New microorganism belonging to lactobacillus genus, and agent for controlling and method for controlling plant disease caused by ralstonia solanacearum or ralstonia pseudosolanacearum

Abstract

Provided herein are bacteria comprising a 16S rRNA gene having a base sequence that is 98.80% or more identical to the base sequence recited in SEQ ID NO: 1, having a mannitol assimilation ability, and having a plant disease control ability, and bacteria having accession numbers NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202, and a control agent for plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosolanacearum, which comprises a bacterial cell or a bacterial cell culture or an extract thereof of the bacteria.

Description

Technical Field

[0001] This invention relates to a novel microorganism belonging to the genus Lactobacillus, and to agents and methods for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis. Background Technology

[0002] Ralstonia solanacearum or Ralstonia pseudosolanacearum infects more than 200 plant species, causing bacterial wilt in plants of the Solanaceae family such as tomatoes, eggplants, bell peppers, and potatoes; plants of the Cucurbitaceae family such as cucumbers; and flowers such as *Lisianthus* and *Staticeus*. It also causes damping-off in tobacco. Plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosolanacearum occur globally, resulting in significant economic losses.

[0003] As methods for controlling plant diseases, for example, Japanese Patent Application Publication No. 2009-201459 (Patent Document 1) discloses a method for controlling soft rot caused by Erwinia carotovora using a composition containing Lactobacillus kyotoensis FERMP-21500 or Lactobacillus plantarum FERMP-21501. J Gen Plant Pathol 70:115-119 (2004) (Non-Patent Document 1) discloses a method for inhibiting Ralstonia solanacearum infection in tomatoes by grafting with tolerant tomatoes. Japanese Patent Application Publication No. 2012-211124 (Patent Document 2) discloses a method for controlling bacterial wilt by allowing target plants such as tomatoes to absorb L-amino acids.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2009-201459

[0007] Patent Document 2: Japanese Patent Application Publication No. 2012-211124

[0008] Non-patent literature

[0009] Non-patent literature 1: J Gen Plant Pathol 70:115-119 (2004) Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] To date, chemical fumigants, fungicides, resistant varieties, and resistance inducers have been used to control plant diseases caused by *Ralstonia solanacearum* and *Ralstonia pseudosporidis*. However, chemical fumigation is limited in use due to its limited effective range, high toxicity to humans, and significant environmental burden. Validamycin can be used as a fungicide against bacterial wilt in potatoes, but no fungicides have been reported for use against bacterial wilt in other plants. Using resistant varieties presents problems with poor taste and productivity, while using rootstocks results in poor control efficacy. Resistance inducers lack direct fungicidal activity against the pathogens, and their effectiveness depends on the crop variety.

[0012] The purpose of this invention is to provide new control agents and methods for plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis.

[0013] Methods for solving problems

[0014] The present invention relates to the following examples [1] to

[14] .

[0015] [1] A bacterium having a 16S rRNA gene containing a base sequence that is more than 98.80% identical to the base sequence described in sequence number 1, having mannitol assimilation ability, and having the ability to control plant diseases.

[0016] [2] A bacterium having a 16S rRNA gene containing a base sequence that is more than 99.90% identical to the base sequence described in sequence number 1, and having the ability to control plant diseases.

[0017] [3] The bacteria described in [1] or [2] have a 16S rRNA gene containing the base sequence described in sequence number 1.

[0018] [4] A bacterium with accession numbers NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 or NITE BP-03202.

[0019] [5] The bacterial cell or culture of any one of [1] to [4] or an extract thereof.

[0020] [6] An agent for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the bacterial cells or bacterial cultures or extracts thereof of any one of [1] to [4].

[0021] [7] A method for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the step of applying the control agent described in [6].

[0022] [8] A disinfectant for plants, nutrient solutions, soil, nutrient solution cultivation materials or soil cultivation materials contaminated with Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the bacterial cells or bacterial cell cultures or extracts thereof of any one of [1] to [4].

[0023] [9] A method for disinfecting plants, nutrient solutions, soil, nutrient solution cultivation materials or soil cultivation materials contaminated with Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the step of using the disinfectant described in [8].

[0024]

[10] A proliferation inhibitor of Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the bacterial cells or bacterial cultures or extracts thereof of any one of [1] to [4].

[0025]

[11] A method for inhibiting the proliferation of Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the step of using the proliferation inhibitor described in

[10] .

[0026]

[12] A method for cultivating a plant, comprising the step of cultivating the plant in a nutrient solution or soil containing the cells or cultures of the bacteria described in any one of [1] to [4] or extracts thereof.

[0027]

[13] An agent for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the cell or cell culture of bacteria belonging to Lactobacillus plantarum or extracts thereof.

