Composition for controlling plant diseases comprising the extract of the culture solution or the strain culture solution of the Streptomyces lidicus JCK-6019 strain, a method for producing the same, and a method for controlling a plant disease

Abstract

The present invention relates to a Streptomyces lydicus JCK-6019 strain, a composition for controlling plant diseases comprising said strain, a culture thereof, or an extract thereof, a method for preparing said composition, and a method for controlling plant diseases using said composition.

Description

Technology Field

[0001] The present invention relates to Streptomyces lidicus ( Streptomyces lydicus The present invention relates to a JCK-6019 strain, a composition for controlling plant diseases comprising said strain, a culture thereof, or an extract thereof, a method for preparing said composition, and a method for controlling plant diseases using said composition. Background Technology

[0002] Plant pathogens cause economically significant diseases in agricultural, horticultural, and ornamental crops, inflicting substantial damage on farms by reducing crop marketability and yield. Fungal plant diseases are more diverse and cause greater damage compared to other pathogens; in fact, fungal causes accounted for 17 out of 33 past cases of severe damage caused by plant diseases. Pathogenic fungi, which make up the majority of plant diseases, form spores or sclerotia to prevent the loss of autonutrients even in the absence of host plants, inhabiting the soil for extended periods and causing severe damage to crops by secreting toxic substances or enzymes.

[0003] In addition, Magnaforte Orise ( Magnaporthe rice ), Botrytis cinerea( Botrytis cinerea ), Foxinia SPP( Powdery mildew spp.), Fusarium graminearum( Fusarium gramineae ), Fusarium oxysporum ( Fusarium oxysporum ), Brumeria gramines ( Blumeria grassinis ), Mycosperella graminicola( Mycosphaerella graminicola ), Coretotrichum spp( Colletotrichum spp.), Ustilago Maydis( May burning ), Melampsora Rini( Melampsora linii ...etc. were reported.

[0004] Plant diseases caused by plant pathogenic fungi are very difficult to control for the following reasons: 1) many plant pathogenic fungi efficiently and continuously produce an excessive number of spores to infect other healthy plants; 2) the time required for spore production on the host after infection is short and the spores are highly mobile, making them highly contagious; and 3) they have a good ability to steal nutrients from the host and may secrete toxic substances or enzymes harmful to the host. Due to these characteristics, despite continuous control efforts, plant pathogenic fungi parasitize host plants every year, causing enormous economic damage.

[0005] Generally, fungal plant diseases have been controlled by spraying chemical fungicides; however, considering issues such as the development of drug resistance in pathogens, residual toxicity, and environmental pollution, it is desirable to maintain continuous control of plant fungal diseases through biological methods.

[0006] Therefore, to address these issues, eco-friendly fungicides utilizing microorganisms and microbial-derived natural substances capable of replacing chemical pesticides have been proposed as an alternative, and related research has been actively conducted to date. However, as the efficacy and variety of developed biological agents are not yet sufficient compared to chemical fungicides, there is an urgent need to develop superior biological agents.

[0007] Although various microorganisms have been considered as potential biological control agents, many recent studies Streptomyces The focus is on the inside. Streptomyces ( StreptomycesThe genus Streptomyces is the largest genus of actinomycetes and consists of Gram-positive bacteria that grow in the form of hyphae, with over 500 known species. Most Streptomyces produce spores and are characterized by complex secondary metabolisms; they are known to produce clinically useful naturally occurring antibiotics such as neomycin, cypamycin, grimemycin, botolomycin, and chloramphenicol. As various physiologically active substances, as well as antibiotics, have been isolated from Streptomyces strains, these strains are treated as highly important microorganisms for both industrial and medical purposes. Research on these antibiotics has advanced from the search for substances effective against bacterial or fungal infections to those effective against viral diseases, but such research is primarily focused on medical applications.

[0008] Using the genus Streptomyces in vitro Studies have reported that this actinomycete inhibits the growth of various plant pathogenic microorganisms, decomposes various organic materials such as chitin and cellulose—components of the cell walls of plant pathogenic fungi—promotes plant growth by secreting plant hormones such as auxin and gibberellin, and induces disease resistance in various plants. Due to these characteristics, the genus Streptomyces has excellent potential as an antimicrobial biological control agent. The problem to be solved

[0009] The inventors of the present invention are Streptomyces lidicus ( Streptomyces lydicus It was confirmed that when the JCK-6019 strain, the strain, its culture, or its extract was treated, the plant disease control activity was significantly superior.

[0010] Accordingly, the object of the present invention is Streptomyces redicus (deposited under accession number KCTC 15107BP) having plant disease control activity Streptomyces lydicus ) It is to provide the JCK-6019 strain.

[0011] Another objective of the present invention is to provide Streptomyces redicus (deposited under accession number KCTC 15107BP) having plant disease control activity. Streptomyces lydicus The present invention provides a composition for controlling plant diseases comprising the JCK-6019 strain, a culture thereof, or an extract thereof.

[0012] Another objective of the present invention is to provide a composition for controlling plant diseases comprising natamycin.

[0013] Another objective of the present invention is to provide Streptomyces redicus (deposited under accession number KCTC 15107BP) having plant disease control activity. Streptomyces lydicus The present invention provides a method for manufacturing a composition for controlling plant diseases, comprising a culture step of culturing the JCK-6019 strain.

[0014] Another object of the present invention is Streptomyces redicus (deposited under accession number KCTC 15107BP) having antifungal activity Streptomyces lydicus The present invention provides a method for controlling plant diseases comprising a treatment step of treating a plant or soil with a composition for controlling plant pathogenic fungal diseases, the composition comprising the JCK-6019 strain, a culture solution thereof, or an extract thereof.

[0015] Another objective of the present invention is to provide a method for controlling plant diseases comprising a treatment step of treating a plant or soil with a composition for controlling plant diseases containing natamycin.

[0016] Another objective of the present invention is to provide Streptomyces redicus (deposited under accession number KCTC 15107BP) having plant disease control activity. Streptomyces lydicus This concerns the use of the JCK-6019 strain for controlling plant diseases.

[0017] Another objective of the present invention is to provide Streptomyces redicus (deposited under accession number KCTC 15107BP) having plant disease control activity. Streptomyces lydicusThis relates to the use of the JCK-6019 strain, its culture, or its extract for controlling plant diseases.

[0018] Another objective of the present invention is to use natamycin for controlling plant diseases. means of solving the problem

[0019] The present invention relates to Streptomyces lidicus ( Streptomyces lydicus The present invention relates to a JCK-6019 strain, a composition for controlling plant diseases comprising said strain, a culture thereof, or an extract thereof, a method for preparing said composition, and a method for controlling plant diseases using said composition. It was confirmed that when the composition for controlling plant diseases according to the present invention is applied, excellent antibacterial activity and resistance-inducing activity in treated plants are significantly superior.

[0020] The present invention will be described in more detail below.

[0021] One aspect of the present invention is Streptomyces redicus having plant disease control activity ( Streptomyces lydicus This concerns the JCK-6019 strain.

[0022] In the present invention, the Streptomyces redicus JCK-6019 strain may be the Streptomyces redicus JCK-6019 strain deposited under accession number KCTC 15107BP.

[0023] In the present invention, the Streptomyces redicus JCK-6019 strain may contain 16S rRNA containing the nucleotide sequence of SEQ ID NO. 3.

[0024] The Streptomyces redicus JCK-6019 strain of the present invention was deposited with the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Biotechnology and Bioengineering on September 26, 2022, under accession number KCTC 15107BP.

[0025] Another aspect of the present invention is Streptomyces redicus having plant disease control activity ( Streptomyces lydicus The present invention relates to a composition for controlling plant pathogenic fungi comprising the JCK-6019 strain, a culture thereof, or an extract thereof.

[0026] In the present invention, the Streptomyces redicus JCK-6019 strain may be the Streptomyces redicus JCK-6019 strain deposited under accession number KCTC 15107BP.

[0027] In the present invention, the Streptomyces redicus JCK-6019 strain may contain 16S rRNA containing the nucleotide sequence of SEQ ID NO. 3.

[0028] In the present invention, the Streptomyces redicus JCK-6019 strain, the culture thereof, or the extract thereof may contain an antifungal substance, an antimicrobial substance and / or an induced resistance substance as an active ingredient.

[0029] In the present invention, the antifungal substance, antimicrobial substance and / or induced resistance substance may be a volatile organic compound and / or a non-volatile organic compound.

[0030] In the present invention, the non-volatile organic compound may be natamycin, but is not limited thereto.

[0031] In the present invention, natamycin may have the structure of the following structural formula I, with a molecular weight of 665, and the molecular formula C 33 H 47 NO 13 am.

[0032] [Structural Formula I]

[0033]

[0034] In the present invention, the volatile organic compound may be one or more selected from the group consisting of volatile organic compounds 2-methyl-2-bornen, 1,2,7,7-tetramethylbicyclo(2.2.1)heptan-2-ol, 1,3-diisopropyl-1,3-cyclopentadiene and 2-methylenebornan, and may be, for example, 2-methyl-2-bornen, but is not limited thereto.

[0035] In the present invention, 2-methyl-2-bornene may have the structure of the following structural formula II, the molecular weight is 150, and the molecular formula is C 11 H 18 am.

[0036] [Structural Formula II]

[0037]

[0038] In the present invention, the plant disease may be a plant pathogenic fungal disease or a plant pathogenic bacterial disease.

[0039] In the present invention, the plant pathogenic fungus is Rhizoctonia solani ( Rhizoctonia solani AG-4, AG2-2(IV) Large patch, AG2-2(IV) Brown patch), Fusarium oxysporum f. sp. cucuminum( Fusarium oxysporum f. sp. cucumber ), Clarididia Jacksoni( Clarireedia jacksonii , previously Sclerotinia homoeocarpa (called), *Gymanomyces graminis* ( Gaeumannomyces graminis ), Botrytis cinerea( Botrytis cinerea ), Fusarium graminearum( Fusarium gramineae ), Fusarium oxysporum F. SP. Lycopacific( Fusarium oxysporum f. sp. tomatoes ), Phytophsora Infestance( Phytophthora infestans ), Phytophthora capsicum ( Phytophthora capsicum ), Pythium aphanidermatum( Pythium aphanidermatum ), Pythium Ultimum( The last Pythium ), and Pseudocercospora sulfumsisa ( Pseudocercospora circumcisaIt may be one or more selected from a group consisting of ), but is not limited thereto.

[0040] In the present invention, the plant pathogenic bacterium is *Pectobacterium carotobora subspecies carotobora* ( Pectobacterium carotovora subsp. carrot-eating It may be ), but is not limited to this.

[0041] In the present invention, the plant pathogenic fungal disease is Rhizoctonia solani ( Rhizoctonia solani Cucumber damping-off caused by ), Fusarium oxysporum f. sp. cucuminum ( Fusarium oxysporum f. sp. cucumber Cucumber wilt caused by ) and Clariredia jacksonii ( Clarireedia jacksonii It may be turf coin blight caused by ), but is not limited to this.

