A composition for controlling fungal, bacterial or nematode diseases in plants, comprising a culture broth of Lysobacter enzymogenes JCK-1421 strain or an extract of the culture broth, a method for producing the composition, and a method for controlling fungal, bacterial or nematode diseases in plants

Abstract

The present invention relates to the Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases or nematode diseases comprising the strain, a culture thereof, or an extract thereof, a method for producing the composition, and a method for controlling plant fungal diseases, bacterial diseases or nematode diseases using the composition.

Description

[Technical Field]

[0001] This patent application claims priority to Korean Patent Application No. 10-2022-0151546 filed with the Korean Intellectual Property Office on November 14, 2022, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to the Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases or nematode diseases comprising the strain, a culture thereof, or an extract thereof, a method for producing the composition, and a method for controlling plant fungal diseases, bacterial diseases or nematode diseases using the composition. [Background technology]

[0003] BACKGROUND ART In agriculture, various plant diseases caused by fungi, bacteria, nematodes, etc. cause deterioration in crop quality and yield, resulting in enormous economic losses, and therefore, it is very important to control them.

[0004] Until now, chemical pesticides have been used repeatedly for a long period of time to prevent plant diseases, but the emergence of pests that are highly resistant to chemical pesticides has ultimately led to a decline in the effectiveness of chemical pesticides in preventing plant diseases.In addition, the misuse and overuse of chemical pesticides has led to the emergence of environmental pollution problems such as residual pesticides, and regulations on the use of chemical pesticides are becoming stricter.

[0005] Therefore, to solve these problems, biological control of plant diseases using antagonistic microorganisms has emerged as an environmentally friendly alternative.

[0006] Biological control of plant pathogens can be categorized into direct antagonism, direct-indirect antagonism, and indirect antagonism. Direct antagonism includes the dual parasitism and predation mechanisms of various fungi and bacteria, while direct-indirect antagonism includes the use of antibiotics and decomposing enzymes.

[0007] Indirect antagonism includes competition and induced resistance. In particular, induced resistance is a phenomenon in which resistance to pathogen invasion is induced in host plants by specific biological or non-biological factors. It improves plant defense capabilities against microbial invasion or similar stresses and, once manifested, is maintained for a long period of time. In recent years, induced resistance has attracted increasing attention in terms of improving agricultural ecosystems and biological control of various plant diseases.

[0008] Lysobacter enzymogenes is a Gram-negative bacterium that does not form spores, exists in the form of rod-shaped bacilli, and moves by gliding motility. Colonies are usually cream, pink, or yellowish-brown in color and exhibit very mucous properties. Lysobacter enzymogenes produces extracellular enzymes such as protease, chitinase, and β-1,3-glucanase, which degrade the structural components of various plant pathogenic microorganisms, including fungi and nematodes, thereby exhibiting high growth inhibitory activity.

[0009] Although direct in vitro antibacterial activity studies of Lysobacter enzymogenes against various plant pathogenic microorganisms have been reported, there is still insufficient research on the control of various plant diseases using induced resistance mechanisms. Summary of the Invention [Problem to be solved by the invention]

[0010] Therefore, the present inventors have confirmed that when a composition containing the Lysobacter enzymogenes JCK-1421 strain, its culture solution, or an extract thereof is applied to plant pathogenic fungal diseases, bacterial diseases, and nematode diseases, the composition exhibits significantly superior resistance induction activity in host plants.

[0011] Therefore, it is an object of the present invention to provide a Lysobacter enzymogenes strain JCK-1421 having antifungal, antibacterial and antinematode activities.

[0012] Another object of the present invention is to provide a composition for controlling plant fungal diseases, bacterial diseases or nematode diseases, which comprises the Lysobacter enzymogenes JCK-1421 strain, a culture broth thereof, or an extract thereof.

[0013] It is still another object of the present invention to provide a method for producing a composition for controlling a plant fungal disease, bacterial disease or nematode disease, which comprises a culturing step of culturing the Lysobacter enzymogenes JCK-1421 strain.

[0014] It is still another object of the present invention to provide a method for controlling fungal diseases, bacterial diseases or nematode diseases in plants, which comprises treating the Lysobacter enzymogenes JCK-1421 strain, a culture broth thereof, or an extract thereof.

[0015] A further object of the present invention relates to use of the Lysobacter enzymogenes JCK-1421 strain, a culture broth thereof, or an extract thereof for controlling fungal diseases, bacterial diseases, or nematode diseases in plants. [Means for solving the problem]

[0016] The present invention relates to the Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases or nematode diseases comprising the strain, a culture thereof, or an extract thereof, a method for producing the composition, and a method for controlling plant fungal diseases, bacterial diseases or nematode diseases using the composition.

[0017] The present invention will now be described in more detail.

[0018] One aspect of the present invention relates to a Lysobacter enzymogenes strain JCK-1421 having antifungal, antibacterial and antinematode activities.

[0019] In the present invention, the Lysobacter enzymogenes JCK-1421 strain may be the Lysobacter enzymogenes JCK-1421 strain deposited under accession number KCTC 15126BP.

[0020] The Lysobacter enzymogenes JCK-1421 strain of the present invention may contain 16S rRNA comprising the nucleotide sequence of SEQ ID NO:3.

[0021] The Lysobacter enzymogenes JCK-1421 strain of the present invention was deposited at the Korean Collection for Type Cultures (KCTC) of the Korea Institute of Bioscience and Biotechnology on October 6, 2022 under accession number KCTC 15126BP.

[0022] Another aspect of the present invention relates to a composition for controlling fungal, bacterial or nematode diseases in plants, comprising the Lysobacter enzymogenes JCK-1421 strain, a culture thereof, or an extract thereof, which has antifungal, antibacterial and antinematode activities.

[0023] In the present invention, the Lysobacter enzymogenes JCK-1421 strain may be the Lysobacter enzymogenes JCK-1421 strain deposited under accession number KCTC 15126BP.

[0024] The Lysobacter enzymogenes JCK-1421 strain of the present invention may contain 16S rRNA comprising the nucleotide sequence of SEQ ID NO:3.

[0025] In the present invention, the fungus may be one or more species selected from the group consisting of Fusarium graminearum, Fusarium oxysporum f.sp. cucumerinum, Rhizoctonia solani AG2-2 Brown patch, Rhizoctonia solani AG2-2 Large patch, Rhizoctonia solani AG-4, Sclerotinia homoeocarpa, and Pythium aphanidermatum, but is not limited thereto.

[0026] In the present invention, the bacterium may be one or more species selected from the group consisting of Ralstonia solanacearum, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas euvesicatoria, and Erwinia amylovora, but is not limited thereto.

[0027] In the present invention, the nematode may be one or more species selected from the group consisting of Meloidogyne incognita and Bursaphelenchus xylophilus, but is not limited thereto.

[0028] In the present invention, the plant fungal disease may be one or more selected from the group consisting of cucumber seedling damping-off caused by Rhizoctonia solani, cucumber fusarium wilt caused by Fusarium oxysporum f.sp. cucumerinum, dollar spot of turf caused by Sclerotinia homoeocarpa, Pythium wilt of turf caused by Pythium aphanidermatum, and rice head blight caused by Fusarium graminearum, but is not limited thereto.

[0029] In the present invention, the bacterial plant disease may be one or more selected from the group consisting of Chinese cabbage soft rot caused by Pectobacterium carotovora subsp. carotovora, tomato bacterial wilt caused by Ralstonia solanacearum, pepper bacterial leaf spot caused by Xanthomonas euvesicatoria, and apple fire blight caused by Erwinia amylovora, but is not limited thereto.

[0030] In the present invention, the plant nematode disease may be one or more selected from the group consisting of tomato root-knot nematode disease caused by Meloidogyne incognita and pine wilt disease caused by Bursaphelenchus xylophilus, but is not limited thereto.

[0031] As used herein, the term "culture" refers to a substance containing a microorganism after the microorganism has been cultivated.

[0032] The term "culture supernatant" as used herein means the upper layer obtained by removing most of the microorganisms from the culture medium by centrifugation, and is also called "supernatant."

[0033] The term "culture filtrate" as used herein refers to the liquid remaining after removing the bacterial cells from the culture medium by centrifugation and filtration. The culture filtrate contains substances formed and excreted during the growth of the microorganisms, and these substances may be purified or extracted.

[0034] The composition of the present invention may contain, in addition to the strain as an active ingredient, its culture, a concentrate of the culture, a dried culture, and / or a culture supernatant of the strain, a culture containing the bacterial cells, an extract of the bacterial cells, a concentrate, concentrate, or dried product thereof, and, if necessary, a diluted solution or dilution, and may include any state obtained by processing a culture solution or a culture.

[0035] The composition of the present invention can be formulated by a conventional method and can be produced in the form of a dry powder or a liquid fertilizer. Specifically, the microbial formulation of the present invention can be produced in a liquid form, to which a bulking agent can be added and used in the form of a powder, or this can be formulated into granules. However, the formulation is not particularly limited.

[0036] In the present invention, the composition may be prepared with the addition of additives such as additives, bulking agents, nutrients, and the like.

[0037] In the present invention, the additive may be one or more selected from the group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkylaryl sulfonate, polyoxyethylene alkylphenyl ether, sodium tripolyphosphate, polyoxyethylene alkylaryl phosphate ester, polyoxyethylene alkylaryl ether, polyoxyethylene alkylaryl polymer, polyoxyalkylone alkylphenyl ether, polyoxyethylene nonylphenyl ether, sodium sulfonate naphthalene formaldehyde, Triton 100, and Tween 80, but is not limited thereto.

[0038] In the present invention, the bulking agent and nutrient may be one or more selected from the group consisting of skim milk (culture medium), soybean flour, rice, wheat, loess, diatomaceous earth, bentonite, dextrin, glucose, and starch, but is not limited thereto.

[0039] In the present invention, the disintegrant may be one or more selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate, but is not limited thereto.

[0040] Yet another aspect of the present invention relates to a method for producing a composition for controlling fungal diseases, bacterial diseases or nematode diseases in plants, which comprises a culturing step of culturing Lysobacter enzymogenes JCK-1421 strain.

[0041] In the present invention, the Lysobacter enzymogenes JCK-1421 strain may be the Lysobacter enzymogenes JCK-1421 strain deposited under accession number KCTC 15126BP.

[0042] The Lysobacter enzymogenes JCK-1421 strain of the present invention may contain 16S rRNA comprising the nucleotide sequence of SEQ ID NO:3.

[0043] In the present invention, the fungus may be one or more species selected from the group consisting of Fusarium graminearum, Fusarium oxysporum f.sp. cucumerinum, Rhizoctonia solani AG2-2 Brown patch, Rhizoctonia solani AG2-2 Large patch, Rhizoctonia solani AG-4, Sclerotinia homoeocarpa, and Pythium aphanidermatum, but is not limited thereto.

[0044] In the present invention, the bacterium may be one or more species selected from the group consisting of Ralstonia solanacearum, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas euvesicatoria, and Erwinia amylovora, but is not limited thereto.

[0045] In the present invention, the nematode may be one or more species selected from the group consisting of Meloidogyne incognita and Bursaphelenchus xylophilus, but is not limited thereto.

[0046] In the present invention, the plant fungal disease may be one or more selected from the group consisting of cucumber seedling damping-off caused by Rhizoctonia solani, cucumber fusarium wilt caused by Fusarium oxysporum f.sp. cucumerinum, dollar spot of turf caused by Sclerotinia homoeocarpa, Pythium wilt of turf caused by Pythium aphanidermatum, and rice head blight caused by Fusarium graminearum, but is not limited thereto.

