Bacillus subtilis jck-1398 strain inducing resistance in various plants, composition for preventing and treating pine wood nematode disease using the same, and prevention method

Abstract

The present application relates to Bacillus subtilis JCK-1398 strain (accession number KCTC 14084BP) having an induced resistance activity against pine trees and various plants, a pesticide or antibacterial agent composition comprising the same as an effective ingredient, a composition for controlling plant diseases or pests, and a control method using the same, and it has been confirmed through experiments that the Bacillus subtilis JCK-1398 strain of the present application has a control activity against pests, nematodes, and pathogens causing various plant diseases by inducing resistance in a host. Accordingly, the Bacillus subtilis JCK-1398 strain of the present application can be effectively used to control related plant diseases, and can be sprayed on a wide range of areas through foliar spraying, and thus is expected to prevent the spread of pine wood nematode disease at a low cost.

Description

[0001] This application was completed under the support of the Korea Forest Service under the subject number FE0702-2016-02, the research management agency of which is the Korea National Forest Service, the research project name is "Service Research Project", the research subject name is "Development and identification of an environmentally friendly pine wood nematode disease control agent using induced resistance", and the research period is from April 19, 2019 to November 29, 2019.

[0002] This patent application claims priority to Korean Granted Patent No. 10-2020-0017232, filed on February 12, 2020, with the Korean Intellectual Property Office, the disclosure of which is incorporated herein by reference.

[0003] The present application relates to Bacillus subtilis JCK-1398 strain inducing resistance in pine trees and various plants, a composition for preventing plant diseases using the same, and a prevention method. BACKGROUND

[0004] Pine wood nematode disease caused by Bursaphelenchus xylophilus causes a serious disaster to pine trees worldwide, and in recent years, with the change in the forest ecosystem environment caused by climate change, it has shown a complex state and rapidly spread.

[0005] It is known that the main cause of pine wood nematode disease is pine wood nematode, which is a difficult-to-control forest pest that causes pine trees to die as a result of a complex interaction between hosts, pathogens, vector insects, and environmental factors, and once infected, it cannot be treated, and almost all pine trees die.

[0006] Various methods have been proposed to prevent such pine wood nematode disease, and the prevention method by trunk injection is almost the only method as a preventive method. However, in the case of trunk injection, the currently widely used abamectin and emamectin benzoate have a short duration of efficacy of only two years, and the injection period is also limited, and Japan's Green Guard is limited in widespread use due to high price.

[0007] ​Also, in the case of trunk injection, not only is there a problem of the price of the agent, but also there is a problem in that it is difficult to prevent a wide range of areas, and in the case of pine forests in high mountainous areas, it is difficult to access, so this method cannot be an effective method. Furthermore, Kuroda and Kenmochi (2016, Pine Wilt Disease Conference - International Union of Forest Research Organizations (IUFRO)) warned that repeated trunk injection of nematicides for the control of pine wood nematode disease is a cause of killing uninfected pines.

[0008] Therefore, there is an urgent need to develop an environmentally friendly agent that can effectively control a wide range of areas through soil irrigation or foliar spraying, etc. SUMMARY

[0009] PROBLEM TO BE SOLVED BY THE INVENTION

[0010] The present inventors tried to develop a microbial agent that induces resistance in pines and various plants. As a result, it was found that the novel Bacillus subtilis JCK-1398 strain of the present invention has a control effect on various plant pathogens including nematodes that cause pine wood nematode disease, and this effect is caused by the resistance-inducing activity of the strain, thereby completing the present invention.

[0011] Therefore, the object of the present invention is to provide Bacillus subtilis JCK-1398 strain having the accession number KCTC 14084BP, which activates the induced resistance of plants against disease pests.

[0012] Still another object of the present invention is to provide an insecticide or antibacterial agent composition comprising one or more selected from the group consisting of Bacillus subtilis JCK-1398 strain, culture of the above strain, concentrate of the above culture, dried product of the above culture, culture supernatant of the above strain, and combinations thereof, as an effective ingredient.

[0013] Another object of the present invention is to provide a composition for controlling plant diseases or pests, comprising one or more selected from the group consisting of Bacillus subtilis JCK-1398 strain, culture of the above strain, concentrate of the above culture, dried product of the above culture, culture supernatant of the above strain, and combinations thereof, as an effective ingredient.

[0014] Still another object of the present application is to provide a method for controlling plant diseases or pests using one or more selected from the group consisting of a Bacillus subtilis JCK-1398 strain, a culture of the above strain, a concentrate of the above culture, a dried product of the above culture, a culture supernatant of the above strain, and combinations thereof.

[0015] Means for solving the problem

[0016] The present inventors have attempted to develop a microbial preparation that induces resistance in pine trees and various plants. As a result, the novel Bacillus subtilis JCK-1398 strain of the present application has been found to have an induced resistance activity against nematodes and pathogens that cause diseases in various plants, including pine wood nematodes.

[0017] The present application relates to an insecticide or antibacterial agent composition and a composition for controlling plant diseases or pests, which contain a Bacillus subtilis JCK-1398 strain having accession number KCTC 14084BP that activates induced resistance of plants against disease pests, a culture of the above strain, etc., as an effective ingredient.

[0018] Hereinafter, the present application will be described in more detail.

[0019] One aspect of the present application relates to a Bacillus subtilis JCK-1398 strain having accession number KCTC 14084BP.

[0020] The above strain is characterized by comprising a gyrA base sequence of SEQ ID NO: 33.

[0021] The base sequence of the above SEQ ID NO: 33 has 99% homology with CP021892, which is a test strain, and it was identified that there is a 1% difference between the two strains, and thus it was proved to be a novel strain.

[0022] The gyrA (Gyrase A subunit) base sequence of the above Bacillus subtilis JCK-1398 strain was confirmed through a Blast search in the GenBank database and the NCBI database.

[0023] The above-mentioned new strain was named Bacillus subtilis JCK-1398 strain, and was deposited in the Korean Collection for Type Cultures on December 19, 2019, and was given a deposit number KCTC 14084BP.

[0024] The above-mentioned Bacillus subtilis JCK-1398 strain can be isolated and identified from a tomato plant body, and the cell properties of the above-mentioned strain are as follows:

[0025] As a rod-shaped cylindrical bacillus, it has a flagellum and moves actively, and forms a round or oval spore in the center of the cell. It develops well in a general incubator and forms a large grayish white colony with a radial pattern around it. Since it has spores, it has very strong resistance to drying or high temperatures, and the cell, as a gram-positive bacterium containing glycogen, can decompose many carbohydrates to produce acid. D-glucose, L-arabinose, sucrose, and the like are used as energy sources.

[0026] According to an embodiment of the present application, the induced resistance activity of the above-mentioned Bacillus subtilis JCK-1398 strain was confirmed through experiments using pine trees and various plant bodies (FIGS. 3 to Figure 9 ).

[0027] The above-mentioned Bacillus subtilis JCK-1398 strain has a disease pest control activity through induced resistance activity.

[0028] The above-mentioned Bacillus subtilis JCK-1398 strain has a plant disease or pest control activity caused by a plant pathogenic nematode, a pest, or a plant pathogenic bacterium and fungus (mold).

[0029] The above-mentioned nematode can be Bursaphelenchus xylophilus, but is not limited thereto.

[0030] The above-mentioned pest can be Aculops lycopersici, but is not limited thereto.

