Composition for controlling plant pathogenic fungi or reducing mycotoxins, containing an extract from a culture broth or strain culture broth of Bacillus veresensis JCK-7158, a method for producing the same, and a method for controlling plant pathogenic fungi or reducing mycotoxins

Abstract

The present invention relates to a composition for controlling plant pathogenic fungi, which contains the Bacillus velezensis JCK-7158 strain, a culture solution thereof, or an extract thereof, a method for producing the composition, and a method for controlling plant pathogenic fungi. The culture solution or extract thereof of the strain has remarkably excellent antifungal activity and induced resistance, and is excellent in the ability to reduce toxins produced by fungi, and therefore can be used to control plant pathogenic fungi.

Description

[Technical Field]

[0001] This invention was made under the support of the Ministry of Agriculture, Food and Rural Affairs under Project ID No. 1545027807 and Detailed Project No. 320036-5. The research management specialist for this project is the Korea Institute for Food and Agriculture Technology Planning and Evaluation, the research project name is "Industrialization Technology Development Project for Crop Virus and Pest Control," the research topic name is "Development of Environmentally Friendly Biological Control Agent for Rice Fusarium Head Blight," the research institution is the Cheongnam University Industry-Academia Collaboration Foundation, and the research period is from 2023.01.01 to 2023.12.31.

[0002] This invention was made under the support of the Ministry of Agriculture, Food and Rural Affairs under Project ID No. 1545025743 and Detailed Project No. 320036-5. The research management specialist organization for this project is the Korea Institute for Food and Agriculture Technology Planning and Evaluation, the research project name is "Industrialization Technology Development Project for Crop Virus and Pest Control," the research topic name is "Development of Environmentally Friendly Biological Control Agent for Rice Fusarium Head Blight," the research organization is the Cheongnam University Industry-Academia Collaboration Foundation, and the research period is from 2022.01.01 to 2022.12.31.

[0003] This patent application claims priority to Korean Patent Application No. 10-2022-0154599, filed with the Korean Intellectual Property Office on November 17, 2022, the disclosure of which is incorporated herein by reference.

[0004] The present invention relates to a composition for controlling plant pathogenic fungi containing the Bacillus velezensis JCK-7158 strain, a culture solution thereof, or an extract thereof, a method for producing the same, a method for controlling plant pathogenic fungi, and a method for reducing mycotoxins. [Background technology]

[0005] Fusarium head blight, caused by various Fusarium species including Fusarium graminearum and Fusarium asiaticum, periodically breaks out in large numbers on wheat, barley, corn, rice, etc., causing significant damage worldwide. In particular, in recent years, the disease has been particularly prevalent in the United States and Canada, causing a surge in global grain prices.

[0006] Fusarium head blight not only reduces grain production but also reduces the quality of agricultural products by leaving mycotoxins such as trichothecene and zearalenone, which are harmful to humans and animals, in the infected areas. In particular, nivalenol (NIV) and deoxynivalenol (DON), which are types of trichothecene, can cause serious poisoning by weakening the immune system and inducing gastrointestinal disorders.

[0007] In addition, since there is a significant correlation between Fusarium contamination rates and trichothecenes detection levels in wheat-growing complexes in Korea, it can be said that contamination with mycotoxins caused by Fusarium head blight poses a significant threat to public health.

[0008] When preventing red mold toxin contamination through chemical control, the timing of fungicide application is very important. For wheat, the application must be made when the anthers first emerge from the ear, and for barley, the application must be made when the ear emerges.

[0009] DMI (Demethylation Inhibitor) fungicides such as metconazole, propiconazole, tebuconazole + prothionazole, and tebuconazole are the most effective, but due to local systemic migration and residue issues with some pesticides, they can only be used up to 30 days before harvest, and their field control rate is less than 50%, meaning they have poor efficacy.

[0010] Furthermore, the misuse and overuse of synthetic pesticides, which account for the majority of pesticides, has raised various problems, including contamination of soil, water and agricultural products, toxicity, ecosystem disruption and the emergence of resistant strains of bacteria.

[0011] Therefore, with OECD countries and other countries trying to reduce the use of synthetic pesticides by more than 40% recently, and with growing interest in well-being and demand for organic produce rapidly increasing, especially in developed countries, there is an urgent need to develop environmentally friendly biological products to control Fusarium head blight. Summary of the Invention [Problem to be solved by the invention]

[0012] Therefore, the present inventors have confirmed that when a composition for controlling plant pathogenic fungi containing the Bacillus velezensis JCK-7158 strain, its culture solution, or an extract thereof is applied to plant pathogenic fungi, the composition has significantly excellent control activity and also effectively reduces the production of mycotoxins produced by the fungi.

[0013] Therefore, an object of the present invention is to provide Bacillus veresensis strain JCK-7158, deposited under accession number KCTC15169BP, which has antifungal activity.

[0014] Another object of the present invention is to provide a composition for controlling plant pathogenic fungi, which contains the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP, a culture broth thereof, or an extract thereof.

[0015] It is still another object of the present invention to provide a composition for controlling plant pathogenic fungi, comprising one or more fungi selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0016] Yet another object of the present invention is to provide a method for producing a composition for controlling plant pathogenic fungi, which comprises a culturing step of culturing the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP to produce a culture solution.

[0017] It is still another object of the present invention to provide a method for controlling plant pathogenic fungi, which comprises treating the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP, its culture broth, or an extract thereof.

[0018] It is still another object of the present invention to provide a method for controlling plant pathogenic fungi, which comprises a treatment step of applying one or more members selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0019] A further object of the present invention is to provide a composition for reducing mycotoxins, which comprises the Bacillus veresensis JCK-7158 strain deposited under accession number KCTC15169BP, a culture broth thereof, or an extract thereof.

[0020] Yet another object of the present invention is to provide a method for producing a composition for reducing mycotoxins, which comprises culturing the Bacillus veresensis JCK-7158 strain deposited under accession number KCTC15169BP to produce a culture broth.

[0021] It is still another object of the present invention to provide a method for reducing mycotoxins, which comprises treating the Bacillus veresensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture broth, or an extract thereof.

[0022] Yet another object of the present invention is to provide use of the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP, its culture broth, or an extract thereof for controlling plant pathogenic fungi or reducing mycotoxins. [Means for solving the problem]

[0023] The present invention will now be described in more detail.

[0024] One aspect of the present invention relates to the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, which has antifungal activity.

[0025] In the present invention, the Bacillus veresensis JCK-7158 strain may contain 16S rRNA comprising the nucleotide sequence of SEQ ID NO:3.

[0026] Another aspect of the present invention relates to a composition for controlling plant pathogenic fungi, which comprises the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP, a culture broth thereof, or an extract thereof.

[0027] As used herein, the term "culture medium" refers to a medium containing a microorganism after the microorganism has been cultured.

[0028] The term "extract" as used herein means something separated from the culture medium of a strain, and the extract includes one or more selected from the group consisting of culture supernatant, culture filtrate, cell suspension, and fractions.

[0029] In the present invention, the extract may contain one or more compounds selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0030] The term "culture supernatant" as used herein refers to the supernatant obtained by removing most of the microorganisms from the culture medium by centrifugation.

[0031] The term "culture filtrate" as used herein refers to the liquid remaining after removing the bacterial cells from the culture medium by centrifugation and filtration. The culture filtrate contains substances formed and excreted during the growth of the microorganisms, and these substances may be purified or extracted.

[0032] In the present invention, the culture filtrate refers to a culture medium from which fungi have been removed by centrifugation and filtration.

[0033] In the present invention, the plant pathogenic fungi include Clarireedia jacksonii (formerly also called Sclerotinia homoeocarpa), Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Rhizoctonia solani AG2-2(IV) Brown patch, Fusarium oxysporum f.sp. cucumerinum, Fusarium oxysporum f.sp. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, and the like. verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytophthora infestans, Botrytis cinerea, and Colletotrichum coccodes, but is not limited thereto.

[0034] In one embodiment of the present invention, a composition for controlling plant pathogenic fungi containing the Bacillus veresensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture solution, or an extract thereof can be used to obtain control activity against Fusarium graminearum or Fusarium asiaticum, which cause rice head blight, a plant pathogenic fungal disease.

