCK-7158 strain

Strain JCK-7158, isolated from rice stems, was identified molecularly through nucleotide sequence analysis of the 16S rRNA gene. The strain was inoculated into TSB medium and cultured with shaking at 1580 rpm for 3 days at 30°C. Then, gDNA (genomic DNA) of the harvested strain was extracted using the I-genomic BYF DNA Extraction Mini kit (iNtRON, Korea) according to the protocol suggested by the manufacturer.

After mixing the extracted gDNA of the strain, iNtRON Biotechnology's PCR-premix (polymerase chain reaction-premix), and a primer set that can amplify the 16S rRNA of the strain, the gene was amplified through PCR. 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 PCR product of the amplified 16S rRNA gene was requested to Genotech (Daejeon, Korea) for base sequence analysis, and a total base sequence of 1352 bp (SEQ ID NO. 3) was obtained as the 16S rRNA coding base sequence of the isolated strain, JCK-7158 strain. got it

sequence number denomination Sequence Listing (5’-> 3’) note One 27F GAG TTT GAT CMT GGC TCA G 2 1492R TAC GGY TAC CTT GTT ACG ACT T 3 16s rRNA GCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACT GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCT CGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGG GGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAAT CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGT 1352 bp

As can be seen in Figure 1, as a result of comparing the base sequences of the GenBank database using NCBI's BlastN search, the JCK-7158 strain was identified as Bacillus velezensis. Therefore, the above strain was named Bacillus belegensis JCK-7158 strain and deposited in the Korean Collection for Type Cultures (KCTC) on November 2, 2022, and given accession number KCTC15169BP.

Example 3. Biochemical characteristics of Bacillus belegensis JCK-7158 strain - evaluation of extracellular enzyme activity

To measure the activities of protease, chitinase, gelatinase, and cellulase of Bacillus velegensis JCK-7158 strain, protease medium (1% skim milk + 1.5% agar, Difco), chitinase medium (1% colloidal chitin + 1.5% agar, Difco), gelatinase medium (10% gelatin + 1.5% agar, Duksan), cellulase medium (0.4% carboxymethyl cellulose sodium + 1.5% agar, Sigma) -Aldrich) was manufactured.

In the case of chitinase medium, to prepare colloidal chitin added to the medium, HCl (hydrochloric acid) was first added to crab shell powder (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 with 0.1 M potassium phosphate buffer (pH 7.0) and used as colloidal chitin. The prepared colloidal chitin was sterilized by high-pressure steam and stored at 4°C until use.

Gelatinase medium was solidified by adding 10% gelatin to LB solid medium, and other media were solidified by adding 1.5% agar.

A sterilized paper disc (0.8 cm, Advantec, Japan) was placed on top of each prepared medium, and 5 μL, 10 μL, and 15 μL of JCK-7158 strain culture filtrate was added to protease medium, chitinase medium, and cellulase medium. , 20 μL, 40 μL, and 60 μL were dispensed into the gelatinase medium, respectively. As a negative control, equal amounts of sterilized LB and medium were dispensed onto paper disks. The experiment was repeated three times, and the plate was maintained at 30°C to observe the clear zone according to extracellular enzyme activity. For visualization, the cellulase medium and chitinase medium were stained by adding 5 mL of Lugol's solution (2.5 g/L iodine and 5 g/L potassium iodide) to the plate and maintained for 10 minutes under dark conditions, and then the clear zone was observed.

As can be seen in Figures 2 and 3, as a result of observing the clear zone by treating the culture filtrate with the medium inducing enzyme activity, the diameter of the clear zone for strain JCK-7158 was 34.87 mm after 9 days of treatment with 15 μL of the sample in the protease medium. , a clear zone of 35.72 mm after 5 days of treatment with 15 μL of sample in cellulose medium, 38.26 mm after 5 days of treatment of 15 μL of sample in chitinase medium, and 19.29 mm after 5 days of treatment of 60 μL of sample in gelatinase medium. could be observed. As a result, it was confirmed that the Bacillus velegensis JCK-7158 strain produces protease, chitinase, gelatinase, and cellulase.

Example 4. IAA production of Bacillus belegensis JCK-7158 strain

To confirm the production of IAA (Indole-3-acetic acid), a plant growth hormone, by Bacillus belegensis JCK-7158 strain, first, JCK was added to TSB medium supplemented with L-tryptophan (150 mg/L). 1% of -7158 strain culture was inoculated and cultured with shaking at 30°C and 150 rpm for 24 hours. Afterwards, the culture was centrifuged at 4°C and 10,000 rpm for 5 minutes and then filtered through a 0.2 μm 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). After maintaining it at room temperature and under dark conditions for 20 minutes, it was checked whether the mixed solution was pink. As a negative control, TSB medium supplemented with L-tryptophan (150 mg/L) was used, and the experiment was repeated three times.

As can be seen in Figure 4, as a result of conducting an experiment to confirm the production of IAA, a plant growth hormone, of the JCK-7158 strain, the JCK-7158 strain culture filtrate treatment group showed color in the test tube, and the TSB treatment group, which was the control group, showed color in the test tube. The original color of the badge was displayed. Therefore, it was confirmed that strain JCK-7158 produces IAA, a plant growth hormone.