[0042] In the present invention, the plant pathogenic bacterial disease is *Pectobacterium carotobora subspecies carotobora* ( Pectobacterium carotovora subsp. carrot-eating It may be cabbage soft rot caused by ), but is not limited to this.

[0043] The term “culture” in this specification refers to a product containing microorganisms after culturing said microorganisms.

[0044] The term “culture supernatant” in this specification refers to the liquid of the upper layer obtained by removing most microorganisms from the culture medium through a centrifugation method, and is also called “supernatant.”

[0045] The term “culture filtrate” in this specification refers to the liquid remaining after removing microbial cells from the culture medium by filtering them out through centrifugation and filtration. The culture filtrate contains substances formed and discharged during the growth of microorganisms, and thus can be purified or extracted.

[0046] Another aspect of the present invention relates to a composition for controlling plant diseases comprising one or more active ingredients selected from the group consisting of natamycin, 2-methyl-2-bornen, 1,2,7,7-tetramethylbicyclo(2.2.1)heptan-2-ol, 1,3-diisopropyl-1,3-cyclopentadiene, and 2-methylenebornan.

[0047] In the present invention, natamycin, 2-methyl-2-bornen, 1,2,7,7-tetramethylbicyclo(2.2.1)heptan-2-ol, 1,3-diisopropyl-1,3-cyclopentadiene, and 2-methylenebornan are used to control plant diseases in Streptomyces redicus ( Streptomyces lydicus It may be derived from the JCK-6019 strain, but is not limited thereto.

[0048] In the present invention, the Streptomyces redicus JCK-6019 strain may be the Streptomyces redicus JCK-6019 strain deposited under accession number KCTC 15107BP.

[0049] In the present invention, the Streptomyces redicus JCK-6019 strain may contain 16S rRNA containing the nucleotide sequence of SEQ ID NO. 3.

[0050] In the present invention, the plant disease may be a plant pathogenic fungal disease or a plant pathogenic bacterial disease.

[0051] In the present invention, the plant pathogenic fungus is Rhizoctonia solani ( Rhizoctonia solani AG-4, AG2-2(IV) Large patch, AG2-2(IV) Brown patch), Fusarium oxysporum f. sp. cucuminum( Fusarium oxysporum f. sp. cucumber ), Clarididia Jacksoni( Clarireedia jacksonii , previously Sclerotinia homoeocarpa (called), *Gymanomyces graminis* ( Gaeumannomyces graminis), Botrytis cinerea( Botrytis cinerea ), Fusarium graminearum( Fusarium gramineae ), Fusarium oxysporum F. SP. Lycopacific( Fusarium oxysporum f. sp. tomatoes ), Phytophsora Infestance( Phytophthora infestans ), Phytophthora capsicum ( Phytophthora capsicum ), Pythium aphanidermatum( Pythium aphanidermatum ), Pythium Ultimum( The last Pythium ), and Pseudocercospora sulfumsisa ( Pseudocercospora circumcisa It may be one or more selected from a group consisting of ), but is not limited thereto.

[0052] In the present invention, the plant pathogenic bacterium is *Pectobacterium carotobora subspecies carotobora* ( Pectobacterium carotovora subsp. carrot-eating It may be ), but is not limited to this.

[0053] In the present invention, the plant pathogenic fungal disease is Rhizoctonia solani ( Rhizoctonia solani Cucumber damping-off caused by ), Fusarium oxysporum f. sp. cucuminum ( Fusarium oxysporum f. sp. cucumber Cucumber wilt caused by ) and Clariredia jacksonii ( Clarireedia jacksonii It may be turf coin blight caused by ), but is not limited to this.

[0054] In the present invention, the plant pathogenic bacterial disease is *Pectobacterium carotobora subspecies carotobora* ( Pectobacterium carotovora subsp. carrot-eating It may be cabbage soft rot caused by ), but is not limited to this.

[0055] Another aspect of the present invention is a method for preparing a composition for controlling plant diseases, comprising a culture step of culturing the Streptomyces redicus JCK-6019 strain.

[0056] In the present invention, the Streptomyces redicus JCK-6019 strain may be the Streptomyces redicus JCK-6019 strain deposited under accession number KCTC 15107BP.

[0057] In the present invention, the Streptomyces redicus JCK-6019 strain may contain 16S rRNA containing the nucleotide sequence of SEQ ID NO. 3.

[0058] In the present invention, the plant disease may be a plant pathogenic fungal disease or a plant pathogenic bacterial disease.

[0059] In the present invention, the plant pathogenic fungus is Rhizoctonia solani ( Rhizoctonia solani AG-4, AG2-2(IV) Large patch, AG2-2(IV) Brown patch), Fusarium oxysporum f. sp. cucuminum( Fusarium oxysporum f. sp. cucumber ), Clarididia Jacksoni( Clarireedia jacksonii , previously Sclerotinia homoeocarpa (called), *Gymanomyces graminis* ( Gaeumannomyces graminis ), Botrytis cinerea( Botrytis cinerea ), Fusarium graminearum( Fusarium gramineae ), Fusarium oxysporum F. SP. Lycopacific( Fusarium oxysporum f. sp. tomatoes ), Phytophsora Infestance( Phytophthora infestans ), Phytophthora capsicum ( Phytophthora capsicum ), Pythium aphanidermatum( Pythium aphanidermatum ), Pythium Ultimum( The last Pythium ), and Pseudocercospora sulfumsisa ( Pseudocercospora circumcisa It may be one or more selected from a group consisting of ), but is not limited thereto.

[0060] In the present invention, the plant pathogenic bacterium is *Pectobacterium carotobora subspecies carotobora* ( Pectobacterium carotovora subsp. carrot-eating It may be ), but is not limited to this.

[0061] In the present invention, the plant pathogenic fungal disease is Rhizoctonia solani ( Rhizoctonia solani Cucumber damping-off caused by ), Fusarium oxysporum f. sp. cucuminum ( Fusarium oxysporum f. sp. cucumber Cucumber wilt caused by ) and Clariredia jacksonii ( Clarireedia jacksonii It may be turf coin blight caused by ), but is not limited to this.

[0062] In the present invention, the plant pathogenic bacterial disease is *Pectobacterium carotobora subspecies carotobora* ( Pectobacterium carotovora subsp. carrot-eating It may be cabbage soft rot caused by ), but is not limited to this.

[0063] In the present invention, the method for preparing a composition for controlling plant diseases may additionally include a fractionation step for obtaining an active fraction.

[0064] In the present invention, the fractionation step may include the following steps:

[0065] A first fractionation step of obtaining a first active fraction from a culture medium using an organic solvent butanol; and

[0066] A second fractionation step of obtaining a second active fraction using high performance liquid chromatography (HPLC).

[0067] Another aspect of the present invention is a method for controlling plant diseases comprising a treatment step of treating a plant or soil with a composition for controlling plant diseases comprising the Streptomyces redicus JCK-6019 strain, a culture solution thereof, or an extract thereof.

[0068] In the present invention, the Streptomyces redicus JCK-6019 strain may be the Streptomyces redicus JCK-6019 strain deposited under accession number KCTC 15107BP.

[0069] In the present invention, the Streptomyces redicus JCK-6019 strain may contain 16S rRNA containing the nucleotide sequence of SEQ ID NO. 3.

[0070] In the present invention, the plant disease may be a plant pathogenic fungal disease or a plant pathogenic bacterial disease.

[0071] In the present invention, the plant pathogenic fungus is Rhizoctonia solani ( Rhizoctonia solani AG-4, AG2-2(IV) Large patch, AG2-2(IV) Brown patch), Fusarium oxysporum f. sp. cucuminum( Fusarium oxysporum f. sp. cucumber ), Clarididia Jacksoni( Clarireedia jacksonii , previously Sclerotinia homoeocarpa (called), *Gymanomyces graminis* ( Gaeumannomyces graminis ), Botrytis cinerea( Botrytis cinerea ), Fusarium graminearum( Fusarium gramineae ), Fusarium oxysporum F. SP. Lycopacific( Fusarium oxysporum f. sp. tomatoes ), Phytophsora Infestance( Phytophthora infestans ), Phytophthora capsicum ( Phytophthora capsicum ), Pythium aphanidermatum( Pythium aphanidermatum ), Pythium Ultimum( The last Pythium ), and Pseudocercospora sulfumsisa ( Pseudocercospora circumcissa It may be one or more selected from a group consisting of ), but is not limited thereto.

[0072] In the present invention, the plant pathogenic bacterium is *Pectobacterium carotobora subspecies carotobora* ( Pectobacterium carotovora subsp. carotovora It may be ), but is not limited to this.

[0073] In the present invention, the plant pathogenic fungal disease is Rhizoctonia solani ( Rhizoctonia solani Cucumber damping-off caused by ), Fusarium oxysporum f. sp. cucuminum ( Fusarium oxysporum f. sp. cucumerinumCucumber wilt caused by ) and Clariredia jacksonii ( Clarireedia jacksonii It may be turf coin blight caused by ), but is not limited to this.

[0074] In the present invention, the plant pathogenic bacterial disease is *Pectobacterium carotobora subspecies carotobora* ( Pectobacterium carotovora subsp. carotovora It may be cabbage soft rot caused by ), but is not limited to this.

[0075] In the present invention, the treatment step may be performed in one or more ways selected from the group consisting of soil drenching, soil irrigation, foliar spraying, trunk injection, stem and foliar treatment, rhizosphere treatment, and seed treatment, and may be, for example, soil drenching, but is not limited thereto. Effects of the invention

[0076] The present invention relates to Streptomyces lidicus ( Streptomyces lydicus The invention relates to a JCK-6019 strain, a composition for controlling plant diseases comprising said strain, a culture thereof, or an extract thereof, a method for preparing said composition, and a method for controlling plant diseases using said composition. When the culture solution of said strain or its active ingredient is applied to a plant or soil, it exhibits excellent antifungal activity and simultaneously induces resistance in the treated plant body. Therefore, it can be effectively used to control various plant pathogenic fungal diseases. In particular, it has a wide range of applications as an eco-friendly biological agent and seed coating agent because it is highly effective in controlling various plant diseases caused by pathogenic fungi and bacteria through seed coating treatment. Brief explanation of the drawing