[0035] Yet another aspect of the present invention is a composition for controlling plant pathogenic fungi, comprising one or more compounds selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0036] In the present invention, the iturin A may be a compound represented by the following formula 1, having a molecular weight of 1,043 and a molecular formula of C 48 H 74 O 14 N 12 is.

[0037] <Chemical formula 1> [ka]

[0038] In the present invention, the surfactin may be a compound represented by the following chemical formula 2, having a molecular weight of 1,036 and a molecular formula of C 53 H 93 O 13 It's N7.

[0039] <Chemical formula 2> [ka]

[0040] In the present invention, the 2,3-butanediol may be a compound represented by the following formula 3, having a molecular weight of 90 and a molecular formula of CH 10 It is O2.

[0041] <Chemical formula 3> [ka]

[0042] In the present invention, the 5-methylhexan-2-one may be a compound represented by the following formula 4, having a molecular weight of 114 and a molecular formula of CH 14It is O.

[0043] <Chemical formula 4> [ka]

[0044] In the present invention, the heptan-2-one may be a compound represented by the following formula 5, having a molecular weight of 114 and a molecular formula of CH 14 It is O.

[0045] <Chemical formula 5> [ka]

[0046] In the present invention, the 2,5-dimethylpyrazine may be a compound represented by the following formula 6, with a molecular weight of 108 and a molecular formula of C6H8N2.

[0047] <Chemical formula 6> [ka]

[0048] In the present invention, the 6-methylheptan-2-one may be a compound represented by the following formula 7, having a molecular weight of 128 and a molecular formula of CH 16 It is O.

[0049] <Chemical formula 7> [ka]

[0050] In the present invention, the 5-methyl-2-heptanone may be a compound represented by the following formula 8, having a molecular weight of 128 and a molecular formula of CH 16 It is O.

[0051] <Chemical formula 8> [ka]

[0052] In the present invention, the plant pathogenic fungus may be one or more species selected from the group consisting of Claryleidaedia jacsoniai, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large Patch, Rhizoctonia solani AG2-2(IV) Brown Patch, Fusarium oxysporum F.SP. cucumerinum, Fusarium oxysporum F.SP. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytofusora infestans, Botrytis cinerea, and Colletotrichum cocodes.

[0053] In the present invention, the composition for controlling plant pathogenic fungi may further contain additional agents such as, but not limited to, additives, extenders, nutrients, or disintegrants.

[0054] In the present invention, the additive may be one or more selected from the group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkylaryl sulfonate, polyoxyethylene alkylphenyl ether, sodium tripolyphosphate, polyoxyethylene alkylaryl phosphate ester, polyoxyethylene alkylaryl ether, polyoxyethylene alkylaryl polymer, polyoxyalkylone alkylphenyl ether, polyoxyethylene nonylphenyl ether, sodium sulfonate naphthalene formaldehyde, Triton 100, and Tween 80, but is not limited thereto.

[0055] In the present invention, the bulking agent may include, but is not limited to, one or more selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate.

[0056] In the present invention, the nutrient may include, but is not limited to, one or more selected from the group consisting of skim milk (culture medium), soybean flour, rice, wheat, loess, diatomaceous earth, bentonite, dextrin, glucose, and starch.

[0057] Yet another aspect of the present invention relates to a method for producing a composition for controlling plant pathogenic fungi, which comprises a culturing step of culturing the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP to produce a culture solution.

[0058] In the present invention, the plant pathogenic fungus may be one or more species selected from the group consisting of Claryleidaedia jacsoniai, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large Patch, Rhizoctonia solani AG2-2(IV) Brown Patch, Fusarium oxysporum F.SP. cucumerinum, Fusarium oxysporum F.SP. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumanomyces graminis, Phytofusora infestans, Botrytis cinerea, and Colletotrichum cocodes, but is not limited thereto.

[0059] In the present invention, the culturing step may include, but is not limited to, a step of preparing a culture filtrate from the culture solution by centrifuging and filtering the culture solution.

[0060] In the present invention, the culture medium may contain one or more selected from the group consisting of milk proteins such as skim milk, whey, and casein, sugars, yeast, and extracts, but is not limited thereto.

[0061] 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 solution.

[0062] 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 solution.

[0063] In the present invention, the method for producing a composition for controlling plant pathogenic fungi may further include an extraction step of extracting components from the fungal cells.

[0064] In the present invention, the method for producing the fungicidal composition may include, but is not limited to, a fractionation step of obtaining an active fraction from the culture broth.

[0065] In the present invention, the method for producing the fungicidal composition may include the following steps:

[0066] a culture filtrate harvesting step for obtaining a culture filtrate from the culture broth;

[0067] a fractionation step in which the culture filtrate is fractionated with a solvent to obtain fractions;

[0068] a concentration step of concentrating the fraction to obtain a concentrate; and

[0069] A purification step in which the concentrate is purified and the active fraction is selected.

[0070] In the present invention, the step of obtaining a culture filtrate may include, but is not limited to, centrifuging the culture solution 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, or 4,500 to 5,000 rpm, for example, 4,500 rpm.

[0071] In the present invention, the fractionation step may include, but is not limited to, fractionation using one or more solvents selected from the group consisting of butanol, ethyl acetate, chloroform, methanol, ethanol, and hexane.

[0072] The fractionation step may consist of the following steps:

[0073] a first fractionation step in which the culture broth or the culture filtrate obtained therefrom is fractionated with butanol to obtain a butanol layer;

[0074] a second fractionation step in which the butanol layer obtained in the first fractionation step is eluted with a mixed solution of chloroform:methanol:water to obtain the fourth or fifth fraction out of five fractions; and

[0075] A third fractionation step is a step of eluting the fractions obtained in the second fractionation step with a mixed solution of chloroform:methanol:water to obtain one or more fractions selected from the group consisting of the first to third fractions among the four fractions.

[0076] In the second or third fractionation step, independently, the volume ratio of chloroform:methanol:water may be 50-60:30-40:5-10, preferably 52-58:35-40:7-9, for example, but is not limited to, 55:36:8.

[0077] In the present invention, the concentration step may include, but is not limited to, concentrating the fraction using a vacuum concentrator.

[0078] In the present invention, the purification step may include, but is not limited to, purification by preparative HPLC (high-performance liquid chromatography).

[0079] In the present invention, the active fraction may include, but is not limited to, one or more selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0080] Yet another aspect of the present invention relates to a method for controlling plant pathogenic fungi, which comprises a treatment step of applying the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP, a culture solution thereof, or an extract thereof to plants or soil.

[0081] In the present invention, the treatment step may be carried out by one or more methods selected from the group consisting of spraying, soil drench, surface spraying, rhizosphere treatment, seed treatment, soaking, poisoning, and fumigation, for example, by soil drench, but is not limited thereto.

[0082] In the present invention, "application" may be carried out by one or more methods selected from the group consisting of spraying, misting, atomizing, powder application, granular application, application to the water surface, and constant use.

[0083] As used herein, the term "drench" refers to a method of chemical application in which holes are drilled into the soil or trees to inject a chemical solution.

[0084] The term "soil drench" as used herein refers to a method of injecting or spraying a chemical solution into the soil for crop cultivation.

[0085] In the present invention, the plant pathogenic fungus may be one or more species selected from the group consisting of Claryleidaedia jacsoniai, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large Patch, Rhizoctonia solani AG2-2(IV) Brown Patch, Fusarium oxysporum F.SP. cucumerinum, Fusarium oxysporum F.SP. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumanomyces graminis, Phytofusora infestans, Botrytis cinerea, and Colletotrichum cocodes, but is not limited thereto.

[0086] Yet another aspect of the present invention is a method for controlling plant pathogenic fungi, which comprises one or more compounds selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0087] Yet another aspect of the present invention relates to a composition for reducing mycotoxins, comprising the Bacillus veresensis JCK-7158 strain deposited under accession number KCTC15169BP, a culture broth thereof, or an extract thereof.

[0088] In the present invention, the extract may contain one or more compounds selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0089] The mycotoxin may be produced by a plant pathogenic fungus, and the plant pathogenic fungus may be one or more species selected from the group consisting of Claryleidaedia jaxonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large Patch, Rhizoctonia solani AG2-2(IV) Brown Patch, Fusarium oxysporum F.sp. cucumerinum, Fusarium oxysporum F.sp. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytofusora infestans, Botrytis cinerea, and Colletotrichum cocodes, and may be, for example, a fungal toxin produced by Fusarium asiaticum, and the mycotoxin may be nivalenol (NIV), but is not limited thereto.