Example 5. Production of 2,3-butanediol by Bacillus belegensis JCK-7158 strain

In order to confirm the production of 2,3-butanediol, a volatile substance, in the Bacillus belegensis JCK-7158 strain, the production of acetoin, a precursor of 2,3-butanediol, was confirmed. To confirm the production of acetoin, MRS-VP (5 g/L Peptone, 5 g/L Dextrose, 5 g/L Dipotassium phosphate) medium was used. Inoculate Bacillus velegensis JCK-7158 in MRS-VP medium and culture with shaking at 30°C and 150 rpm for 3 days. Then, 100 μl of culture medium and 60 μl of 5% Naphtol were added to a 96 well plate. , 20 μl of 40% potassium hydroxide was added in that order. It was maintained for 10-15 minutes at room temperature and checked to see if the mixed solution was pink. The negative control used only MRS-VP medium, and the positive control used MRS-VP medium with acetoin (1 mg/mL) added.

sample 2,3-butanediol production JCK-7158 ++ MRS-VP - Acetoin +++

As can be seen in Table 2 and Figure 2, the production of 2,3-butanediol, a volatile substance, of the Bacillus velegensis JCK-7158 strain was confirmed by the production of acetoin, a precursor. As a result, the Bacillus velegensis JCK-7158 strain treatment group was a positive control. As it turned pink, it was confirmed that acetoin was produced, and through this, it was predicted that the Bacillus belegensis JCK-7158 strain produces 2,3-butanediol. 2,3-Butanediol is known to have resistance-inducing activity in various plants. Therefore, it can be seen that the Bacillus belegensis JCK-7158 strain culture medium also has the potential to control various plant diseases by producing 2,3-butanediol and inducing resistance in the host.

Example 6. Growth inhibitory activity through MIC test of plant pathogen mycelium according to treatment of Bacillus belegensis JCK-7158 strain

In order to determine the effect of the culture filtrate of the Bacillus belegensis JCK-7158 strain of the present invention on the growth of various plant pathogenic fungi, the growth inhibitory activity was measured for eight types of plant pathogens. The JCK-6019 strain was inoculated into TSB liquid medium and then cultured with shaking at 150 rpm for 3 days at 30°C. The culture was centrifuged at 10,000 MIC (Minimum Inhibitory Concentration) test was performed by applying the 96-well microtiter plate method, and the plant pathogenic fungi were Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (Ⅳ) Large patch, Fusarium graminearum, and Fusarium asiaticum. , Gaeumannomyces graminis, Phytophthora infestans, Botrytis cinerea, and Colletotrichum coccodes were used.

The plant pathogenic fungi used in the experiment were inoculated into PDB (potato dextrose broth, Becton, Dickinson and Co., Sparks, MD, USA) medium and cultured in an incubator at 25°C for 7 days. After stationary culture, the weight of the mycelium was measured, sterilized distilled water was added to a concentration of 50 mg/mL, homogenized using a homogenizer at 10,000 rpm for 5 seconds, and then added to PDB medium to obtain a fungal suspension with a 1% concentration.

The phytopathogenic fungal suspension was treated with the culture filtrate sample of the Bacillus velegensis JCK-7158 strain of the present invention to a concentration of 10%, 5%, 2.5%, 1.25%, 0.625%, 0.313%, 0.156%, and 0.078%, respectively. was performed three times. The treated plate was sealed and cultured in an incubator at 25°C for 3-5 days to determine the minimum inhibitory concentration (MIC) of pathogens.

Minimum growth inhibitory concentration (MIC) for various plant pathogenic fungi in culture filtrate samples of Bacillus belegensis JCK-7158 strain No. plant pathogenic fungi MIC value (%) One Clariredia jacksonii 1.25 2 Rhizoctonia solani AG-4 5 3 Rhizoctonia solani AG 2-2 (Ⅳ) Large patch 5 4 Fusarium graminearum 1.25 5 Fusarium asiaticum 10 6 Gaeumannomyces graminis 5 7 Phytophthora infestans 5 8 Colletotrichum coccodes 5 9 Botrytis cinerea 5

As can be seen in Table 3, as a result of performing an MIC test to confirm the antifungal activity of the Bacillus belegensis JCK-7158 strain of the present invention against phytopathogenic fungi, the JCK-7158 strain had an antifungal activity of 1.25 against Clarireedia jacksonii and Fusarium graminearum . It showed the strongest antifungal activity with a MIC value of %, and was also effective against Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2(IV) Large patch, Gaeumannomyces graminis , Botrytis cinerea, Colletotrichum coccodes , and Phytophthora infestans with a MIC value of 5%. It showed strong activity. In addition, it was confirmed that Fusarium asiaticum had antifungal activity as it had an MIC value of 10%.

As a result, it was confirmed that the Bacillus belegensis JCK-7158 strain of the present invention has strong antifungal activity against various plant pathogenic fungi used in the MIC test results.

Example 7. Growth inhibition activity through replacement culture of plant pathogens according to treatment of Bacillus belegensis JCK-7158 strain

To investigate the mycelial growth inhibitory activity of the Bacillus belegensis JCK-7158 strain of the present invention against various plant pathogenic fungi, the growth inhibitory activity was measured against 13 types of plant pathogens by replacement culture.

The target plant pathogenic fungi are Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (Ⅳ) Large patch, Rhizoctonia solani AG 2-2 (Ⅳ) Brown patch, Fusarium oxysporum f. sp. cucumerinum , Fusarium oxysporum f.sp. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytophthora infestans, Botrytis cinerea, and Colletotrichum coccodes were used.