[0031] The above-mentioned bacterium and fungus can be Xanthomonas euvesicatoria and / or Sclerotinia homoeocarpa, but is not limited thereto.

[0032] The plant disease can be pine wood nematode disease, pepper bacterial spot disease, and / or dollar spot disease, but is not limited thereto.

[0033] Accordingly, the Bacillus subtilis JCK-1398 strain of the present application induces resistance in a host, thereby having plant disease control activity by inhibiting the growth of plant pathogenic nematodes, bacteria, fungi, and pests.

[0034] Also, if the above-mentioned Bacillus subtilis JCK-1398 strain is combined with an existing resistance-inducing substance, a synergistic effect is induced, and thus can be used for environmentally friendly control of plant diseases.

[0035] The resistance-inducing substance used in combination with the above-mentioned Bacillus subtilis JCK-1398 strain can be Acibenzolar-S-methyl (ASM) and / or Methyl salicylate (MeSA), but is not limited thereto.

[0036] Also, when the above-mentioned Bacillus subtilis JCK-1398 strain is combined with an existing synthetic fungicide (synthetic pesticide), a synergistic effect is induced, and thus even if a synthetic pesticide is used at a concentration lower than the usual concentration, the plant disease control effect is similar to or higher than that of the usual concentration. Thus, it can reduce the amount of use of the usual concentration of the synthetic fungicide, and can be used for environmentally friendly control of plant diseases.

[0037] The synthetic pesticide used in combination with the above-mentioned Bacillus subtilis JCK-1398 strain can be tebuconazole, iprodione, fludioxonil, benomyl, and / or difenoconazole, but is not limited thereto.

[0038] Still another aspect of the present application relates to an insecticide or antibacterial agent composition, which comprises one or more selected from the group consisting of the Bacillus subtilis JCK-1398 strain having the accession number KCTC 14084BP, a culture of the above-mentioned strain, a concentrate of the above-mentioned culture, a dried product of the above-mentioned culture, a culture supernatant of the above-mentioned strain, and combinations thereof.

[0039] Therefore, the present application can provide an insecticide or antibacterial agent microbial preparation including one or more selected from the group consisting of Bacillus subtilis JCK-1398 strain, culture of the above strain, concentrate of the above culture, dried product of the above culture, culture supernatant of the above strain, and combinations thereof, and can provide an insecticidal or antibacterial method using the above microbial preparation.

[0040] The above microbial preparation can be formulated in a conventional method, and can be prepared in a dry powder form or a liquid fertilizer form. Specifically, the microbial preparation of the present application can be prepared in a liquid form, and can be used by adding an extender to be in a powder form, and can be formulated and granulated. However, the dosage form is not particularly limited.

[0041] In the present application, the above microbial preparation can be prepared by adding an additive, an extender, a nutrient, or the like to the strain or culture thereof. In this case, as the additive, one or more selected from the group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkylsulfosuccinate, sodium alkylarylsulfonate, polyoxyethylene alkylphenyl ether, sodium tripolyphosphate, polyoxyethylene alkylarylphosphate ester, polyoxyethylene alkylaryl ether, polyoxyethylene alkylaryl polymer, polyoxyalkyl ketone alkylphenyl ether, polyoxyethylene nonylphenyl ether, sulfonated formaldehyde naphthyl sodium, triton 100, and tween 80 can be used, as the extender and the nutrient, one or two or more selected from the group consisting of skim milk (medium), soybean powder, rice, wheat, loess, diatomite, bentonite, dextrin, glucose, and starch can be used, and as the disintegrant, one or more selected from the group consisting of bentonite, talc, diatomite, kaolin, and calcium carbonate can be used.

[0042] Further, the present application provides a method of inducing resistance in a plant body by treating soil or a plant with the above microbial preparation. In this case, as the treatment method, a method commonly performed, i.e., spraying (e.g., spraying, atomizing, sprinkling, powder spraying, granule spraying, dusting, conventional application, or the like), soil application (e.g., mixing, watering, or the like), surface use (e.g., coating, daubing, wrapping, or the like), immersion, poisoning, fumigation, or the like can be performed. The amount of use can be appropriately determined depending on the dosage form, the damage situation, the use method, the use site, or the like.

[0043] In the present application, the effective amount of the microorganism contained in the preparation treated according to the above method can be 1 to 1 x 10 2 ) per cultivated area (m 100microorganism. Also, the effective amount of the microorganism contained in the preparation by the spray treatment in the above method can be included at a concentration of 1 to 1 x 10 100 microorganism per 1 ml. The effective amount of the microorganism contained in the composition by the dipping treatment can be included at a concentration of 1 x 10 100 microorganism per 1 ml.

[0044] Another aspect of the present application relates to a composition for controlling plant disease or pests, comprising one or more selected from the group consisting of Bacillus subtilis JCK-1398 strain having accession number KCTC 14084BP, a culture of the above strain, a concentrate of the above culture, a dried product of the above culture, a culture supernatant of the above strain, and combinations thereof.

[0045] Each of the above compositions of the present application can contain, in addition to the above strain, culture thereof, concentrate of the culture, dried product of the culture, and / or culture supernatant of the above strain as an effective ingredient, a culture containing bacterial cells, an extract of the bacterial cells, a concentrate, a concentrate, a dried product thereof, and, if necessary, a diluent, a dilution, etc., and can contain all states of substances obtained by treating with a culture solution, a culture.

[0046] The culture method, extraction method, separation method, concentration method, drying method, dilution method, etc. of the above strain are not particularly limited.

[0047] The culture medium for culturing the above strain generally contains milk proteins such as skim milk, whey, casein, sugars, yeast extract, etc., and as a culture method, various ordinary aerobic or anaerobic methods can be appropriately used.

[0048] After culturing the above strain, the culture or its supernatant can be concentrated, dried, or diluted, if necessary.

[0049] Also, the supernatant and the bacterial cells of the culture can be separated by centrifugation or membrane separation to recover the bacterial cells in a concentrated state. In addition, the bacterial cells can be subjected to ultrasonic treatment or enzyme treatment, etc. to extract components in the bacterial cells, the culture or its supernatant, or the bacterial cells or its extract, etc. can be dried. These can be used as effective ingredients of the above compositions of the present application.

[0050] In consideration of the complexity of the present specification, the repetitive content of the above strain and each composition containing the same is omitted.

[0051]

Effects of the Invention

[0052] The present application relates to Bacillus subtilis JCK-1398 strain (accession number KCTC 14084BP) having an induced resistance activity in pine trees and various plants, a pesticide or antibacterial agent composition comprising the same as an effective ingredient, a composition for controlling plant diseases or pests, and a control method using the same, and it has been confirmed through experiments that the Bacillus subtilis JCK-1398 strain of the present application has a control activity on pests, nematodes, and pathogens causing various plant diseases by inducing resistance in a host. Accordingly, the Bacillus subtilis JCK-1398 strain of the present application can be effectively used to control related plant diseases, and can be sprayed on a wide range of areas through foliar application, and thus is expected to prevent the spread of pine wood nematode disease at a low cost. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 A graph for confirming the expression of an induced resistance-related gene caused by the Bacillus subtilis JCK-1398 strain of an embodiment of the present application.

[0054] Figure 2 A graph showing the results of systematic analysis of the gyrA base sequence of the Bacillus subtilis JCK-1398 strain of an embodiment of the present application.