[0028]

[14] A method for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the step of applying the control agent described in

[13] .

[0029] Invention Effects

[0030] According to the present invention, it is possible to prevent and control plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis.

[0031] Brief description of the attached diagram

[0032] [ Figure 1 [A] Figure showing the colony shape and (B) Gram staining results of NITE BP-03197 in Experiment 2.

[0033] [ Figure 2 [A] Figure showing the colony shape and (B) Gram staining results of NITE BP-03198 in Experiment 3.

[0034] [ Figure 3 [A] Figure showing the colony shape and (B) Gram staining results of NITE BP-03199 in Experiment 4.

[0035] [ Figure 4 [A] Figure showing the colony morphology and (B) Gram staining results of NITE BP-03200 in Experiment 5.

[0036] [ Figure 5 [A] Figure showing the colony shape and (B) Gram staining results of NITE BP-03201 in Experiment 6.

[0037] [ Figure 6 [A] Figure showing the colony shape and (B) Gram staining results of NITE BP-03202 in Experiment 7.

[0038] [ Figure 7 [A diagram illustrating the antibacterial activity evaluation test in Experiment 8.]

[0039] [ Figure 8 [The results are from the antimicrobial activity evaluation tests of NITE BP-03197, NITE BP-04198, NITE BP-03199, NITE BP-03200, NITE BP-03201 or NITE BP-02202 in Experiment 8.]

[0040] [ Figure 9 [A diagram illustrating the antibacterial activity evaluation test in Experiment 9.]

[0041] [ Figure 10 [The results are from the antimicrobial activity evaluation tests of NITE BP-03197, NITE BP-04198, NITE BP-03199, NITE BP-03200, NITE BP-03201 or NITE BP-02202 in Experiment 9.]

[0042] [ Figure 11 [The results are from the antimicrobial activity evaluation tests of NITE BP-03197, NITE BP-04198, NITE BP-03199, NITE BP-03200, NITE BP-03201 or NITE BP-02202 in Experiment 10.]

[0043] [ Figure 12 [A diagram illustrating the antibacterial activity evaluation test of Experiment 11.]

[0044] [ Figure 13 [The results are from the antimicrobial activity evaluation tests of NITE BP-03197, NITE BP-04198, NITE BP-03199, NITE BP-03200, NITE BP-03201 or NITE BP-02202 in Experiment 11.]

[0045] [ Figure 14 [Graphical representation of the effect of NITE BP-03201 culture supernatant on bacterial wilt in Experiment 13.]

[0046] [ Figure 15 [Graphical representation showing the effect of culture supernatant of NITE BP-03199 or NITE BP-03200 on the prevention and control of bacterial wilt in Experiment 13.]

[0047] Detailed Implementation of the Invention

[0048] The following provides a detailed description of the methods for implementing the present invention. It should be noted that the present invention is not limited to the following embodiments.

[0049] [Microorganisms belonging to the genus Lactobacillus]

[0050] One embodiment of the present invention relates to a bacterium a, which has a 16S rRNA gene containing a base sequence having more than 98.80% identity with the base sequence described in Serial No. 1, has mannitol assimilation ability, and has the ability to control plant diseases.

[0051] Bacterium a preferably has a 16S rRNA gene containing a base sequence that has 99.00%, 99.20%, 99.50%, 99.80%, or 99.90% identity with the base sequence described in Serial No. 1. Bacterium a may have a 16S rRNA gene containing the base sequence described in Serial No. 1. Examples of bacteria having a 16S rRNA gene containing a base sequence that has 98.80% or more identity with the base sequence described in Serial No. 1 include bacteria belonging to the genus Lactobacillus (lactic acid bacteria).

[0052] Bacterium a may have a 16S rRNA gene containing a sequence of bases that, compared to the sequence described in Serial No. 1, have undergone substitution, deletion, or addition of one or more bases. The one or more bases may be, for example, 1 to 18 bases, preferably 1 to 10 bases, and more preferably 1 to 5 bases.

[0053] Bacterium a may possess a 16S rRNA gene containing approximately 15 or more bases, preferably from about 18 to about 500 bases, more preferably from about 18 to about 200 bases, and even more preferably a continuous sequence of about 18 to about 50 bases, or a base sequence that hybridizes with its complementary sequence under stringent conditions. Stringent conditions refer to conditions under which nonspecific hybridization does not occur, such as washing at 60°C, 1×SSC, 0.1% SDS, preferably at 68°C, 0.1×SSC, 0.1% SDS at least once.