Specifically, the culture medium of the Bacillus belegensis JCK-7158 strain of the present invention was streaked to a length of 5 cm at a point 2 cm away from the edge of the PDA plate, and the mycelial pieces of each pathogen were cut with a cork borer with a diameter of 6 mm to form JCK-7158. It was inoculated at a distance of 5 cm from the strain. The untreated group was inoculated only with mycelial fragments of the pathogen. After inoculation with fragments of pathogenic fungal hyphae and the culture medium of the Bacillus belegensis JCK-7158 strain of the present invention, the cells were cultured in an incubator at 25°C. The mycelial growth radius of the pathogen was measured according to the growth rate of the pathogen, and the experiment was repeated three times. Antifungal activity was calculated by measuring the radius (R2) of pathogen hyphae in the direction of the JCK-7158 strain and measuring the radius (R1) of pathogen hyphae in control conditions. Both values were converted to percent mycelial growth inhibition using the following formula:

Mycelial growth inhibition rate (%) =

As a result of performing replacement culture of the Bacillus belegensis JCK-7158 strain of the present invention against various plant pathogenic fungi, as can be seen in Figure 6 and Table 4, the Bacillus belegensis JCK-7158 strain was effective against all pathogens used in the experiment. showed growth inhibitory activity. Among them, the growth of all molds used in the experiment except Pythium ultimum was inhibited by about 50%. Therefore, it was confirmed that the JCK-7158 strain had strong antifungal activity against various plant pathogenic fungi used in the experiment even through replacement culture.

Mycelial growth inhibition activity of various plant pathogenic fungi by replacement culture of Bacillus belegensis JCK-7158 strain No. plant pathogenic fungi Mycelial growth inhibition rate (%) One Rhizoctonia solani AG-4 46.04±0.65 2 Rhizoctonia solani AG 2-2 (Ⅳ) Large patch 56.12±1.52 3 Rhizoctonia solani AG 2-2 (Ⅳ) Brown patch 47.27±1.31 4 Fusarium graminearum 51.55±2.19 5 Fusarium asiaticum 56.19±1.79 6 Fusarium oxysporum f. sp. cucumerinum 57.97±2.48 7 Fusarium oxysporum f. sp. lycopersici 53.13±3.94 8 Gaeumannomyces graminis 51.36±0.72 9 Pythium ultimum 19.09±3.81 10 Botrytis cinerea 59.90±0.93 11 Fusarium verticillioides 53.84±3.56 12 Colletotrichum coccodes 50.02±4.74 13 Phytophthora infestans 52.08±1.17

^a Mycelial growth inhibition rate (%) = (1 ^- fungal growth length in treatment/control fungal growth period)

Example 8. Isolation and structural identification of antibacterial active substances of Bacillus belegensis JCK-7158 strain

To isolate the antibacterial active substance produced by the Bacillus belegensis JCK-7158 strain, the strain was inoculated into TSB liquid medium and then cultured with shaking at 150 rpm for 3 days at 30°C. The isolation process of the antibacterial active substance produced by the Bacillus velegensis JCK-7158 strain was performed according to the process shown in FIG. 7.

To explain the separation process in detail, after culturing, the culture medium (total 3 L) was centrifuged at 4500 rpm for 20 minutes to obtain the supernatant of the culture medium. The supernatant (290 mL) was fractionated twice with equal amounts of ethyl acetate (EtOAc) and butanol (BuOH) to obtain an organic solvent layer and an aqueous solution layer of the ethyl acetate layer and the butanol layer, then concentrated under reduced pressure, and each fraction layer was washed with acetone. , re-dissolved in methanol and water. To test the antifungal activity of each fraction , Fusarium graminearum was used as an antifungal activity test pathogen.

As can be seen in Figure 8, as a result of the antifungal activity test, it was confirmed that the butanol fraction layer showed antifungal activity against F. graminearum . The butanol fraction layer that showed activity was developed in preparative thin-layer chromatography under chloroform:methanol:water (14:6:1, v/v/v) solvent conditions and then eluted with methanol to produce five fractions (BF1 to BF5). As a result of performing antifungal activity against Fusarium graminearum on the five obtained fractions, the BF4 fraction had the highest activity with an MIC of 125 ppm, as shown in Table 5 below.

Antifungal activity of five fractions isolated from the butanol fraction of Bacillus belegensis JCK-7158 strain sample Yield (mg) MIC(ppm) BF1 12.4 - BF2 6.5 - BF3 6.7 - BF4 21.4 125 BF5 25.1 250

As a result of the antifungal activity test, the selected active fraction, the BF4 fraction, was developed again in preparative thin layer chromatography under chloroform:methanol:water (55:36:8, v/v/v) solvent conditions, and then eluted with methanol to produce four fractions (BF4). -1~BF4-4) was obtained. These four fractions and the standard substances Iturin, Fengycin, and Surfactin purchased from Sigma-Aldrich were loaded into thin layer chromatography (TLC) and chloroform:methanol:water (55:36). :8, v/v/v) After development in solvent conditions, it was read by UV at 254 and 365 nm wavelengths and visualized by spraying p-anisaldehyde and water.

As a result, as shown in FIG. 9, it was confirmed that all four fractions obtained after visualization contained iturin A. In addition, as a result of testing the antifungal activity of these four fractions, as shown in Table 5, the activity of the active fraction BF4-1 was the best among the four separated fractions with an MIC of 125 ppm.

Antifungal activity of four fractions isolated from BF4 fraction derived from Bacillus belegensis JCK-7158 strain sample Yield (mg) MIC(ppm) BF4-1 3.4 125 BF4-2 4.7 250 BF4-3 3 250 BF4-4 4.3 -

The active fraction BF4-1 was observed to contain iturin as a result of thin layer chromatography, but as a result of LC/MS analysis of the BF4-1 fraction, positive ion mode peaks were found at molecular weights of 1043.55 and 1036.69, respectively, as shown in Figures 10 and 11 below. It was found that these substances were Iturin A (C ₄₈ H ₇₄ O ₁₄ N ₁₂ , molecular weight 1,043) and Surfactin (C ₅₃ H ₉₃ O ₁₃ N ₇ , molecular weight 1,036).