[0055] Figure 3a A graph for confirming the pine wood nematode disease control effect of the Bacillus subtilis JCK-1398 strain of an embodiment of the present application, in which the seedling identification results of the JCK-1398 strain culture solution on Korean pine seedlings are shown through a disease progression curve.

[0056] Figure 3b A graph for confirming the pine wood nematode disease control effect of the Bacillus subtilis JCK-1398 strain of an embodiment of the present application, in which Korean pine seedlings treated with the JCK-1398 strain culture solution (a), an untreated area (b), and an un-inoculated group (c) are shown.

[0057] Figure 4a A graph for confirming the pine wood nematode disease control effect of the Bacillus subtilis JCK-1398 strain of an embodiment of the present application, in which the pine wood nematode disease control effects of the culture solution, culture filtrate, and cell fraction of the JCK-1398 strain at O.D 600 = 0.8 are shown.

[0058] Figure 4bFIG. 1 is a graph for confirming the pine wood nematode disease control effect of the Bacillus subtilis JCK-1398 strain according to an embodiment of the present application, in which red pine seedlings treated with the JCK-1398 strain culture solution (a), the JCK-1398 strain culture filtrate (b), the JCK-1398 strain cell fraction (c), an untreated area (d), and an un-inoculated group (e) are shown.

[0059] Figure 5a FIG. 2 is a graph for confirming the pine wood nematode disease control effect of the treatment time of the Bacillus subtilis JCK-1398 strain culture solution according to an embodiment of the present application, in which the seedling identification results of the JCK-1398 strain culture solution on red pine seedlings are shown by a disease progress curve.

[0060] Figure 5b FIG. 3 is a graph for confirming the pine wood nematode disease control effect of the treatment time of the Bacillus subtilis JCK-1398 strain culture solution according to an embodiment of the present application, in which the JCK-1398 strain culture solution control effect after inoculation of pine wood nematodes for 60 days is shown.

[0061] Figure 5c FIG. 4 is a graph for confirming the pine wood nematode disease control effect of the treatment time of the Bacillus subtilis JCK-1398 strain culture solution according to an embodiment of the present application, in which red pine seedlings treated with the JCK-1398 strain culture solution 2 weeks before-1 week before (a), 3 weeks before-1 week before (b), 4 weeks before-1 week before (c), an untreated area (d), and an un-inoculated group (e) are shown.

[0062] Figure 6 FIG. 5 is a graph for confirming the pine wood nematode disease control effect of the treatment concentration of the Bacillus subtilis JCK-1398 strain culture solution according to an embodiment of the present application.

[0063] Figure 7a FIG. 6 is a graph for confirming the pine wood nematode disease control effect of the Bacillus subtilis JCK-1398 strain according to an embodiment of the present application, in which the seedling identification results of the JCK-1398 strain culture solution on mature red pines are shown by a disease progress curve.

[0064] Figure 7b FIG. 7 is a graph for confirming the pine wood nematode disease control effect of the Bacillus subtilis JCK-1398 strain according to an embodiment of the present application, in which the control effect of the Bacillus subtilis JCK-1398 strain is shown by first and second field tests.

[0065] Figure 7c A graph for confirming the pine wood nematode disease control effect of Bacillus subtilis JCK-1398 strain of an embodiment of the present application, in which mature Korean pines treated with 50-fold diluted JCK-1398 strain spray-dried powder a), milbemycin oxalate b), and an un-inoculated group c) are shown.

[0066] Figure 8 A graph for confirming the pine wood nematode disease control effect of Bacillus subtilis JCK-1398 20% SC (suspension concentrate) formulation and ASM (azoxystrobin) and MeSA (exogenous salicylic acid methyl ester) mixture as a resistance inducing substance of an embodiment of the present application.

[0067] Figure 9 A graph for confirming the pine wood nematode disease control effect of Bacillus subtilis JCK-1398 20% SC formulation and JCK-1398 10% SC formulation at various concentrations of an embodiment of the present application.

[0068] Figure 10a A graph for confirming the pine wood nematode disease control effect of Bacillus subtilis JCK-1398 10% SC formulation on 30-year-old pine trees of an embodiment of the present application, in which the practical evaluation results using unmanned helicopters are shown by disease progress curves.

[0069] Figure 10b A graph for confirming the pine wood nematode disease control effect of Bacillus subtilis JCK-1398 10% SC formulation on 30-year-old pine trees of an embodiment of the present application, in which the incidence rate after inoculation of nematodes for 6 months is shown.

[0070] Figure 11 A graph for confirming the pepper bacterial spot disease control effect of Bacillus subtilis JCK-1398 strain culture solution of an embodiment of the present application.

[0071] Figure 12 A graph for confirming the turf dollar spot disease control effect of Bacillus subtilis JCK-1398 strain culture solution of an embodiment of the present application.

[0072] Figure 13FIG. 1 is a graph showing the tomato spider mite control effect of a 20% SC preparation of the Bacillus subtilis JCK-1398 strain according to an embodiment of the present application.

DETAILED DESCRIPTION

[0073] The present application relates to a Bacillus subtilis JCK-1398 strain, which has a preservation number of KCTC 14084BP, and activates the induced resistance of a plant against a disease insect.

[0074]

Embodiment of the Invention

[0075] Hereinafter, the present application will be described in more detail by examples. These examples are only for illustrating the present application, and the scope of the present application should not be construed as being limited by these examples, which will be apparent to those skilled in the art.

[0076]

Example 1. Screening of a strain inducing induced resistance

[0077] In order to find a strain inducing induced resistance against pine wood nematode disease, more than 500 plant-derived microbial strains were isolated from various plants. The strain inducing induced resistance (25 strains) was screened from more than 500 plant-derived microbial strains isolated from Arabidopsis thaliana transformed with a vector labeled with GUS (β-glucuronidase) on a PR-1 promoter, and the results are shown in Table 1 below.

[0078]

Table 1

[0079] Strain Activity Strain Activity Strain Activity Strain Activity JCK-757 * JCK-1180 * JCK-1266 * JCK-1320 * JCK-758-1 * JCK-1182 * JCK-1287 * JCK-1328 * JCK-758-2 * JCK-1187 * JCK-1288 * JCK-1333 * JCK-761 * JCK-1217 * JCK-1307 * JCK-1398 * JCK-767 * JCK-1222 * JCK-1308 * JCK-947 * JCK-1229 * JCK-1309 * JCK-1005 * JCK-1233 * JCK-1318 *

[0080]

Example 2. In vitro induced resistance identification using pine callus

[0081] For the 25 strains screened by Example 1 described above, the expression of induced resistance-related genes of red pine callus was confirmed.

[0082] Specifically, in a 12-well microplate, Pinus taeda-induced resistance strains were cultured in 500 μl of LM medium to reach 0.8 at an optical density (OD 600) of 600 nm, and then 100 mg of Pinus densiflora callus was treated with 500 μl of the culture solution on the surface, and the callus surface and the Pinus taeda-induced resistance strain culture solution were allowed to react for 24 hours in a 25°C dark condition, 50 rpm shaking incubator. Then, the reacted callus was separated by centrifugation at 600 x g for 5 to 10 minutes using a centrifugal separator. After extracting RNA (ribonucleic acid) from the separated callus, cDNA (complementary deoxyribonucleic acid) was synthesized, and the expression of the specifically increased induced resistance genes was confirmed by RT-PCR (reverse transcription-polymerase chain reaction) or qRT-PCR (quantitative real-time PCR), and the results are shown in Table 2. Figure 1 and Table 2. The induced resistance-related genes used in the qRT-PCR are shown in Table 3 below.