[0047] In the present invention, the bacterial plant disease may be one or more selected from the group consisting of Chinese cabbage soft rot caused by Pectobacterium carotovora subsp. carotovora, tomato bacterial wilt caused by Ralstonia solanacearum, pepper bacterial leaf spot caused by Xanthomonas euvesicatoria, and apple fire blight caused by Erwinia amylovora, but is not limited thereto.

[0048] In the present invention, the plant nematode disease may be one or more selected from the group consisting of tomato root-knot nematode disease caused by Meloidogyne incognita and pine wilt disease caused by Bursaphelenchus xylophilus, but is not limited thereto.

[0049] In the present invention, the culturing step may include, but is not limited to, a step of producing a culture filtrate from the culture solution by centrifuging and filtering the culture solution.

[0050] In the present invention, the culture medium may contain one or more selected from the group consisting of milk proteins such as skim milk, whey, and casein, sugars, yeast, and extracts, but is not limited thereto.

[0051] In the present invention, the method for producing a composition for controlling a plant fungal disease, bacterial disease or nematode disease may further include a concentration step of concentrating the culture solution.

[0052] In the present invention, the method for producing a composition for controlling a plant fungal disease, bacterial disease or nematode disease may further include a dilution step of diluting the culture solution.

[0053] In the present invention, the method for producing a composition for controlling plant fungal, bacterial or nematode diseases may further comprise an extraction step of extracting components within the fungal cells.

[0054] In the present invention, the production method may include, but is not limited to, a fractionation step for obtaining an active fraction from the culture medium.

[0055] In the present invention, the fractionation step may comprise the following steps:

[0056] a culture filtrate harvesting step for obtaining a culture filtrate from the culture broth;

[0057] a fractionation step in which the culture filtrate is fractionated with a solvent to obtain fractions;

[0058] a concentration step of concentrating the fraction to obtain a concentrate; and

[0059] A purification step in which the concentrate is purified and the active fraction is selected.

[0060] In the present invention, the step of obtaining a culture filtrate may include, but is not limited to, centrifuging the culture solution at 2000 to 5000 rpm, 2500 to 5000 rpm, 3000 to 5000 rpm, 3500 to 5000 rpm, 4000 to 5000 rpm, or 4500 to 5000 rpm, for example, 4500 rpm.

[0061] In the present invention, the fractionation step may include, but is not limited to, fractionation using one or more solvents selected from the group consisting of butanol, ethyl acetate, methanol, ethanol, and hexane.

[0062] In the present invention, the concentration step may include, but is not limited to, concentrating the fraction using a vacuum concentrator.

[0063] In the present invention, the purification step may include, but is not limited to, purification by preparative HPLC (high-performance liquid chromatography).

[0064] Yet another embodiment of the present invention relates to a method for controlling fungal diseases, bacterial diseases, or nematode diseases in plants, which comprises treating a culture broth of Lysobacter enzymogenes JCK-1421 strain or an extract thereof with a composition.

[0065] In the present invention, the composition treatment step may be carried out by one or more methods selected from the group consisting of spraying (e.g., spraying, misting, atomizing, powder application, granular application, water application, box application, etc.), soil drenching (e.g., mixing, drenching, etc.), surface application (e.g., painting, smearing, coating, etc.), dipping, poisoning, fumigation, and seed treatment, but is not limited thereto.

[0066] In the present invention, "application" may be carried out by one or more methods selected from the group consisting of spraying, misting, atomizing, powder application, granular application, water surface application, and box application.

[0067] The term "drench" as used herein refers to a method of spraying chemicals by drilling holes in the soil or trees and injecting a chemical solution.

[0068] As used herein, the term "soil drench" refers to a method of injecting or spraying a chemical solution into crop-growing soil.

[0069] In the present invention, the amount of the composition to be used may be appropriately determined depending on the dosage form, the condition of the damage, the application method, the application site, and the like.

[0070] In the method for producing a composition for controlling a plant fungal disease, bacterial disease or nematode disease and the method for controlling a plant fungal disease, bacterial disease or nematode disease, content that overlaps with the composition for controlling a plant fungal disease, bacterial disease or nematode disease will be omitted in consideration of the complexity of this specification. [Effects of the Invention]

[0071] The present invention relates to a Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases, which comprises the strain, a culture thereof, or an extract thereof, a method for producing the composition, and a method for controlling plant fungal diseases, bacterial diseases, or nematode diseases using the composition, which, when applied, promotes plant growth and induces resistance in host plants, thereby effectively controlling various plant diseases and nematode diseases simultaneously. [Brief explanation of the drawings]

[0072] [Figure 1] 1 shows the results of testing for resistance induction activity by GUS gene expression analysis following treatment with JCK-1421 strain in Arabidopsis thaliana seedlings transformed with a PR-1 promoter-labeled GUS vector according to one embodiment of the present invention.

[0073] [Figure 2] 1 shows the results of a phylogenetic analysis of the 16S rRNA gene sequence of Lysobacter enzymogenes JCK-1421 strain according to one embodiment of the present invention.

[0074] [Figure 3] 1 shows the extracellular enzyme activity of Lysobacter enzymogenes JCK-1421 strain according to one embodiment of the present invention.

[0075] [Figure 4] 1 shows the plant growth hormone IAA-producing activity of Lysobacter enzymogenes JCK-1421 strain according to one embodiment of the present invention.

[0076] [Figure 5] 1 shows the plant growth-promoting activity of turf two weeks after treatment with a culture solution of Lysobacter enzymogenes JCK-1421 strain according to one example of the present invention.

[0077] [Figure 6] 1 shows the in vitro antifungal activity of Lysobacter enzymogenes JCK-1421 strain in dual culture according to one embodiment of the present invention.

[0078] [Figure 7A] 1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on cucumber damping-off disease according to an example of the present invention.

[0079] [Figure 7B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on cucumber damping-off disease according to an example of the present invention.

[0080] [Figure 8A] 1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on cucumber fusarium wilt according to an example of the present invention.

[0081] [Figure 8B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on cucumber Fusarium wilt according to one example of the present invention.

[0082] [Figure 9A]1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on dollar spot disease in turf according to an embodiment of the present invention.

[0083] [Figure 9B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on dollar spot disease in turf according to an embodiment of the present invention.

[0084] [Figure 10A] 1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on Pythium disease in turf according to an embodiment of the present invention.

[0085] [Figure 10B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on Pythium disease in turf according to an embodiment of the present invention.

[0086] [Figure 11A] 1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on rice Fusarium head blight according to an example of the present invention.

[0087] [Figure 11B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on rice Fusarium head blight according to one example of the present invention.

[0088] [Figure 12A] 1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on soft rot of Chinese cabbage according to an example of the present invention.

[0089] [Figure 12B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on soft rot of Chinese cabbage according to an example of the present invention.

[0090] [Figure 13A] 1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on tomato bacterial wilt according to an example of the present invention.

[0091] [Figure 13B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on tomato bacterial wilt according to one example of the present invention.

[0092] [Figure 14A] 1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on pepper bacterial leaf spot according to an embodiment of the present invention.

[0093] [Figure 14B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on pepper bacterial leaf spot according to an embodiment of the present invention.

[0094] [Figure 15] 1 shows the control effect of Lysobacter enzymogenes strain JCK-1421 on apple fire blight in Chinese pearleaf crabapple seedlings 10 days after inoculation with the pathogen according to one embodiment of the present invention.

[0095] [Figure 16A] 1 is a graph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on apple fire blight in M9 seedlings 7 days and 10 days after inoculation with the pathogen according to an example of the present invention.

[0096] [Figure 16B] 1 is a photograph showing the control effect of Lysobacter enzymogenes JCK-1421 strain on apple fire blight in M9 seedlings 7 and 10 days after inoculation with the pathogen according to an example of the present invention.

[0097] [Figure 17A] According to one embodiment of the present invention, the control effect of Lysobacter enzymogenes strain JCK-1421 against tomato root-knot nematode disease is shown in terms of a) the inhibitory effect on the galling index and b) the inhibitory effect on the number of egg masses 6 weeks after inoculation with the pathogen. [Figure 17B] This is a continuation of Figure 17A.

[0098] [Figure 17C] 1 shows the control effect of Lysobacter enzymogenes strain JCK-1421 against tomato root-knot nematode disease using photographs of roots 6 weeks after inoculation with the pathogen according to one example of the present invention.

[0099] [Figure 18] 1 shows the pine wilt disease control effect after 4 weeks when the Lysobacter enzymogenes JCK-1421 strain was applied to Japanese black pine seedlings according to one embodiment of the present invention.

[0100] [Figure 19] 1 shows the pine wilt disease control effect after 6 weeks when Lysobacter enzymogenes JCK-1421 strain was applied to pine seedlings according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention relates to the Lysobacter enzymogenes strain JCK-1421, deposited under accession number KCTC 15126BP, which has antifungal, antibacterial and antinematode activities. [Example]

[0102] The present invention will now be described in more detail with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0103] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, or (vol / vol)% for liquid / liquid, unless otherwise specified.

[0104] Example 1. Selection of strains with plant resistance induction activity

[0105] To select endophytic fungal strains isolated from conifers that have the ability to induce resistance in plants, we investigated the expression of the PR-1 gene, a gene related to induced resistance, using Arabidopsis thaliana transformed with a PR-1 promoter-GUS-tagged vector. Resistance is induced in plants by the salicylic acid signaling system, which in turn expresses the PR-1 protein. The PR-1 gene is used as a marker gene to test whether resistance is induced in plants by utilizing this signaling system.

[0106] Transformed Arabidopsis seeds were surface-sterilized for 30 seconds with 70% ethanol, followed by a second surface-sterilization step with bleach solution (2% NaOCl + 0.05% Tween-20) for 5 minutes. The remaining bleach solution on the seed surface was then washed 3-4 times with sterile water and soaked at 4°C for 2 days. The soaked seeds were individually placed on 1 / 2 Murashige-Skoog solid medium (MS, 2.2g MS salts, 10g sucrose, 8g phyto agar / L, Duchefa) supplemented with 50μg / mL kanamycin, sealed with 3M tape, and cultured in a plant growth incubator at 25°C (16-hour photoperiod, 80% relative humidity). After 12 days of incubation, 2 mL of the bacterial culture medium, culture filtrate, or cell fraction was added to each well of a 24-well plate (500x, 1,000x, 2,000x, or 4,000x dilutions). Two plantlets were placed in each well and incubated on an orbital shaker at room temperature for 2 days. After 2 days, the plants were immersed in 90% acetone at -20°C for 1 hour to fix the reaction. After washing twice with 0.1 M sodium phosphate buffer (pH 7.0), the plants were then immersed in a staining solution (100 mM sodium phosphate buffer, 0.1% Triton X-100, 2 mM X-GlcA (Duchefa, X1405), 2.5 mM potassium ferricyanide, and 2.5 mM potassium ferrocyanide). The cells were then placed in a 37°C water bath for 12 hours, and then immersed in 70% ethanol for 1 hour to stop the reaction. Then, unnecessary pigments such as chlorophyll were removed by immersing the cells in 90% ethanol several times. Finally, PR-1 gene expression was examined by confirming the blue coloration under a microscope (Stemi 508, Carl Zeiss, Germany).

[0107] In order to select strains with resistance-inducing activity against endophytic fungi isolated from pine and other conifers, a GUS staining assay was performed and the JCK-1421 strain was selected, which showed GUS activity due to the expression of the PR-1 gene.