[0077] FIG. 1 is a Streptomyces redicus according to one embodiment of the present invention ( Streptomyces lydicus This is the result of phylogenetic analysis based on the 16S rRNA gene sequence of the JCK-6019 strain. Figure 2 is the result of measuring the extracellular enzyme activity of the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. Figure 3 is the result of measuring the production of the plant growth hormone IAA (Indole-3-acetic acid) by the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. Figure 4 is the result of measuring the growth inhibitory activity of plant pathogenic mycelia following treatment with the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. Figure 5 is the result of measuring the degree of inhibition of mycelial growth of plant pathogenic fungi by the antifungal activity of a volatile substance produced by the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. FIG. 6 is a schematic diagram illustrating the extraction method of a natamycin active fraction (BF15-3) purified from a culture medium of Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. Figure 7 is the result of TLC analysis of the natamycin active fraction (BF15-3) purified from a culture medium of Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. Figure 8a is the HPLC analysis result of a natamycin fraction (BF15-3) isolated from a culture medium of Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. FIG. 8b is the result of UV spectrum analysis of a natamycin fraction (BF15-3) isolated from a culture medium of Streptomyces redicicus JCK-6019 strain according to one embodiment of the present invention. Figure 9a is the result of LC / MS cation mode analysis of natamycin (BF15-3) isolated from a culture medium of Streptomyces redicicus JCK-6019 strain according to one embodiment of the present invention. FIG. 9b is the chemical structure of natamycin (BF15-3) isolated from a culture medium of Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. FIG. 10a is the result of GC-MS analysis of volatile substances produced by the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. FIG. 10b is a mass spectrum of 2-methyl-2-bornene, a major volatile substance produced by the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. FIG. 10c is the chemical structure of 2-methyl-2-bornene, a major volatile substance produced by the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. FIG. 11a is a graph showing the control effect against cucumber wilt disease 30 days after inoculation with the Streptomyces redicus JCK-6019 strain, according to one embodiment of the present invention, when treated twice: once 1 day before inoculation and once 1 week after inoculation. FIG. 11b is a photograph showing the control effect against cucumber wilt disease 30 days after inoculation with the Streptomyces redicus JCK-6019 strain, according to one embodiment of the present invention, when treated twice: once 1 day before inoculation and once 1 week after inoculation. FIG. 12a is a graph showing the control effect against cucumber damping-off disease after 5 days of inoculation with the Streptomyces redicus JCK-6019 strain, according to one embodiment of the present invention, when treated 1 day prior to inoculation with the causative agent of cucumber damping-off disease. FIG. 12b is a photograph showing the control effect against cucumber damping-off disease 5 days after inoculation with the Streptomyces redicus JCK-6019 strain, according to one embodiment of the present invention, when treated 1 day prior to inoculation with the causative agent of cucumber damping-off disease. FIG. 13a is a graph showing the control effect against turf coin blight after 7 days of inoculation with the Streptomyces redicus JCK-6019 strain, the causative agent of turf coin blight, when treated 1 hour after inoculation according to one embodiment of the present invention. FIG. 13b is a photograph showing the control effect against turf coin blight 7 days after inoculation with the Streptomyces redicus JCK-6019 strain, the causative agent of turf coin blight, 1 hour after inoculation according to one embodiment of the present invention. Figure 14 is the result of testing the presence or absence of PR-1 gene expression according to the presence or absence of GUS expression in the culture filtrate, cell suspension, positive control salicylic acid (SA), and untreated group of the Streptomyces redicicus JCK-6019 strain according to one embodiment of the present invention. Figure 15a is a graph showing the control effect against cucumber wilt disease by inducing resistance when a culture of the Streptomyces redicus JCK-6019 strain is treated 4 days prior to inoculation with the causative agent of cucumber wilt disease according to one embodiment of the present invention, 30 days after inoculation with the pathogen. FIG. 15b is a photograph taken 30 days after inoculation of the causative agent of cucumber wilt disease, showing the control effect of the Streptomyces redicus JCK-6019 strain culture solution on cucumber wilt disease by inducing resistance when treated 4 days prior to inoculation according to one embodiment of the present invention. Figure 16 is a graph showing the control effect against cucumber damping-off by inducing resistance when a culture of the Streptomyces redicus JCK-6019 strain is treated 4 days prior to inoculation with the causative agent of cucumber damping-off according to one embodiment of the present invention, 5 days after inoculation with the pathogen. Figure 17a is a graph showing the control effect against cucumber wilt disease by seed coating with the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention 30 days after inoculation with the pathogen. FIG. 17b is a photograph taken 30 days after inoculation with the pathogen showing the control effect against cucumber wilt by seed coating with the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. Figure 18a is a graph showing the control effect against cucumber damping-off by seed coating with the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention, 5 days after inoculation with the pathogen. FIG. 18b is a photograph taken 5 days after inoculation of the pathogen showing the control effect against cucumber damping-off by seed coating with the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. Figure 19 is a photograph taken 8 days after inoculation with the pathogen showing the control effect against cabbage soft rot by seed coating with the Streptomyces redicus JCK-6019 strain according to one embodiment of the present invention. Specific details for implementing the invention

[0078] The present invention will be explained in more detail below through the following examples. However, these examples are merely illustrative of the invention, and the scope of the invention is not limited by these examples.

[0079] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise noted.

[0081] Example 1. Streptomyces lidicus ( Streptomyces lydicus Molecular biological analysis and phylogenetic analysis of the JCK-6019 strain

[0082] The strain JCK-6019 isolated from soil through the present invention was identified molecularly through sequencing analysis of the 16S rRNA gene. The strain was inoculated into ISP2 broth (Yeast extract 4 g / L, Malt extract 10 g / L, Dextrose 4 g / L, pH 7.2) and cultured at 28°C for 4 days with shaking at 180 rpm.

[0083] Genomic DNA (gDNA) of the harvested strain was extracted according to the protocol using iNtRON’s I-genomic BYF DNA Extraction Mini Kit. The extracted gDNA of 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 (Sequence No. 1 / Sequence No. 2), and the gene was amplified via PCR. The PCR process began 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, after which the amplification was stopped at 72°C for 10 minutes and 4°C. The amplified 16S rRNA gene PCR product was sent to Genotech (Daejeon, South Korea) for sequencing analysis.

[0084] Sequence number denomination Sequence list (5'-->3') note 1 9F GAGTTTGATCCTGGCTCAG 2 1512R ACGGCTACCTTGTTACGACTT 3 GACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCAACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTTTGAGATTCGCTCCACCTCGCGGTATCGCAGCTCATTGTACCGGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGTGAGTCCCCATCACCCCGAAGGGCATGCTGGCAACACAGAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGGGACCCTGTCTCCAGGGTTTTCCGGTGTATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCTGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGAACTTAATGCGTTAGCTGCGGCACGGACGACGTGGAATGTCGCCCACACCTAGTTCCCAACGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTATCGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTACCGAACTCTAGCCTGCCCGTATCGAATGCAGACCCGGGGTTAAGCCCCGGGCTTTCACATCCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTCTCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTAGGCCATCACCCCACCAACAAGCTGATAGGCCGCGGGCTCATCCTTCACCGCCGGAGCTTTCCACACGGAGGTCATGCGACCCCGTGTCGTATCCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGAGTGAAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAAT

[0085] As a result, a total nucleotide sequence of 1316 bp (Sequence No. 3) was obtained as the 16S rRNA coding sequence of the isolated strain JCK-6019. As a result of comparing the nucleotide sequences in the GenBank database using the NCBI BlastN search, as disclosed in Fig. 1, the JCK-6019 strain is Streptomyces redicus ( Streptomyces lydicus It was identified as ) and named Streptomyces redicus JCK-6019, and was deposited with the Korean Collection for Type Cultures (KCTC) on September 26, 2022, and assigned accession number KCTC 15107BP.

[0087] Example 2. Streptomyces lidicus ( Streptomyces lydicus ) Elucidation of the Biochemical Characteristics of the JCK-6019 Strain - Extracellular Enzyme Activity

[0088] To confirm the extracellular enzyme activity of the Streptomyces redicus JCK-6019 strain, experiments were performed on protease, chitinase, gelatinase, and cellulose.

[0089] Specifically, 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% carboxymethyl cellulose sodium + 1.5% agar, Sigma-Aldrich) were prepared, and the chitinase medium was prepared by adding colloidal chitin.

[0090] A sterile paper disc (0.8 cm, Advantec, Japan) was placed on each prepared medium, and 30 µl, 60 µl, and 90 µl of the JCK-6019 strain culture supernatant were dispensed, respectively. As a negative control, an equal amount of sterile GSS medium was dispensed onto the paper disc.

[0091] The experiment was performed in triplicate, and the plates were maintained at 30°C to observe the clear zone according to extracellular enzyme activity. To visualize the 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 for staining, and after maintaining them in the dark for 10 minutes, the clear zone was observed. The culture supernatant was treated with the media inducing enzyme activity to observe the clear zone, and the results are shown in Figure 2.

[0092] As can be seen in Figure 2, the JCK-6019 strain produced protease, chitinase, gelatinase, and cellulase, and it was confirmed that it produced large amounts of chitinase and cellulase.

[0094] Example 3. Streptomyces lidicus ( Streptomyces lydicus ) Elucidation of the Biochemical Characteristics of the JCK-6019 Strain - Production of the Plant Growth Hormone Indole-3-acetic Acid (IAA)

[0095] To confirm the production of the plant growth hormone IAA by the Streptomyces redicus JCK-6019 strain, 1% of the JCK-6019 strain culture was first inoculated into 5 ml of TSB medium supplemented with L-tryptophan (150 mg / l) and cultured with shaking at 28°C and 180 rpm for 7 days. Subsequently, the culture medium was centrifuged at 4°C and 10,000 rpm for 5 minutes, and 1 ml of the culture filtrate obtained by filtering through a 0.2 µm sterile filter 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 maintaining the mixture under dark conditions at room temperature for 20 minutes, it was checked whether the mixture turned pink. The negative control used TSB medium supplemented with L-tryptophan (150 mg / l), and the experiment was performed in triplicate. The results are shown in Figure 3.

[0096] As can be seen in Figure 3, the treatment group of the JCK-6019 strain culture supernatant showed a pink color in the test tube, while the control group of the TSB treatment group showed the natural color of the medium. Therefore, it was confirmed that the JCK-6019 strain produces the plant growth hormone IAA.

[0098] Example 4. Streptomyces lidicus ( Streptomyces lydicus Growth inhibitory activity of plant pathogen mycelia treated with JCK-6019 strain via MIC test

[0099] To determine the effect of the culture supernatant of Streptomyces redicus JCK-6019 strain on the growth of various plant pathogenic fungi, growth inhibitory activity was measured against 14 types of plant pathogens.

[0100] Specifically, the JCK-6019 strain was inoculated into GSS liquid medium (Soluble starch 10 g / l, Glucose 20 g / l, Soybean meal 25 g / l, Beef extract 1 g / l, Yeast extract 4 g / l, NaCl 2 g / l, K2HPO4 0.25 g / l, CaCO3 2 g / l) and cultured at 28°C for 5 days with shaking at 180 rpm. The culture medium was centrifuged at 8000 xg for 20 minutes, and the supernatant was filtered through a 0.2 μm sterile filter to harvest the culture filtrate (hereinafter referred to as 'sample'). An MIC (Minimum Inhibitory Concentration) test was performed using the 96-well microtiter plate method.

[0101] plant pathogenic fungi Clariredia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (₃) Large patch, Rhizoctonia solani AG 2-2 (₃) Brown patch, Fusarium oxysporum f. sp. cucumerinum , Fusarium oxysporum f.sp. lycopersici, Fusarium graminearum, Gaeumannomyces graminis, Phytophthora infestans, Phytophthora capsici, Pythium aphanidermatum, Pythium ultimum, Botrytis cinerea, Pseudocercospora circumcissa used.