[0083] [Table 2]

[0084] Order Designation Expression level 1 PR-1 b family 1.35 2 PR-2 family 1.02 3 PR-3 family class 1 0.70 4 PR-3 family class 4 2.84 5 PR-4 family 10.20 6 PR-5 family 2.21 7 PR-9 family 1.49 8 PR-10 family 1.56 9 Antimicrobial peptide 11.91 10 Cytochrome P450 1.06 11 Auxin 0.84 12 Hydroxyproline-rich glycoprotein precursor 0.50 13 Metallothionein-like protein 1.59 14 Xyloglucan endotransglycosylase 0.72

[0085] [Table 3]

[0086]

[0087]

[0088] As can be confirmed from Figure 1 and Table 2, it was confirmed that among the 25 strains screened, the Bacillus subtilis JCK-1398 strain of the present application specifically increased the production of genes such as the PR-1b family (PR-1b family), the PR-3 family 4th class (PR-3 family 4th class), the PR-4 family (PR-4 family), the PR-5 family (PR-5 family), the PR-9 family (PR-9 family), the PR-10 family (PR-10 family), the antimicrobial peptide (antibacterial peptide), and the metallothionein-like protein (metallothionein).

[0089] [Example 3. Molecular biological identification and systematic classification]

[0090] The Bacillus subtilis JCK-1398 strain was streaked on a TSA (Tryptic soy agar, medium (Difco)) solid medium and incubated at 30°C for 1 day. The obtained single colony was inoculated in a TSB (Tryptic soy broth, Difco) liquid medium using a sterile loop and then incubated at 30°C for 1 day with shaking at 150 rpm. Then, gDNA (genomic DNA) of the strain was extracted according to the protocol using a DOKDO-prep bacterial genomic DNA purification kit of ELPIS-Biotech. The extracted gDNA was mixed with a PCR-premix and a primer set capable of amplifying the gyrA (Gyrase A subunit) gene of iNtRON Biotechnology, and the gyrA gene of the above JCK-1398 strain was amplified by PCR. The primer set used for PCR is shown in Table 4.

[0091] [Table 4]

[0092] Sequence number Designation Sequence (5'→3') 31 gyrA For CAG TCA GGA AAT GCG TAC GTC CTT 32 gyrA Rev CAA GGT AAT GCT CCA GGC ATT GCT

[0093] PCR was repeated 30 times, starting from 5 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 55°C, 30 seconds at 72°C, and then 7 minutes at 72°C, and the amplification was completed at 12°C. Genotech (Daejeon, Korea) was commissioned to perform base sequence analysis on the amplified PCR product, and the base sequence of the gyrA (SEQ ID NO: 33) gene of the JCK-1398 strain was obtained. The obtained base sequence of the gyrA gene was compared with the base sequences of the GenBank database using nBlast search of NCBI. Also, the gyrA gene base sequences having high similarity were obtained from the GenBank database, the gyrA gene base sequence of the JCK-1398 strain was aligned with all the base sequences using BioEdit Sequence Alignment Editor, and the system analysis was performed based on the NJ (neighbour-joining) algorithm using the mega program version 6.0 under the condition of 1000 boot-strap trial sets, and the results are shown in Figure 2

[0094] [Table 5]

[0095] ​

[0096] It was confirmed that the JCK-1398 strain was identified as a Bacillus subtilis strain. Figure 2

[0097] [Example 4. Confirmation of pine wood nematode disease control effect]

[0098] In order to confirm the pine wood nematode disease control effect of the Bacillus subtilis JCK-1398 strain culture solution, a seedling identification was performed using Korean red pine (Pinus densiflora) 3- to 4-year-old seedlings (about 5.5 mm in diameter, about 35 to 45 cm in height from the soil).

[0099] [4-1. Preparation of JCK-1398 strain culture solution]

[0100] The JCK-1398 strain suspended in a 20% glycerol solution and stored in a -80°C deep freezer was statically cultured in TSA at 30°C for about 24 hours. Then, the TSB was put into a test tube, the inlet was blocked with a mask and sterilized, and then one colony of the cultured JCK-1398 strain was scraped with a platinum tooth and inoculated into the TSB, and then further shake-cultured at 30°C, 150 rpm, and aerobic conditions for about 24 hours. The JCK-1398 strain culture solution was put into a 250 ml Erlenmeyer flask containing 50 ml of sterilized TSB to prepare a suspension so that the optical density value at 600 nm in the UV spectrophotometer was 1.0. Then, it was shake-cultured at 30°C, 150 rpm, and aerobic conditions for about 24 hours.

[0101] [4-2. Spraying of JCK-1398 strain culture solution]

[0102] First, in order for the JCK-1398 strain culture solution to be well adsorbed to the leaves, each pine tree was pretreated with 5 ml of 250 μg / ml Tween 20 and dried for 2 hours. Then, the JCK-1398 strain culture solution cultured in the above Experimental Example 4-1 was dissolved in an aqueous solution containing 250 μg / ml Tween 20 so that the optical density value was about 0.8, and then put into a micro-sprayer, and each pine tree was foliar-sprayed in an amount of 5 ml.

[0103] [4-3. Culture of Bursaphelenchus xylophilus]

[0104] ​Botrytis cinerea, a food source for pine wood nematodes, was inoculated into PDA (Potato dextrose agar, Difco) medium and incubated statically at 25°C for 7 days. Pine wood nematodes (Bursaphelenchus xylophilus, Korea National Forest Research Institute) were then inoculated onto the cultured Botrytis cinerea and incubated statically at 25°C for 7 days. The cultured pine wood nematodes were harvested using the Baermann funnel method, and their numbers were adjusted to approximately 20,000 nematodes / ml under an optical microscope.

[0105] [4-4. Confirmation of the effectiveness of pine wilt disease control]

[0106] Using a foliar spray of JCK-1398 strain culture solution (Example 4-2), a pre-treated pine tree xylem was cut approximately 1 cm deep into the inner bark using a sterilized knife. A 0.5 cm wide and 1 cm long piece of absorbent cotton was then inserted into the cut. 2000 isolated pine wood nematodes were inoculated with 100 μl of the solution (treated with JCK-1398 strain culture solution and other agents for one week before inoculation). The inoculation site was sealed with sealing film to prevent drying (bark peeling inoculation method). The degree of drying was observed 45 days after inoculation with pine wood nematodes. The results are as follows... Figure 3a to 3b As shown in Table 6.

[0107] Table 6

[0108]

[0109] Available from Figure 3a to 3b As confirmed in Table 6, the culture medium of strain JCK-1398 showed a low incidence of pine wilt disease of 24% in Korean pine seedlings, and a control value of 71.4% compared with the untreated area.

[0110] Example 5. Evaluation of in vitro nematicidal activity

[0111] [5-1. Ensure the JCK-1398 strain culture filtrate and the nematodes to be controlled are properly stored]

[0112] After culturing strain JCK-1398 in the same manner as in Example 4-1, the culture filtrate was harvested by centrifuging at 13,000 rpm for 30 minutes using a centrifuge, and then sterilized using a sterile 0.2 μm microsyringe filter.

[0113] As Meloidogyne incognita, eggs were obtained from tomato roots artificially infected with M. incognita in a greenhouse of the Green Agriculture Center of Chonnam National University in Gwangju Metropolitan City, Korea, and the larvae (2nd instar) hatched from the eggs were isolated using the Bellman funnel method for experiments. Bursaphelenchus xylophilus was harvested in the same manner as in Example 4-3.