[0108] As can be seen in Figure 1, the JCK-1421 strain turned blue in all treatments: culture broth, culture filtrate, and cell suspension. Of these, the culture broth showed a darker color over a wider area. In contrast, the medium used as a negative control showed no GUS activity. This confirmed that the JCK-1421 strain expresses the resistance marker gene PR-1 and has resistance-inducing activity.

[0109] Example 2. Molecular biological analysis and phylogenetic analysis of JCK-1421 strain

[0110] Strain JCK-1421, selected for its plant resistance-inducing activity, was identified molecularly by 16S rRNA gene sequence analysis. The strain was inoculated into LB liquid medium (Luria-Bertani, Becton, Dickinson and Co., Sparks, MD, USA) and cultured at 30°C for 24 hours with shaking at 150 rpm. Genomic DNA (gDNA) was extracted from the harvested strain using iNtRON's I-genomic BYF DNA Extraction Mini Kit according to the protocol. The extracted gDNA was mixed with iNtRON Biotechnology's PCR premix and a primer set capable of amplifying the strain's 16S rRNA, and the gene was amplified by PCR.

[0111] [Table 1]

[0112] PCR was performed starting with 5 minutes at 95°C, followed by 30 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 72°C for 90 seconds, followed by 10 minutes at 72°C and 4°C for amplification. The amplified 16S rRNA gene PCR product was sequenced by Genotech (Daejeon, Korea), and a 1388-bp sequence (SEQ ID NO: 3) was obtained as the 16S rRNA coding sequence of the isolated strain, JCK-1421. As shown in Figure 2, a comparison of the sequence with the GenBank database using NCBI's BlastN search identified JCK-1421 as Lysobacter enzymogenes. The 16S rRNA sequence of the strain was uploaded to the GenBank database as OP420512, and the selected strain was named Lysobacter enzymogenes JCK-1421 strain and deposited at the Korea Collection for Type Cultures (KCTC) of the Korea Institute of Bioscience and Biotechnology on October 6, 2022, with the accession number KCTC 15126BP.

[0113] Example 3. Extracellular enzyme activity of Lysobacter enzymogenes JCK-1421 strain

[0114] To confirm the extracellular enzyme activities of Lysobacter enzymogenes JCK-1421, experiments were conducted on proteases, chitinases, gelatinases, and cellulases. Protease medium (1% skim milk + 1.5% agar, Difco), chitinase medium (1% colloidal chitin + 1.5% agar, Difco), gelatinase medium (10% gelatin + 1.5% agar, Duksan), and cellulase medium (0.4% sodium carboxymethylcellulose + 1.5% agar, Sigma-Aldrich) were prepared. Chitinase medium was prepared by adding colloidal chitin to XL1 medium (5 g / L peptone (Difco), 5 g / L yeast extract (Difco), 5 g / L NaCl), and this medium was designated XL1+C. Colloidal chitin was prepared by adding crab shell powder to hydrochloric acid (HCl) (10 g crab shell powder / 150 mL HCl) and stirring for 6 hours. After 6 hours, 1 L of cold ethanol (99.9%) was added, mixed thoroughly using a stirrer, and centrifuged at 4°C and 4500 rpm for 20 minutes. The supernatant was then removed, and the chitin precipitate was washed 3-4 times with 0.1 M potassium sodium phosphate buffer (pH 7.0). The prepared colloidal chitin was autoclaved and stored at 4°C until use. Gelatin medium was prepared by adding 10% gelatin to LB solid medium, while other media were solidified by adding 1.5% agar.

[0115] Sterilized paper discs (0.8 cm, Advantec, Japan) were placed on each medium, and the JCK-1421 culture filtrate was dispensed at 2.5 μL, 5 μL, and 10 μL for protease, 30 μL, 60 μL, and 90 μL for chitinase and cellulase, and 10 μL, 20 μL, and 30 μL for gelatinase. As negative controls, equal volumes of sterilized LB medium and medium were dispensed onto the paper discs. The experiment was performed in triplicate, and the plates were kept at 30°C. Clear zones due to extracellular enzyme activity were observed. To visualize the cellulase medium, 5 mL of Lugol's solution (2.5 g / L iodine and 5 g / L potassium iodide) was added to the plates, and the plates were kept in the dark for 10 minutes before being observed for clear zones.

[0116] As can be seen in Figure 3, the culture filtrate was treated with a medium that induces enzyme activity, and the clear zone was observed. The JCK-1421 strain produced a clear zone diameter of 24.61 mm after 9 days of treatment with 10 μL of protease, 17.25 mm after 7 days of treatment with 90 μL of chitinase, 29.42 mm after 4 days of treatment with 30 μL of gelatinase, and 28.67 mm after 5 days of treatment with 90 μL of cellulase. The Lysobacter enzymogenes JCK-1421 strain produced all of the enzymes: protease, chitinase, gelatinase, and cellulase, with gelatinase being the most abundant.

[0117] Example 4. Production of indole-3-acetic acid (IAA) by Lysobacter enzymogenes JCK-1421 strain

[0118] To confirm the production of the plant growth hormone IAA by Lysobacter enzymogenes strain JCK-1421, a 1% culture of JCK-1421 was inoculated into three media (LB, TSB, and XL1+C) supplemented with L-tryptophan (150 mg / L) and cultured at 30°C for 24 hours with shaking at 150 rpm. The culture was then centrifuged at 10,000 rpm for 5 minutes at 4°C and filtered through a 0.2 μm sterile filter. 1 mL of the resulting culture filtrate was mixed with 2 mL of Salkowski's reagent (150 mL H2SO4, 250 mL sterile water, 7.5 mL 0.5 M FeCl3·6H2O). After 20 minutes at room temperature in the dark, the mixture was checked for a pink color. As negative controls, three types of media, LB, TSB, and XL1+C media supplemented with L-tryptophan (150 mg / L), were used, and the experiment was repeated three times.

[0119] As shown in Figure 4, an experiment was conducted to confirm the production of IAA, a plant growth hormone, by JCK-1421. The culture filtrate-treated test tubes showed a pink color, while the control treatments (LB, TSB, and XL1+C) showed the original color of the medium. This confirmed that JCK-1421 produces IAA, a plant growth hormone.

[0120] Example 5. Plant growth promoting effect of Lysobacter enzymogenes JCK-1421 strain on turf

[0121] After confirming the Lysobacter enzymogenes JCK-1421 strain's ability to produce the plant growth hormone IAA, a lawn test was conducted to confirm whether this was linked to plant growth-promoting effects. Two grams of creeping bentgrass (Agrostis stolonifera cv. Penncross) seeds, a cool-season grass species, were soaked in distilled water at 4°C for two days. Creeping bentgrass seeds were then sown (2 g seeds / 500 mL soil) in a plastic pot (7 cm diameter, 6 cm height) filled with a mixture of soil and sand (Songin General Development Co., Ltd.) (1:1 sand:soil, v / v). After sowing, the seeds were grown in a constant temperature room at 25°C under dark conditions for three days, then incubated under a 16-hour photoperiod for three weeks.

[0122] Lysobacter enzymogenes JCK-1421 strain was inoculated into LB agar medium using a disposable loop and cultured at 30°C for 2 days. A single colony of the cultured strain was inoculated into 5 mL of LB liquid medium and cultured at 30°C with shaking at 150 rpm for 24 hours. The optical density of the culture was measured at 600 nm using a UV-VIS spectrophotometer, and the OD was calculated. 600 The value of is 0.1 (1.0x10 8 The strain suspension was adjusted to a concentration of 1000 CFU / mL and inoculated into LB liquid medium at 1%. After that, the culture was cultured at 30°C and 150 rpm for 24 hours with shaking, and the culture was used as the main culture medium.

[0123] Before treatment, the grass was cut to the same length to create identical conditions. JCK-1421 culture solution was diluted 500-fold, 1,000-fold, and 2,000-fold, and 20 mL of the solution was drenched in the soil per pot. Horiqua (ai 25% tebuconazole EC, Farm Hannon) was used as a control, diluted 2,000-fold (the usual concentration). The negative control (untreated area) was treated with LB liquid medium without the strain, diluted 500-fold. The plants were then placed in a 25°C constant temperature room with a 16-hour photoperiod. The experiment was repeated three times, with two plants per treatment area. Two weeks after treatment, grass growth was visually assessed.

[0124] As can be seen in Figure 5, when the plant growth-promoting effect of Lysobacter enzymogenes JCK-1421 strain on creeping bentgrass was examined, treatment with JCK-1421 culture solution showed a higher growth effect than the untreated area, and treatment with the triazole fungicide Horiqua actually suppressed plant growth.

[0125] Example 6. In vitro antifungal activity of Lysobacter enzymogenes JCK-1421 strain against plant pathogenic fungi

[0126] The direct antifungal activity of JCK-1421 was evaluated by dual culture and minimum inhibitory concentration (MIC) tests against various plant pathogenic fungi, examining its inhibitory activity against fungal mycelia. Seven plant pathogenic fungi were used in the in vitro experiments to examine direct antifungal activity: Rhizoctonia solani AG-4 (cucumber seedling damping-off), Fusarium oxysporum f.sp. cucumerinum (cucumber fusarium wilt), Sclerotinia homoeocarpa (turf dollar spot), Rhizoctonia solani AG2-2 (turf large patch), Rhizoctonia solani AG2-2 (turf brown patch), Pythium aphanidermatum (turf Pythium wilt), and Fusarium graminearum (rice head blight). For their cultivation, P. aphanidermatum was inoculated onto cornmeal agar (CMA, Sigma-Aldrich, India) medium, and the remaining strains onto potato dextrose agar (PDA, Becton, Dickinson and Co., Sparks, MD, USA) medium, and then cultured statically in an incubator at 25°C.

[0127] For dual culture, agar plugs of plant pathogens grown at 25°C were isolated using a cork borer (0.6 cm diameter) and inoculated 2 cm from the edge of the PDA. A colony of JCK-1421 strain statically cultured on LB solid medium was inoculated opposite the agar plug (5 cm long). The agar plugs were then statically cultured in an incubator at 25°C, and the degree of mycelial growth of the pathogen was observed after 2 to 7 days.

[0128] For the MIC test, agar plugs were isolated from the pathogen grown at 25°C using a cork borer (0.5 cm diameter), inoculated into 48-well plates, and treated with the culture filtrate at final concentrations of 10%, 5%, 2.5%, 1.25%, and 0.625%. PDB medium was used as a negative control and was incubated at 25°C. The experiment was repeated three times to confirm the MIC required for complete inhibition of mycelial growth.

[0129] [Table 2]

[0130] As can be seen from Figure 6 and Table 2, to examine the antifungal activity of JCK-1421 strain, dual culture and MIC tests were performed against various plant pathogenic fungi. The results showed that the strain showed no antifungal activity against any of the plant pathogenic fungi used in the experiment. Therefore, it was confirmed that Lysobacter enzymogenes JCK-1421 strain does not have direct antifungal activity against the seven plant pathogenic fungi used in the experiment.