[0102] The plant pathogenic fungi used in the experiment were inoculated onto potato dextrose agar (PDA, Becton, Dickinson and Co., Sparks, MD, USA) medium and cultured statically in a 25°C incubator. After static culture, the mycelium was weighed, sterile distilled water was added to achieve a concentration of 50 mg / ml, and the mixture was homogenized using a homogenizer at 10,000 rpm for 5 seconds before being added to the PDA medium to obtain a 1% fungal suspension. The samples were added to the plant pathogenic fungal suspension at concentrations of 10%, 5%, 2.5%, 1.25%, 0.625%, 0.313%, 0.156%, and 0.078%, respectively, and the experiment was performed in triplicate. The treated plates were sealed and incubated in a 25°C incubator for 3 to 5 days to determine the minimum inhibitory concentration (MIC) of the pathogen, and the results are shown in Table 2.

[0103] No. Phytopathogenic fungi MIC value (%) 1 Clarireedia jacksonii 0.078 2 Rhizoctonia solani AG-4 0.156 3 Rhizoctonia solani AG 2-2 (₃) Large patch 0.078 4 Rhizoctonia solani AG 2-2 (₃) Brown patch 0.078 5 Fusarium oxysporum f. sp. cucumber 1.25 6 Fusarium oxysporum f. sp. tomatoes 0.625 7 Fusarium gramineae 0.625 8 Gaeumannomyces graminis 0.625 9 Phytophthora infestans 2.5 10 Phytophthora capsicum 0.625 11 Pythium aphanidermatum 10 12 The last Pythium 10 13 Botrytis cinerea 0.625 14 Pseudocercospora circumcissa 5

[0104] As can be seen in Table 2, the JCK-6019 strain is Clarireedia jacksonii , Rhizoctonia solani It exhibited the strongest antifungal activity with an MIC value of 0.078%, and Fusarium oxysporum , Fusarium gramineae , Gaeumannomyces graminis , Phytophthora capsicum It also showed strong activity with an MIC value of 0.625%. In addition, the MIC test results confirmed that the JCK-6019 strain has strong antifungal activity against various plant pathogenic fungi used in the experiment.

[0106] Example 5. Streptomyces lydicus JCK-6019

[0107] To investigate the mycelial growth inhibitory activity of the Streptomyces redicus JCK-6019 strain against various plant pathogenic fungi, growth inhibitory activity was measured by replacement culture against 10 types of plant pathogens.

[0108] Target plant pathogenic fungi Clarireedia jacksonii, Rhizoctonia solani AG-4, Rhizoctonia solani AG 2-2 (₃) Large patch, Rhizoctonia solani AG 2-2 (₃) Brown patch, Fusarium oxysporum f. sp. cucumber , Fusarium graminearum, Phytophthora infestans, Phytophthora capsici, Pythium aphanidermatum, Pythium ultimum used.

[0109] Specifically, three days prior to pathogen inoculation, a 5 cm streak of JCK-6019 culture was applied at a point 2 cm from the edge of the PDA plate, and fragments of the mycelium of each pathogen were cut using a 6 mm diameter cork borer and inoculated at a distance of 5 cm from the JCK-6019 strain. For the control group, only fragments of the pathogen's mycelium were inoculated. The samples were incubated in a 25°C incubator, and the mycelial growth radius of the pathogen was measured according to the growth rate; the experiment was performed in triplicate. Antifungal activity was calculated by measuring the radius of the pathogen's mycelium (R2) in the direction of the JCK-6019 strain and the radius of the pathogen's mycelium (R1) under the control condition. Both values ​​were converted into a percentage of mycelial growth inhibition using the following formula. The results are shown in Figure 4 and Table 3.

[0110] [Calculation Formula]

[0111] Inhibition (%) = (1 - R2 / R1)

[0112] No. Phytopathogenic fungi Inhibition rate (%) a JCK-6019 1 Clarireedia jacksonii 88.77±2.36 b 2 Rhizoctonia solani AG-4 83.14±5.93 3 Rhizoctonia solani AG 2-2 (₃) Large patch 84.57±0.15 4 Rhizoctonia solani AG 2-2 (₃) Brown patch 74.65±1.99 5 Fusarium oxysporum f. sp. cucumber 100±0.00 6 Fusarium gramineae 90.91±0.01 7 Phytophthora infestans 81.9±5.08 8 Phytophthora capsicum 100±0.00 9 Pythium aphanidermatum 56.63±1.99 10 The last Pythium 52.30±7.01

[0113] a Mycelial growth inhibition rate (%) = (1- the length of the fungal growth in treatment / the length of the fungal growth in control) x 100 b Each value represents the mean ± standard deviation with 3 replicates.

[0115] As can be seen in Figure 4 and Table 3, the Streptomyces redicus JCK-6019 strain exhibited more than 50% inhibitory activity against all pathogens used in the experiment. Among them Phytophthora capsici, Fusarium oxysporum f. sp. cucumerinum Regarding, it showed 100% growth inhibitory activity, and Clarireedia jacksonii and Rhizoctonia solani It also exhibited more than 80% inhibitory activity against. In addition, when the growth inhibitory activity of the JCK-6019 strain was measured by replacement culture, it was confirmed that it possessed strong antifungal activity exceeding at least 52.3% against various plant pathogenic fungi used in the experiment.

[0117] Example 6. Streptomyces lidicus ( Streptomyces lydicus ) Evaluation of antifungal activity by volatile organic compounds produced by the JCK-6019 strain

[0118] Plant pathogenic fungi of volatile organic compounds produced by the JCK-6019 strain C. jacksonii, 5 types R. solani , F. oxysporum f. sp. cucumerinum , P. ultimum , P. infestans Antifungal activity was evaluated against [the substance]. To prevent direct contact, a Bi-Petri Dish (SPL Life Sciences Co., Ltd., Pocheon, Gyeonggi-do, Korea) with a split center was used. ISP2 agar was inoculated onto one side of the plate and PDA onto the other; after inoculation, the culture of the JCK-6019 strain was spread and incubated at 28°C for 3 days. After 3 days of incubation, a 6 mm diameter fragment of pathogenic mycelium was inoculated onto the PDA on the other side. For the control group, only the pathogen was inoculated onto the PDA without spreading the JCK-6019 strain culture. The experiment was performed in triplicate; all plates were sealed twice with Parafilm and incubated at 25°C to measure the diameter of the pathogenic mycelial growth. The results confirming the antifungal activity of volatile organic compounds produced by the JCK-6019 strain against plant pathogenic fungi are shown in Figure 5 and Table 4.

[0119] No. Phytopathogenic fungi Inhibition of growth (%) a JCK-6019 1 Clarireedia jacksonii 64.97±4.45 b 2 Rhizoctonia solani AG-4 55.34±7.98 3 Rhizoctonia solani AG 2-2 (₃) Large patch 76.97±4.26 4 Rhizoctonia solani AG 2-2 (₃) Brown patch 93.05±4.51 5 Rhizoctonia solani DJ 64.27±13.26 6 Rhizoctonia solani AT 2-2 (₃) 55.30±6.38 7 Fusarium oxysporum f. sp. cucumerinum 35.28±3.59 8 Phytophthora infestans 56.69±22.3 9 Pythium ultimum 61.17±4.45

[0120] For the colony area measurement, colony area (㎟) = a: the length of colony (㎜), b: the width of colony (㎜) a Inhibition rate (%) = [(Colony of area control, ㎟ - Colony of area treated, ㎟) / (Colony of area control, ㎟)] x 100

[0121] b Each value represents the mean ± standard deviation with 3 replicates.

[0123] As can be seen in Figure 5 and Table 4, the volatile substances of the JCK-6019 strain are the pathogen of turfgrass dollar spot, C. jacksonii It was confirmed that it inhibited mycelial growth by approximately 64.97%. In addition, the cucumber damping-off pathogen and the turf fungal pathogen R. solani It exhibited mycelial growth inhibitory activity of 55.34 to 93.05% against various strains, and the cucumber wilt pathogen F. oxysporum f. sp. cucumerinum and potato late blight pathogen P. infestans It also showed pathogen hyphae inhibition effects of 35.28% and 56.69%, respectively. Therefore, it was confirmed that the JCK-6019 strain produces volatile substances that exhibit antifungal activity against various plant pathogenic fungi.

[0125] Example 7. Isolation, structural identification, and MIC determination of antifungal active substances

[0126] To isolate the antifungal active substance of the Streptomyces redicus JCK-6019 strain, solvent fractionation was performed as schematically illustrated in Fig. 6.

[0127] Specifically, JCK-6019 culture medium (1 L) was solvent-fractionated twice for each solvent using ethyl acetate (EtOAC) and butanol (BuOH), and each fraction was concentrated at 45°C using a rotary vacuum concentrator (N-1110, EYELA Co., Tokyo, Japan). Subsequently, each fraction was analyzed by the 96-well microtiter plate method R. solani The antifungal activity against AG-4 was tested. As a result of the antifungal activity test, the antifungal active substance was isolated from the BuOH layer exhibiting strong antifungal activity. The BuOH layer (1.8776 g) was dissolved in a minimum volume of methanol and loaded onto a preparative TLC (20 cm x 20 cm, 0.5 cm thickness, Merck, Darmstadt, Germany). The sample was then developed in a chloroform:methanol:water (14:6:1, v / v / v) solvent system and eluted with methanol to obtain 15 fractions. Of the 15 fractions R. solani Antifungal activity against AG-4 was tested, and the active fraction BF15 was loaded onto a preparative TLC (20 cm x 20 cm, 0.5 cm thickness, Merck, Darmstadt, Germany). The mixture was developed in a chloroform:methanol:water (55:36:8, v / v / v) solvent system and eluted with methanol to obtain four fractions. The antifungal activity of the four fractions was tested, and the active BF15-3 fraction was obtained. The BF15-3 fraction was loaded onto thin-film chromatography (TLC) (20 cm x 20 cm, 0.2 cm thickness, Merck, Darmstadt, Germany) and analyzed by developing with a solvent of chloroform:methanol:water (55:36:8, v / v / v). The TLC plates were read by UV at wavelengths of 254 and 365 nm and visualized by spraying p-anisaldehyde reagent.

[0128] High-performance liquid chromatography (HPLC) analysis of the active fraction BF15-3 was performed using a Waters 996 Photodiode Array Detector and an Atlantis T3 column (5 µm pore size, 4.6 x 250 mm; Waters). The analysis was conducted under the solvent conditions shown in Table 3 using a gradient system with a mobile phase consisting of distilled water containing 0.1% trifluoroacetic acid (TFA) (Pump A) and acetonitrile containing 0.1% TFA (ACN, Pump B). The sample injection volume was maintained at 10 µl, the column temperature at 40°C, and the flow rate at 1 ml / min, and a peak was detected at 304 nm. In addition, mass spectrometry was performed to identify the chemical structure of the active fraction (BF15-3). Ultra-performance liquid chromatography (UPLC; ACQUITY UPLC) TM Analysis was performed using a quadrupole time-of-flight (QTOF-MS; Xevo G2-XS QToF, Waters MS Technologies, Manchester, UK) system, Waters Co., Milford, MA USA), and the analysis conditions are as shown in Table 5 (mobile phase conditions for HPLC analysis of active fraction BF15-3), Table 6 (UPLC analysis conditions for active fraction BF15-3), and Table 7 (QTOF-MS analysis conditions for active fraction BF15-3).