[0090] Yet another aspect of the present invention relates to a method for producing a composition for reducing mycotoxins, which comprises culturing the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP to produce a culture broth.

[0091] In the present invention, the extract may contain one or more compounds selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0092] The mycotoxin may be produced by a plant pathogenic fungus, and the plant pathogenic fungus may be one or more species selected from the group consisting of Claryleidaedia jaxonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large Patch, Rhizoctonia solani AG2-2(IV) Brown Patch, Fusarium oxysporum F.SP. cucumerinum, Fusarium oxysporum F.SP. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytofusora infestans, Botrytis cinerea, and Colletotrichum cocodes, and may be, for example, produced by Fusarium asiaticum, and the mycotoxin may be, but is not limited to, nivalenol.

[0093] Yet another aspect of the present invention relates to a method for reducing mycotoxins, which comprises treating the Bacillus veresensis JCK-7158 strain deposited under Accession No. KCTC15169BP, its culture broth, or an extract thereof.

[0094] In the present invention, the extract may contain one or more compounds selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

[0095] The mycotoxin may be produced by a plant pathogenic fungus, and the plant pathogenic fungus may be one or more species selected from the group consisting of Claryleidaedia jacsoniai, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large Patch, Rhizoctonia solani AG2-2(IV) Brown Patch, Fusarium oxysporum F.sp. cucumerinum, Fusarium oxysporum F.sp. lycopersici, Fusarium graminearum, Fusarium asiaticum, Fusarium verticillioides, Pythium ultimum, Gaeumannomyces graminis, Phytofusora infestans, Botrytis cinerea, and Colletotrichum cocodes, and may be, for example, produced by Fusarium asiaticum, and the mycotoxin may be, but is not limited to, nivalenol.

[0096] In the method for producing a composition for controlling plant pathogenic fungi and the method for controlling plant pathogenic fungi, the content that overlaps with the composition for controlling plant pathogenic fungi will be omitted in consideration of the complexity of this specification. [Effects of the Invention]

[0097] The present invention relates to a composition for controlling plant pathogenic fungi or reducing mycotoxins, which contains the Bacillus velezensis JCK-7158 strain, a culture solution thereof, or an extract thereof, a method for producing the composition, and a method for controlling plant pathogenic fungi or reducing mycotoxins. The culture solution or extract thereof of the strain has significantly excellent antifungal activity and induced resistance, and can therefore be used to control plant pathogenic fungi or reduce mycotoxins. [Brief explanation of the drawings]

[0098] [Figure 1] 1 is a phylogenetic diagram of Bacillus velezensis JCK-7158 strain analyzed by the 16S rRNA gene sequence according to one embodiment of the present invention.

[0099] [Figure 2A]1 is a graph showing extracellular protease activity of Bacillus veresensis JCK-7158 strain over time according to an embodiment of the present invention.

[0100] [Figure 2B] 1 is a graph showing extracellular chitinase activity of Bacillus veresensis JCK-7158 strain over time according to an embodiment of the present invention.

[0101] [Figure 2C] 1 is a graph showing extracellular cellulase activity of Bacillus veresensis JCK-7158 strain over time according to an embodiment of the present invention.

[0102] [Figure 2D] 1 is a graph showing extracellular gelatinase activity of Bacillus veresensis JCK-7158 strain over time according to an embodiment of the present invention.

[0103] [Figure 3] 1 is a photograph showing the extracellular enzyme activity of Bacillus veresensis JCK-7158 strain according to one embodiment of the present invention.

[0104] [Figure 4] 1 is a photograph showing the IAA production activity of Bacillus veresensis JCK-7158 strain according to one embodiment of the present invention.

[0105] [Figure 5] 1 is a photograph showing the acetoin-producing activity of Bacillus veresensis JCK-7158 strain according to one embodiment of the present invention.

[0106] [Figure 6A] 6A and 6B are photographs showing the mycelial growth inhibitory activity of various plant pathogenic fungi in dual culture of Bacillus veresensis JCK-7158 strain according to one embodiment of the present invention. [Figure 6B] 6A and 6B are photographs showing the mycelial growth inhibitory activity of various plant pathogenic fungi in dual culture of Bacillus veresensis JCK-7158 strain according to one embodiment of the present invention.

[0107] [Figure 7] 1 is a diagram illustrating a process for fractionating antifungal active substances from Bacillus veresensis JCK-7158 strain according to one embodiment of the present invention.

[0108] [Figure 8] 1 is a photograph showing the antifungal activity of a butanol fraction of a culture filtrate of Bacillus veresensis JCK-7158 strain, which was analyzed by thin layer chromatography (TLC) against a pathogenic fungus, Fusarium graminearum, according to an embodiment of the present invention.

[0109] [Figure 9] 1 is a thin-layer chromatography photograph of four fractions separated from the BF4 fraction of Bacillus veresensis JCK-7158 strain according to an embodiment of the present invention.

[0110] [Figure 10] 1 is a graph showing the results of a positive ion mode LC / MS analysis of iturin A isolated from the BF4-1 fraction of Bacillus veresensis JCK-7158 strain according to an embodiment of the present invention.

[0111] [Figure 11] 1 is a graph showing the results of a positive ion mode LC / MS analysis of surfactin isolated from the BF4-1 fraction of Bacillus veresensis JCK-7158 strain according to an embodiment of the present invention.

[0112] [Figure 12A] 12A and 12B are photographs showing the mycelial growth inhibitory activity of plant pathogenic fungi due to the antifungal activity of volatile substances produced by Bacillus veresensis JCK-7158 strain according to one embodiment of the present invention. [Figure 12B] 12A and 12B are photographs showing the mycelial growth inhibitory activity of plant pathogenic fungi due to the antifungal activity of volatile substances produced by Bacillus veresensis JCK-7158 strain according to one embodiment of the present invention.

[0113] [Figure 13] 1 is a graph illustrating the results of solid phase microextraction (SPME) GC-MS analysis of volatile organic compounds produced by Bacillus veresensis JCK-7158 strain according to an embodiment of the present invention.

[0114] [Figure 14] 1 shows photographs of the PR-1 gene expression activity assayed in transformed Arabidopsis thaliana plants according to an embodiment of the present invention, depending on whether or not GUS expression is present in Bacillus veresensis JCK-7158 strain.

[0115] [Figure 15] 1 is a graph showing the control effect of induced resistance of Bacillus veresensis JCK-7158 strain against Fusarium head blight of rice under greenhouse conditions according to an embodiment of the present invention.

[0116] [Figure 16] 1 shows photographs of the control effect of induced resistance of Bacillus veresensis JCK-7158 strain against rice Fusarium head blight observed under greenhouse conditions 7 days after inoculation according to an embodiment of the present invention.

[0117] [Figure 17] 1 is a graph showing the control effect of two formulations of Bacillus veresensis JCK-7158 strain on rice Fusarium head blight under greenhouse conditions according to an embodiment of the present invention.

[0118] [Figure 18]1 shows photographs of the control effect against rice Fusarium head blight caused by induced resistance of two formulations using Bacillus veresensis JCK-7158 strain under greenhouse conditions, observed 7 days after inoculation, according to an embodiment of the present invention.

[0119] [Figure 19] 1 is a graph showing the control effect of a Bacillus veresensis JCK-7158 strain formulation on rice Fusarium head blight through induced resistance under field conditions according to an embodiment of the present invention.

[0120] [Figure 20] 1 shows photographs of the control effect of a Bacillus veresensis JCK-7158 strain formulation on rice Fusarium head blight disease through induced resistance under field conditions, observed two weeks after inoculation, according to an example of the present invention.

[0121] [Figure 21] 1 shows photographs of the control effect of a Bacillus veresensis JCK-7158 strain formulation on rice Fusarium head blight caused by induced resistance, observed under field conditions 4 weeks after inoculation, according to an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0122] The present invention relates to the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, which has antifungal activity. [Example]

[0123] The present invention will be described in more detail below with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0124] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, or (vol / vol)% for liquid / liquid, unless otherwise specified.