[0114] [5-2. Confirmation of JCK-1398 strain culture filtrate and nematodes]

[0115] Each of the nematode suspensions confirmed in Example 5-1 was placed in each well of a 96-well microplate, and then treated with the JCK-1398 strain culture filtrate to a concentration of 0.625 to 20% so that the final volume of the well was 100 μl. After the sample treatment, the 96-well plate was stirred for 30 seconds and stored at room temperature in a plastic bucket with a relative humidity of 100%. After 72 hours of sample treatment, the nematocidal rate was confirmed under an optical inverted microscope using the following calculation formula. All experiments were repeated three times, and the results are shown in Table 7 below.

[0116] [Calculation formula]

[0117] Nematocidal rate (%) = [(mortality rate of treated area - mortality rate of control group) / (100 - mortality rate of control group)] x 100

[0118] [Table 7]

[0119]

[0120] As can be confirmed from Table 7 above, in the case of M. incognita, the 20% culture filtrate-treated area showed a nematocidal activity of 97.45%, and the 10% culture filtrate-treated area showed a nematocidal activity of 13.15%, and it can be seen that no nematocidal activity occurs at a concentration of 5% or less.

[0121] On the other hand, in the case of B. xylophilus, no nematocidal activity occurred in any of the treated areas, which indicates that the control effect of B. thuringiensis JCK-1398 on pine wilt disease is an effect due to induced resistance, not due to direct nematocidal activity.

[0122] [Example 6. Confirmation of induced resistance substance showing pine wilt disease control effect]

[0123] Based on the results of Example 5, it was confirmed that the B. thuringiensis JCK-1398 strain has an induced resistance substance (culture broth, culture filtrate, and cell fraction) showing a pine wilt disease control effect. The culture of the JCK-1398 strain was performed by adjusting the optical density value to 0.8 in the same manner as in Example 4-1.

[0124] The culture solution of the JCK-1398 strain was centrifuged (1300 rpm, 15 minutes) to separate into a culture filtrate and a cell fraction. The precipitated cell fraction was suspended by adding the same volume of phosphate-buffered saline as used before centrifugation. In order to allow each sample (culture solution, culture filtrate, and cell fraction) to be well adsorbed onto the leaves, each pine was pretreated with 5 ml of 250 μg / ml Tween 20 and dried for 2 hours. Then, the culture solution, culture filtrate, and cell fraction were dissolved in an aqueous solution containing 250 μg / ml Tween 20 to have an optical density of about 0.8, and then put into a micro-sprayer to spray the leaves of each pine in an amount of 5 ml. In this case, as a control group, emamectin benzoate, which is a nematicidal substance, was dissolved in an aqueous solution containing 10% methanol to have a concentration of about 20 mg / ml, and then injected into the trunk. The results are shown in Table 7 and FIG. 8. Figure 4a to 4b and Table 8.

[0125] [Table 8]

[0126] Treatment zone Culture broth Culture filtrate Cell fraction EB Control value (%) 61.7 64.2 42.0 97.0

[0127] As can be confirmed from Figure 4a to 4b and Table 8, the culture solution and the culture filtrate of the JCK-1398 strain showed control effects of 61.7% and 64.2%, respectively, but the cell fraction showed a slightly lower control effect of 42%.

[0128] This result indicates that the pine wood nematode control effect of the JCK-1398 strain of the present application is an effect caused by the secretion secreted outside the cell.

[0129] [Example 7. Confirmation of optimal treatment time showing pine wood nematode control effect]

[0130] Based on the results of Example 6, the optimal treatment time was confirmed through in vivo identification of the pine wood nematode using the culture solution of Bacillus subtilis JCK-1398 strain. The in vivo identification was performed using Korean red pine 3- to 4-year-old seedlings (about 5.5 mm in diameter, about 35 to 45 cm in height from the soil).

[0131] The JCK-1398 strain was cultured in the same manner as in Example 4-1 and the optical density value was adjusted to about 0.8, and then the culture solution was foliar-sprayed in an amount of 5 ml per pine tree. In this case, the culture solution treatment was performed twice at different times, 2 weeks before and 1 week before inoculation of the pine wood nematode, 4 weeks before and 1 week before, and 4 weeks before and 1 week before, and 7 days after the second culture solution treatment, the pine wood nematode was inoculated in the same manner as in Example 4-4 described above. Finally, the degree of drying was observed 60 days after inoculation of the pine wood nematode, and the results are shown in Table 8 and Table 9. Figure 5a to 5c and Table 9 to Table 10.

[0132] [Table 9]

[0133]

[0134] [Table 10]

[0135] Treatment zone 2 weeks before - 1 week before 3 weeks before - 1 week before 4 weeks before - 1 week before Control value (%) 59.2 20.4 38.8

[0136] It can be confirmed from Figure 5a to 5c and Table 9 to Table 10 that the incidence of pine wood nematode disease remained at a similar level regardless of the treatment time until the 49th day of inoculation, but it was known that it significantly changed Figure 5a thereafter depending on the treatment time. As a result, when treated 2 weeks before-1 week before inoculation (59.2%), it showed an excellent control effect compared to when treated 4 weeks before-1 week before (20.4%) and 4 weeks before-1 week before (38.8%).

[0137] [Example 8. Confirmation of optimal treatment concentration showing pine wood nematode disease control effect]

[0138] Based on the results of Example 7, the optimal treatment concentration was confirmed through in vivo identification of the pine wood nematode disease by Bacillus subtilis JCK-1398 strain spray-dried powder. Also, in vivo identification was performed using Korean pine 3- to 4-year-old seedlings.

[0139] Specifically, the spray-dried powder of the JCK-1398 strain prepared by the method of Table 11 was prepared into 50-fold, 500-fold, and 5000-fold dilutions with sterile water, and then foliar-sprayed on pine trees twice at 7-day intervals in the same manner as in Example 4-2 described above. Finally, the pine wood nematode was inoculated 7 days after the second treatment of the above dilutions in the same manner as in Example 4-4 described above, and the degree of drying was observed 55 days after inoculation of the pine wood nematode, and the results are shown in Table 12 and Table 13. Figure 6 and Table 12.

[0140] [Table 11]

[0141] Spray drying process JCK-1398 Inlet and outlet temperature 190～198℃ / 90～98℃ Spray rate 10000 rpm Carrier Culture broth 70 L Spray dried powder 11 kg QC 2.4 x 10 9 cfu / g CFU (x 50) 4.8 x 10 7 cfu / g]]

[0142] [Table 12]

[0143] Treatment zone 50-fold dilution 500-fold dilution 5000-fold dilution Control value (%) 85.5 60.5 69.8

[0144] Available from Figure 6 As confirmed in Table 12, treatment with a 50-fold dilution (85.5%) of the spray-dried powder of strain JCK-1398 showed superior control effects compared to dilutions of 500 times (60.47%) and 5000 times (69.77%).