[0131] Example 7. In vitro antibacterial activity of Lysobacter enzymogenes JCK-1421 strain against plant pathogenic bacteria

[0132] We attempted to examine the direct antibacterial activity of JCK-1421 strain by conducting minimum inhibitory concentration (MIC) tests against various plant pathogenic bacteria to determine their growth inhibitory activity. Three plant pathogenic bacteria were used to examine the direct antibacterial activity: Ralstonia solanacearum (Rs, tomato bacterial wilt), Pectobacterium carotovorum subsP. carotovorum (Pcc, Chinese cabbage soft rot), and Xanthomonas euvesicatoria (Xe, pepper bacterial spot). A single colony of the pathogen grown in tryptic soy agar (TSA, Becton, Dickinson and Co., Sparks, MD, USA) was inoculated into tryptic soy broth (TSB, Becton, Dickinson and Co., Sparks, MD, USA) and cultured at 30°C with shaking at 150 rpm for 3 days for Rs, 2 days for Xe, and 1 day for Pcc. The optical density of the pathogen culture was then measured at 600 nm using a UV-VIS spectrophotometer, and the OD 600 The value is 0.1 (1.0x10 8 The bacterial suspension was adjusted to a concentration of 10%, 5%, 2.5%, 1.25%, or 0.625%. The positive control was streptomycin sulfate, and the negative control was an untreated control treated with the bacterial suspension alone. All plates were incubated in a 30°C incubator to examine bacterial growth, and each experiment was performed in triplicate.

[0133] [Table 3]

[0134] As can be seen from Table 3, MIC tests were performed on various plant pathogenic bacteria to examine the direct antibacterial activity of the JCK-1421 strain. The results showed that the JCK-1421 strain showed no antibacterial activity against any of the plant pathogenic bacteria used in the experiment. Therefore, it was confirmed that the Lysobacter enzymogenes JCK-1421 strain does not have direct antibacterial activity against the three plant pathogenic bacteria.

[0135] Example 8. In vitro nematicidal activity of Lysobacter enzymogenes strain JCK-1421 against plant pathogenic nematodes

[0136] The direct nematicidal activity of strain JCK-1421 was investigated by in vitro assays to examine its growth inhibitory activity against various plant pathogenic nematodes. Two plant pathogenic nematodes, the sweet potato root-knot nematode (Meloidogyne incognita) and the pinewood nematode (Bursaphelenchus xylophilus), were used to examine the direct nematicidal activity.

[0137] The in vitro nematicidal activity of JCK-1421 against the sweet potato root-knot nematode (M. incognita) was assessed by measuring egg hatch inhibition and nematicidal activity. Sweet potato root-knot nematodes were grown on the susceptible species, Lycopersicon esculentum Mill. cv. Seokwang, Farm Hannon, Korea, in a constant temperature room at 25°C for 2–3 months. First, artificially infected tomato roots were washed with running water and cut into 1 cm pieces and placed in a grinder (HM-2100S; Hanil, Korea). After grinding for 1 minute in 1% sodium hypochlorite (v / v), root-knot nematode eggs were collected using a 45 μm sieve followed by a 25 μm sieve. The collected nematode eggs were used in the in vitro egg hatch inhibition experiment. Nematode eggs were incubated at 28°C for 3 days using a modified Baermann funnel method to separate second-instar larvae. The collected nematode egg and larval suspensions were adjusted to contain 150 nematode eggs and 50 second-instar larvae per 100 μL, respectively, and then injected into each well of a 96-well plate. The culture filtrate was treated to a final concentration of 20% and 10%. Experiments were performed in triplicate. After treatment, the nematodes were kept at room temperature, in the dark, and at 100% relative humidity for 3 days. After treatment, the nematodes were observed under a microscope (Leica DM IL LED; Leica Microsystems CMS GmbH, Wetzlar, Germany). Nematodes were considered dead if their bodies were straight and motionless. Each experiment was performed in triplicate. The mortality rate (%) and egg hatching inhibition rate of sweet potato root-knot nematodes were calculated using the following formula:

[0138] [Formula 1]

[0139] Mortality rate (%) = [(mortality rate in the control area - mortality rate in the treatment area) / (mortality rate in the control area)] × 100

[0140] [Formula 2]

[0141] Egg hatch inhibition rate (%) = [(Egg hatch rate in the control group - Egg hatch rate in the treated group) / Egg hatch rate in the control group] x 100

[0142] To test the in vitro nematicidal activity of strain JCK-1421 against the pinewood nematode (Bursaphelenchus xylophilus), pathogenic pinewood nematodes were artificially cultured in the laboratory. PDA medium was inoculated with Botrytis cinerea (a pinewood nematode prey species) and incubated statically at 25°C for 7 days. Afterwards, the pinewood nematode (B. xylophilus, National Academy of Forestry Science) was inoculated onto the cultured Botrytis cinerea and incubated statically at 25°C for 7 days. Cultured pinewood nematodes were harvested using the Baermann funnel method, counted under a light microscope, and adjusted to 50 nematodes per 100 μL. The culture filtrate was then injected into each well of a 96-well plate to achieve final concentrations of 20% and 10%. The experiment was performed in triplicate. After treatment, the plants were kept at room temperature, in the dark, and at 100% relative humidity for three days. After that, the plants were observed under a microscope (Leica DM IL LED; Leica Microsystems CMS GmbH, Wetzlar, Germany). Nematodes were considered dead if their bodies were straight and motionless. Each experiment was performed in triplicate. The mortality rate (%) of pinewood nematodes was calculated using the following formula:

[0143] [Formula 3]

[0144] Mortality rate (%) = [(mortality rate in the control area - mortality rate in the treatment area) / (mortality rate in the control area)] × 100

[0145] [Table 4]

[0146] As can be seen from Table 4, the JCK-1421 strain was tested for its egg hatching inhibitory and nematicidal activity against the sweet potato root-knot nematode and pinewood nematode, and did not exhibit any egg hatching inhibitory or nematicidal activity against the sweet potato root-knot nematode or pinewood nematode. This confirms that the Lysobacter enzymogenes JCK-1421 strain does not have direct nematicidal activity against the two plant pathogenic nematodes.

[0147] Example 9. Cultivation and formulation production of Lysobacter enzymogenes JCK-1421 strain for controlling various plant diseases

[0148] To investigate the disease control activity of JCK-1421 strain against various plant diseases through its resistance induction activity, the culture solution of JCK-1421 strain and JCK-1421 20 SC (Suspension concentrate, liquid wettable powder) prepared by adding 20% ​​spray-dried culture solution were used in this experiment. The culture solution was inoculated into LB agar medium using a disposable loop and incubated at 30°C for 2 days. A single colony of the cultured strain was inoculated into 5 mL of LB liquid medium and incubated at 30°C and 150 rpm for 24 hours. The optical density of the pre-culture solution was measured at 600 nm using a UV-VIS spectrophotometer, and the OD 600 The value of is 0.1 (1.0x10 8 The strain suspension was adjusted to a concentration of 1000 CFU / mL and inoculated into LB liquid medium at 1%. After that, the culture was cultured at 30°C and 150 rpm for 24 hours with shaking, and the culture was used as the main culture medium.

[0149] To formulate JCK-1421, a total of 3 L of JCK-1421 culture broth was cultured at 30°C and 150 rpm for 24 hours with shaking, and then spray-dried. The 3 L culture broth was mixed with 600 g of excipient (oxidized starch:maltodextrin = 3:1) and then spray-dried. The spray-drier inlet temperature was 195°C, and the outlet temperature was 93°C or higher. The spray-dried product was then formulated in the form of JCK-1421 20 SC, which contains 20% spray-dried JCK-1421 culture broth.

[0150] Example 10: Control effect of Lysobacter enzymogenes JCK-1421 strain on cucumber seedling damping-off

[0151] To investigate the in vivo efficacy of JCK-1421 strain in controlling damping-off disease in cucumber seedlings, 'Nebakja' seeds (Cucumis sativus L. cv. Nebakja, Shinzenta Korea) were immersed in filter paper containing distilled water (in a 9-cm diameter Petri dish) and germinated in an incubator at 28°C for 24 hours. The seeds were then sown in horticultural soil in plastic pots (6 cm diameter, 6.5 cm height) and grown in a constant temperature room at 25°C under 16 hours of light per day for 6 days.

[0152] R. solani AG-4, the pathogen of cucumber damping-off disease, was inoculated onto brown rice (Dongdae Agricultural Corporation, Jeollanam-do, Korea) that had been soaked in distilled water for 24 hours and then autoclaved three times. The inoculation was performed at 25°C for three weeks. After three weeks of incubation, the pathogen was placed in an aluminum dish, covered with gauze, and dried at room temperature for three days. The dried material was ground in a blender and sieved through a 0.5-0.85 mm sieve to be used as inoculum. Six days after sowing, 0.75 g of inoculum was uniformly mixed with 1 L of horticultural soil, which was then placed in a plastic pot (7 cm diameter, 6 cm height). After six days of cultivation, cucumber seedlings were transplanted.

[0153] To conduct an in vivo pot experiment to examine the control effect of JCK-1421 strain culture solution on cucumber seedling damping-off disease through induced resistance, JCK-1421 strain culture solution and formulation (JCK-1421 20 SC) were prepared as described in Example 9. JCK-1421 strain culture solution was diluted with distilled water containing 250 ppm of Tween 20 (Duksan Science, Seoul, Korea) to prepare formulations at 500-fold, 1,000-fold, and 2,000-fold concentrations, respectively, and JCK-1421 20 SC formulation was prepared by diluting it at the same dilution ratio as the JCK-1421 strain culture solution. For the positive controls, Zaroquen (dispersible concentrate, DC: 30% hymexazole + 5% penthiopyrad, Samgon, Korea) and Gajilan (WP: 10% etridiazole + 55% thiophanate methyl, Farm Hannon) were diluted 1,000-fold and 2,000-fold, and 10 mL of each solution was drenched in the soil one hour after inoculation. The JCK-1421 culture solution and JCK-1421 20 SC samples were drenched in the soil at 10 mL per pot four days before inoculation with the cucumber damping-off pathogen. After inoculation and treatment, the pots were placed in a constant temperature room at 25°C and exposed to a light source for 12 hours per day. The experiment was repeated three times with three plants per treatment area. Four days after inoculation, the disease incidence rate (%) of cucumber damping-off disease was determined, and the control value (%) relative to the untreated area was calculated using the following formula.

[0154] [Formula 4]

[0155] Control value (%) = 100 × [(disease incidence rate in untreated area - disease incidence rate in treated area) / disease incidence rate in untreated area]

[0156] As shown in Figure 7, the JCK-1421 culture medium showed 67%, 83%, and 79% control values ​​compared to the untreated control at 500-, 1,000-, and 2,000-fold dilutions, respectively. JCK-1421 20 SC also showed 75%, 88%, and 75% control values ​​at 500-, 1,000-, and 2,000-fold dilutions, respectively. Zaroquen, used as a control, showed 100% and 83% control values ​​at 1,000- and 2,000-fold dilutions, and Gadjilan showed 92% and 75% control values ​​at 1,000- and 2,000-fold dilutions, respectively. The JCK-1421 culture medium and JCK-1421 20 SC formulation showed the highest control efficacy at a 1:1000 dilution. In particular, the 1:1000 dilution of JCK-1421 20 SC showed no statistically significant difference from the control agent, Gadjiran. Therefore, JCK-1421, which has no direct antifungal activity against the causative agent of cucumber damping-off, R. solani AG-4, can be said to have induced resistance in cucumbers and controlled damping-off disease. This suggests that it may be possible to develop a highly effective cucumber damping-off disease control agent using this method.