Example 9. Antifungal activity assay using volatile organic compounds produced by Bacillus velegensis JCK-7158 strain

The phytopathogenic fungi Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (Ⅳ) Large patch, Rhizoctonia solani AG 2-2 (Ⅳ) of volatile organic compounds produced by the Bellezensis JCK-7158 strain of the present invention Brown patch, Fusarium oxysporum f. sp. cucumerinum , Fusarium oxysporum f. sp. Antifungal activity was tested against lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytophthora infestans, Botrytis cinerea, and Colletotrichum coccodes .

To prevent direct contact, a Bi-Petri Dish (SPL Life Sciences Co., Ltd., Pocheon, Gyeonggi-do, Korea) with a separated center was used. TSA was dispensed on one side of the plate, and PDA was dispensed on the other side. After dispensing, the culture medium of the JCK-7158 strain was smeared and cultured, and a piece of pathogen mycelium with a diameter of 6 mm was inoculated into the PDA on the other side and cultured at 25°C. In the untreated group, only the pathogen was inoculated into the PDA without smearing the culture medium of the JCK-7158 strain. The experiment was performed in three repetitions, and all plates were sealed twice with parafilm, cultured at 25°C, and the mycelial growth diameter of the pathogen was measured. In the untreated group, only pathogens were inoculated without smearing the culture medium of the strain.

Antifungal activity was calculated by measuring the length and width (R1, R2) of pathogen hyphae and measuring the length and width (R3, R4) of pathogen hyphae in control conditions. Both values were converted to percent mycelial growth inhibition using the following formula: The results are shown in the table below.

Inhibition of mycelial growth of plant pathogenic fungi by antifungal activity of volatile substances produced by Bacillus belegensis JCK-7158 strain. No. plant pathogenic fungi Mycelial growth inhibition rate (%) One Rhizoctonia solani AG-4 9.39±1.56 2 Clariredia jacksonii 7.54±12.7 3 Rhizoctonia solani AG 2-2 (Ⅳ) Large patch 38.57±17.69 4 Rhizoctonia solani AG 2-2 (Ⅳ) Brown patch 31.45±6.88 5 Fusarium asiaticum 10.78±18.30 6 Fusarium graminearum 48.66±25.43 7 Fusarium oxysporum f. sp. cucumerinum 29.48±2.13 8 Fusarium oxysporum f. sp. lycopersici 7.26±1.11 9 Gaeumannomyces graminis 21.29±9.26 10 Pythium ultimum 8.94±3.46 11 Botrytis cinerea 60.50±8.85 12 Fusarium verticillioides 12.51±22.20 13 Colletotrichum coccodes 27.57±4.85 14 Phytophthora infestans 4.53±3.53

As can be seen in Table 7 and Figure 12, the antifungal activity of volatile organic compounds produced by the JCK-7158 strain against plant pathogenic fungi was confirmed, and the volatile substances produced by the JCK-7158 strain inhibited the growth of various plant pathogenic fungi. However, especially Botrytis cinerea, which causes gray mold disease on fruits and crops, and Fusarium graminearum, which causes red mold disease on grains. They showed excellent pathogenic mycelia inhibition effects of 60.50% and 48.66%, respectively. Therefore, it was confirmed that the JCK-7158 strain of the present invention produces volatile substances showing antifungal activity against various plant pathogenic fungi.

Example 10. Analysis of volatile organic compounds using GC-MS

SPME (solid phase microextraction) GC-MS (Gas chromatography-mass spectrometry) analysis was performed to analyze volatile organic compounds produced by the JCK-7158 strain of the present invention. The JCK-7158 strain was inoculated into 5 mL of TSB medium and cultured with shaking for 3 days at 30°C and 150 rpm. Volatile organic compounds produced by JCK-7158 were collected using SPME fiber (Supelco, Bellefonte, PA, USA) for 30 minutes at 50°C in the headspace.

The material adsorbed on the SPME fiber was injected and exposed for 1 minute to be desorbed and then analyzed using a GC-MS (Shimadzu GC-MS QP2010, Shimadzu) equipped with a DB-5MS capillary column (30 m co., Kyoto, Japan). He was used as the mobile phase, and the flow rate was maintained at 1.0 mL/min. The inlet temperature was set at 250°C, and the column temperature was maintained at 60°C for 2 minutes, then increased to 250°C at a rate of 10°C/min and maintained at 250°C for 20 minutes. The ionization voltage of the mass spectrometer was 70 eV, and the analysis was performed in positive ion mode with a scan of 50 to 400 m/z at 200°C.

The obtained mass spectrum was accurately identified by comparison with data from the WILEY8 Library, and the content of each substance was expressed as the area ratio of the TIC (total ion chromatogram) peak.

SPME GC-MS analysis results for volatile organic compounds produced by Bacillus belegensis JCK-7158 strain No. Retention time (min) Relative peak area (%) Possible compound One 4.158 4.36 5-Methylhexan-2-one 2 4.700 24.06 Heptan-2-one 3 5.095 15.94 2,5-Dimethylpyrazine 4 5.770 34.89 6-Methylheptan-2-one 5 5.930 20.75 5-Methyl-2-heptanone

As can be seen in Table 8 and Figure 13, as a result of the analysis, the volatile organic compounds produced by the JCK-7158 strain of the present invention were detected as 5 substances within 7 minutes. The chemical structures of the five substances were confirmed by comparing their mass spectrum with those in the library, and as a result of GC-MS analysis and mass spectrum analysis, the five volatile compounds were 5-methylhexan-2-one, heptan- They were identified as 2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanon. It was thought that these substances produced by the JCK-7158 strain of the present invention inhibit the growth of the plant pathogenic fungi used in the experiment.