[0054] In this specification, the analysis of the 16S rRNA gene base sequence can be performed, for example, by the following method. First, genomic DNA is extracted from the target microorganism using a known method, and the 16S rRNA gene is amplified. There are no particular limitations on the method for amplifying the 16S rRNA gene; examples include PCR using universal primers, which are commonly used by those skilled in the art. The amplification product obtained by the PCR method can be purified as needed and used with a DNA sequencer or similar instrument to determine the base sequence. The obtained base sequence is then compared with the sequence described in Serial No. 1.

[0055] In this specification, the 16S rRNA gene is preferably an endogenous 16S rRNA gene naturally present in bacteria, but it can also be an artificially mutated 16S rRNA gene. The mutation of the 16S rRNA gene is a mutation that does not result in the loss of 16S rRNA expression and function.

[0056] Bacterium a possesses mannitol assimilation capacity. Mannitol assimilation capacity can be determined based on sugar assimilation capacity tests commonly performed in microbial identification assays. For example, if the target microorganism is cultured in an anaerobic environment using a medium containing mannitol and fermentation occurs, the microorganism's mannitol assimilation capacity can be determined. Commercially available kits can be used for sugar assimilation capacity tests, such as API50CHB (manufactured by bioMerieux, France).

[0057] Bacteria a possesses the ability to control plant diseases. This ability includes preventing pathogenic microorganisms from infecting plants, preventing plant diseases, preventing the spread of plant diseases, and killing, degrading, and inhibiting the proliferation of pathogenic microorganisms.

[0058] In this specification, plant diseases are, for example, those caused by Ralstonia solanacearum or Ralstonia pseudosporidis. Plants infected with Ralstonia solanacearum or Ralstonia pseudosporidis will suffer from bacterial wilt or damping-off and die.

[0059] Whether a target microorganism possesses the ability to control plant diseases can be verified, for example, by culturing pathogenic microorganisms in the presence of the target microorganism or by culturing pathogenic microorganisms in the presence of a culture of the target microorganism. If, under these conditions, pathogenic microorganisms are killed or their proliferation is inhibited, then the target microorganism can be considered to have the ability to control plant diseases. Even when plant pathogens are present in cultivated plants, and the infection or onset of disease is inhibited by the target microorganism, it can still be determined that the target microorganism has the ability to control plant diseases.

[0060] One embodiment of the present invention relates to a bacterium b that has a 16S rRNA gene containing a base sequence having at least 99.90% identity with the base sequence described in Serial No. 1, and has the ability to control plant diseases. Bacterium b may have a 16S rRNA gene containing the base sequence described in Serial No. 1. Examples of bacteria having a 16S rRNA gene containing a base sequence having at least 99.90% identity with the base sequence described in Serial No. 1 include bacteria belonging to the genus *Lactobacillus* (lactic acid bacteria).

[0061] Bacterium b may have a 16S rRNA gene containing a sequence of bases that has been deleted or added by one base compared to the sequence described in sequence number 1.

[0062] Bacterium b may possess a 16S rRNA gene containing approximately 15 or more bases, preferably from about 18 to about 500 bases, more preferably from about 18 to about 200 bases, and even more preferably a continuous sequence of about 18 to about 50 bases, or a base sequence that hybridizes with its complementary sequence under stringent conditions. Stringent conditions refer to conditions under which nonspecific hybridization does not occur, such as washing at 60°C, 1×SSC, 0.1% SDS, preferably at 68°C, 0.1×SSC, 0.1% SDS at least once.

[0063] Bacteria b possesses the ability to control plant diseases. Examples of plant diseases include those caused by *Ralstonia solanacearum* or *Ralstonia pseudosporidis*. The ability to control plant diseases can be evaluated using the methods described above.

[0064] Representative bacteria of bacteria a and b include *Lactobacillus* sp. SC-2001. *Lactobacillus* sp. SC-2001 is a new isolate from the genus *Lactobacillus*, which is closely related to *Lactobacillus mali*. *Lactobacillus* sp. SC-2001, with accession number NITEBP-03197 (original accession date: April 9, 2020), is internationally deposited under the Budapest Treaty at the Patent and Microbiological Collection Center of the Technical Base for Evaluation of Products (NPMD, Room 2-5-8122, Kamisae, Kisarazu City, Chiba Prefecture 292-0818). The bacteriological properties of this strain are shown in Tables 1 and 2 below. Figure 1 middle.

[0065] Lactobacillus bacteria, including bacteria a and b, exist in the natural environment and are therefore considered highly safe as food and feed. Bacteria a and b can be isolated bacteria. Bacteria a and b can also be the same bacteria.