[0157] Example 11: Control effect of Lysobacter enzymogenes JCK-1421 strain on cucumber Fusarium wilt

[0158] To investigate the in vivo control effect of induced resistance of JCK-1421 against cucumber fusarium wilt disease, 'Jungboksamcheok' seeds (Cucumis sativus L. cv. Jungboksamcheok, Shinzenta Korea) were immersed in filter paper containing distilled water (9 cm diameter Petri dish) and germinated in an incubator at 28°C for 24 hours. They were then sown in long square slit pots (7 cm diameter, 9.5 cm height) using bed soil (Horticultural Bed Soil No. 2, Bunon). The seeds were then grown in a constant temperature room at 25°C under 16 hours of light per day for 7 days.

[0159] To induce cucumber fusarium wilt disease, cultured mycelia of the pathogenic fungus F. oxysporum f.sp. cucumerinum were cut using a cork borer (0.6 cm diameter), and five agar plugs were inoculated onto PDB medium and cultured at 25°C and 150 rpm for 7 days with shaking. The culture medium was then harvested and filtered through four layers of gauze to remove mycelia. 2.5 × 10 cells were analyzed under a microscope (Axio Imager. A2, Carl Zeiss, Germany) using a hemocytometer. 6 A pathogen spore suspension was prepared to a concentration of 100 spores / mL. The prepared spore suspension was used as an inoculum to investigate its efficacy in controlling cucumber Fusarium wilt disease. 7 days after sowing, the prepared pathogen spore suspension inoculum was inoculated into the soil by drenching 10 mL of the inoculum per pot.

[0160] To conduct an in vivo pot experiment to examine the control effect of JCK-1421 strain culture on cucumber Fusarium wilt disease through induced resistance, the JCK-1421 strain culture solution and formulation (JCK-1421 20 SC) were prepared as described in Example 9. The JCK-1421 strain culture solution was diluted with distilled water containing 250 ppm of Tween 20 (Duksan Science, Seoul, Korea) to prepare formulations at 500-fold, 1,000-fold, 2,000-fold, 4,000-fold, and 8,000-fold concentrations, respectively, and the JCK-1421 20 SC formulation was prepared by diluting it at the same dilution ratio as the JCK-1421 strain culture solution. As positive controls, Zaroquen (dispersible concentrate, DC: 30% hymexazole + 5% penthiopyrad, Samgon, Korea) and Gajiran (WP: 10% etridiazole + 55% thiophanate methyl, Farm Hannon) were diluted 1:1000 and drenched in 10 mL of soil one hour after inoculation. The prepared JCK-1421 culture solution and formulation were drenched in soil at 10 mL per pot four days before inoculation. After inoculation and treatment, the pots were placed in a constant temperature room at 25°C and exposed to light for 12 hours per day. The experiment was performed three times, with three pots per treatment. 30 days after inoculation, the severity of disease was evaluated using a disease index ranging from 0 to 4. The disease index was calculated on a five-point scale, with 0 being healthy, 1 being browned roots and leaves, and some suppression of seedling growth, 2 being markedly suppressed seedling growth, 3 being very suppressed seedling growth, and 4 being dead, and the control effect was calculated based on this.

[0161] As can be seen from Figure 8, the control effect of Lysobacter enzymogenes JCK-1421 strain against cucumber Fusarium wilt was tested. The 2,000-fold diluted JCK-1421 culture solution and the 500-fold diluted JCK-1421 20 SC showed high control values ​​of 77% and 88%, respectively. In addition, the 1,000-fold diluted JCK-1421 culture solution showed a 69% control value, and both the 1,000-fold and 2,000-fold diluted JCK-1421 20 SC formulations showed 77% control values. As a result, it was confirmed that the JCK-1421 strain exhibits excellent control effects against cucumber Fusarium wilt. As with cucumber seedling damping-off, this can be said to be due to the fact that the JCK-1421 strain, which has no direct antifungal activity against the causative agent of cucumber Fusarium wilt, F. oxysporum f.sp. cucumerinum, induces resistance in cucumbers, thereby controlling cucumber Fusarium wilt. It is anticipated that in the future, it will be possible to develop an effective cucumber Fusarium wilt control agent by utilizing the induced resistance of the JCK-1421 strain.

[0162] Example 12: Control effect of Lysobacter enzymogenes JCK-1421 strain on dollar spot disease in turfgrass

[0163] To examine the in vivo efficacy of JCK-1421 in controlling dollar spot disease in turfgrass, creeping bentgrass (Agrostis stolonifera cv. Penncross), a cool-season grass species, was soaked in distilled water for 2 days at 4°C. The soaked seeds were then sown in plastic pots (7 cm diameter, 6 cm height) filled with a 60% mixture of sand and horticultural soil (1:1 sand:soil, v / v). After sowing, the seeds were grown at 25°C under dark conditions and 50% relative humidity for 2 days, followed by 3 weeks of growth at 25°C under a 16 / 8 h light / dark regime with 50% relative humidity.

[0164] Sclerotinia homoeocarpa, the pathogen of dollar spot disease in turfgrass, was inoculated into a bran-rice husk medium (9 g bran, 1.5 g rice husk, 10 mL distilled water; 250 mL Erlenmeyer flask) that had been autoclaved twice and incubated statically at 25°C for 7 days. After incubation, 110 mL of distilled water containing 200 ppm streptomycin sulfate was added and the fungal cells were ground. The resulting fungal suspension was used as an inoculum to investigate the efficacy of Sclerotinia homoeocarpa in controlling dollar spot disease in turfgrass. 3.5 mL of Sclerotinia homoeocarpa inoculum was inoculated into each pot via a 1 cm deep hole drilled in the center.

[0165] To investigate the efficacy of JCK-1421 culture against dollar spot disease in turf due to induced resistance, JCK-1421 culture and JCK-1421 20 SC were prepared as described in Example 9. JCK-1421 culture was diluted with distilled water containing 250 ppm Tween 20 (Duksan Science, Seoul, Korea) to prepare 500-fold, 1,000-fold, and 2,000-fold dilutions, respectively. JCK-1421 20 SC formulations were prepared by diluting the JCK-1421 culture at the same dilution ratio. As a positive control, Horikuo (EC:ai 25% tebuconazole, Farm Hannon) was diluted 2,000-fold and drenched in soil at 20 mL per pot 1 hour after inoculation. The prepared JCK-1421 culture and formulation were applied by soil drench treatment at 20 mL per pot 4 days before inoculation. Three weeks after inoculation, creeping bentgrass was treated with JCK-1421 culture and formulation by soil drench treatment at 20 mL per pot. Four days after treatment, 3.5 mL of the prepared S. homoeocarpa mycelial suspension was inoculated per pot. After inoculation and treatment, the pots were placed in trays filled with water and covered with a plastic chamber (35 cm wide, 28 cm long, 16 cm high) to maintain 100% relative humidity. The pots were placed in a constant temperature room at 25°C and exposed to 12 hours of light per day. The experiment was conducted in two replicates, with three pots per treatment. Thirteen days after inoculation, the diseased area rate was determined, and the control value (%) relative to the untreated control was calculated using the following formula:

[0166] [Formula 5]

[0167] Control value (%) = 100 × [(disease incidence in untreated area - disease incidence in treated area) / disease incidence in untreated area]

[0168] As shown in Figure 9, the control efficacy of Lysobacter enzymogenes strain JCK-1421 against dollar spot disease in turf was tested 13 days after inoculation. The JCK-1421 culture solution showed 86%, 92%, and 85% control compared to the untreated control at 500-, 1000-, and 2000-fold dilutions, respectively. JCK-1421 20 SC showed 66%, 83%, and 55% control at 500-, 1000-, and 2000-fold dilutions, respectively. Horiqua, used as a control, showed 94% control at a 2000-fold dilution. JCK-1421 culture solution and JCK-1421 20 SC each showed the highest control efficacy at a 1:1000 dilution. In particular, all dilutions of JCK-1421 culture solution used in the experiment, as well as the 1:1000 dilution of JCK-1421 20 SC, demonstrated excellent activity in controlling dollar spot disease in turf, with no statistically significant difference from the control, Horiqua. Therefore, JCK-1421 demonstrated excellent control efficacy against dollar spot disease in turf. Since it lacked direct antifungal activity against S. homoeocarpa, the causative agent of dollar spot disease, it appears that dollar spot disease was controlled by inducing resistance in turf. It is believed that the induced resistance of JCK-1421 may be used to develop highly effective dollar spot control agents for turf.

[0169] Example 13: Control effect of Lysobacter enzymogenes JCK-1421 strain against Pythium disease in turfgrass

[0170] To examine the in vivo control of Pythium disease in turfgrass by induced resistance of strain JCK-1421, creeping bentgrass (Agrostis stolonifera cv. Penncross), a cool-season grass species, was soaked in distilled water for 2 days at 4°C. The soaked seeds were then sown in plastic pots (7 cm diameter, 6 cm height) filled with 60% sand and horticultural soil (1:1 sand:soil, v / v). After sowing, the seeds were grown at 25°C under dark conditions and 50% relative humidity for 2 days, followed by 3 weeks of growth at 25°C under a 16 / 8 h light / dark regime at 50% relative humidity.

[0171] A strain of Pythium aphanidermatum, the causal agent of Pythium wilt of turfgrass, was inoculated onto cornmeal agar medium (CMA, Sigma-Aldrich, India) and incubated statically at 30°C for 3 days. Agar plugs were isolated from the cultured P. aphanidermatum mycelium using a cork borer (0.5 cm diameter), and the isolated agar plugs (2 plugs / pot) were used as inoculum.

[0172] To investigate the efficacy of JCK-1421 culture medium in controlling Pythium disease in turf due to induced resistance, JCK-1421 culture medium and JCK-1421 20 SC were prepared as described in Example 9. JCK-1421 culture medium was diluted with distilled water containing 250 ppm Tween 20 (Duksan Science, Seoul, Korea) to prepare 500-fold, 1,000-fold, and 2,000-fold dilutions, respectively. JCK-1421 20 SC formulations were prepared by diluting the JCK-1421 culture medium to the same dilution ratio. As a positive control, Heritiji (al-Azoxystrobin 50% WG) was diluted 10,000-fold and 1 hour after inoculation, 10 ml of the dilution was drenched in the soil per pot. Three weeks after sowing, creeping bentgrass was treated with JCK-1421 by soil drench treatment with 10 ml of JCK-1421 culture medium and formulation per pot. Four days after treatment, two pathogen agar plugs per pot were inoculated by turning them over so that the mycelium was in contact with the grass surface. After inoculation and treatment, the pots were placed in trays filled with water and covered with a plastic chamber (35 cm wide, 28 cm long, 16 cm high) to maintain 100% relative humidity. The pots were then placed in a constant temperature room at 25°C, exposed to 12 hours of light per day. The experiment was conducted in two replicates, with three pots per treatment. Seven days after inoculation, the diseased area rate was measured, and the control value (%) relative to the untreated control was calculated using the following formula:

[0173] [Formula 6]

[0174] Control value (%) = 100 × [(disease incidence in untreated area - disease incidence in treated area) / disease incidence in untreated area]

[0175] As shown in Figure 10, the control effect of Lysobacter enzymogenes strain JCK-1421 against Pythium disease in turf was tested 7 days after inoculation. The JCK-1421 culture solution showed 71%, 84%, and 59% control compared to the untreated control at 500-, 1,000-, and 2,000-fold dilutions, respectively. The JCK-1421 20 SC formulation showed 53%, 78%, and 56% control at 500-, 1,000-, and 2,000-fold dilutions, respectively. Heritige, used as the control, showed 88% control at a 10,000-fold dilution. JCK-1421 culture broth and JCK-1421 20 SC each showed the highest control efficacy at a 1:1000 dilution. In particular, all dilutions of JCK-1421 culture broth and the 1:1000 dilution of JCK-1421 20 SC formulation used in the experiment demonstrated excellent control of turf Pythium disease with no statistically significant difference compared to the control agent, Heritige. These results confirmed that JCK-1421 exhibits excellent control of turf Pythium disease. Since it lacks direct antifungal activity against P. aphanidermatum, the causative agent of turf Pythium disease, it appears that it controlled turf Pythium disease by inducing resistance in turf. Therefore, it is believed that the induced resistance of JCK-1421 may be useful for the development of turf dollar spot control agents as well as highly effective turf Pythium disease control agents.