[0125] Example 1. Isolation of strain JCK-7158 with antibacterial activity against red mold

[0126] A fungal strain with antifungal activity against Fusarium graminearum (F. graminearum), which causes head blight in crops, was isolated from the stalks of Shindongjin rice cultivated in Jeongnam Gokseong, South Korea. Collected rice stalks (10 g) were ground in 100 mL of sterile water. The ground stalk sample was diluted 40-fold with sterile water and smeared on a TSA (Tryptic Soy Agar) plate containing a 1% suspension of F. graminearum mycelium. The plate was then incubated at 30°C for 3-7 days or more. Strain JCK-7158, which produced a clear zone, was isolated.

[0127] Example 2. Molecular biological analysis and phylogenetic analysis of JCK-7158 strain

[0128] Strain JCK-7158, isolated from rice stalks, was identified molecularly by 16S rRNA gene sequence analysis. The strain was inoculated into TSB medium and cultured at 30°C for 3 days with shaking at 1,580 rpm. Genomic DNA (gDNA) was extracted from the harvested strain using the I-genomic BYF DNA Extraction Mini kit (iNtRON, Korea) according to the manufacturer's protocol.

[0129] The extracted gDNA from the strain was mixed with iNtRON Biotechnology's PCR premix (polymerase chain reaction premix) and a primer set capable of amplifying the strain's 16S rRNA, and the gene was amplified by PCR. PCR was performed starting at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 90 seconds, and then completed at 72°C for 10 minutes and 4°C.

[0130] The PCR product of the amplified 16S rRNA gene was subjected to sequence analysis by Genotech (Daejeon, Korea), and a total of 1352 bp of the 16S rRNA coding sequence of the isolated strain, JCK-7158, was obtained (SEQ ID NO: 3).

[0131] [Table 1]

[0132] As can be seen in Figure 1, the JCK-7158 strain was identified as Bacillus velezensis by comparing its nucleotide sequence with that of the GenBank database using NCBI's BlastN search. Therefore, the strain was designated Bacillus velezensis JCK-7158 and deposited with the Korean Collection for Type Cultures (KCTC) on November 2, 2022, with the accession number KCTC15169BP.

[0133] Example 3. Biochemical characteristics of Bacillus veresensis JCK-7158 strain - evaluation of extracellular enzyme activity

[0134] To measure the activity of the proteolytic enzymes protease, chitinase, gelatinase, and cellulase of Bacillus veresensis JCK-7158, 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), and cellulase medium (0.4% sodium carboxymethylcellulose + 1.5% agar, Sigma-Aldrich) were prepared.

[0135] To prepare colloidal chitin, crab shell powder was first added to the chitinase medium and stirred with hydrochloric acid (HCl) (10 g crab shell powder / 150 mL HCl) for 6 hours. After 6 hours, 1 L of cold ethanol (99.9%) was added, mixed thoroughly with a stirrer, and centrifuged at 4°C and 4500 rpm for 20 minutes. The supernatant was then removed, and the chitin precipitate was washed 3-4 times with 0.1 M potassium phosphate buffer (pH 7.0) before use. The prepared colloidal chitin was stored at 4°C after autoclaving and before use.

[0136] The gelatinase medium was prepared by adding 10% gelatin to the LB solid medium, and the other media were solidified by adding 1.5% agar.

[0137] Sterilized paper discs (0.8 cm, Advantec, Japan) were placed on each medium, and 5 μL, 10 μL, and 15 μL of JCK-7158 culture filtrate were dispensed onto the protease, chitinase, and cellulase media, respectively, and 20 μL, 40 μL, and 60 μL onto the gelatinase medium. Equal volumes of sterilized LB medium and medium were dispensed onto the paper discs as negative controls. Experiments were performed in triplicate, and plates were maintained at 30°C. Clear zones due to extracellular enzyme activity were observed for each. To visualize cellulase and chitinase media, 5 mL of Lugol's solution (2.5 g / L iodine and 5 g / L potassium iodide) was added to the plates, and the plates were incubated in the dark for 10 minutes before being observed for clear zones.

[0138] As can be seen from Figures 2 and 3, the culture filtrate was treated with a medium that induces enzyme activity, and the clear zones were observed. The JCK-7158 strain formed clear zones with diameters of 34.87 mm after 9 days of treatment with 15 μL of sample in protease medium, 35.72 mm after 5 days of treatment with 15 μL of sample in cellulose medium, 38.26 mm after 5 days of treatment with 15 μL of sample in chitinase medium, and 19.29 mm after 5 days of treatment with 60 μL of sample in gelatinase medium. These results confirmed that the Bacillus veresensis JCK-7158 strain produces all of protease, chitinase, gelatinase, and cellulase.

[0139] Example 4. IAA production by Bacillus veresensis JCK-7158 strain

[0140] To confirm the production of the plant growth hormone IAA (indole-3-acetic acid) by Bacillus veresensis JCK-7158, a 1% culture of JCK-7158 was inoculated into TSB medium supplemented with L-tryptophan (150 mg / L) and cultured at 30°C for 24 hours with shaking at 150 rpm. The culture was then centrifuged at 10,000 rpm for 5 minutes at 4°C and filtered through a 0.2 μm sterile filter. 1 mL of the resulting culture filtrate was mixed with 2 mL of Salkowski's reagent (150 mL H2SO4, 250 mL sterile water, 7.5 mL 0.5 M FeCl3·6H2O). After 20 minutes at room temperature in the dark, the mixture was checked for a pink color. The negative control was TSB medium supplemented with L-tryptophan (150 mg / L), and the experiment was repeated three times.

[0141] As shown in Figure 4, an experiment was conducted to confirm the production of IAA, a plant growth hormone, by JCK-7158. The JCK-7158 culture filtrate-treated group showed color in the test tube, while the control group, TSB-treated group, showed the natural color of the medium. This confirmed that JCK-7158 produces IAA, a plant growth hormone.

[0142] Example 5. 2,3-butanediol production by Bacillus veresensis JCK-7158 strain

[0143] To confirm the production of the volatile compound 2,3-butanediol by Bacillus verezensis JCK-7158, we also examined the production of acetoin, a precursor of 2,3-butanediol. To confirm acetoin production, we used MRS-VP medium (5 g / L peptone, 5 g / L dextrose, 5 g / L dipotassium phosphate). Bacillus verezensis JCK-7158 was inoculated into MRS-VP medium and cultured at 30°C and 150 rpm for 3 days. Then, 100 μl of the culture medium, 60 μl of 5% naphthol, and 20 μl of 40% potassium hydroxide were added to a 96-well plate in that order. The mixture was then incubated at room temperature for 10–15 minutes, and the resulting pink color was observed. As a negative control, MRS-VP medium alone was used, and as a positive control, MRS-VP medium supplemented with acetoin (1 mg / mL) was used.

[0144] [Table 2]

[0145] As can be seen from Table 2 and Figure 5, the production of 2,3-butanediol, a volatile compound of Bacillus verezensis JCK-7158, was confirmed by examining the production of its precursor, acetoin. The Bacillus verezensis JCK-7158-treated plots turned pink, similar to the positive control plots, confirming the production of acetoin. This suggests that Bacillus verezensis JCK-7158 produces 2,3-butanediol. 2,3-butanediol is known to induce resistance in various plants. Therefore, the culture solution of Bacillus verezensis JCK-7158 also produces 2,3-butanediol, which has the potential to induce resistance in host plants and thus control various plant diseases.

[0146] Example 6. Growth inhibition activity of plant pathogenic fungal mycelia by treatment with Bacillus veresensis JCK-7158 strain in MIC test

[0147] To confirm the effect of the culture filtrate of the present invention's Bacillus veresensis strain JCK-7158 on the growth of various plant pathogenic fungi, the growth inhibitory activity was measured against eight plant pathogenic fungi. The JCK-6019 strain was inoculated into TSB liquid medium and cultured at 30°C for 3 days with shaking at 150 rpm. The culture was centrifuged at 10,000 x g for 5 minutes, and the supernatant was filtered through a 0.2 μm sterile filter to harvest the culture filtrate (hereinafter referred to as "sample"). MIC (Minimum Inhibitory Concentration) tests were performed using the 96-well microtiter plate method, and the plant pathogenic fungi used were Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Fusarium graminearum, Fusarium asiaticum, Gaeumannomyces graminis, Phytophthora infestans, Botrytis cinerea, and Colletotrichum coccodes.