[0129] Time (min.) 0.1% TFA in H2O 0.1% TFA in ACN 0 80 20 30 40 60 33 0 100 40 0 100 43 80 20 50 80 20

[0130] Item Condition Column ACQUITY UPLC HSS T3 (1.7 Åm, 2.1 mm Flow rate 0.35 ml / min Injection volume 1 ㎕ Mobile phase A; H2O containing 0.1% formic acidB; Acetonitrile containing 0.1% formic acid Column temp. (℃) 40 Gradient elution (0-2 min, 0% B; 2-11 min, 50% B;11-13 min 2% 13-15min, 2% B)

[0131] Item Condition Ionization mode ESI (positive) Capillary voltage 3.0 kV Scan range m / z 50-1200 Cone voltage 40 V Desolvation N2 gas flow 600 L / h Desolvation temperature 250℃ Ion source temperature 100℃ Collision energy low: 6, high: 20 - 45 eV Calibrator 0.5 mM sodium formate Lock mass leucine - enkephalin(m / z 556.2771)

[0132] As can be seen in Fig. 7, the contents of the BF15-3 fraction, composed of pure compounds, were confirmed by the TLC results of the BF15-3 fraction isolated from the culture supernatant of the Streptomyces redicus JCK-6019 strain, and the pure substances of the BF15-3 fraction could be confirmed in Figs. 8a and 8b, which show the chromatogram and UV spectrum through HPLC analysis of the BF15-3 fraction.

[0133] In addition, as can be seen in Fig. 9a, the LC / MS analysis results showed 666.3133 in cation mode, so the molecular weight was estimated to be 665, and as can be seen in Fig. 9b, the chemical composition is C 33 H 47 NO 13 It was identified as such. Consequently, the UV spectrum, molecular weight, and chemical composition of the BF15-3 fraction were confirmed to be consistent with the UV spectrum, molecular weight, and chemical composition of natamycin.

[0134] No. Phytopathogenic fungi MIC value JCK - 6019 CF(%) Natamycin(ppm) 1 Clarireedia jacksonii 0.078 0.39 2 Rhizoctonia solani AG-4 0.156 0.39 3 Rhizoctonia solani AG 2-2 (₃) Large patch 0.078 0.39 4 Rhizoctonia solani AG 2-2 (₃) Brown patch 0.078 0.39 5 Fusarium oxysporum f. sp. cucumerinum 1.25 12.48 6 Fusarium graminearum 0.625 0.78 7 Gaeumannomyces graminis 0.625 1.56 8 Phytophthora infestans 2.5 3.12 9 Phytophthora capsici 0.625 1.56 10 Pythium aphanidermatum 10 12.48 11 Pythium ultimum 10 3.12

[0135] As shown in Table 8, the results of testing the mycelial growth inhibitory activity of natamycin from the BF15-3 fraction isolated from the JCK-6019 strain culture supernatant against various plant pathogenic fungi showed that natamycin also [did not show] the same as the MIC value of the JCK-6019 strain culture supernatant. C. jacksonii and R. solani It exhibited the lowest MIC value for [specific factor], demonstrating strong antifungal activity, and also showed excellent antifungal activity against various plant pathogenic fungi used in the experiment.

[0137] Example 8. Analysis of Volatile Organic Compounds Using Gas Chromatography - Mass Spectrometry

[0138] Solid phase microextraction (SPME) GC-MS analysis was performed to analyze volatile organic compounds produced by the Streptomyces redicus JCK-6019 strain. The JCK-6019 strain was inoculated into 5 ml of ISP2 broth and pre-cultured for 2 days at 28°C and 180 rpm, and 1% of the pre-culture solution was inoculated into 20 ml of ISP2 broth and cultured at 28°C for 5 days. Volatile organic compounds produced by JCK-6019 were collected in the headspace using an SPME fiber (Supelco, Bellefonte, PA, USA) at 50°C for 30 minutes. After injecting the substance adsorbed onto the SPME fiber and exposing it for 1 minute to desorb it, the sample was 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 injection port 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 the analysis was performed in cation mode at 200°C with a scan of 50 to 400 m / z. The acquired mass spectra were accurately identified by comparing them with data from the WILEY8 Library, and the content of each substance was expressed as the ratio of the area of ​​the total ion chromatogram (TIC) peaks. The results of solid phase microextraction (SPME) GC-MS analysis of volatile organic compounds produced by the Streptomyces redicus JCK-6019 strain are shown in Table 8 below.

[0139] No. Retention time (min) Relative peak area (%) Possible compound 1 7.230 5.61 1,3 - Diisopropyl - 1,3 - cyclopentadiene 2 7.360 4.48 2 - Methylenebornane 3 7.945 72.86 2 - Methyl - 2 - bornene 4 10.775 13.75 1,2,7,7, - Tetramethylbicyclo(2,2,1)heptan - 2 - ol

[0140] As can be seen in Table 9, four volatile organic compounds produced by the JCK-6019 strain were detected within 15 minutes. The chemical structures of the four substances were identified by comparing their mass spectra with those in the library, and as a result of GC-MS analysis and mass spectrum analysis, as can be seen in Figure 10a, they were identified as 2-methyl-2-bornene (72.86%), 1,2,7,7-tetramethylbicyclo(2,2,1)heptan-2-ol (13.75%), 1,3-diisopropyl-1,3-cyclopentadiene (5.61%), and 2-methylenebornane (4.48%), respectively.

[0141] Among these, the JCK-6019 strain produced 2-methyl-2-bornene, which is schematically represented in Figures 10b and 10c, as the main volatile substance, and it was found that 1,2,7,7-tetramethylbicyclo(2.2.1)heptan-2-ol, 1,3-diisopropyl-1,3-cyclopentadiene, and 2-methylenebornane, including 2-methyl-2-bornene, inhibited the growth of plant pathogenic fungi used in the experiment.

[0143] Example 9. Against Cucumber Vine Wilt in vivo Control effect investigation

[0144] On cucumber wilt with Streptomyces redicus JCK-6019 strain in vivo In order to investigate the pest control effect, 'Jungbok Samcheok' seeds ( Cucumis sativus L. cv. Jungboksamcheok (Syngenta Korea) was immersed in filter paper (9 cm diameter Petri dish) containing distilled water and germinated in a 28°C incubator for 24 hours, after which it was sown in long square slit pots (7 cm diameter, 9.5 cm height) using potting soil (Horticultural Potting Soil No. 2, Bunong). Subsequently 25 It was grown for 7 days by irradiating with light for 16 hours a day in a constant temperature and humidity room.

[0145] To cause cucumber wilt, the pathogen F. oxysporum f. sp. cucumerinum It was inoculated into PDB medium and cultured with shaking at 25°C and 150 rpm for 7 days. Afterward, the culture medium was harvested, filtered through four layers of cheese cloth to remove the mycelia, and 2.5 x 10⁶ were examined under a microscope (Axio Imager. A2, Carl Zeiss, Germany) using a hemocytometer. 6 A pathogen spore suspension was prepared to a concentration of spore / ml. The prepared spore suspension was used as an inoculum to investigate the control effect of cucumber wilt disease. Seven days after sowing, 10 ml of the prepared pathogen spore suspension was applied as a soil drench per pot.

[0146] To investigate the control effects of JCK-6019 strain culture (CB), BuOH wettable powder (WP; BuOH WP20), and natamycin, an antifungal active substance produced by JCK-6019, on cucumber wilt disease in vivoPot experiments were performed. The BuOH layer wettable powder was prepared by mixing 0.8 g of extract with 0.6 g of white carbon, evaporating the methanol used to dissolve the BuOH layer, and then adding 0.2 g of CR-SDS, 0.2 g of CS-WP100, and 0.2 g of kaolin. The JCK-6019 strain culture solution was diluted with distilled water containing 250 ppm of Tween 20 (Duksan Science, Seoul, Korea) to prepare formulations at dilutions of 10, 30, and 100, respectively, and the JCK-6019 BuOH layer wettable powder (BuOH WP20) was diluted with distilled water to prepare formulations at dilutions of 250, 500, and 1,000, respectively. Natamycin was dissolved in methanol (MeOH) and then diluted in distilled water containing 250 ppm Tween 20 to prepare formulations with final concentrations of 150 ppm, 50 ppm, and 16.7 ppm, respectively. The final concentration of MeOH was 5%. As positive controls, Jalokend (dispensable concentrate (DC): ai 30% Hymexazol + 5% Penthiopyrad, Hankook Samgong) and Kajiran (WP: ai 10% Etridiazole + 55% Thiophanate-methyl, Farm Hannong) were diluted 1000-fold and administered as a 20 ml soil drench treatment one hour after pathogen inoculation. The prepared formulations were applied as a soil drench treatment at a rate of 20 ml per pot twice: one day before and one week after pathogen inoculation. Pots that had undergone both inoculation and drug treatment were exposed to light for 12 hours a day in a 25°C, constant temperature and humidity chamber. The experiment was performed in triplicate with three samples per treatment group, and 30 days after inoculation with the pathogen, the disease was evaluated using a disease index ranging from 0 to 4 based on the severity of the disease.The disease index was assessed on five levels, with 0 = healthy, 1 = browning of roots or leaves and slight inhibition of seedling growth, 2 = significant inhibition of seedling growth, 3 = severe inhibition of seedling growth, and 4 = death, and the control effect was calculated accordingly.

[0147] Treatment group Control rate (%) JCK - 6019 strain culture solution (JCK - 6019 CB) X10 85.19±16.97 JCK - 6019 CB X30 96.3±6.42 JCK - 6019 CB X100 55.56±11.11 JCK - 6019 BuOH layer hydrating agent (BuOH WP20) X250 44.44±19.25 BuOH WP20 X500 85.19±6.42 BuOH WP20 X1000 74.07±12.83 Natamycin 150 ppm 100.00±0.00 Natamycin 50 ppm 81.48±12.83 Natamycin 16.7 ppm 55.56±11.11 Jalokend X1000 100.00±0.00 Kajiran X1000 96.3±6.42

[0148] As can be seen in Table 10 and Figure 11, high control efficacy of approximately 85.19% and 96.30% was observed in the treatment groups with 10x and 30x dilutions of the culture solution, respectively, and a high control efficacy of approximately 85.19% was also observed in the treatment group with 500x dilution of the BuOH extract WP20 formulation. In addition, it was confirmed that natamycin, an antifungal active substance produced by the JCK-6019 strain, inhibited cucumber wilt by 100% at 150 ppm and showed a concentration-dependent control effect. It was confirmed that Jalok-en and Gajiran, used as positive controls, inhibited cucumber wilt by 100% and 96.30%, respectively, when treated with a 1000x dilution. Therefore, it was confirmed that the culture solution of the JCK-6019 strain, the BuOH extract WP20 preparation, and natamycin effectively suppressed cucumber wilt when applied as a soil drench a total of two times, one day before and one week after inoculation with the pathogen, and in particular, natamycin was confirmed to exhibit strong control activity against cucumber wilt.