[0176] Example 14: Control effect of Lysobacter enzymogenes JCK-1421 strain against rice head blight

[0177] Samkwang rice seeds were used to examine the in vivo control activity of induced resistance to rice head blight by strain JCK-1421. Ten grams of Samkwang rice seeds (Oryza sativa cv. Samkwang) provided by the National Institute of Food Science, Rural Development Administration, Korea, were placed in a plastic container and disinfected with a 2,000-fold dilution of Spotak emulsion (25% prochloraz, Kyungnon Co., Ltd.) for one day. After germination for two days, the seeds were sown at 10 seeds per pot in plastic pots (6 cm diameter, 6.5 cm height) filled with 80% paddy rice soil (heavy soil, Bunon Co., Ltd.). After sowing, the pots were placed on yellow trays with holes at the bottom, submerged in water, and then cultured in a 30°C constant-temperature room, covered with nonwoven seedling cloth. Once the rice seeds germinated to approximately 1 cm, the nonwoven fabric was removed and the seeds were grown under a 16-hour photoperiod for 4 weeks before being transplanted into Wagner pots (top diameter 17.5 cm, bottom diameter 16 cm, height 19.8 cm, NF-5). Seven days before transplanting, a base fertilizer compound (Huksaran 21, Namhae Chemical) was dissolved in water and applied evenly to the flooded paddy soil. The seedlings were then grown in a constant temperature and humidity room and transplanted into the center of Wagner pots filled with 70% paddy soil. Before transplanting, the drainage holes at the bottom of the pots were sealed with silicone plugs, and the pots were lined with polystyrene foam balls (4 cm diameter, 10 balls per pot) and a 14 cm diameter net. After transplanting, the Wagner pots were filled with water and grown in a glass greenhouse (minimum 20–25°C, maximum 30–35°C) for the experiments. 20 days after transplanting into pots, the first fertilizer was applied with Super Arari (14 kg / 10 a, Namhae Chemical), and two months after transplanting into pots, the second fertilizer was applied with NK24 (N:P:K, 24-0-12, Farm Hannon).

[0178] To prepare the rice head blight pathogen inoculum, Fusarium graminearum, the causative agent of rice head blight, was inoculated onto PDA medium and incubated statically at 25°C for 6 days. Five agar plugs (0.1 cm x 0.1 cm) covered with cultured F. graminearum mycelia were cut and inoculated into carboxymethylcellulose medium (CMC, 15 g carboxymethylcellulose (Sigma-Aldrich), 1 g yeast extract, 0.5 g MgSO 7H O, 1 g NH NO , and 1 g KH PO , 1 L distilled water) and incubated at 25°C for 4 days with shaking at 150 rpm. The pathogen culture was then filtered through four layers of gauze to remove mycelia. The filtered spore suspension was analyzed under a microscope (Axio Imager A2, Carl Zeiss, Germany) using a hemocytometer to measure 5 x 10 spores. 5 The spore suspension was centrifuged at 4,000 rpm for 10 minutes at 4°C, and the supernatant was discarded to remove medium components. The spores were then suspended in an equal volume of 0.05% Tween-80 and used as the inoculum for this experiment.

[0179] We investigated the efficacy of JCK-1421 strain in controlling rice Fusarium head blight caused by F. graminearum through induced resistance. JCK-1421 strain culture solution and JCK-1421 20 SC formulation were diluted 1,000-fold and 2,000-fold and sprayed onto rice plants 1 week and 2 weeks before inoculation, respectively, to thoroughly wet the ears and stems. JCK-1421 strain culture solution was diluted in distilled water containing 250 ppm Tween 20 (Duksan Science, Seoul, Korea). As a control, Nonbra (30% Ferimzone + 10% Tricyclazole WP, Kennon), a known fungicide for rice Fusarium head blight, was diluted 2,000-fold and applied 1 day before inoculation. The pathogen was inoculated with a suspension of F. graminearum spores (5 × 10 5The same amount of 1000μg / mL of the fusarium spores was sprayed uniformly on the leaves of each treatment group. During the formulation process, the wetting agent Tween-20 was added to the JCK-1421 strain culture solution at approximately 500μg / mL. Immediately after inoculation, the plants were treated in a moist room using vinyl bags for three days. The experiment was repeated five times per treatment group, and seven days after inoculation, the severity of rice Fusarium head blight disease based on the percentage of diseased area and the control value relative to the untreated group were calculated.

[0180] [Formula 7]

[0181] Control value (%) = 100 × [(disease incidence in untreated area - disease incidence in treated area) / disease incidence in untreated area]

[0182] As shown in Figure 11, the control efficacy of Lysobacter enzymogenes strain JCK-1421 against rice Fusarium head blight was examined 7 days after inoculation. The JCK-1421 culture solution showed 92% and 21% control, respectively, compared to the untreated control at 1:1,000 and 2:1,000 dilutions. The JCK-1421 20 SC formulation showed 34% and 47%, respectively, at 1:1,000 and 2:1,000 dilutions. Nonbra, used as the control, showed 46% control at 1:1,000 dilution. The JCK-1421 culture solution and JCK-1421 20 SC showed the highest control efficacy at 1:1,000 dilution and 2:1,000 dilution, respectively. In particular, a 1,000-fold diluted solution of the JCK-1421 culture solution showed greater control activity than the control agent, the synthetic pesticide Nonbra. As a result, it was confirmed that the JCK-1421 strain exhibits excellent control effects against rice Fusarium head blight. Since it lacks direct antifungal activity against the causative agent of Fusarium head blight, F. graminearum, it can be said that it controls Fusarium head blight by inducing resistance in rice. Therefore, it was confirmed that the resistance-inducing activity of the JCK-1421 strain can be used to develop a highly effective rice Fusarium head blight control agent.

[0183] Example 15. Control effect of Lysobacter enzymogenes JCK-1421 strain on soft rot of Chinese cabbage

[0184] To investigate the in vivo control activity of induced resistance to Chinese cabbage soft rot by strain JCK-1421, Chungwang spring cabbage seeds (Brassica campestris L. ssp. pekinensis (Lour.) Rupr. cv. Chungwang, Sakata Korea) were used. Black square connecting pots (4x8 square; 32 openings, 4.2cm bottom diameter, 5.8cm top diameter, 6.3cm height, Bomnong) were filled with soil and placed under a cutting tray. Chungwang spring cabbage seeds were then sown using tweezers. After sowing, the seeds were grown in a constant temperature room at 25°C under a 16-hour photoperiod for three weeks. After sown, cabbage plants at the five-leaf and six-leaf stages were transplanted into larger pots (7cm diameter, 6cm height) 24 hours before sample treatment.

[0185] To investigate the efficacy of JCK-1421 culture medium in controlling Chinese cabbage soft rot disease through induced resistance, JCK-1421 culture medium and JCK-1421 20 SC were prepared as described in Example 9. JCK-1421 culture medium was diluted with distilled water containing 250 ppm Tween 20 (Duksan Science, Seoul, Korea) to prepare formulations at 500x, 1,000x, 2,000x, and 4,000x concentrations, respectively. JCK-1421 20 SC formulations were prepared by diluting the JCK-1421 culture medium to the same dilution ratio. For the positive control, songbocycline (also known as oxytetracycline 17% WP, SeongBo Chemical) was diluted 1,000x and drenched in the soil at 10 ml per pot one day before inoculation. The treatment with JCK-1421 strain was carried out by transplanting Chinese cabbages at the 5- or 6-leaf stage three weeks after sowing into larger pots, and 24 hours later, 10 ml of the prepared JCK-1421 strain culture solution and formulated pesticide per pot was drenched into the soil.Four days after pesticide treatment, 10 ml of the pathogen was inoculated per pot.

[0186] Pectobacterium carotovorum subsP. carotovorum (Pcc), the causative agent of soft rot of Chinese cabbage, was inoculated into TSA medium and cultured at 30°C for 24 hours. Colonies of the cultured Pcc were harvested into 50 mL conical tubes using distilled water and a cell scraper. The optical density was then measured at 600 nm using a UV-VIS spectrophotometer, and the OD was calculated. 600 The value is 0.1 (1.0x10 7 After adjusting the bacterial count to 100 CFU / mL, 10 ml of the bacterial suspension containing 10 mM magnesium chloride was drenched into the soil per pot. The inoculated plants were placed in a constant temperature, high humidity chamber at 30°C and kept dark for 24 hours. After that, the photoperiod was maintained at 12 hours and the relative humidity was 100%, and the disease severity was examined after 10 days.

[0187] The severity of the disease was expressed as an index from 0 to 5 (0: no symptoms, 1: one or two thin lesions, 2: two or more thin lesions, 3: leaf chlorosis, 4: leaf necrosis, 5: complete withering and death), and the control value (control value, %) relative to the untreated plot was calculated using the following formula.

[0188] [Formula 8]

[0189] Control value (%) = 100 × [(disease incidence rate in untreated area - disease incidence rate in treated area) / disease incidence rate in untreated area]

[0190] As shown in Figure 12, the control effect of Lysobacter enzymogenes strain JCK-1421 against Chinese cabbage soft rot was tested 10 days after inoculation. The JCK-1421 culture solution showed 65%, 25%, 0%, and 38% control compared to the untreated control at 500-, 1,000-, 2,000-, and 4,000-fold dilutions, respectively. The JCK-1421 20 SC formulation showed 70%, 95%, 93%, and 7% control at 500-, 1,000-, 2,000-, and 4,000-fold dilutions, respectively. Songbocycline, used as a control, showed 27% control at a 1,000-fold dilution. Therefore, the JCK-1421 culture medium showed the highest control efficacy at a 500x dilution, and the JCK-1421 20 SC formulation showed the highest control efficacy at a 1,000x dilution. The JCK-1421 culture medium showed higher control activity than the control agent, songbocycline, at 500x and 4,000x dilutions, and the JCK-1421 20 SC formulation showed higher control activity than the control agent, songbocycline, at all treatments except for the 4,000x dilution. These results confirm that JCK-1421 exhibits excellent control efficacy against Chinese cabbage soft rot. Since it lacks direct antibacterial activity against P. carotovorum subs. carotovorum, the causative agent of Chinese cabbage soft rot, it appears that it controlled Chinese cabbage soft rot by inducing resistance in Chinese cabbage. Therefore, we believe that the resistance-inducing activity of JCK-1421 can be used to develop a highly effective Chinese cabbage soft rot control agent.

[0191] Example 15. Control effect of Lysobacter enzymogenes JCK-1421 strain against bacterial wilt of tomato

[0192] Tomato seeds were used to examine the in vivo control of tomato bacterial wilt caused by induced resistance of JCK-1421. Tomato seeds (Lycopersicon esculentum Mill. cv. Seokwang, Farhannong Co., Seoul, Korea) were sown using tweezers in square-shaped connecting pots filled with soil. The seeds were then grown in a 25°C, constant-temperature room under a 16-hour photoperiod for 4 weeks. The plants used for bacterial wilt testing were then transplanted into pots (7 cm diameter, 6 cm height) using soil.