[0148] The plant pathogenic fungi used in the experiments were inoculated into PDB (potato dextrose broth, Becton, Dickinson and Co., Sparks, MD, USA) medium and cultured statically in an incubator at 25°C for 7 days. After static culture, the mycelium was weighed and homogenized in sterilized distilled water at 10,000 rpm for 5 seconds to a concentration of 50 mg / mL. The mycelium was then added to PDB medium to obtain a 1% fungal suspension.

[0149] Plant pathogenic fungal suspensions were treated with the culture filtrate samples of the present Bacillus verezensis JCK-7158 strain at concentrations of 10%, 5%, 2.5%, 1.25%, 0.625%, 0.313%, 0.156%, and 0.078%, respectively, and the experiment was repeated three times. The treated plates were sealed and cultured in an incubator at 25°C for 3 to 5 days, and the pathogen minimum inhibitory concentration (MIC) was determined.

[0150] [Table 3]

[0151] As can be seen from Table 3, MIC tests were conducted to confirm the antifungal activity of the Bacillus verezensis JCK-7158 strain of the present invention against plant pathogenic fungi. The strain JCK-7158 exhibited the strongest antifungal activity with an MIC value of 1.25% against Clarireedia jacksonii and Fusarium graminearum, and also exhibited strong activity with an MIC value of 5% against Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Gaeumannomyces graminis, Botrytis cinerea, Colletotrichum coccodes, Phytophthora infestans, etc. Furthermore, the strain exhibited antifungal activity with an MIC value of 10% against Fusarium asiaticum.

[0152] As a result, it was confirmed that the Bacillus veresensis JCK-7158 strain of the present invention has strong antifungal activity against various plant pathogenic fungi used in the experiments as a result of the MIC test.

[0153] Example 7. Growth inhibition activity of plant pathogenic fungi in dual culture by treatment with Bacillus veresensis JCK-7158 strain

[0154] In order to examine the mycelial growth inhibitory activity of the Bacillus verezensis JCK-7158 strain of the present invention against various plant pathogenic fungi, the growth inhibitory activity was measured by dual culture against 13 kinds of plant pathogenic fungi.

[0155] Target plant pathogenic fungi include Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Rhizoctonia solani AG2-2(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, Colletotrichum coccodes.

[0156] Specifically, a 5 cm long streak of the inventive Bacillus verezensis JCK-7158 culture solution was applied 2 cm from the edge of a PDA plate. Mycelial fragments of each pathogen were cut with a 6 mm diameter cork borer and inoculated 5 cm away from the JCK-7158 strain. The control group was inoculated with only the pathogenic fungal mycelial fragments. After inoculation of the pathogenic fungal mycelial fragments and the inventive Bacillus verezensis JCK-7158 culture solution, the pathogenic fungal mycelial growth radius was measured according to the pathogenic fungal growth rate while the inoculation was performed in an incubator at 25°C. The experiment was repeated three times. The antifungal activity was calculated by measuring the radius of the pathogenic fungal mycelia (R2) in the direction of the JCK-7158 strain and the radius of the pathogenic fungal mycelia (R1) under control conditions. Both values ​​were converted to the percentage of mycelial growth inhibition using the following formula:

[0157] [Mycelium growth inhibition rate (%) = (1-R2 / R1) x 100]

[0158] The Bacillus verezensis JCK-7158 strain of the present invention was cultured against various plant pathogenic fungi. As can be seen from FIG. 6 and Table 4, the Bacillus verezensis JCK-7158 strain exhibited growth inhibitory activity against all pathogenic fungi used in the experiment. Among these, the JCK-7158 strain inhibited the growth of all fungi used in the experiment by approximately 50%, except for Pythium ultimum. Therefore, the JCK-7158 strain was confirmed to have strong antifungal activity against the various plant pathogenic fungi used in the experiment, even through the culture.

[0159] [Table 4]

[0160] a1 [Mycelium growth inhibition rate (%) = (1 - fungal growth length at treatment / control fungal growth length) x 100 b ] Each value represents the mean ± standard deviation of three replicates.

[0161] Example 8. Isolation and structural identification of antibacterial substances from Bacillus veresensis JCK-7158 strain

[0162] To isolate the antibacterial substance produced by Bacillus verezensis JCK-7158, the strain was inoculated into a TSB liquid medium and cultured at 30°C for 3 days with shaking at 150 rpm. The isolation process of the antibacterial substance produced by Bacillus verezensis JCK-7158 was carried out in the same manner as in Figure 7.

[0163] To explain the isolation process in detail, after cultivation, the culture medium (3 L total) was centrifuged at 4500 rpm for 20 minutes to obtain the supernatant. The supernatant (290 mL) was fractionated twice with equal volumes of ethyl acetate (EtOAc) and butanol (BuOH) to obtain organic solvent and aqueous layers, respectively, and then concentrated under reduced pressure. Each fraction was redissolved in acetone, methanol, and water. To test the antifungal activity of each fraction, Fusarium graminearum was used as the test pathogen.

[0164] As can be seen from Figure 8, the results of the antifungal activity test confirmed that the butanol fraction exhibited antifungal activity against F. graminearum. The butanol fraction that exhibited activity was developed on a preparative thin-layer chromatography column using a solvent mixture of chloroform, methanol, and water (14:6:1, v / v / v), and then eluted with methanol to obtain five fractions (BF1 to BF5). The five fractions obtained were tested for antifungal activity against Fusarium graminearum. As shown in Table 5 below, the BF4 fraction had the highest activity, with an MIC of 125 ppm.

[0165] [Table 5]

[0166] The active BF4 fraction, selected as a result of the antifungal activity test, was again subjected to preparative thin-layer chromatography using a solvent mixture of chloroform, methanol, and water (55:36:8, v / v / v) and eluted with methanol to obtain four fractions (BF4-1 to BF4-4). These four fractions, along with the standards iturin, fengycin, and surfactin purchased from Sigma-Aldrich, were loaded onto a thin-layer chromatography (TLC) column and developed using a solvent mixture of chloroform, methanol, and water (55:36:8, v / v / v). The columns were then read by UV at wavelengths of 254 nm and 365 nm and visualized by spraying with p-anisaldehyde and water.

[0167] As a result, as shown in Figure 9, all four fractions obtained after visualization were confirmed to contain iturin A. Furthermore, the four fractions were tested for antifungal activity, and as shown in Table 5, the active fraction BF4-1 had an MIC of 125 ppm, making it the most potent of the four isolated fractions.

[0168] [Table 6]

[0169] The active fraction BF4-1 was observed to contain iturin as a result of thin-layer chromatography. LC / MS analysis of the BF4-1 fraction revealed positive ion mode peaks at molecular weights of 1043.55 and 1036.69, respectively, as shown in Figures 10 and 11. These peaks were identified as iturin A (iturin A, C). 48 H 74 O 14 N 12 , molecular weight 1,043) and surfactin (surfactin, C 53 H 93 O 13 N7, molecular weight 1,036).

[0170] Example 9. Antifungal activity assay of volatile organic compounds produced by Bacillus veresensis JCK-7158 strain

[0171] The antifungal activities of the volatile organic compounds produced by the Rhizoctonia berezensis strain JCK-7158 of the present invention were assayed against the plant pathogenic fungi Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Rhizoctonia solani AG2-2(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.

[0172] To prevent direct contact, bi-Petri dishes (SPL Life Sciences Co., Ltd., Pocheon, Gyeonggi-do, Korea) with a center separation were used. TSA was dispensed onto one side of the plate, and PDA onto the other. After dispensing, a culture of the JCK-7158 strain was smeared and incubated. The other PDA was inoculated with 6 mm-diameter mycelial fragments of the pathogen and incubated at 25°C. For the untreated control group, the PDA was inoculated with the pathogen alone, without being inoculated with the JCK-7158 culture. The experiment was performed in triplicate, and all plates were sealed twice with parafilm and incubated at 25°C to measure the mycelial growth diameter of the pathogen. For the untreated control group, the pathogen alone was inoculated without being inoculated with the culture of the strain.

[0173] The antifungal activity was calculated by measuring the length and width of the pathogen's hyphae (R1, R2) and the length and width of the pathogen's hyphae under control conditions (R3, R4). Both values ​​were converted into the percentage of mycelium growth inhibition using the following formula. The results are shown in the table below.