[0145] [Example 9. Confirmation of the control effect of pine wilt disease]

[0146] Based on the above results, to confirm the efficacy of Bacillus subtilis strain JCK-1398 in controlling pine wilt disease in mature trees, a field trial was conducted using 15-year-old Korean pine trees (root diameter 8-11 cm, height 6-8 m). The field trial was conducted twice, in 2018 (first field trial) and 2019 (second field trial). The first field trial tested the spray-dried powder of JCK-1398 strain culture solution (prepared using the same method as in Example 8). The second field trial formulated the spray-dried powder of JCK-1398 strain culture solution into JCK-1398 20% SC (emulsion) for use. The JCK-1398 20% SC formulation was prepared with the following composition: 20.0% JCK-1398 spray-dried powder, 4.5% CR-NF 135B, 0.03% xanthan gum, 0.1% defoamer, and 75.37% water.

[0147] The spray-dried powder of JCK-1398 strain culture and the JCK-1398 20% SC formulation were diluted 50-fold and 2000-fold respectively in an aqueous solution containing 250 μg / ml Tween 20, and then applied as foliar sprays at a rate of approximately 1 L per pine tree. Foliar spraying was performed twice, one month apart. One week after the two treatments, trunk injection was used. In the first field trial, 20,000 pine wood nematodes were inoculated per pine tree, and in the second field trial, 10,000 pine wood nematodes were inoculated per pine tree. As a control group, anionic polyacrylamide (2.15% emamectin benzoate, emulsion, Syngenta) was injected into the trunk at a rate of 1 ml per breast height diameter. The anionic polyacrylamide treatment was performed one week before pine wood nematode inoculation. As an untreated area, the same treatment was performed using only 250 μg / ml Tween 20 without any other pesticides. The results are as follows... Figure 7a to 7c As shown in Tables 13 and 14.

[0148] Table 13

[0149]

[0150] Table 14

[0151]

[0152] Available from Figure 7a to 7c As confirmed in Tables 13 and 14, compared with the untreated area, the 50-fold dilution of the JCK-1398 strain culture broth spray-dried powder (JCK-1398) and the JCK-1398 20% SC formulation (20% SC formulation) showed control effects of 65.5% and 68.8%, respectively. Two field trials confirmed the reproducibility of the JCK-1398 strain's control effect on pine wilt disease.

[0153] [Example 10. Confirmation of the synergistic effect of the compound in the control of pine wilt disease]

[0154] Based on the results of the examples, in order to confirm the efficacy of the mixture of acibenzolar-S-methyl (ASM) and methyl salicylate (MeSA), which are the inducible resistance substances of Bacillus subtilis strain JCK-1398, in controlling pine wilt disease in mature trees, a field test was also conducted using 15-year-old Korean pine (root diameter 8-11 cm, height 6-8 m).

[0155] JCK-1398 20% SC formulation, ASM 5% SC formulation, and MeSA 20% EC-LV formulation were diluted 2000 times and prepared into 0.1 mm and 0.5 mm samples, respectively. JCK-1398 20% SC + ASM 5% SC mixture and JCK-1398 20% SC + MeSA 20% EC-LV mixture were prepared to a final concentration containing half the concentration of each single-agent treatment area. Each sample was placed in a sprayer and treated twice with approximately 1 L per pine tree, one month apart. One week after the two treatments, 10,000 pine wood nematodes were inoculated into each pine tree using a trunk injection method. In this case, as a control group, anionic polyacrylamide (2.15% emamectin benzoate, emulsion, Syngenta) was injected into the trunk at a rate of 1 ml per breast height diameter. The anionic polyacrylamide treatment was performed one week before pine wood nematode inoculation. As an untreated area, the same treatment was performed using only 250 μg / ml Tween20 without any reagent. Results are as follows: Figure 8 As shown in Table 15.

[0156] Table 15

[0157]

[0158] Available from Figure 8As confirmed in Table 15, compared with the untreated area, the control efficacy of JCK-1398 20% SC, ASM 5% SC, and MeSA 20% EC-LV formulations was 23.1%, 27.3%, and 33.9%, respectively. However, compared with the untreated area, the control efficacy of the JCK-1398 20% SC + ASM 5% SC mixture and the JCK-1398 20% SC + MeSA 20% EC-LV combination was improved by 46.2% and 53.8%, respectively.

[0159] Therefore, it was confirmed that Bacillus subtilis strain JCK-1398, when treated together with ASM and MeSA as resistance-inducing substances, can more effectively control pine wilt disease through a synergistic effect.

[0160] [Example 11. Preparation of aerial spraying formulation and confirmation of its control effect on pine wilt disease]

[0161] To confirm the efficacy of aerial spraying of Bacillus subtilis strain JCK-1398 in controlling pine wilt disease in mature trees, a novel formulation was prepared. The JCK-1398 10% SC formulation was prepared with the following composition: 10.0% JCK-1398 spray-dried powder, 3.0% CR-NF 135B, 0.01% xanthan gum, 0.1% defoamer, 5.0% propylene glycol, 0.2% sodium benzoate, and 81.69% water.

[0162] After preparing 10-fold, 100-fold, and 500-fold dilutions of JCK-1398 10% SC formulation and JCK-1398 20% SC formulation (existing formulation, Example 9), foliar sprays were applied twice to each 3-year-old pine tree at a dose of 100 μl (1 / 50 of the existing treatment amount) 7 days before and 1 week before inoculation. Finally, pine wood nematodes were inoculated 7 days after the second treatment with the above dilutions, using the same method as in Examples 4-4 above. The degree of drying was observed 40 days after inoculation with pine wood nematodes, and the results were as follows. Figure 9 As shown in Table 16.

[0163] Table 16

[0164]

[0165] Available from Figure 9 As confirmed in Table 16, compared with the JCK-1398 20% SC formulation, the JCK-1398 10% SC showed stable control efficacy at all concentrations of 10x dilution (87.9%), 100x dilution (79.3%), and 500x dilution (86.2%). Therefore, JCK-1398 10% SC was selected as the final formulation for subsequent aerial spraying.

[0166] Example 12. Field evaluation of pine wood nematode control using JCK-1398 10% SC formulation by aerial spraying

[0167] Based on the above results, in order to confirm the pine wood nematode disease control effect of JCK-1398 10% SC formulation by aerial spraying on mature trees, a field test was performed using 30-year-old Korean pines.

[0168] After preparing 50 times diluted solution of JCK-1398 10% SC formulation, 450M 2 3.6L (80L / 1ha level) was sprayed by aerial spraying. The treatment was performed three times by aerial spraying 2 months before, 1 month before and 1 month after inoculation of pine wood nematode. 20000 pine wood nematodes were inoculated into each pine tree 1 month after the second treatment in the same manner as Example 9. The results are shown in Figure 10a 10b and Table 17.

[0169] [Table 17]

[0170]

[0171] It can be confirmed from Figure 10a 10b and Table 17 that, after 6 months of inoculation of nematodes, JCK-1398 10% SC formulation (20.0%) showed a very low incidence rate compared to the untreated area (73.3%). That is, it showed a control effect of 72.7%, and thus it was confirmed that JCK-1398 10% SC formulation can very effectively control pine wood nematode disease even in the field as in the seedling identification.

[0172] Example 13. Confirmation of control effect on various plant diseases

[0173] Based on the induced resistance effect of Bacillus subtilis JCK-1398 strain, in order to perform an application expansion experiment, the control effect on various plant diseases was confirmed using spray-dried powder of JCK-1398 strain culture solution.