Example 11. Preparation of formulation using culture medium of Bacillus belegensis JCK-7158 strain

In order to prepare a powder hydrate and liquid hydrate using the JCK-7158 strain and culture medium of the present invention, the Bacillus belegensis JCK-7158 strain was inoculated into 20 mL of TSB medium, and then cultured with shaking at 30°C and 150 rpm for 3 days. did. 20% of Flocet Lite (oxidized starch) was added to the culture solution harvested after culturing for 3 days and spray-dried at 140°C while stirring. The spray-dried samples obtained as a result of spray-drying were prepared as described in Tables 9 and 10 below to prepare JCK-7158 strain suspension concentrate (SC) and wettable powder (WP).

Composition of liquid moisturizer using spray-dried sample of Bacillus belegensis JCK-7158 strain number ingredient Quantity One Spray dried sample 100g 2 CR-NF135B 30mL 3 Propylene glycol 50mL 4 Xanthan Gum 1.0% solid 100mL 5 Sodium benzoate 2g 6 Defoamer (CR-SAG672) 1mL 7 water 719mL 8 total 1,000mL

Composition of powdered wettable powder using spray-dried sample of Bacillus belegensis JCK-7158 strain number ingredient Quantity One Spray dried sample 10g 2 White carbon 15g 3 CR-SDS 12.5g 4 CR-WP100 12.5g 5 kaoline 500g 6 total 100g

Example 12. Resistance induction activity assay of JCK-7158 strain in Arabidopsis thaliana

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. Therefore, in order to test the resistance-inducing activity of Bacillus belegensis JCK-7158 strain culture using the PR-1 gene system, Arabidopsis thaliana transformed with a GUS-labeled vector in the PR-1 promoter was used. .

The surface of Arabidopsis seeds was sterilized using 95% ethanol, and secondary surface sterilization was performed using a bleach solution prepared with 2% NaOCl and 0.05% Tween-20. The bleaching solution remaining on the seeds was washed with sterile distilled water and soaked at 4°C for 48 hours.

The soaked seeds were placed on Murashige-Skoog Agar (MS agar) supplemented with 50 ppm kanamycin using a sterilized toothpick and cultured in a plant growth incubator at 25°C. After 12 days of culture, the culture medium, culture filtrate, cell suspension, liquid hydrate (SC), and powder hydrate (WP) of the Bacillus belegensis JCK-7158 strain of the present invention were added to each well of a 24-well plate in sterile water at 500, 1,000, It was diluted 2,000- and 4,000-fold and treated at 2.5 mL each. Two Arabidopsis plants were submerged in each well and maintained at room temperature for 48 hours in an orbital shaker.

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). After washing , prepare a staining solution (100mM Sodium phosphate buffer, 0.1% Triton X-100, 2mM and maintained in a constant temperature water bath at 37°C for 24 hours.

After 24 hours, the staining solution was removed, immersed in 70% ethanol for 1 hour, and then immersed in 90% ethanol several times at hourly intervals to remove unnecessary pigments such as chlorophyll. After immersing several times and all the pigment was removed, the expression of the PR-1 gene was confirmed by checking whether it was blue using a microscope.

Through the Arabidopsis assay system, the expression of the PR-1 gene was confirmed by the presence or absence of GUS expression, and the induction resistance and induction activity were tested.

PR-1 gene expression inducing activity of Bacillus belegensis JCK-7158 strain Sample Partition Concentration X 500 X 1,000 X 2,000 X 4,000 JCK-7158 CB ++ +++ +++ ++ CF ++ ++ ++ ++ Cell + ± ++ ++ S.C. ++ + + ++ WP ++ ++ +++ + TSB - Sample Concentration 0.1mM SA +++

As can be seen in Table 11 and Figure 14, the culture broth (CB), culture filtrate (CF), and cell suspension (Culture Broth (CB)) of the Bacillus belegensis JCK-7158 strain were the same as the salicylic acid (SA) treatment group, which is a positive control. Cell), and the two types of preparations prepared in Example 11, liquid hydrate (SC) and powder hydrate (WP), were confirmed to induce expression of the PR-1 gene by expressing GUS, and through this, Bacillus belegensis JCK It was confirmed that strain -7158 can control pathogenic fungi by inducing resistance in plants through a mechanism similar to salicylic acid.

Example 13. Control effect on rice red mold disease by induced resistance of Bacillus belegensis JCK-7158 strain in greenhouse conditions

Samkwang rice seeds were used to investigate the in vivo control activity of rice red mold disease by induced resistance of the JCK-7158 strain of the present invention under greenhouse conditions. Put 10 g of 'Samkwang rice' seeds ( Oryza sativa cv. Samkwang) distributed from the National Institute of Crop Science of the Rural Development Administration into a plastic container, dilute 2,000 times with Sportak emulsion (25% prochloraz, Kyungnong Co., Ltd.), and sterilize the seeds for 1 day. Afterwards, it was immersed in water for 2 days under dark conditions.

For 2 days, 10 seeds per pot were sown in plastic pots (6 cm in diameter, 6.5 cm in height) filled with 80% water-soluble topsoil (heavy soil, Bunong). After sowing, 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. When the rice germinated by about 1 cm, 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).

Seven days before transplanting, 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℃, maximum temperature of 30-35℃) and used in experiments. 20 days after pot transplantation, the first fertilization was done with SuperAli (14 kg/10 a, Namhae Chemical), and 2 months after pot transplantation, the second fertilization was done with NK 24 (N:P:K, 24-0-12, FarmHannong). Additional fertilization was carried out.

To prepare rice red mold pathogen inoculum , Fusarium asiaticum strain, one of the causative bacteria of rice red mold disease, was inoculated into PDA medium and cultured for 5 days at 25°C. Five _agar plugs (0.1 cm ·7H ₂ O, 1 g NH ₄ NO ₃ , and 1 g KH ₂ PO ₄ , 1 L distilled water) and cultured with shaking at 25°C and 150 rpm for 4 days.