[0174] [Mycelium growth inhibition rate (%) = (1-(R1 x R2) / (R3 x R4)) x 100]

[0175] [Table 7]

[0176] As can be seen from Table 7 and Figure 12, the antifungal activity of volatile organic compounds produced by the JCK-7158 strain against plant pathogenic fungi was examined. The volatile compounds produced by the JCK-7158 strain inhibited the growth of various plant pathogenic fungi, and in particular, they showed excellent inhibitory effects against Botrytis cinerea, which causes gray mold in fruits and crops, and Fusarium graminearum, which causes Fusarium head blight in grains, by 60.50% and 48.66%, respectively. Therefore, it was confirmed that the JCK-7158 strain of the present invention produces volatile compounds that exhibit antifungal activity against various plant pathogenic fungi.

[0177] Example 10. Analysis of Volatile Organic Compounds Using GC-MS

[0178] To analyze the volatile organic compounds produced by the JCK-7158 strain of the present invention, solid phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) analyses were performed. JCK-7158 was inoculated into 5 mL of TSB medium and cultured at 30°C and 150 rpm for 3 days. Volatile organic compounds produced by JCK-7158 were collected in the headspace using an SPME fiber (Supelco, Bellefonte, PA, USA) at 50°C for 30 minutes.

[0179] The adsorbed material was injected onto the SPME fiber and exposed for 1 minute to desorb. It was then analyzed using a GC-MS (Shimadzu GC-MS QP2010, Shimadzu Co., Kyoto, Japan) equipped with a DB-5MS capillary column (30 m x 0.25 mm id x 0.25 μm film thickness, Agilent). He was used as the mobile phase, and the flow rate was maintained at 1.0 mL / min. The inlet temperature was set to 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 analysis was performed in positive ion mode by scanning from 50 to 400 m / z at 200°C.

[0180] The acquired mass spectra were compared with the data of the WILEY8 library for accurate identification, and the content of each substance was expressed as the area ratio of the TIC (total ion chromatogram) peak.

[0181] [Table 8]

[0182] As can be seen from Table 8 and Figure 13, the analysis revealed that five volatile organic compounds produced by the JCK-7158 strain of the present invention were detected within seven minutes. The chemical structures of the five compounds were confirmed by comparing their mass spectra with those of library compounds. GC-MS and mass spectrometry confirmed that the five volatile compounds were 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone. These compounds produced by the JCK-7158 strain of the present invention were determined to inhibit the growth of the plant pathogenic fungi used in the experiments.

[0183] Example 11. Preparation of a dosage form using a culture medium of Bacillus veresensis JCK-7158 strain

[0184] To prepare wettable powder and wettable liquid powder formulations using the JCK-7158 strain and culture broth of the present invention, the Bacillus veresensis JCK-7158 strain was inoculated into 20 mL of TSB medium and cultured at 30°C and 150 rpm for 3 days with shaking. 20% Flosetrite® (oxidized starch) was added to the culture broth harvested after 3 days of culture, and the mixture was spray-dried at 140°C with stirring. The spray-dried samples were prepared as shown in Tables 9 and 10 below to produce JCK-7158 strain suspension concentrate (SC) and wettable powder (WP).

[0185] [Table 9]

[0186] [Table 10]

[0187] Example 12: Resistance induction activity assay of JCK-7158 strain in Arabidopsis thaliana

[0188] The PR-1 gene is used as a marker gene to test for resistance induction in plants by utilizing a series of signal transduction systems that induce the expression of the PR-1 protein. Therefore, to test the resistance induction activity of the culture medium of Bacillus veresensis JCK-7158 using the PR-1 gene system, Arabidopsis thaliana transformed with a vector in which the PR-1 promoter is tagged with GUS was used.

[0189] Arabidopsis seeds were surface-sterilized with 95% ethanol and then subjected to secondary surface sterilization using a bleach solution made of 2% NaOCl and 0.05% Tween-20. The remaining bleach solution on the seeds was washed with sterile distilled water, and the seeds were then soaked at 4°C for 48 hours.

[0190] The soaked seeds were placed on MS agar (Murashige-Skoog Agar) supplemented with 50 ppm kanamycin using sterilized toothpicks and cultured in a plant growth incubator at 25°C. After 12 days of culture, each well of a 24-well plate was treated with 2.5 mL of the culture broth, culture filtrate, cell suspension, wettable powder (SC), and wettable powder (WP) of the present Bacillus verezensis JCK-7158 strain of the present invention, diluted 500, 1,000, 2,000, or 4,000 times in sterilized water. Two Arabidopsis plants were placed in each well so that they were submerged, and the wells were maintained at room temperature for 48 hours on an orbital shaker.

[0191] After 48 hours, the plates were immersed in 90% acetone at -20°C for 1 hour to fix the reaction, and then washed twice with 0.1 M sodium phosphate buffer (pH 7.0). After washing, the plates were immersed in staining solution (100 mM sodium phosphate buffer, 0.1% Triton X-100, 2 mM X-GlucA, 2.5 mM potassium ferricyanide, 2.5 mM potassium ferrocyanide, sterile distilled water) in the dark and then kept in a 37°C water bath for 24 hours.

[0192] After 24 hours, the staining solution was removed, and the cells were immersed in 70% ethanol for one hour, followed by multiple immersions in 90% ethanol at one-hour intervals to remove unwanted pigments such as chlorophyll. Once all the pigments had been removed, the cells were examined under a microscope to confirm the blue coloration, confirming the expression of the PR-1 gene.

[0193] The expression of PR-1 gene was confirmed by the presence or absence of GUS expression using an Arabidopsis thaliana assay system, and the resistance induction activity was assayed.

[0194] [Table 11]

[0195] As can be seen from Table 11 and Figure 14, GUS was expressed and PR-1 gene expression was induced in the culture broth (CB), culture filtrate (CF), cell suspension (Cell), and two formulations (SC and WP) of Bacillus verezensis JCK-7158, as well as in the positive control (SA) treatment group. This confirms that Bacillus verezensis JCK-7158 can induce resistance in plants and control pathogenic fungi through a mechanism similar to that of salicylic acid.

[0196] Example 13. Control effect of induced resistance of Bacillus veresensis JCK-7158 strain against rice head blight under greenhouse conditions

[0197] To investigate the in vivo control activity of rice Fusarium head blight caused by induced resistance of the present strain JCK-7158 under greenhouse conditions, Samkwang rice seeds were used. Ten grams of Samkwang rice seeds (Oryza sativa cv. Samkwang) provided by the National Institute of Food Science, Korea Rural Development Administration, were placed in a plastic container and disinfected with Spotak emulsion (25% prochloraz, Kyung Nong Co., Ltd.) diluted 2,000 times. After sterilization for one day, the seeds were soaked in water for two days in the dark.

[0198] The rice seeds were germinated for two days and then sown at 10 seeds per pot in plastic pots (6 cm diameter, 6.5 cm height) filled with 80% paddy rice soil (heavy-duty soil, Bunon Co.). After sowing, the pots were placed on yellow trays with holes at the bottom so that the bottoms of the pots were submerged in water, and then cultured in a 30°C constant temperature room covered with nonwoven fabric for seedling support. When the rice seeds germinated to about 1 cm, the nonwoven fabric was removed and the seeds were grown under a 16-hour photoperiod for four weeks before being transplanted into Wagner pots (top diameter 17.5 cm, bottom diameter 16 cm, height 19.8 cm, NF-5).

[0199] Seven days before transplanting, a compound fertilizer (Huksaran 21, Namhae Chemical) was dissolved in water and applied evenly to the flooded paddy field soil. The seedlings were then grown in a constant temperature and humidity room and transplanted into the center of a Wagner pot filled with 70% paddy field soil. Before transplanting, the drainage holes at the bottom of the pots were sealed with silicone plugs, and the pots were lined with polystyrene foam balls (4 cm diameter, 10 balls per pot) and a pot bottom net (14 cm diameter).

[0200] After transplanting, the Wagner pots were filled with water and grown in a glass greenhouse (minimum 20-25°C, maximum 30-35°C) for use in the experiment. 20 days after transplanting, the plants were top-dressed with Super Arari (14 kg / 10a, Namhae Chemical), and two months after transplanting, they were top-dressed with NK 24 (N:P:K, 24-0-12, Farm Hannon).