[0150] Example 10. Regarding cucumber damping-off disease in vivo Investigation of pest control effectiveness

[0151] On cucumber damping-off with Streptomyces redicus JCK-6019 strain in vivo To investigate the pest control effect, 'Four Beats' seeds ( Cucumis sativusCucumber seeds (L. cv. Nebakja, Syngenta Korea) were immersed in filter paper (9 cm diameter Petri dish) containing distilled water and germinated in a 28°C incubator for 24 hours, then placed in plastic pots (6 cm diameter, 6.5 cm height) with horticultural potting soil and sown, and grown for 6 days in a 25°C constant temperature and humidity chamber with light for 16 hours a day.

[0152] The pathogen of cucumber damping-off R. solani AG-4 was inoculated onto brown rice (Deungdae Agricultural Corporation, Jeollanam-do, Korea) that had been soaked in distilled water for 24 hours and then sterilized three times using high-pressure steam, and was cultured statically at 25°C for 3 weeks. The pathogen cultured for 3 weeks was placed in an aluminum dish, covered with cheese cloth, and dried at room temperature for 3 days. The dried material was ground with a blender, sieved through a 0.5 to 0.85 mm screen, and used as an inoculum. Six days after sowing, 0.75 g of the inoculum was evenly mixed into 1 L of horticultural potting soil, placed in plastic pots (7 cm in diameter, 6 cm in height), and cucumber seedlings grown for 6 days after sowing were transplanted.

[0153] To investigate the control effects of JCK-6019 strain culture solution, BuOH layer WP20 formulation, and natamycin, an antifungal active substance produced by JCK-6019, on cucumber damping-off disease in vivo Pot experiments were conducted. The fungicide was prepared using the same method as the sample treated for cucumber vine splitting disease described in Example 9 and used in the experiment. One day prior to pathogen inoculation, 10 ml was applied as a soil drench per pot. After pathogen inoculation and fungicide treatment, the pots were irradiated with a light source for 12 hours a day in a 25°C, constant temperature, and humidity chamber. The experiment was performed in triplicate with three samples per treatment group. Five days after inoculation, the disease incidence (%) of cucumber damping-off was investigated, and the control value (%) compared to the control group was calculated according to the following formula.

[0154] [Calculation Formula]

[0155] Control value (%) = 100 X [(Incidence rate of untreated group - Incidence rate of treated group) / Incidence rate of untreated group]

[0156] Treatment area Pest control (%) JCK-6019 Strain Culture Solution (JCK-6019 CB) X10 100.00±0.00 JCK-6019 CB X30 100.00±0.00 JCK-6019 CB X100 88.89±19.25 JCK-6019 BuOH Layer Hydrating Powder (BuOH WP20) X250 88.89±19.25 BuOH WP20 X500 66.67±0.00 BuOH WP20 X1000 88.89±19.25 Natamycin 150 ppm 88.89±19.25 Natamycin 50 ppm 66.67±0.00 Natamycin 16.7 ppm 55.56±19.25 Jalokend X1000 100.00±0.00 Kajiran X1000 100.00±0.00

[0157] As can be seen in Table 11 and Figure 12, the treatment groups with 10x and 30x dilutions of the culture solution showed 100% control activity, and the 100x treatment group also exhibited a high control activity of 88.89%. The treatment groups for the BuOH extract WP20 formulation also showed a high control effect of 88.89% at 250x and 1000x dilutions. In addition, natamycin, an antifungal active substance produced by the JCK-6019 strain, showed a high control effect of 88.89% at 150 ppm, confirming that it exhibits a concentration-dependent control effect. It was confirmed that Jaloken and Gajiran, used as positive controls, inhibited cucumber damping-off by 100% when treated with a 1000x dilution. Therefore, it was confirmed that the culture solution of the JCK-6019 strain, the BuOH extract WP20 preparation, and natamycin effectively suppressed cucumber damping-off when applied as a soil drench one day before inoculation with the pathogen.

[0159] Example 11. Regarding turf coin blight in vivo Investigation of pest control effectiveness

[0160] On turf coin blight with the Streptomyces lidicus JCK-6019 strain in vivo To investigate the control effect, creeping bentgrass seeds were soaked in distilled water at 4°C for 2 days and sown in plastic pots (diameter 7 cm, height 6 cm) filled with 60% of a mixture of sand and horticultural potting soil (sand:potting soil, 3:1, v / v). After sowing, the plants were grown for 2 days under 25°C dark conditions at 50% relative humidity, followed by 3 weeks of growth under 16 / 8 hour light / dark conditions at 25°C and 50% relative humidity.

[0161] The pathogen of lawn coin blight C. jacksoniiThe bacteria were inoculated into a wheat bran-rice husk medium (9 g wheat bran, 1.5 g rice husk, 10 ml distilled water; 250 ml Erlenmeyer flask) that had been steam-sterilized twice by autoclaving, and incubated statically at 25°C for 7 days. After incubation, 110 ml of distilled water containing 200 ppm streptomycin sulfate was added to pulverize the cells. The prepared cell suspension was used as an inoculum to investigate the control effect of turfgrass dollar spot. C. jacksonii A 1 cm deep hole was made in the center of the pot, and 3.5 ml of inoculum was inoculated per pot.

[0162] To investigate the control effects of JCK-6019 strain culture solution and BuOH wettable powder on turf silver blight in vivo Pot experiments were conducted. The JCK-6019 strain culture solution was diluted with distilled water containing 250 ppm of Tween 20 (Duksan Science, Seoul, Korea) to prepare formulations at dilutions of 9 and 27, respectively, while the JCK-6019 BuOH wettable powder was diluted with distilled water to prepare formulations at dilutions of 500 and 1,000, respectively. Horikure (EC: ai 25% Tebuconazole, Farm Hannong) was used as a positive control after being diluted 2,000 times. The prepared formulations were applied as soil drenching at a rate of 10 ml per pot one hour after pathogen inoculation. After pathogen inoculation and formulation, the pots were filled with water in trays and covered with a plastic chamber (35 cm wide, 28 cm deep, 16 cm high) to maintain 100% relative humidity, and light was irradiated for 12 hours a day in a 25°C constant temperature and humidity chamber. The experiment was performed in 2 replicates with 3 samples per treatment plot. The disease area percentage was investigated 7 days after inoculation, and the control value (%) compared to the control plot was calculated according to the following formula.

[0163] [Calculation Formula]

[0164] Control value (%) = 100 X [(Disease incidence in untreated group - Disease incidence in treated group) / Disease incidence in untreated group]

[0165] Treatment area Pest control (%) JCK-6019 Strain Culture Solution (JCK-6019 CB) X9 77.89±1.49 JCK-6019 CB X27 64.21±2.98 JCK-6019 BuOH Layer Hydrating Powder (BuOH WP20) X500 46.32±1.49 BuOH WP20 X1000 20.00±0.00 Horikuro X2000 100.00±0.00

[0166] As shown in Table 12 and Figure 13, the disease incidence was investigated 7 days after pathogen inoculation. The treatment groups diluted 9-fold and 27-fold with the JCK-6019 culture solution showed control effects of 77.89% and 64.21%, respectively, compared to the control group, while the treatment groups with the BuOH extract WP20 formulation showed control effects of 46.32% and 20.00%, respectively, confirming a concentration-dependent control effect. Horikure emulsion, used as a positive control, was confirmed to inhibit turfgrass dollar spot by 100% when applied as a soil drench after being diluted 2000-fold. In this experiment, the BuOH extract WP20 formulation showed a control effect approximately 2-3 times lower than that of the culture solution; this was attributed to the fact that the culture solution treatment groups contained various substances, such as volatile compounds, which likely influenced the control effect. Therefore, it was confirmed that the JCK-6019 strain effectively suppressed turfstone disease when the culture solution was applied as a soil drench 1 hour after inoculation with the pathogen, compared to the BuOH extract WP20 preparation.

[0168] Example 12. Assay of resistance-inducing activity of JCK-6019 strain in Arabidopsis thaliana

[0169] As a marker gene for verifying the induction of resistance in plants by utilizing a series of signaling pathways in which resistance is induced in plants through the salicylic acid signaling pathway and the PR-1 protein is expressed as a result PR-1 The gene is being used. Therefore, the resistance-inducing activity of the Streptomyces redicus JCK-6019 strain culture medium PR-1To test using a genetic system, Arabidopsis thaliana transformed with a vector labeled with GUS on the PR-1 promoter was used. The presence or absence of GUS expression and the activity of inducing resistance were tested using the Arabidopsis assay system, and the results are as shown in Figure 14 below. It was confirmed that GUS was expressed in the culture supernatant and cell suspension of the Streptomyces redicus JCK-6019 strain, just as in the positive control treated with salicylic acid (SA). Through this, it was confirmed that the Streptomyces redicus JCK-6019 strain can control pathogenic fungi by inducing resistance in plants through a mechanism similar to that of salicylic acid.

[0171] Example 13. For cucumber wilt disease caused by induced resistance of the JCK-6019 strain in vivo Investigation of pest control effectiveness

[0172] For cucumber wilt caused by the induced resistance of the JCK-6019 strain in vivo In order to investigate the pest control effect, 'Jungbok Samcheok' seeds ( Cucumis sativus L. cv. Jungboksamcheok, Syngenta Korea) was immersed in filter paper (9 cm diameter Petri dish) containing distilled water and 28 After germination in an incubator for 24 hours, seeds were sown in long square slit pots (diameter 7 cm, height 9.5 cm) using potting soil (Horticultural Potting Soil No. 2, Bunong). Subsequently, the plants were grown for 7 days in a 25℃ constant temperature and humidity chamber under 16 hours of light per day.

[0173] To cause cucumber wilt, the pathogen F. oxysporum f. sp. cucumerinum It was inoculated into PDB medium and cultured with shaking at 25°C and 150 rpm for 7 days. Afterward, the culture medium was harvested, filtered through four layers of cheese cloth to remove the mycelia, and 2.5 x 10⁶ were examined under a microscope (Axio Imager. A2, Carl Zeiss, Germany) using a hemocytometer. 6A pathogen spore suspension was prepared to a concentration of spore / ml. The prepared spore suspension was used as an inoculum to investigate the control effect of cucumber wilt disease. Seven days after sowing, 10 ml of the prepared pathogen spore suspension was applied as a soil drench per pot.