[0193] To prepare the tomato bacterial wilt inoculum, the causative agent of tomato bacterial wilt, Ralstonia solanacearum, was inoculated into TSA medium and incubated at 30°C for 2-3 days. Cultured R. solanacearum colonies were harvested into 50 mL conical tubes using distilled water and a cell scraper. The optical density was then measured at 600 nm using a UV-VIS spectrophotometer, and the OD was calculated. 600 The value is 0.1 (1.0x10 8 The suspension was adjusted to a concentration of 0.1 CFU / mL and the resulting suspension was used as the inoculum.

[0194] To investigate the efficacy of JCK-1421 strain in controlling tomato bacterial wilt caused by R. solanacearum through induced resistance, the JCK-1421 culture solution and formulation (JCK-1421 20 SC) were prepared as described in Example 9. Four weeks after sowing, Sokang tomato seedlings were treated with JCK-1421 culture solution and JCK-1421 20 SC formulation diluted 500-fold, 1,000-fold, 2,000-fold, and 4,000-fold, and 20 mL of each solution was drenched in the soil per pot. The JCK-1421 culture solution was diluted in distilled water containing 250 ppm Tween 20 (Duksan Science, Seoul, Korea). The control pesticide was Sungbocycline (aiOxytetracycline 17% WP, SeongBo Chemical) diluted 2,000-fold and 4,000-fold, and 10 ml per pot was drenched in the soil one day before inoculation. In the case of the JCK-1421 strain culture solution and the formulated pesticide treatment, the R. solanacearum bacterial suspension (OD ) per pot was measured 4 days after pesticide treatment. 600 0.1, 1.0x10 8 Ten mL of the pathogen inoculum (CFU / mL) was inoculated. The inoculated pots were placed in closed yellow trays, filled with water, and exposed to light with a 12-hour photoperiod per day in a 30°C constant temperature room. The experiment was conducted three times with three pots per treatment. Nine days after inoculation, disease symptoms were observed and assessed for severity. Disease severity was assessed using a disease index ranging from 0 to 5 based on the severity of symptoms. The disease index was scored using six levels: 0 = no symptoms, 1 = 1-2 leaf withering, 2 = 3-4 leaf withering, 3 = 5-6 leaf withering, 4 = most leaves withering, and 5 = death. The control efficacy was calculated based on this. The control value (%) relative to the untreated control was calculated using the following formula:

[0195] [Formula 9]

[0196] Control value (%) = 100 × [(disease incidence rate in untreated area - disease incidence rate in treated area) / disease incidence rate in untreated area]

[0197] As shown in Figure 13, the control efficacy of Lysobacter enzymogenes strain JCK-1421 against tomato bacterial wilt was tested 9 days after inoculation. The JCK-1421 culture solution showed 15%, 76%, 59%, and 29% control compared to the untreated control at dilutions of 500:1, 1,000:1, 2,000:1, and 4,000:1, respectively. The JCK-1421 20 SC formulation showed 54%, 66%, 88%, and 78% control at dilutions of 500:1, 1,000:1, 2,000:1, and 4,000:1, respectively. Songvocycline, used as a control, showed 80% and 54% control at dilutions of 2,000:1 and 1,000:1, respectively. The JCK-1421 culture medium showed the highest control efficacy at a 1:1000 dilution, and the JCK-1421 20 SC formulation showed the highest control efficacy at a 2:2000 dilution. However, there were no statistically significant differences between the 1:1000 and 2:2000 dilutions of the JCK-1421 culture medium and the 2:2000 dilution of the control, songbocycline, in any of the dilutions used in the experiment, except for the 5:100 dilution. This confirms the excellent antibacterial activity of the JCK-1421 culture medium at the same level as chemical pesticides. Consequently, JCK-1421 demonstrated excellent control efficacy against tomato bacterial wilt. Since it lacked direct antibacterial activity against R. solanacearum, the causative agent of tomato bacterial wilt, it appears that it induced resistance in tomatoes to control the disease. Therefore, it was confirmed that it will be possible to develop a highly effective agent for controlling bacterial wilt of tomato using the resistance induction activity of the JCK-1421 strain.

[0198] Example 16: Control effect of Lysobacter enzymogenes JCK-1421 strain on bacterial leaf spot of pepper

[0199] Pepper Josaengshintap (Nongwoo Bio, Korea) seeds were used to examine the in vivo control activity of JCK-1421 strain against pepper bacterial leaf spot caused by induced resistance.

[0200] To prepare the plants for the experiment, Josaeng Shintaap (Nongwoo Bio, Korea) chili pepper seeds were sown in plastic cups (6 cm diameter) filled with commercial horticultural soil produced by Bunong (Gyeongju, Korea) and grown under a 12-hour photoperiod. Chili peppers at the 5- or 6-leaf stage were transplanted into 7.5-cm diameter plastic cups 24 hours before treatment.

[0201] To investigate the effectiveness of JCK-1421 strain in controlling pepper bacterial spot disease caused by Xanthomonas euvesicatoria through induced resistance, JCK-1421 culture solution and JCK-1421 20 SC were prepared as described in Example 9. Treatments were applied to pepper seedlings at the 5-leaf or 6-leaf stage 24 hours after transplanting. JCK-1421 culture solution and JCK-1421 20 SC formulations were diluted 2,000-fold and 4,000-fold, and 5 mL of each solution was sprayed onto the leaves of the seedlings. The JCK-1421 culture solution was diluted in distilled water containing 250 ppm Tween 20 (Duksan Science, Seoul, Korea). As a control, Sungbocycline (aiOxytetracycline 17% WP, SeongBo Chemical) was diluted 2,000 times and sprayed on the leaves at 5 mL per seedling one day before inoculation. The prepared JCK-1421 culture solution and the formulation were analyzed for the X. euvesicatoria bacterial suspension (OD ) per pot four days after the treatment. 600 0.1, 1.0x10 7 The pathogen was inoculated by foliar spraying of 5 mL of inoculum (CFU / mL). The inoculated plants were covered with plastic covers and maintained at 100% relative humidity, and placed in a constant temperature and humidity chamber at 25°C and kept in darkness for 24 hours. After a 12-hour photoperiod, the disease severity was examined 11 days later.

[0202] The severity of disease was expressed as an index ranging from 0 to 7, with 0 indicating no symptoms, 1 indicating no symptoms, 2 indicating necrotic lesions on some leaflets, 3 indicating lesions on some leaves, 4 indicating lesions on many leaves, 5 indicating lesions on many leaflets, 6 indicating severe lesions and leaf drop, and 7 indicating plant death. The disease control value was calculated using the following formula:

[0203] [Formula 10]

[0204] Control value (%) = 100 × [(disease incidence rate in untreated area - disease incidence rate in treated area) / disease incidence rate in untreated area]

[0205] As can be seen in Figure 14, the control effect of Lysobacter enzymogenes strain JCK-1421 against pepper bacterial leaf spot was examined 11 days after inoculation. The culture medium of JCK-1421 reduced the incidence of pepper bacterial leaf spot, and the treatment with 4,000x JCK-1421 20 SC formulation (control value: 60%) showed a control effect similar to that of the control agent songbocycline (control value: 55%). The present invention confirmed that JCK-1421 induces resistance in pepper and has excellent disease control activity against pepper bacterial leaf spot caused by X. euvesicatoria.

[0206] Example 17: Control effect of Lysobacter enzymogenes JCK-1421 strain on apple fire blight

[0207] To examine the in vivo control activity of JCK-1421 strain against apple fire blight by induced resistance, apple seedlings (M9 and Chinese pearleaf crabapple, 15±5 cm in height) were used.

[0208] Five mL of the sample was sprayed onto the leaves of apple seedlings, followed by inoculation of 5 mL of the apple fire blight pathogen (Erwinia amylovora TS3128). To examine the efficacy of the JCK-1421 strain in controlling apple fire blight caused by E. amylovora TS3128 through induced resistance, a JCK-1421 formulation, JCK-1421 20 SC, was prepared as described in Example 9. The JCK-1421 formulation was applied to apple seedlings approximately 15 cm tall by foliar spraying 5 mL of a 1:1000 diluted JCK-1421 20 SC formulation per seedling. As a control agent, streptomycin sulfate was diluted to 100 μg / ml, and Seripel biopesticide (active ingredient: Bacillus amyloliquefaciens MBI600 (11%) WP, Farm Hannon, Korea) was diluted 2,000 times, the usual concentration, and 5 mL of each was sprayed on the leaves of each seedling. The JCK-1421 strain formulation and the control agent treatment were sprayed on the leaves twice, 10 days and 3 days before inoculation with the pathogen. Three days after the second agent treatment, E. amylovora TS3128 bacterial suspension (OD ) was applied to each apple seedling as an inoculum for inducing apple fire blight. 600 0.3, 3.0x10 7 Inoculation was performed by foliar spray of 5 mL (CFU / mL). Streptomycin sulfate was applied once one day before inoculation, and seripel was applied twice, 10 and 3 days before inoculation. The inoculated plants were covered with a plastic cover to maintain 100% relative humidity and placed in a constant temperature and humidity room at 25°C in the dark for two days, after which the disease severity was examined while maintaining a 12-hour photoperiod and 75% relative humidity. Each treatment was repeated three times, each consisting of three pots, and the same experiment was repeated three times to evaluate efficacy. The disease severity index was examined in four groups. The disease severity was also divided into the following indices:

[0209] Index 0 = no symptoms

[0210] Index 1 = partial necrosis of the stem tip, which is the growing meristem of the stem

[0211] Index 2 = complete necrosis of the stem end

[0212] Index 5 = petiole necrosis of terminal leaves

[0213] Index 10 = necrosis of leaf and stem petioles

[0214] The disease control value was calculated using the following formula:

[0215] [Formula 11]

[0216] Control value (%) = (disease incidence in untreated area - disease incidence in treated area) / disease incidence in untreated area × 100%

[0217] As shown in Figure 15, the control effect of Lysobacter enzymogenes strain JCK-1421 against apple fire blight was examined on Chinese pearleaf crabapple seedlings 10 days after inoculation. A 1,000-fold dilution of JCK-1421 20 SC formulation demonstrated 90% control compared to the untreated control, demonstrating excellent disease control activity. The control agent, streptomycin sulfate (100 ppm), demonstrated 100% control.

[0218] Furthermore, as shown in Figure 16, the control effect of Lysobacter enzymogenes strain JCK-1421 against apple fire blight was examined on M9 seedlings 7 and 10 days after inoculation. The results showed that treatment with a 1:1000 diluted solution of JCK-1421 20 SC formulation demonstrated 100% control compared to the untreated control at all times 7 and 10 days after inoculation. Therefore, treatment with a 1:1000 diluted solution of JCK-1421 20 SC formulation demonstrated significantly superior disease control activity compared to the control biological pesticide, Seripel (48% control 7 days after inoculation, 40% control 10 days after inoculation). These results demonstrate that pre-treatment of JCK-1421 prior to inoculation induces resistance, resulting in excellent control of apple fire blight. Therefore, it was confirmed that it will be possible to develop a highly effective agent for controlling fruit tree stem disease using the control activity of the JCK-1421 strain.