[0174] [13-1. Confirmation of control effect on pepper bacterial spot disease (pathogenic bacteria: Xanthomonas euvesicatoria (Xanthomonas))]

[0175] ​​In the same manner as in Example 8, a solution of spray-dried powder of JCK-1398 strain diluted 500 times and 5000 times, respectively, was treated with 250 μg / ml of Tween 20 as a surfactant, and was applied to 8-9 leaf peppers (Heungnong seeds, Bultap pepper) by soil drenching and foliar spraying in an amount of 20 ml per pot. All samples were treated 4 days before inoculation with a bacterial spot of pepper strain source. In this case, as a control group, a wettable powder of Seobang Cefriaxone (oxytetracycline, Korea Seobang Chemical Co.) and a wettable powder of Ipink (oxolinic acid 20% WP, Dongbang Agro Co., Ltd.) as synthetic pesticides fungicides were treated in a constant amount (1000 times dilution), and as an untreated area, the same treatment was performed with only 250 μg / ml of Tween 20 without the agent.

[0176] Then, the bacterial spot of pepper strain (Xanthomonas euvesicatoria) was incubated at 30°C for about 48 hours in TSA, and then the cells were harvested using 2 ml of sterile water. The harvested bacterial spot of pepper pathogen was diluted to an optical density value of 0.1 at 600 nm using a UV spectrophotometer, thereby preparing an inoculum source. The inoculum source was foliar sprayed in an amount of 5 ml per seedling. The inoculated seedlings were maintained in a growth chamber at 25±2°C for 3 days with 95% humidity. Then, the incidence of disease was observed under constant irrigation and foliar watering, and the results are shown in Table 18. Figure 11

[0177] All treatments were repeated 3 times, and all experiments were repeated 2 times. The incidence of bacterial spot of pepper formed on the pepper leaves was confirmed by measuring the degree of disease in an index of 0-6 according to Abbasi. The index standard of the degree of disease is as follows:

[0178] 0 = no disease,

[0179] 1 = 1-2 small spots on 1-2 leaves,

[0180] 2 = multiple spots on multiple leaves,

[0181] 3 = many spots gathered and appeared on the leaves,

[0182] 4 = many spots gathered and appeared on many leaves,

[0183] 5 = severe disease with defoliation,

[0184] 6 = plant death.

[0185] Table 18​

[0186]

[0187] Available from Figure 11 As confirmed in Table 18, soil irrigation with a 5000-fold dilution of Bacillus subtilis JCK-1398 spray-dried powder showed a control effect of 52.1%, while foliar spraying showed a control effect of 31.3%. Furthermore, soil irrigation with a 500-fold dilution showed a control effect of 37.5%, and foliar spraying showed a control effect of 20.8%. Therefore, treating peppers with JCK-1398 strain culture may effectively induce resistance, thus contributing to the control of bacterial spot disease in peppers.

[0188] [13-2. Confirmation of the control effect on lawn coin spot disease (pathogen: Sclerotinia homoeocarpa)]

[0189] As diluents for JCK-1398 culture medium, a 100-fold dilution was prepared by culturing JCK-1398 strain in the same manner as in Example 4-1 and then diluting it to a density of 0.08 at 600 nm using a UV spectrophotometer. A 1000-fold dilution was prepared by diluting the JCK-1398 culture medium 100-fold and 1000-fold with sterile water. JCK-1398 20% SC was prepared as the untreated zone using a solution containing Tween-20 at a level of 250 μg / ml. As the control group, 2000-fold and half-fold (4000-fold dilution) of 25% Tebuconazole EC (Horikuo emulsion) were used.

[0190] All samples were treated with soil irrigation 4 days before inoculation with the turfgrass spot strain inoculum. JCK-1398 culture medium was treated as a single agent with 100-fold and 1000-fold dilutions, or in combination with 4000-fold dilution of 25% EC Tebuconazole. JCK-1398 20% SC was treated as a single agent with 100-fold and 1000-fold dilutions, or in combination with 4000-fold dilution of 25% EC Tebuconazole.

[0191] Then, the agar plug (2 x 2 mm in width x length) was used to remove the colony of Sclerotinia homoeocarpa and inoculated in PDA medium, and incubated in a thermostat at 25°C for 5 days. The colony of the strain cultured in 2 times sterilized bran-rice hull medium (bran 9 g, rice hull 1.5 g, distilled water 10 ml) was cut into 1 x 1 cm in width x length, and 5 of each were transplanted, and only the entrance was closed with bio-silicon rubber, and incubated in a thermostat at 25°C for 7 days. Then, the cultured bacteria were put in a grinder together with the medium, and ground after adding 110 ml of distilled water and 1.1 ml of streptomycin sulfate 200 μg / ml.

[0192] Finally, a hole of about 1 cm was drilled in the center of a pot in which creeping bentgrass was grown for about one month after sowing, and 3.3 ml of the inoculum was inoculated per pot. The inoculated grass was observed at 95% humidity and 25°C until the difference between the untreated area and the treated area was observed. The disease severity was calculated by investigating the disease area relative to the total area of the pot, and the result of 3 pots per treated area was repeated twice, and the result is shown in Table 19. Figure 12 and Table 19.

[0193] [Table 19]

[0194]

[0195]

[0196] It can be confirmed from Figure 12 and Table 19 that, in the case of treating the JCK-1398 culture solution (100-fold dilution and 1000-fold dilution) and the JCK-1398 20% SC (100-fold dilution) as a single agent, the disease severity similar to that of the untreated area was exhibited. In contrast, in the case of treating the JCK-1398 culture solution (100-fold dilution) in combination with Tebuconazole 25% EC 4000-fold dilution, a high control effect of 97.26% was exhibited, and in the case of treating the JCK-1398 20% SC (100-fold dilution and 1000-fold dilution) in combination with Tebuconazole 25% EC 4000-fold dilution, a control effect of 84.93% and 73.97%, respectively, was exhibited, and it can be seen that the control effect appears in a concentration-dependent manner. Therefore, when the JCK-1398 strain culture solution of the present application is used, it is expected that the amount of chemical pesticide used for the prevention and treatment of Sclerotinia homoeocarpa can be significantly reduced.

[0197] [13-3. Confirmation of the control effect of tomato russet mite (Pest: Aculops lycopersici)

[0198] JCK-1398 20% SC was prepared using JCK-1398 culture solution diluted 250 times, 500 times and 1000 times with sterile water. As an untreated area, a solution in which Tween-20 was added at a level of 250 μg / ml was used, and as a control group, a nematicide (Fosthiazate (thiazophos) 30% SL, Farm Hannong Co., Ltd.) was used in a quantity (4000 times dilution).

[0199] Each sample was soil drench treated in 4-5 leaf Seogwang tomato (Lycopersicon esculentum Mill) in an amount of 20 ml per plant. In this case, JCK-1398 20% SC was treated twice with an interval of 3 days, and the nematicide was treated once. Then, the tomato russet mite was naturally infected by maintaining the appropriate temperature and humidity (25-28°C 30%) at which the tomato russet mite occurs. The tomato russet mite occurred 2 weeks after the second treatment with the agent, and the length of the aboveground part was measured 2 weeks after the occurrence of the tomato russet mite, and the control effect on the tomato russet mite was investigated, and the results are shown in Table 19 Figure 13 and Table 20.