[0174] To investigate the control effect of JCK-6019 strain culture solution on cucumber wilt due to induced resistance in vivo To conduct the pot experiment, the JCK-6019 strain culture medium (FB) was diluted with distilled water containing 250 ppm of Tween 20 (Duksan Science, Seoul, Korea) to prepare pesticides at dilutions of 500-fold, 1000-fold, 2000-fold, and 4000-fold, respectively. As positive controls, Jalok-en (dispensable concentrate (DC): ai 30% Hymexazol + 5% Penthiopyrad, Hankook Samgong) and Gajiran (WP: ai 10% Etridiazole + 55% Thiophanate-methyl, Farm Hannong) were diluted 1000-fold and administered as a soil drench treatment of 20 ml one hour after pathogen inoculation. The prepared pesticides were applied as a soil drench treatment of 20 ml per pot four days prior to pathogen inoculation. After completing both inoculation and pesticide treatment, the pots were exposed to light for 12 hours a day in a 25°C, constant temperature and humidity chamber. The experiment was conducted in triplicate with three samples per treatment group, and 30 days after inoculation with the pathogen, the disease was evaluated using a disease index ranging from 0 to 4 based on the severity of the disease. The disease index was assessed on five levels: 0 = healthy, 1 = browning of roots or leaves and slight inhibition of seedling growth, 2 = significant inhibition of seedling growth, 3 = severe inhibition of seedling growth, and 4 = death; the control effect was calculated accordingly.

[0175] As can be seen in Figure 15, control effects of approximately 45.83% and 37.5% were observed in the treatment groups at 500x and 1000x dilution of the culture solution (FB), respectively, and control effects of 33.33% were observed in the treatment groups at 2000x and 4000x dilution of the culture solution (FB). It was confirmed that Jaloken and Gajiran, used as positive controls, inhibited cucumber wilt by 54.17% and 100.0%, respectively, when treated with a 1000x dilution. Therefore, it was confirmed that applying the culture solution of the JCK-6019 strain as a soil drench 4 days prior to pathogen inoculation induces resistance in cucumbers and effectively inhibits cucumber wilt.

[0177] Example 14. For cucumber damping-off caused by the induced resistance of the JCK-6019 strain in vivo Investigation of pest control effectiveness

[0178] For cucumber damping-off caused by induced resistance of the Streptomyces redicus JCK-6019 strain in vivo To investigate the pest control effect, 'Four Beats' seeds ( Cucumis sativus Cucumber seeds (L. cv. Nebakja, Syngenta Korea) were immersed in filter paper (9 cm diameter Petri dish) containing distilled water and germinated in a 28°C incubator for 24 hours, then placed in plastic pots (6 cm diameter, 6.5 cm height) with horticultural potting soil and sown, and grown for 6 days in a 25°C constant temperature and humidity chamber with light exposure for 16 hours a day.

[0179] The pathogen of cucumber damping-off R. solaniAG-4 was inoculated onto brown rice (Deungdae Agricultural Corporation, Jeollanam-do, Korea) that had been soaked in distilled water for 24 hours and then sterilized three times using high-pressure steam, and was cultured statically at 25°C for 3 weeks. The pathogen cultured for 3 weeks was placed in an aluminum dish, covered with cheese cloth, and dried at room temperature for 3 days. The dried material was ground with a blender, sieved through a 0.5 to 0.85 mm screen, and used as an inoculum. Six days after sowing, 0.75 g of the inoculum was evenly mixed into 1 L of horticultural potting soil, placed in plastic pots (7 cm in diameter, 6 cm in height), and cucumber seedlings grown for 6 days after sowing were transplanted.

[0180] To investigate the control effect against cucumber damping-off disease by induced resistance of the JJCK-6019 strain culture solution in vivo To conduct the pot experiment, the fungicide was prepared using the same method as the sample treated for cucumber vine splitting disease described in Example 13, and 10 ml was applied as a soil drench per pot 4 days prior to pathogen inoculation. After pathogen inoculation and fungicide treatment, the pots were irradiated with a light source for 12 hours a day in a 25°C, constant temperature and humidity chamber. The experiment was performed in triplicate with 3 samples per treatment group. Five days after inoculation, the disease incidence (%) of cucumber damping-off disease was investigated, and the control value (%) compared to the control group was calculated according to the following formula.

[0181] [Calculation Formula]

[0182] Control value (%) = 100 X [(Incidence rate of untreated group - Incidence rate of treated group) / Incidence rate of untreated group]

[0183] As can be seen in Figure 16, excellent control effects of approximately 66.67% and 83.33% were observed in the treatment groups diluted 1,000 times and 4,000 times, respectively, and a control effect of 33.33% was observed in the treatment groups diluted 500 times and 2,000 times. Although there was no statistically significant difference in control efficacy among the 500, 1,000, 2,000, and 4,000 times treatment groups, the highest control efficacy was observed at 4,000 times. In the case of the 4,000-fold dilution treatment group, the control efficacy was found to be statistically similar to that of the positive controls, Jaloken and Gajiran (100.0% and 83.33%), indicating very high potential for development. It is believed that if resistance-related substances are identified in the future and optimal fermentation and formulation technologies utilizing them are developed, it will be possible to develop a highly effective control agent for cucumber damping-off disease.

[0185] Example 15. Cucumber wilt disease by seed coating with JCK-6019 strain in vivo Investigation of pest control effectiveness

[0186] Cucumber wilt disease induced by cucumber seed coating with Streptomyces lidicus JCK-6019 strain in vivo To investigate the control effect, the JCK-6019 strain was cultured in ISP2 medium for 5 days, and then 10 8 A spore suspension at a concentration of CFU / ml was prepared. Seed coating with the spore suspension was 10 8 cucumber 'Jungbok Samcheok' seeds (using the spore suspension itself at a concentration of CFU / ml) Cucumis sativusThe seeds were coated by immersion with L. cv. Jungboksamcheok (Syngenta Korea), and the spore microencapsulation formulation was prepared by adding sodium alginate to a spore suspension, after which the cucumber 'Jungboksamcheok' seeds were immersed and coated. The coated seeds and untreated 'Jungboksamcheok' seeds were immersed in filter paper (9 cm diameter Petri dish) containing distilled water and germinated in a 28°C incubator for 24 hours. Subsequently, they were sown in long square slit pots (7 cm diameter, 9.5 cm height) using potting soil (Horticultural Potting Soil No. 2, Bunong). Afterward, the seeds were grown for 2 weeks in a 25°C, constant temperature and humidity chamber under 16 hours of light per day. Two weeks after sowing, the cucumber seedlings were inoculated with the pathogen causing cucumber wilt disease at the 2-leaf stage. For the positive control group, Jaloen and Gajiran were diluted 1,000-fold and applied as a 20 ml soil drench one hour after pathogen inoculation. After completing both inoculation and the positive control treatment, the pots were exposed to light for 12 hours a day in a 25°C, constant temperature and humidity chamber. The experiment was conducted in triplicate with three specimens per treatment group, and the disease was evaluated using a disease index ranging from 0 to 4 based on the severity of the disease 30 days after pathogen inoculation. The disease index was assessed on a five-stage scale: 0 = healthy, 1 = browning of roots or leaves and slight inhibition of seedling growth, 2 = significantly inhibition of seedling growth, 3 = severe inhibition of seedling growth, and 4 = death; the control effect was calculated based on this scale.

[0187] As can be seen in Figure 17, the JCK-6019 spore suspension seed coating treatment group and the JCK-6019 spore microencapsulation treatment group showed excellent control effects of 53.3% and 80.0%, respectively, for cucumber wilt disease. In particular, it was confirmed that seed treatment with the JCK-6019 spore microencapsulation formulation showed a much superior control effect compared to the positive control groups, Jalok-en and Gajiran.

[0188] Therefore, it was confirmed that the JCK-6019 strain is a candidate strain with excellent potential as an active strain for the development of a seed coating agent for controlling cucumber wilt, a plant fungal disease, and the mechanism of this plant disease control effect is presumed to be due to induced resistance.

[0190] Example 16. Cucumber damping-off caused by seed coating with JCK-6019 strain in vivo Investigation of pest control effectiveness

[0191] Cucumber damping-off caused by cucumber seed coating with Streptomyces lidicus JCK-6019 strain in vivo To investigate the control effect, the JCK-6019 strain was cultured in ISP2 medium for 5 days, and then 10 8 A spore suspension at a concentration of CFU / ml was prepared. Seed coating with the spore suspension was 10 8 cucumber 'Nebakja' seeds (using the spore suspension itself at a concentration of CFU / ml) Cucumis sativus The seeds were coated by immersion in L. cv. Nebakja (Syngenta Korea). The coated seeds and untreated 'Nebakja' seeds were immersed in filter paper (9 cm diameter Petri dish) containing distilled water and germinated in a 28°C incubator for 24 hours. Then, cucumber seeds were sown in long horticultural potting soil placed in plastic pots (6 cm diameter, 6.5 cm height) and grown for 6 days in a 25°C constant temperature and humidity chamber under 16 hours of light per day.

[0192] The pathogen of cucumber damping-off R. solaniAG-4 was inoculated onto brown rice (Deungdae Agricultural Corporation, Jeollanam-do, Korea) that had been soaked in distilled water for 24 hours and then sterilized three times using high-pressure steam, and was cultured statically at 25°C for 3 weeks. The pathogen cultured for 3 weeks was placed in an aluminum dish, covered with cheese cloth, and dried at room temperature for 3 days. The dried material was ground with a blender, sieved through a 0.5 to 0.85 mm screen, and used as an inoculum. Six days after sowing, 0.75 g of the inoculum was evenly mixed into 1 L of horticultural potting soil, placed in plastic pots (7 cm in diameter, 6 cm in height), and cucumber seedlings grown for 6 days after sowing were transplanted.

[0193] To investigate the control effect of seed coating with JJCK-6019 strain culture solution on cucumber damping-off disease in vivo To conduct the pot experiment, the sample JCK-6019 strain culture solution (FB) and the BuOH-ext WP20 wettable powder (WP) were prepared using the same method as the sample treated for cucumber wilt disease described in Example 9, excluding the seed coating. The samples were then applied as a soil drench at a rate of 10 ml per pot one day prior to pathogen inoculation. After pathogen inoculation and treatment, the pots were irradiated with a light source for 12 hours a day in a 25°C, constant temperature, and humidity chamber. The experiment was performed in triplicate with three samples per treatment group. Five days after inoculation, the disease incidence (%) of cucumber damping-off disease was investigated, and the control value (%) compared to the control group was calculated according to the following formula.

[0194] [Calculation Formula]

[0195] Control value (%) = 100 X [(Incidence rate of untreated group - Incidence rate of treated group) / Incidence rate of untreated group]

[0196] As can be seen in Figure 18, the treatment group coated with JCK-6019 spore suspension showed an excellent 73% control effect against cucumber damping-off. Therefore, since the seed coating results of the JCK-6019 strain showed excellent preventive effects against cucumber damping-off, it was confirmed once again that it is a candidate strain with outstanding potential as an active strain for the development of seed coating agents for controlling plant pathogenic fungal diseases, and the mechanism of this plant disease control effect is presumed to be due to induced resistance.