[0201] To prepare the rice head blight pathogen inoculum, Fusarium asiaticum, the causative agent of rice head blight, was inoculated onto PDA medium and incubated statically at 25°C for 6 days. Five agar plugs (0.1 cm × 0.1 cm) covered with cultured F. asiaticum mycelia were cut and inoculated into carboxymethylcellulose medium (CMC, 15 g carboxymethylcellulose (Sigma-Aldrich), 1 g yeast extract, 0.5 g MgSO 7H O, 1 g NH NO , and 1 g KH PO in 1 L distilled water) and incubated at 25°C with shaking at 150 rpm for 4 days.

[0202] The pathogen culture was then filtered through four layers of gauze to remove mycelia, and the filtered spore suspension was analyzed under a microscope (Axio Imager. A2, Carl Zeiss, Germany) using a hemocytometer to measure 5 × 10 5 The spore suspension was centrifuged at 4,000 rpm for 10 minutes at 4°C, and the supernatant was discarded to remove medium components. The spores were then suspended in an equal volume of 0.05% Tween-80 and used as the inoculum for this experiment.

[0203] We investigated the efficacy of JCK-7158 in controlling rice Fusarium head blight caused by F. asiaticum through induced resistance. JCK-7158 was applied to rice plants 11 weeks after transplantation into pots. Two and one weeks before inoculation, JCK-7158 culture solution, culture filtrate, cell suspension, wettable powder (SC), and wettable powder (WP) were diluted 1,000-fold and 2,000-fold, respectively, and sprayed onto the leaves of the plants until the rice ears and stems were thoroughly wetted. As a control, a 2,000-fold diluted prey emulsion (Peulrei, AI 13% difenoconazole + 13% propiconazole EC, Shinzenta Korea), a known fungicide for rice Fusarium head blight, was applied one day before inoculation.

[0204] Pathogen inoculation was performed seven days after the second fungicide treatment by uniformly spraying the leaves with the same amount of F. asiaticum spore suspension (2 x 10 spores / mL) for each treatment group. During fungicide manufacturing, the wetting agent Tween-20 was added to the JCK-7158 strain culture medium, culture filtrate, and cell suspension at approximately 250 μg / mL. Immediately after inoculation, the plants were treated in a moist chamber using vinyl bags for three days. The experiment was repeated five times per treatment group, and seven days after inoculation, the disease severity of rice Fusarium head blight was calculated based on the diseased area percentage, and the control value compared to the untreated group was calculated.

[0205] [Control value (%) = (1 - (disease incidence in treated area / disease incidence in untreated area)) x 100]

[0206] As can be seen from Figures 15 and 16, the control effect of Bacillus veresensis JCK-7158 strain on rice Fusarium head blight due to induced resistance was examined 7 days after inoculation of the pathogen. As a result, the incidence of rice Fusarium head blight induced by F. asiaticum was reduced in all treatments with JCK-7158 strain culture broth (CB), culture filtrate (CF), and cell suspension.

[0207] In particular, a 1,000-fold dilution of JCK-7158 strain culture broth showed 49.88% and a 2,000-fold dilution showed 34.71% control activity compared to the untreated control group, a 1,000-fold dilution of JCK-7158 strain culture filtrate showed 43.88% and a 2,000-fold dilution showed 38.82% control activity, and a 1,000-fold dilution of JCK-7158 strain cell suspension showed 47.88% and a 2,000-fold dilution showed 42.59% control activity. Concentration-dependent effects were observed in all treatment groups. Prey, used as a control agent, showed a 34.41% control rate in a 2,000-fold diluted solution, and the control activity of the treated area was slightly superior to that of the control agent, confirming that the JCK-7158 strain effectively controls rice Fusarium head blight through induced resistance.

[0208] In addition, as can be seen from FIGS. 17 and 18, the control effect of the Bacillus veresensis JCK-7158 strain prepared in Example 11 in the form of a wettable powder (SC) and a wettable powder (WP) against rice head blight due to induced resistance was examined 7 days after inoculation of the pathogen. As a result, both the treatments with the JCK-7158 strain in the form of a wettable powder (SC) and a wettable powder (WP) reduced the occurrence of rice head blight induced by F. asiaticum.

[0209] In particular, a 1:1000 dilution of JCK-7158 SC demonstrated 63.87% and a 2:1000 dilution demonstrated 35.43% control activity compared to the control. A 1:1000 dilution of JCK-7158 WP demonstrated 34.40% and a 2:1000 dilution demonstrated 30.42% control activity, respectively. Concentration-dependent control was observed in all treatments. Prey, used as the control, demonstrated a 44.87% control rate at a 2:1000 dilution. The 1:1000 dilution of JCK-7158 SC (63.87%) demonstrated superior control activity compared to the control, confirming its effective control of rice Fusarium head blight. Therefore, it was confirmed that the resistance induction activity of the JCK-7158 strain can be used to develop an effective rice Fusarium head blight control agent.

[0210] Example 14. Control effect of Bacillus veresensis JCK-7158 strain formulation on rice head blight by induced resistance under field conditions

[0211] To examine the in vivo control activity of rice Fusarium head blight disease (FBD) caused by induced resistance of the JCK-7158 strain of the present invention under field conditions, Samkang rice seeds were treated with the Bacillus veresensis JCK-7158 liquid dispersible powder (SC) described in Example 11. The Bacillus veresensis JCK-7158 liquid dispersible powder (SC) was diluted 1,000-fold and 2,000-fold and sprayed onto the target Samkang rice plants, 2 weeks and 1 week prior to inoculation with the pathogen. The control efficacy of the Bacillus veresensis JCK-7158 formulation was then evaluated by inoculation with F. asiaticum. As a control, a 2,000-fold diluted pre-emulsifiable concentrate (13% propiconazole + 13% difenconazole, Shinzenta Korea Co., Ltd.) was applied 1 day prior to inoculation with the pathogen.

[0212] The inoculum used to inoculate rice plants under field conditions was 2.0×10 F. asiaticum, which causes rice head blight, in the same manner as described in Example 13. 5The spore suspension was prepared by adding 500 μg / mL Tween 20 to the spores, adjusting the concentration to spores / mL. To maintain humidity similar to that in a greenhouse, the treated rice ears were placed in a zippered plastic bag (8 cm x 20 cm) and sprayed with distilled water once. The ears and leaves were then placed in a sealed bag and treated with humidity for three days. Three days after inoculation, the zippered plastic bag was removed and the rice was sprayed with water twice daily. Treatments were performed three times, with 30 rice ears per treatment area.

[0213] Seven days after inoculation, the ears were cut and the disease severity was assessed. The disease severity was calculated from 0% to 100% at 5% intervals. The control value was calculated using the following formula:

[0214] [Control value (%) = (1 - (disease incidence in treated area / disease incidence in untreated area)) x 100]

[0215] As can be seen from Figures 19 to 21, the wettable powder (SC) of Bacillus veresensis JCK-7158 strain prepared in Example 11 was applied two weeks and one week prior to inoculation of the pathogen to induce resistance in rice plants, and the control effect against rice Fusarium head blight was then examined under field conditions. As a result, the incidence of rice Fusarium head blight induced by F. asiaticum was reduced in the plots treated with the JCK-7158 wettable powder (SC) two and four weeks after inoculation of the pathogen.

[0216] In particular, two weeks after inoculation, a 1:1000 diluted JCK-7158 SC solution showed 48.81% and a 2:2000 diluted JCK-7158 SC solution showed 20.56% control activity compared to the untreated control. Four weeks after inoculation, a 1:1000 diluted JCK-7158 SC solution showed 50.28% and a 2:2000 diluted JCK-7158 SC solution showed 17.5% control activity compared to the untreated control. The control values ​​were concentration-dependent in the treated areas, and the results were similar to those of the greenhouse experiment. Prey, used as the control agent, showed a control value of 32.57% and 35.21% at a 2,000-fold dilution two and four weeks after inoculation of the pathogen, respectively.It was confirmed that the control value of JCK-7158 liquid dispersible agent (SC) diluted 1,000 times was superior to that of the control agent at both times when the rice head blight control value was measured.