Afterwards, the pathogen culture was filtered through 4 layers of gauze to remove hyphae, and the filtered spore suspension was adjusted to a concentration of 2 did. The adjusted spore suspension was centrifuged at 4°C and 4,000 rpm for 10 minutes, the supernatant was discarded to remove the medium components, and then suspended in the same amount of 0.05% Tween-80 and used as an inoculum in this experiment.

We aimed to investigate the disease control effect of strain JCK-7158 on induced resistance against rice red mold disease caused by F. asiaticum . Treatment of the JCK-7158 strain was performed on 11-week-old rice plants after pot transplantation, using JCK-7158 strain culture medium, culture filtrate, cell suspension, liquid hydrate (SC), and powder hydrate (WP) 1,000 times 2 weeks and 1 week before pathogen inoculation. , was diluted 2,000 times and applied as a foliar spray to saturate the rice ears and stems. As a control agent, Play Emulsion (Peulrei, ai 13% Difenoconazole + 13% Propiconazole EC, Syngenta Korea), a publicly available drug for rice red mold disease, was diluted 2,000 times 1 day before pathogen inoculation.

Pathogen inoculation was carried out 7 days after the second drug treatment, by spraying the same amount of F. asiaticum spore suspension (2 When manufacturing the drug, Tween-20, an electrodeposition agent, was added to the JCK-7158 strain culture medium, culture filtrate, and cell suspension at a level of 250 μg/mL. Immediately after inoculation, it was treated in a wet room using a plastic bag for 3 days, and the experiment was repeated 5 times per treatment group. Seven days after inoculation, the severity of rice red mold disease according to the affected area and the control value compared to the untreated group were calculated.

As can be seen in Figures 15 and 16, the control effect on rice red mold disease due to the induced resistance of Bacillus belegensis JCK-7158 strain was examined 7 days after inoculation with the pathogen, and the JCK-7158 strain culture broth (CB) , Culture Filtrate (CF), and cell suspension (Cell) treatments all reduced the occurrence of rice red mold disease caused by F. asiaticum .

In particular, the 1,000-fold dilution of the JCK-7158 strain culture showed a control activity of 49.88% and 34.71% in the 2,000-fold dilution compared to the untreated group, and the 1,000-fold dilution of the JCK-7158 strain culture filtrate showed 43.88% and 38.82% in the 2,000-fold dilution. It showed control activity of 47.88% at 1,000-fold dilution and 42.59% at 2,000-fold dilution of JCK-7158 strain cell suspension. It was observed that the control agent was effective in a concentration-dependent manner in all treatments. Play used as a control agent showed a control value of 34.41% at a 2,000-fold dilution, and the control activity of the treatment was superior to that of the control agent, confirming that the JCK-7158 strain effectively controls rice red mold disease through induced resistance. there was.

In addition, as can be seen in Figures 17 and 18, the control effect on rice red mold disease by induced resistance of the liquid wetting agent (SC) and powdered wetting agent (WP) of the Bacillus belegensis JCK-7158 strain prepared in Example 11 was confirmed as a pathogen. As a result of investigation 7 days after inoculation, both the liquid wetting agent (SC) and powdered wetting agent (WP) treatments of JCK-7158 strain reduced the occurrence of rice red mold disease caused by F. asiaticum .

In particular, the 1,000-fold dilution of JCK-7158 liquid wetting agent (SC) showed a control activity of 63.87% and 35.43% at a 2,000-fold dilution, and 34.40% and a 2,000-fold dilution of JCK-7158 strain powder wetting agent (WP). Each pear diluted solution showed a control activity of 30.42%. It was observed that the control agent was effective in a concentration-dependent manner in all treatments. Play, which was used as a control agent, showed a control value of 44.87% at a 2,000-fold dilution, and the control value of JCK-7158 liquid hydrate (SC) at a 1,000-fold dilution (63.87%) was superior to the control agent, effectively controlling rice red mold disease. Control was confirmed. Therefore, it was confirmed that a highly effective rice red mold disease control agent could be developed in the future by using the resistance-inducing activity of the JCK-7158 strain.

Example 14. Control effect on rice red mold disease by induced resistance of Bacillus belegensis JCK-7158 strain preparation under field conditions

In order to investigate the in vivo control activity of rice red mold disease caused by the induced resistance of the JCK-7158 strain of the present invention under field conditions, the Bacillus belegensis JCK-7158 liquid moisturizer (SC) described in Example 11 was applied to Samkwang rice seeds. was processed. Bacillus belegensis JCK-7158 liquid moisturizer (SC) was diluted 1,000 times and 2,000 times and sprayed on the ears of Samkwang rice, the target plant, 2 weeks and 1 week before inoculation with the pathogen, and F. asiaticum was inoculated to inoculate Bacillus belegensis. The control effect of the JCK-7158 agent was investigated. The control agent was Play emulsion (13% propiconazole + 13% difenoconazole, Syngenta Korea Co., Ltd.) diluted 2,000 times and used 1 day before pathogen inoculation.

The inoculant to be inoculated into rice under field conditions was F. asiaticum, which causes rice ^red mold disease, by adjusting it to 2.0 It was prepared by adding. In order to maintain humidity, the rice ears treated with the inoculum were sprayed with distilled water once in a zipper bag (8 cm was removed and water was sprayed twice daily. Treatment was repeated 3 times based on 30 rice heads per treatment group.

Seven days after inoculation with the pathogen, ears were cut and a virulence survey was conducted. Incidence was calculated from 0% to 100% at 5% intervals. The control value was calculated using the following formula.