[0198] Example 17. For cabbage soft rot by seed coating with JCK-6019 strain in vivo Investigation of pest control effectiveness

[0199] Cabbage soft rot caused by seed coating with JCK-6019 strain (Pathogen: Pectobacterium cerotoborum subspecies cerotoborum, Pectobacterium carotovorum subsp. carotovorum ) in vivo To investigate the control effect, the JCK-6019 strain was cultured in ISP2 medium for 5 days, and then 10 8 A spore suspension at a concentration of CFU / ml was prepared. Seed coating with the spore suspension was 10 8 Using the spore suspension itself at a concentration of CFU / ml, cabbage 'Chungwang' seeds ( Brassica rapa subsp. pekinensisThe seeds were coated by immersion in L. cv. Chunkwnag (Sakata Korea). The coated seeds and untreated 'Chunkwang' seeds were immersed in filter paper (9 cm diameter Petri dish) containing distilled water and germinated in a 28°C incubator for 24 hours. Then, the cabbage seeds were sown in long horticultural potting soil placed in plastic pots (6 cm diameter, 6.5 cm height) and cultivated in a 25°C constant temperature and humidity chamber under 16 hours of light per day. Cabbage plants at the 5-leaf or 6-leaf stage were transplanted into 7.5 cm diameter plastic cups 24 hours prior to treatment. For the untreated group, cabbage grown from seeds that had not been pretreated was used, and for the positive control group, Ilpum pesticide (Ilpum, ai. oxolinic acid 20% WP, Dongbang Agro) was applied to cabbage grown from seeds from the untreated group by soil drenching at a rate of 20 ml per pot after being diluted 1,000 times one day before inoculation.

[0200] Colonies of the pathogen *Pectobacterium cerotoborum* subspecies, cultured on TSA medium for 3 days after 24 hours of positive control treatment, were harvested with sterile water to prepare a bacterial suspension, and an OD value of 0.1 (10 at 600 nm) was measured using a spectrophotometer (Bio-Rad). 7 After adjusting the concentration to CFU / ml, a bacterial suspension containing 10 mM magnesium chloride was inoculated via soil drench at a rate of 20 ml per pot. The inoculated plants were placed in a constant temperature and humidity room at 30°C and kept under dark conditions for 24 hours. Subsequently, a photoperiod of 12 hours and a relative humidity of 100% were maintained, and the disease level was assessed after 8 days.

[0201] The incidence was expressed as an index from 0 to 5 (0: no symptoms, 1: one or two fine lesions, 2: two or more fine lesions, 3: chlorosis on leaves, 4: necrosis of leaves, 5: complete death), and the control value (Control value, %) compared to the control group was calculated according to the following formula.

[0202] [Calculation Formula]

[0203] Control value (%) = 100 X [(Incidence rate of untreated group - Incidence rate of treated group) / Incidence rate of untreated group]

[0204] Treatment area Pest control (%) JCK-6019 Seed Treatment 72.50±20.43 article par excellence 50.00±47.85

[0205] As can be seen in Table 13, the JCK-6019 strain showed a high control efficacy of 72.50%, which was higher than that of the positive control, the antibiotic Ilpum. As shown in Figure 19, all the cabbages in the untreated group withered and died, whereas when the seeds were coated with the JCK-6019 strain, most of the cabbages grew well with almost no damage. Thus, it can be seen that when the JCK-6019 strain is applied to seeds, it effectively controls not only fungal plant diseases but also bacterial plant diseases, and the mechanism of this plant disease control effect is presumed to be due to induced resistance.

[0206] Name of Depositing Institution: Korea Research Institute of Bioscience and Biotechnology Biological Resource Center Trustee Number: KCTC15107BP Date of Deposit: 2022-09-26

Claims

Claim 1 Streptomyces redicus deposited under accession number KCTC 15107BP having plant disease control activity ( Streptomyces lydicus ) JCK-6019 strain. Claim 2 In claim 1, the strain comprises 16S rRNA containing the nucleotide sequence of SEQ ID NO.

3. Claim 3 Streptomyces redicus deposited under accession number KCTC 15107BP having plant disease control activity ( Streptomyces lydicus A composition for controlling plant diseases comprising the JCK-6019 strain, a culture thereof, or an extract thereof. Claim 4 In paragraph 3, the plant disease control composition comprises one or more selected from the group consisting of natamycin, 2-methyl-2-bornene, 1,2,7,7-tetramethylbicyclo(2.2.1)heptan-2-ol, 1,3-diisopropyl-1,3-cyclopentadiene, and 2-methylenebornan. Claim 5 In paragraph 3, the above plant disease is Rhizoctonia solani AG-4 ( Rhizoctonia solani AG-4), Rhizoctonia Solani AG2-2(IV) Large Patch( Rhizoctonia solani AG2-2(IV) Large patch), Rhizoctonia solani AG2-2(IV) Brown patch( Rhizoctonia solani AG2-2(IV) Brown patch), Fusarium oxysporum f. sp. cucuminum ( Fusarium oxysporum f. sp. cucumerinum ), Clarididia Jacksoni( Clarireedia jacksonii ), Gaeummanomyces graminis( Gaeumannomyces graminis ), Botrytis cinerea( Botrytis cinerea ), Fusarium graminearum( Fusarium graminearum ), Fusarium oxysporum F. SP. Lycopacific( Fusarium oxysporum f. sp. lycopersici ), Phytophsora Infestance( Phytophthora infestans ), Phytophthora capsicum ( Phytophthora capsici ), Pythium aphanidermatum( Pythium aphanidermatum ), Pythium Ultimum( Pythium ultimum ), Pseudocercospora sulfumsisa( Pseudocercospora circumcissa ) and Pectobacterium carotobora subspecies carotobora( Pectobacterium carotovora subsp. carotovora A composition for controlling plant diseases, wherein one or more causes are selected from the group consisting of ). Claim 6 A composition for controlling plant diseases comprising one or more selected from the group consisting of 1,2,7,7-tetramethylbicyclo(2.2.1)heptan-2-ol, 1,3-diisopropyl-1,3-cyclopentadiene and 2-methylenebornan. Claim 7 In paragraph 6, the above plant disease is Rhizoctonia solani AG-4 ( Rhizoctonia solani AG-4), Rhizoctonia Solani AG2-2(IV) Large Patch( Rhizoctonia solani AG2-2(IV) Large patch), Rhizoctonia solani AG2-2(IV) Brown patch( Rhizoctonia solani AG2-2(IV) Brown patch), Fusarium oxysporum f. sp. cucuminum ( Fusarium oxysporum f. sp. cucumerinum ), Clarididia Jacksoni( Clarireedia jacksonii ), Gaeummanomyces graminis( Gaeumannomyces graminis ), Botrytis cinerea( Botrytis cinerea ), Fusarium graminearum( Fusarium graminearum ), Fusarium oxysporum F. SP. Lycopacific( Fusarium oxysporum f. sp. lycopersici ), Phytophsora Infestance( Phytophthora infestans ), Phytophthora capsicum ( Phytophthora capsici ), Pythium aphanidermatum( Pythium aphanidermatum ), Pythium Ultimum( Pythium ultimum ), Pseudocercospora sulfumsisa( Pseudocercospora circumcissa ) and Pectobacterium carotobora subspecies carotobora( Pectobacterium carotovora subsp. carotovora A composition for controlling plant diseases, wherein one or more causes are selected from the group consisting of ). Claim 8 Streptomyces redicus deposited under accession number KCTC 15107BP having plant disease control activity ( Streptomyces lydicus A method for preparing a composition for controlling plant diseases, comprising a culture step of culturing the JCK-6019 strain. Claim 9 In paragraph 8, the above plant disease is Rhizoctonia solani AG-4 ( Rhizoctonia solani AG-4), Rhizoctonia Solani AG2-2(IV) Large Patch( Rhizoctonia solani AG2-2(IV) Large patch), Rhizoctonia solani AG2-2(IV) Brown patch( Rhizoctonia solani AG2-2(IV) Brown patch), Fusarium oxysporum f. sp. cucuminum ( Fusarium oxysporum f. sp. cucumerinum ), Clarididia Jacksoni( Clarireedia jacksonii ), Gaeummanomyces graminis( Gaeumannomyces graminis ), Botrytis cinerea( Botrytis cinerea ), Fusarium graminearum( Fusarium graminearum ), Fusarium oxysporum F. SP. Lycopacific( Fusarium oxysporum f. sp. lycopersici ), Phytophsora Infestance( Phytophthora infestans ), Phytophthora capsicum ( Phytophthora capsici ), Pythium aphanidermatum( Pythium aphanidermatum ), Pythium Ultimum( Pythium ultimum ), Pseudocercospora sulfumsisa( Pseudocercospora circumcissa ) and Pectobacterium carotobora subspecies carotobora( Pectobacterium carotovora subsp. carotovora A method for manufacturing a composition for controlling plant diseases, wherein one or more causes are selected from the group consisting of ). Claim 10 A method for manufacturing a composition for controlling plant diseases according to claim 8, wherein the manufacturing method additionally includes a fractionation step for obtaining an active fraction. Claim 11 A method for preparing a plant disease control composition according to claim 8, wherein the plant disease control composition comprises one or more selected from the group consisting of natamycin, 2-methyl-2-bornene, 1,2,7,7-tetramethylbicyclo(2.2.1)heptan-2-ol, 1,3-diisopropyl-1,3-cyclopentadiene, and 2-methylenebornan. Claim 12 Streptomyces redicus deposited under accession number KCTC 15107BP having antifungal activity ( Streptomyces lydicus A method for controlling plant diseases comprising a treatment step of treating a plant or soil with a composition for controlling plant diseases comprising the JCK-6019 strain, a culture solution thereof, or an extract thereof. Claim 13 In Clause 12, the above plant disease is Rhizoctonia solani AG-4 ( Rhizoctonia solani AG-4), Rhizoctonia Solani AG2-2(IV) Large Patch( Rhizoctonia solani AG2-2(IV) Large patch), Rhizoctonia solani AG2-2(IV) Brown patch( Rhizoctonia solani AG2-2(IV) Brown patch), Fusarium oxysporum f. sp. cucuminum ( Fusarium oxysporum f. sp. cucumerinum ), Clarididia Jacksoni( Clarireedia jacksonii ), Gaeummanomyces graminis( Gaeumannomyces graminis ), Botrytis cinerea( Botrytis cinerea ), Fusarium graminearum( Fusarium graminearum ), Fusarium oxysporum F. SP. Lycopacific( Fusarium oxysporum f. sp. lycopersici ), Phytophsora Infestance( Phytophthora infestans ), Phytophthora capsicum ( Phytophthora capsici ), Pythium aphanidermatum( Pythium aphanidermatum ), Pythium Ultimum( Pythium ultimum ), Pseudocercospora sulfumsisa( Pseudocercospora circumcissa ) and Pectobacterium carotobora subspecies carotobora( Pectobacterium carotovora subsp. carotovora A method for controlling plant diseases, wherein one or more of the causes are selected from a group consisting of ). Claim 14 A method for controlling plant diseases according to claim 12, wherein the treatment step is performed in one or more ways selected from the group consisting of soil drenching, soil irrigation, foliar spraying, trunk injection, stem and foliar treatment, rhizosphere treatment, and seed treatment.

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