[0217] As a result, the treatment with a 1,000-fold diluted solution of JCK-7158 SC effectively controlled rice Fusarium head blight compared to the control treatment with chemical pesticides. Therefore, it was confirmed that rice Fusarium head blight control using the resistance induction activity of JCK-7158 strain works efficiently even under field conditions.

[0218] Example 15. Reduction of toxin production in rice Fusarium head blight by induced resistance to Bacillus veresensis JCK-7158 strain formulation under field conditions

[0219] To examine the effect of the treatment of the JCK-7158 strain liquid wettable powder of the present invention on reducing the production of nivalenol (NIV), a mycotoxin produced by Fusarium asiaticum (F. asiaticum), a pathogen that causes rice head blight, under field conditions, the Bacillus verezensis JCK-7158 liquid wettable powder (SC) described in Example 11 was applied to Samkang rice seeds.

[0220] Bacillus verezensis JCK-7158 wettable powder (SC) was diluted 1,000-fold and 2,000-fold and sprayed onto the target rice plants, Samkang rice, two weeks and one week before inoculation. Fusarium asiaticum was then inoculated to examine the control efficacy of the Bacillus verezensis JCK-7158 formulation. The control agent was a 2,000-fold diluted spray emulsion (13% propiconazole + 13% difenconazole, Shinzenta Korea Co., Ltd.) applied one day before inoculation.

[0221] The inoculum used to inoculate rice plants under field conditions was Fusarium asiaticum, which causes rice head blight, at 2.0×10 5The spore suspension was prepared by adding 500 μg / mL Tween 20 to the spores, which were adjusted to 100 spores / mL. To maintain humidity, the treated rice ears were placed in a resealable plastic bag (8 cm x 20 cm) and sprayed with distilled water once. The ears and leaves were then placed in a sealed bag and treated with humidity for three days. Three days after inoculation, the resealable plastic bag was removed and the ears were sprayed with water twice daily. Thirty rice ears per treatment area were treated three times, with three replicates.

[0222] Six weeks after inoculation, seeds were harvested from the field and dried in sunlight and an oven for three days. The dried sample was crushed, and 5 g of the crushed sample was added to 25 mL of sterile water and extracted by shaking at 300 rpm for one hour. The sample was then centrifuged at 3,600 rpm for 10 minutes at 4°C, and 5 mL of the supernatant was diluted with 20 mL of phosphate-buffered saline (PBS).

[0223] 25 mL of the diluted solution was loaded onto a DON-NIV WB (Vicam) column. After the diluted solution had completely drained, 5 mL of PBS and 5 mL of sterile water were added to wash the column. Pressure was then applied to the column to remove the solution, and the column was then eluted with 0.5 mL of methanol and 1.5 mL of acetonitrile (MeCN), followed by drying with nitrogen.

[0224] The sample was then reconstituted in 1 mL of sterile water:MeCN:MeOH (90:5:5, v / v / v). It was then filtered through a 0.22 μm membrane filter and analyzed by UPLC-PDA. The instrument used was a WATRERS ACQUITY UPLC H Class with a PDA (218 nm) detector. The column was an Xselect CSH C18 (2.1 x 100 mm, 2.5 μm, Waters, Scotland), and the temperature was maintained at 40°C. The flow rate was 0.3 mL / min, and 10 μL was injected and analyzed for 10 minutes. Nivalenol (NIV) was detected as a result of the analysis.

[0225] [Table 12]

[0226] Furthermore, as can be seen from Table 12, treatment with JCK-7158 liquid wettable powder reduced nivalenol by 40.62% compared to the untreated area, demonstrating a superior effect in reducing the mycotoxin nivalenol compared to the chemical fungicide, play (toxin reduction effect: 21.12%).

[0227] As a result, the treatment with a 1:1000 diluted solution of JCK-7158 SC not only effectively controlled rice Fusarium head blight compared to the control chemical pesticide treatment, but also more effectively reduced the amount of mycotoxins produced by the pathogen compared to the chemical control. Therefore, it was confirmed that rice Fusarium head blight control using the resistance-inducing activity of JCK-7158 strain works efficiently even under field conditions.

[0228] Therefore, JCK-7158 is an environmentally friendly strain with excellent pesticidal activity that can be used as an alternative to chemical pesticides, which have the drawback of only being able to be used up to 30 days before harvest due to residue issues.If used as a preventative agent, it is likely to become a cornerstone in the development of control agents for various plant diseases, including rice Fusarium head blight, which has been difficult to control. [Industrial Applicability]

[0229] The present invention relates to a composition for controlling plant pathogenic fungi containing the Bacillus velezensis JCK-7158 strain, a culture solution thereof, or an extract thereof, a method for producing the same, a method for controlling plant pathogenic fungi, and a method for reducing mycotoxins. [Accession number]

[0230] Name: Bacillus velezensis JCK-7158 Accession number: KCTC15169BP TIFF2025539800000022.tif203170

Claims

1. Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, which has antifungal activity.

2. A composition for controlling plant pathogenic fungi, comprising the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, a culture solution thereof, or an extract thereof.

3. The plant pathogenic fungi include Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Rhizoctonia solani AG2-2(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 cocodes. The composition for controlling plant pathogenic fungi according to claim 2, wherein the fungal species is one or more selected from the group consisting of phytopathogenic fungi.

4. A composition for controlling plant pathogenic fungi, comprising one or more compounds selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexan-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

5. The plant pathogenic fungi include Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Rhizoctonia solani AG2-2(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 cocodes. The composition for controlling phytopathogenic fungi according to claim 4, wherein the fungal species is one or more selected from the group consisting of phytopathogenic fungi (coccodes).

6. A method for producing a composition for controlling plant pathogenic fungi, comprising culturing Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP to produce a culture solution.

7. The plant pathogenic fungi include Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Rhizoctonia solani AG2-2(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 cocodes.

7. The method for producing a composition for controlling phytopathogenic fungi according to claim 6, wherein the composition is one or more selected from the group consisting of phytopathogenic fungi.

8. The method for producing a composition for controlling plant pathogenic fungi according to claim 6, further comprising a first fractionation step of fractionating the culture broth or a culture filtrate obtained therefrom with butanol to obtain a butanol layer.

9. 9. The method for producing a composition for controlling plant pathogenic fungi according to claim 8, further comprising a second fractionation step of eluting the butanol layer obtained in the first fractionation step with a mixed solution of chloroform:methanol:water to obtain the fourth or fifth fraction out of the five fractions.

10. The method for producing a composition for controlling plant pathogenic fungi according to claim 9, wherein the mixing volume ratio of chloroform:methanol:water is 50-60:30-40:5-10.

11. 10. The method for producing a composition for controlling plant pathogenic fungi according to claim 9, further comprising a third fractionation step of eluting the fractions obtained in the second fractionation step with a mixed solution of chloroform:methanol:water to obtain one or more fractions selected from the group consisting of the first to third fractions among the four fractions.

12. The method for producing a composition for controlling plant pathogenic fungi according to claim 11, wherein the mixing volume ratio of chloroform:methanol:water is 50-60:30-40:5-10.

13. A method for controlling plant pathogenic fungi, comprising treating Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, a culture solution thereof, or an extract thereof.

14. 14. The method for controlling plant pathogenic fungi according to claim 13, wherein the treatment step is carried out by one or more methods selected from the group consisting of spraying, soil drench, surface spraying, rhizosphere treatment, seed treatment, immersion, poisoning, and fumigation.

15. The plant pathogenic fungi include Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG2-2(IV) Large patch, Rhizoctonia solani AG2-2(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 cocodes. The method for controlling plant pathogenic fungi according to claim 13, wherein the fungal pathogenic fungus is one or more selected from the group consisting of phytopathogenic fungi (coccodes).

16. A method for controlling plant pathogenic fungi, comprising a treatment step of treating a plant with one or more compounds selected from the group consisting of iturin A, surfactin, 2,3-butanediol, 5-methylhexane-2-one, heptan-2-one, 2,5-dimethylpyrazine, 6-methylheptan-2-one, and 5-methyl-2-heptanone.

17. A composition for reducing mycotoxins, comprising the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, a culture solution thereof, or an extract thereof.

18. A method for producing a composition for reducing mycotoxins, comprising culturing Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP to produce a culture broth.

19. A method for reducing mycotoxins, comprising treating the Bacillus velezensis JCK-7158 strain deposited under accession number KCTC15169BP, its culture broth, or an extract thereof.

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