As can be seen in FIGS. 19 to 21, resistance was induced in rice by pre-treating the liquid hydrate (SC) of the Bacillus belegensis JCK-7158 strain prepared in Example 11 2 weeks and 1 week before inoculation with the pathogen, followed by rice red mold disease. As a result of examining the control effect under field conditions, the JCK-7158 strain liquid hydrate (SC) treatment reduced the occurrence of rice red mold disease caused by F. asiaticum 2 and 4 weeks after inoculation with the pathogen.

In particular, 2 weeks after inoculation, the 1,000-fold dilution of JCK-7158 liquid hydration agent (SC) showed 48.81% of the control activity compared to the untreated group, and 20.56% of the control activity at 2,000-fold dilution, and 4 weeks after inoculation, the 1,000-fold dilution of JCK-7158 liquid hydration agent (SC) The control activity was 50.28% compared to the untreated group and 17.5% in the 2,000-fold diluted solution, respectively. The control effect was concentration-dependent in the treatment group, and the results of the greenhouse experiment were similar. Play, which was used as a control agent, showed control values of 32.57% and 35.21% in a 2,000-fold dilution 2 and 4 weeks after inoculation with the pathogen, respectively, and JCK-7158 liquid moisturizer showed control values for rice red mold disease at both times. (SC) It was confirmed that the control value of the 1,000-fold diluted solution was superior to the control drug.

As a result, the 1,000-fold dilution treatment with JCK-7158 liquid hydrant (SC) controlled rice red mold disease more effectively than the control chemical pesticide treatment. Therefore, it was confirmed that rice red mold disease control using the resistance-inducing activity of the JCK-7158 strain works efficiently even under field conditions.

Example 15. Effect of reducing toxin production in rice red mold disease by induced resistance of Bacillus belegensis JCK-7158 strain preparation under field conditions

The effect of treatment with the liquid moisturizer for the JCK-7158 strain of the present invention is to reduce the production of nivalenol (NIV), a fungal toxin produced by F. asiaticum , a pathogen causing rice red mold disease, under packaging conditions. To investigate, Samgwang rice seeds were treated with the Bacillus velegensis JCK-7158 liquid hydrator (SC) described in Example 11 above.

Bacillus belegensis JCK-7158 liquid moisturizer (SC) was diluted 1,000 times and 2,000 times and sprayed on the ears of Samkwang rice, the target plant, 2 weeks and 1 week before inoculation with the pathogen, and Fusarium asiaticum was inoculated to inoculate Bacillus belle. The control effect of Gensis JCK-7158 preparation was investigated. The control agent was Play emulsion (13% propiconazole + 13% difenoconazole, Syngenta Korea Co., Ltd.) diluted 2,000 times and used 1 day before pathogen inoculation.

The inoculant to be inoculated into rice under field conditions is Fusarium asiaticum ^, which causes rice red mold disease, adjusted to 2.0 It was prepared by adding. In order to maintain humidity, the rice ears treated with the inoculum were sprayed with distilled water once in a zipper bag (8 cm was removed and water was sprayed twice daily. Treatment was repeated 3 times based on 30 rice heads per treatment group.

Six weeks after pathogen inoculation, grains were harvested from the field and dried in the sun and oven for 3 days. After grinding the dried sample, 25 mL of sterilized water was added to 5 g of the ground sample, and the mixture was shaken and extracted at 300 rpm for 1 hour. Afterwards, it was centrifuged at 3,600 rpm and 4°C for 10 minutes, and 20 mL of PBS (Phosphate-Buffered Saline) was added to 5 mL of the supernatant to dilute it.

25 mL of the diluted solution was loaded onto the DON-NIV WB (Vicam) column, and when all of the diluted solution flowed out, it was washed with 5 mL of PBS and 5 mL of sterile water in that order. Afterwards, the solution in the column was removed by applying pressure within the column, and then eluted with 0.5 mL of methanol and 1.5 mL of acetonitrile (MeCN) and dried under nitrogen.

Afterwards, it was re-dissolved in 1 mL of sterile water: MeCN:MeOH (90:5:5, v/v/v). Afterwards, it was filtered with a 0.22 μm membrane filter and then analyzed by UPLC-PDA. The instrument used was a WATRERS ACQUITY UPLC H Class equipped with a PDA (218 nm) detector, the column used was Xselect CSH C18 (2.1 The flow rate was 0.3 mL/min and 10 μL was injected and analyzed for 10 minutes. As a result of the analysis, nivalenol (NIV) was detected.

Fungal toxin reduction effect according to treatment of Bacillus belegensis JCK-7158 strain under packaging conditions treatment area　 NIV (ug/kg) Toxin reduction rate (%) JCK-7158 SC x1,000 169.29±17.73 40.62 JCK-7158 SC x2,000 343.59±96.50 0.00 Peulrei 224.89±5.24 21.12 Non-inoculating sphere N.D. 　- Non-processed area 285.09±73.19 0.00

Additionally, as can be seen in Table 12, treatment with JCK-7158 liquid moisturizer had the effect of reducing nivalenol by 40.62% compared to the untreated treatment, and reduced nivalenol, a fungal toxin, compared to Play, a chemical disinfectant (toxin reduction effect of 21.12%). It was confirmed that the effect was excellent.

As a result, the 1,000-fold dilution treatment with JCK-7158 liquid hydratant (SC) not only controlled rice red mold disease more effectively than the control chemical pesticide treatment, but also reduced the amount of mycotoxins produced by pathogens more effectively than the chemical pesticide treatment. Therefore, it was confirmed that rice red mold disease control using the resistance-inducing activity of the JCK-7158 strain works efficiently even under field conditions.