[0219] Example 18: Control effect of Lysobacter enzymogenes JCK-1421 strain against tomato root-knot nematode disease

[0220] To investigate the in vivo control activity of tomato root-knot nematode disease induced by strain JCK-1421, 'Samkang' tomato seeds were used. Tomato seeds (Lycopersicon esculentum Mill. cv. Seokwang, Farhannong Co., Seoul, Korea) were sown using tweezers in square-shaped connecting pots filled with soil. They were then grown for four weeks in a 25°C, constant-temperature greenhouse under a 16-hour photoperiod. Plants for root-knot nematode disease control were then transplanted into pots (12 cm diameter, 11 cm height) using a soil mixture (sand:soil, 1:1, v / v).

[0221] To inoculate tomato plants with root-knot nematodes, the pathogenic sweet potato root-knot nematode (M. incognita) was grown on the susceptible species, Sokwang tomato (Lycopersicon esculentum Mill. cv. Seokwang), for 2–3 months in a constant temperature room at 25°C. First, artificially infected tomato roots were washed with running water and cut into 1 cm pieces and placed in a grinder (HM-2100S; Hanil, Gimpo, Korea). After grinding for 1 minute in 1% sodium hypochlorite (NaOCl, v / v), root-knot nematode eggs were collected using a 45 μm sieve followed by a 25 μm sieve. The collected root-knot nematode eggs were adjusted to 8,000 eggs per 10 mL of egg suspension and used as the inoculum.

[0222] To investigate the efficacy of induced resistance of JCK-1421 against tomato root-knot nematode disease caused by M. incognita, the JCK-1421 culture solution and formulation (JCK-1421 20 SC) were prepared as described in Example 9. Four weeks after sowing, Sokang tomato seedlings were treated with JCK-1421 culture solution and JCK-1421 20 SC diluted 500-fold, 1,000-fold, and 2,000-fold, and 20 mL of each solution was drenched in the soil per pot. As a control, Terranova (a 1.68% Abamectin SC, Shinzenta Korea), a known root-knot nematode treatment agent, was diluted 5,000-fold (its usual concentration) and drenched in the soil at 10 mL per pot one day before inoculation. Four days after treatment with the JCK-1421 culture medium and the formulated fungicide, 10 mL of M. incognita nematode egg suspension was inoculated into each pot as the pathogenic nematode. The inoculated pots were placed in a constant temperature room at 25°C under a 12-hour photoperiod per day. The experiment was repeated three times, with two pots per treatment. Six weeks after inoculation, the degree of gall formation on the tomato roots was evaluated using a galling index ranging from 0 to 5. The galling index (GI) was calculated using six levels: 0 = 0–10% gall formation, 1 = 11–20%, 2 = 21–50%, 3 = 51–80%, 4 = 81–90%, and 5 = 91–100% (Kim et al., 2018). Egg masses were measured after staining tomato roots by immersing them in 0.0015% phloxine B (Daejong Hwa Kim Co., Ltd.) for 1 hour, and the control efficacy was calculated based on this data.

[0223] [Formula 12]

[0224] Control value (%) = 100 × [(disease incidence rate in untreated area - disease incidence rate in treated area) / disease incidence rate in untreated area]

[0225] As shown in Figure 17, the control efficacy of Lysobacter enzymogenes strain JCK-1421 against tomato root-knot nematode disease was examined 6 weeks after inoculation. The JCK-1421 culture solution and JCK-1421 20 SC, at 500-fold, 1,000-fold, and 2,000-fold dilutions, demonstrated gall index (%) control of 65%, 39%, and 17%, and 58%, 61%, and 70%, respectively, compared to the control. The egg mass count (%) control values ​​were 72%, 39%, and 0%, and 74%, 67%, and 78%, respectively. Terranova, used as a control, demonstrated gall index (%) and egg mass count (%) control values ​​of 87% and 96%, respectively, at a 5,000-fold dilution. Therefore, JCK-1421 20 SC formulation showed the highest egg hatch inhibition and gall formation inhibition effects at a 2,000-fold dilution. In particular, in the treatments with a 500-fold dilution of JCK-1421 culture solution and all treatments with JCK-1421 20 SC formulation, it showed excellent tomato root-knot nematode control activity that was not statistically significantly different from the control, Terranova. Therefore, it can be concluded that JCK-1421 did not have direct nematicidal activity against M. incognita, the nematode that causes tomato root-knot nematode disease, but rather induced resistance in tomatoes to control the disease. Therefore, it was confirmed that the resistance-inducing activity of JCK-1421 could be used to develop a highly effective tomato root-knot nematode control agent.

[0226] Example 19. Control effect of Lysobacter enzymogenes JCK-1421 strain against pine wilt disease

[0227] To investigate the in vivo control effect of induced resistance of JCK-1421 strain against pine wilt disease, a seedling test was conducted using two-year-old seedlings (approximately 5.5 mm in diameter and approximately 35-45 cm tall from the soil) of two different susceptible tree species: Japanese red pine (Pinus densiflora) and Japanese black pine (Pinus thunbergii).

[0228] The JCK-1421 culture solution for sample treatment was prepared as described in Example 9. The prepared JCK-1421 culture solution was dissolved in an aqueous solution containing 250 μg / mL Tween 20 to an optical density of approximately 0.8, and then placed in a fine sprayer and sprayed twice, 5 mL per pine tree, on the leaves two weeks and one week before inoculation with the pinewood nematode. For the positive control, the nematicide EB (emamectin benzoate) was dissolved at a level of 10 mg / mL in an aqueous solution containing 10% methanol, and 100 μL of this solution was injected into the trunk one week before inoculation with the pinewood nematode.

[0229] To induce pine wilt disease in Japanese red pine and Japanese black pine seedlings, pine wood nematodes were cultured as follows. First, Botrytis cinerea, the pine wood nematode's food source, was inoculated and cultured statically in an incubator at 25°C for 7 days. Pine wood nematodes (Bursaphelenchus xylophilus, National Academy of Forestry Science) were inoculated onto the cultured Botrytis cinerea and then cultured statically in an incubator at 25°C for 7 days. The cultured pine wood nematodes were harvested using the Baermann funnel method and counted under a light microscope to adjust the number to 20,000 / ml. After preparing the inoculum, approximately 1cm of the wood of the red pine and black pine trees treated with the JCK-1421 culture medium and the control was incised with a sterilized blade down to the deep endobarbital layer. A 0.5cm x 1cm strip of sterilized absorbent cotton was inserted into the incision and 2,000 isolated pine wood nematodes were inoculated in 100μL portions (inoculation one week after treatment with the JCK-1421 culture medium and the positive control). The inoculated area was sealed with parafilm to prevent drying (bark peeling inoculation method). The red pine seedlings were observed for the extent of pine wood nematode inoculation six weeks after inoculation, and the black pine seedlings were observed for the extent of pine wood nematode inoculation four weeks after inoculation.

[0230] [Table 5]

[0231] As can be seen from Table 5 and Figure 18, the control effect of Lysobacter enzymogenes strain JCK-1421 against pine wilt disease in Japanese black pine was examined after inoculation with the pathogen. As a result, 6 weeks after inoculation, the culture solution of JCK-1421 strain showed excellent disease control activity, with a pine wilt disease control rate of 57%.

[0232] [Table 6]

[0233] Furthermore, as can be seen from Table 6 and Figure 19, the control effect of Lysobacter enzymogenes strain JCK-1421 against pine wilt disease in Japanese red pine was examined 4 weeks after inoculation with the pathogen. The culture medium of JCK-1421 strain demonstrated excellent disease control activity, with a pine wilt disease control rate of 79%. Therefore, it can be said that JCK-1421 strain did not have direct nematicidal activity against B. xylophilus, the nematode that causes pine wilt disease, but rather induced resistance in Japanese red pine and Japanese black pine to control pine wilt disease. This confirms that the resistance-inducing activity of JCK-1421 strain may be utilized to develop highly effective pine wilt disease control agents in the future. [Industrial Applicability]

[0234] The present invention relates to the Lysobacter enzymogenes JCK-1421 strain, a composition for controlling plant fungal diseases, bacterial diseases or nematode diseases comprising the strain, a culture thereof, or an extract thereof, a method for producing the composition, and a method for controlling plant fungal diseases, bacterial diseases or nematode diseases using the composition. [Accession number]

[0235] Name: Lysobacter enzymogenes JCK-1421 Accession number: KCTC 15126BP JPEG2025537567000008.jpg215170

Claims

1. Lysobacter enzymogenes strain JCK-1421, deposited under accession number KCTC 15126BP, has antifungal, antibacterial and antinematode activities.

2. A composition for controlling plant fungal diseases, bacterial diseases or nematode diseases, comprising a culture broth or an extract thereof of Lysobacter enzymogenes JCK-1421 strain deposited under accession number KCTC 15126BP.

3. The fungi include Fusarium graminearum, Fusarium oxysporum f. sp. cucumerinum (Fusarium oxysporum f.sp.cucumerinum), Rhizoctonia solani AG2-2 Brown patch (Rhizoctonia solani AG2-2 Brown patch), Rhizoctonia solani AG2-2 Large patch (AG2-2 Large patch), Rhizoctonia solani AG-4 (Rhizoctonia solani AG-4), Sclerotinia homoeocarpa (Sclerotinia homoeocarpa), and Pythium aphanidermatum (Pythium aphanidermatum). The composition for controlling fungal diseases, bacterial diseases, or nematodes of plants according to claim 2, which is one or more selected from the group consisting of:

4. 3. The composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases according to claim 2, wherein the bacterium is one or more selected from the group consisting of Ralstonia solanacearum, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas euvesicatoria, and Erwinia amylovora.

5. 3. The composition for controlling a plant fungal disease, bacterial disease, or nematode disease according to claim 2, wherein the nematode is one or more species selected from the group consisting of Meloidogyne incognita and Bursaphelenchus xylophilus.

6. A method for producing a composition for controlling plant fungal diseases, bacterial diseases or nematode diseases, comprising a culturing step of culturing the Lysobacter enzymogenes JCK-1421 strain deposited under accession number KCTC 15126BP.

7. The fungi include Fusarium graminearum, Fusarium oxysporum f. sp. cucumerinum (Fusarium oxysporum f.sp.cucumerinum), Rhizoctonia solani AG2-2 Brown patch (Rhizoctonia solani AG2-2 Brown patch), Rhizoctonia solani AG2-2 Large patch (AG2-2 Large patch), Rhizoctonia solani AG-4 (Rhizoctonia solani AG-4), Sclerotinia homoeocarpa (Sclerotinia homoeocarpa), and Pythium aphanidermatum (Pythium aphanidermatum). The method for producing a composition for controlling fungal diseases, bacterial diseases or nematodes of plants according to claim 6, which is at least one selected from the group consisting of.

8. 7. The method for producing a composition for controlling plant fungal diseases, bacterial diseases, or nematode diseases according to claim 6, wherein the bacterium is one or more selected from the group consisting of Ralstonia solanacearum, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas euvesicatoria, and Erwinia amylovora.

9. 7. The method for producing a composition for controlling a plant fungal disease, bacterial disease or nematode disease according to claim 6, wherein the nematode is one or more species selected from the group consisting of Meloidogyne incognita and Bursaphelenchus xylophilus.

10. A method for controlling fungal, bacterial or nematode diseases in plants, comprising a step of treating the plants with a composition comprising a culture broth of the Lysobacter enzymogenes JCK-1421 strain deposited under accession number KCTC 15126BP or an extract thereof.

11. 11. The method for controlling plant fungal diseases, bacterial diseases, or nematode diseases according to claim 10, wherein the composition treatment step is carried out by one or more methods selected from the group consisting of spraying, soil drench, immersion, poisoning, fumigation, and seed treatment.

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