[0200] [Table 20]

[0201]

[0202] As can be confirmed from Table 19 Figure 13 and Table 20, the length of the aboveground part of the area treated with JCK-1398 20% SC 250 times, 500 times and 1000 times dilution was 26.13 cm, 21.75 cm, 15.88 cm, respectively, and it can be seen that the length of the aboveground part increases as the concentration increases. On the other hand, the length of the aboveground part of the area treated with Fosthiazate 30% SL 4000 times dilution, which was used as a control group, was the longest at 32 cm, and the length of the aboveground part of the untreated area was the shortest at 16.83 cm. Therefore, when tomato is treated with the JCK-1398 strain culture solution, it is expected that the tomato russet mite can be controlled by effectively inducing resistance.

[0203] [Industrial applicability]

[0204] The present application relates to Bacillus subtilis JCK-1398 strain inducing resistance in pine trees and various plants, a composition for controlling plant diseases using the same, and a control method.

[0205] [International recognition of the Budapest Treaty on the Microorganism Deposit for Patent Procedure]

[0206] International form

[0207] Receipt in the original deposit

[0208] Rule 7.1 issued to

[0209] To: Chonnam National University Industry-University Cooperation Foundation

[0210] Chonnam National University Industry-University Cooperation Foundation

[0211] 77, Yongbong-ro, Buk-gu, Gwangju, Republic of Korea

[0212]

[0213] BP / 4 form (KCTC form 17) (single page). SEQUENCE LISTING <110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY National Institute of Forest Science Pusan National University Industry‑University Cooperation Foundation <120> Bacillus subtilis JCK-1398 strain inducing resistance in various plants, composition for preventing and treating pine wood nematode disease using the same, and prevention method <130> PP200080 <150> KR 10‑2020‑0017232 <151> 2020‑02‑12 <160> 33 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PR-1b family For <400> 1 tgccccttca ggtaaatcgt 20 <210> 2 <211> 21 <212> DNA <213> Artificial sequence <220> <223> PR-1b family Rev <400> 2 gcgggtcgta gttgcagata a 21 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PR-2 family For <400> 3 cgacaacatt cgccccttct 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PR-2 family Rev <400> 4 ctgcagcgcg gtttgaatat 20 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <220> <223> PR-3 family Class 1 For <400> 5 acctacagcg cttcattgc 19 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> PR-3 family Class 1 Rev <400> 6 tgtggtttca tgcgacgttt 20 <210> 7 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> PR-3 family, Class 4 For <400> 7 ccatcgaagc ccaggtaatt t 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PR-3 family class 4 Rev <400> 8 agccgggaag caatattatg gt 22 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PR-4 family For <400> 9 ccccgttact gtcaattgca t 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PR-4 family Rev <400> 10 aaagcgtgac ggtgcgtatt 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PR-5 family For <400> 11 gaaccagtgc ccatacacag tct 23 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PR-5 family Rev <400> 12 cctgcggcaa cgttaaaagt c 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PR-9 family For <400> 13 acaccaccgt gctggacatt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PR-9 family Rev <400> 14 gtgcgggagt cggtgtagag 20 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PR-10 family For <400> 15 tgtctcaagt ggaggcaagg a 21 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PR-10 family Rev <400> 16 aagcgacaat ttcaggcaaa ac 22 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antimicrobial peptide For <400> 17 gcgttgctca tacccgtttt 20 <210> 18 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Antimicrobial peptide Rev <400> 18 gcagcactta gcactggatg aa 22 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Cytochrome P450 For <400> 19 aacatgtcct gcagcacgaa 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Cytochrome P450 Rev <400> 20 gtgcaccgca agtaaaccaa 20 <210> 21 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Auxin For <400> 21 cgaatgtaat tccgaagttg ca 22 <210> 22 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Auxin Rev <400> 22 ccatcccaaa ccaccagtct 20 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Hydroxyproline-rich glycoprotein precursor For <400> 23 gagaaactgg caccgtctta gga 23 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Hydroxyproline-rich glycoprotein precursor Rev <400> 24 acctccccct ccatctcaca 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Metallothionein-like protein For <400> 25 tcaggctgct gcgttatttg 20 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Metallothionein-like protein Rev <400> 26 tgtcagcgca gtcacaattt g 21 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Xyloglucan endotransglycosylase For <400> 27 tctgcgcccc tacttttcc 19 <210> 28 <211> 20 <212> DNA <213> Artificial sequences <220> <223> Wood glucan endotransglycosylase Rev <400> 28 agctgggcga ttgatcatgt 20 <210> 29 <211 > 20 <212> DNA <213> Artificial sequences <220> <223> Elongation factor-1 alpha For <400> 29 gggaagccac ccaaagtttt 20 <210> 30 <211 > 20 <212> DNA <213> Artificial sequences <220> <223> Elongation factor-1 alpha Rev <400> 30 tacatgggaa gacgccgaat 20 <210> 31 <211 > 24 <212> DNA <213> Artificial sequences <220> <223> gyrA For <400> 31 cagtcaggaa atgcgtacgt cctt 24 <210> 32 <211 > 24 <212> DNA <213> Artificial sequences <220> <223> gyrA Rev <400> 32 caaggtaatg ctccaggcat tgct 24 <210> 33 <211 > 856 <212> DNA <213> Artificial sequences <220> <223> gyrA sequence of Bacillus subtilis JCK-1398 strain <400> 33 gatgtttgta gagcagtttg tttgtacaga ttgtttaaga tgacattcgc attggcatcg 60 cgtctgattt caatgacaat tctcatacct gtacgatctg actcatcacg cagatctgtg 120 ataccctcta tctttttgtc ccttacgaga tcagcaattt tctcaattaa tttcgcctta 180 tttacttggt aaggtaactc tgtaacgata attctttctt tacccgaaga tgtttgttcg 240 atctcagctt ttgcccggat cgtgatagag cctcggcctg attcgtatgc tttccggata 300 ccgctgcggc ccaagatttg acccgcagtc gggaaatcag gtcctggaat gacttccata 360 agctctggaa tggtaatgtc cggattctca ctgacagcaa gtactccgtc aatgatttct 420 cccagctggt gcggaggaat gtttgttgcc atacctaccg caatgccggc agcaccgttc 480 acgagcagat tcgggaacct tgaaggcata acgacaggtt ctctttctga cccgtcatag 540 ttatcctggt aatcgattgt gtcttttgtg atgtcacgaa gaatctccat tgagatttta 600 gacattcttg cttctgtata acgcatggcc gccgctgagt ctccgtcaac agaaccgaag 660 tttccgtgac cgtcaacgag catataacgg tagttgaaat cctgagccat tctgaccatg 720 gattcatata ccgctgaatc accgtgcggg tggtatttcc cgataacttc tccaacgata 780 cgcgcggatt ttttataagg cttgtcactt gtcatgccta aatcattcat tgcatacaaa 840 atccgtctat gaactg 856

Claims

1.A Bacillus subtilis JCK-1398 strain having accession number KCTC 14084BP for activating induced resistance of a plant against a disease. 2.Use of the strain according to claim 1 in the manufacture of a composition for controlling a plant disease selected from one or more of pine wood nematode disease, bacterial spot of pepper and dollar spot of turf. 3.A composition for controlling a plant disease, comprising one or more selected from the group consisting of the strain according to claim 1, a culture of the strain, a concentrate of the culture, a dry of the culture and a culture supernatant of the strain. 4.The composition according to claim 3, wherein the plant disease is selected from the group consisting of pine wood nematode disease, bacterial spot of pepper and dollar spot of turf. 5.The composition according to claim 3, wherein the composition is used to make a mixture with a synthetic pesticide. 6.The composition according to claim 4, wherein the synthetic pesticide is tebuconazole.

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