Through this, the JCK-7158 strain is an eco-friendly strain with excellent control activity as an alternative to the use of chemical pesticides, which has the problem that it can only be used up to 30 days before harvest due to residual problems. It is expected to become a cornerstone in the development of control agents for various plant diseases.

Korea Research Institute of Bioscience and Biotechnology (KCTC) KCTC15169BP 20221102

Sequence list electronic file attached

Claims (19)

Bacillus velezensis JCK-7158 strain deposited with accession number KCTC15169BP, which has antifungal activity. A composition for controlling phytopathogenic fungi comprising Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract. The method of claim 2, wherein the plant pathogenic fungi are Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG-2 (Ⅳ ) Large patch, Rhizoctonia solani Aju 2-2 (Ⅳ) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum ( Fusarium oxysporum f. sp. cucumerinum ), Fusarium oxysporum f. Esp. Lycopersici ( Fusarium oxysporum f. sp. lycopersici ), Fusarium graminearum , Fusarium asiaticum , Fusarium verticillioides , Pythium ultimum , Gaeumannomyces graminis , Phytophthora infestans , Botrytis Cinerea, and Colletotrichum coccodes. A composition for controlling phytopathogenic fungi, which is one or more selected species. Iturin A, Surfactin, 2,3-butanediol, 5-Methylhexan-2-one, Heptan-2-one (heptan-2-one), 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone (5 A composition for controlling plant pathogenic fungi containing at least one compound selected from the group consisting of -methyl-2-heptanone). The method of claim 4, wherein the plant pathogenic fungi are Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG-2 (Ⅳ ) Large patch, Rhizoctonia solani Aju 2-2 (Ⅳ) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum ( Fusarium oxysporum f. sp. cucumerinum ), Fusarium oxysporum f. Esp. Lycopersici ( Fusarium oxysporum f. sp. lycopersici ), Fusarium graminearum , Fusarium asiaticum , Fusarium verticillioides , Pythium ultimum , Gaeumannomyces graminis , Phytophthora infestans , Botrytis Cinerea, and Colletotrichum coccodes. A composition for controlling phytopathogenic fungi, which is one or more selected species. A method for producing a composition for controlling plant pathogenic fungi, comprising a culturing step of preparing a culture solution by culturing the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP. The method of claim 6, wherein the phytopathogenic fungi are Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG-2-2 (Ⅳ ) Large patch, Rhizoctonia solani Aju 2-2 (Ⅳ) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum ( Fusarium oxysporum f. sp. cucumerinum ), Fusarium oxysporum f. Esp. Lycopersici ( Fusarium oxysporum f. sp. lycopersici ), Fusarium graminearum , Fusarium asiaticum , Fusarium verticillioides , Pythium ultimum , Gaeumannomyces graminis , Phytophthora infestans , Botrytis Cinerea, and Colletotrichum coccodes. A method for producing a composition for controlling phytopathogenic fungi, which is one or more selected types. The method of claim 6, wherein the production method additionally includes a first fractionation step of obtaining a butanol layer by fractionating the culture medium or the culture filtrate obtained therefrom with butanol. The method of claim 8, wherein the butanol layer obtained in the first fractionation step is eluted with a mixed solution of chloroform: methanol: water, and additionally includes a second fractionation step of obtaining the fourth or fifth fraction among the five fractions. A method for producing a composition for controlling plant pathogenic fungi. The method of claim 9, wherein the mixing volume ratio of chloroform:methanol:water is 50 to 60:30 to 40:5 to 10. The method of claim 9, wherein in the production method, the fraction obtained in the second fractionation step is eluted with a mixed solution of chloroform: methanol: water, and one or more fractions selected from the group consisting of the first to third fractions among the four fractions. A method for producing a composition for controlling phytopathogenic fungi, which additionally includes a third fractionation step to obtain. The method of claim 11, wherein the mixing volume ratio of chloroform:methanol:water is 50 to 60:30 to 40:5 to 10. A method for controlling phytopathogenic fungi, comprising a treatment step of treating the Bacillus velezensis JCK-7158 strain, its culture, or its extract, deposited under accession number KCTC15169BP. The method of claim 13, wherein the treatment step is performed by one or more methods selected from the group consisting of spraying, soil drench, surface spraying, root zone treatment, seed treatment, soaking, poisoning, and smoking. Control of phytopathogenic fungi. method. The method of claim 13, wherein the plant pathogenic fungi are Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG-2-2 (Ⅳ ) Large patch, Rhizoctonia solani Aju 2-2 (Ⅳ) Brown patch, Fusarium oxyforme F. Esp. Cucumerinum ( Fusarium oxysporum f. sp. cucumerinum ), Fusarium oxysporum f. Esp. Lycopersici ( Fusarium oxysporum f. sp. lycopersici ), Fusarium graminearum , Fusarium asiaticum , Fusarium verticillioides , Pythium ultimum , Gaeumannomyces graminis , Phytophthora infestans , Botrytis Cinerea, and Colletotrichum coccodes. A method for controlling phytopathogenic fungi, wherein at least one species is selected. Iturin A, Surfactin, 2,3-butanediol, 5-Methylhexan-2-one), Heptan-2-one ( heptan-2-one), 2,5-dimethylpyrazine, 6-methylheptan-2-one and 5-methyl-2-heptanone (5- A method for controlling phytopathogenic fungi comprising a treatment step of treating one or more compounds selected from the group consisting of methyl-2-heptanone). A composition for reducing fungal toxins comprising the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture medium, or its extract. A method for producing a composition for reducing fungal toxins, comprising a culturing step of preparing a culture medium by culturing the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP. A method for reducing fungal toxins comprising a treatment step of treating the Bacillus velezensis JCK-7158 strain, its culture, or its extract, deposited under accession number KCTC15169BP.