[0066] In one embodiment of the present invention, the bacteria c can be any one of the bacteria listed under accession numbers NITEBP-03198 (original accession date: April 9, 2020), NITEBP-03199 (original accession date: April 9, 2020), NITEBP-03200 (original accession date: April 9, 2020), NITEBP-03201 (original accession date: April 9, 2020), and NITEBP-03202 (original accession date: April 9, 2020), deposited internationally under the Budapest Treaty at the Patent and Microbiological Collection Center of the Technical Base Center for Product Evaluation (NPMD, Room 2-5-8122, Kamisae, Kisarazu City, Chiba Prefecture 292-0818, Japan). All bacteria c belong to the Lactobacillus plantarum family. The bacteriological properties of this strain are shown in Tables 3–12 below. Figures 2-6 middle.

[0067] Bacterium c possesses the ability to control plant diseases. These plant diseases include those caused by *Ralstonia solanacearum* or *Ralstonia pseudosporidis*. The ability to control plant diseases can be evaluated using the methods described above. Furthermore, it is believed that *Lactobacillus plantarum*, to which bacterium c belongs, also possesses the ability to control plant diseases caused by *Ralstonia solanacearum* or *Ralstonia pseudosporidis*.

[0068] Lactobacillus bacteria, including bacterium c, exist in the natural environment and are therefore considered highly safe as food and feed. Bacterium c can be an isolated bacterium.

[0069] [Bacterial cells or cell cultures or extracts thereof]

[0070] One embodiment of the present invention relates to bacterial cells of any one of bacteria, namely bacteria a, bacteria b, and bacteria c. The bacterial cells can be bacterial cells isolated from the environment or cultured bacterial cells. The bacterial cells can be dead or live bacteria. The bacterial cells can be bacterial cells present in culture media, buffer solutions, etc., or bacterial cells that have been concentrated and had the liquid removed, or their freeze-dried products. The bacterial cells of bacteria a, b, and c each possess the ability to control plant diseases.

[0071] One embodiment of the present invention relates to a bacterial cell culture of any one of bacteria, namely bacteria a, bacteria b, and bacteria c. The cell culture contains bacterial secretions, metabolites, etc., including peptides, proteins, sugars, enzymes, organic acids produced by the bacteria, and a culture medium (liquid or solid) containing them. The cell culture can be the supernatant from a bacterial culture. The culture supernatant can be obtained, for example, by removing bacteria from a liquid culture medium through centrifugation, filtration, or other operations. Cell cultures of bacteria a, b, and c each possess the ability to control plant diseases.

[0072] Bacteria a, b, and c can be cultured using standard methods for bacteria belonging to the genus *Lactobacillus*. A representative method is culturing them using MRS (de Man, Rogosa, and Sharpe) liquid medium or MRS agar medium at 30°C.

[0073] One embodiment of the present invention relates to an extract of the bacterial cells or cultures of any one of bacteria, namely bacteria a, b, and c. The extract is prepared in a manner that does not lose the plant disease control capabilities possessed by the bacterial cells or cultures. The extract can be obtained, for example, by treating the bacterial cells or cultures with ultrasonic disruption, bead milling, freeze-thaw cycles, or chemical dissolution. The extract can also be obtained by salting out, ultrafiltration, ion exchange chromatography, or liquid-phase extraction using organic solvents. These treatments can be appropriately combined. The extract may contain bacterial cell fragments, nucleic acids, peptides, proteins, and enzymes.

[0074] [Control agents and methods for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis]

[0075] In this specification, bacterial cells, bacterial cultures, or their extracts are also referred to as "bacterial cell preparations." A bacterial cell preparation can be any one, or multiple, selected from the group consisting of bacterial cells, bacterial cultures, and their extracts. One embodiment of the present invention relates to a control agent comprising a bacterial cell preparation of any one of bacteria a to c. Another embodiment of the present invention relates to a control agent comprising a bacterial cell preparation of a bacterium belonging to *Lactobacillus plantarum* (hereinafter also referred to as "bacterium d"). The control agent may comprise two or more bacterial cell preparations selected from the group consisting of bacteria a to d. The control agent of the present invention has the ability to control plant diseases caused by *Ralstonia solanacearum* or *Ralstonia pseudosobacterium*. The control ability can be evaluated by the methods described above.

[0076] The form of the control agent can be any solid, liquid, or gaseous form that is commonly used for pesticides, such as powder, DL (driftless) powder, granules, tablets, hydration agents, granular hydration agents, dry suspensions, emulsions, liquids, oils, microcapsules, suspensions, emulsions, microemulsions, AL (applicable liquid) agents, fumigants, etc. There are no particular limitations; it can be prepared into any formulation and used according to the purpose.

[0077] When formulated, pesticides can include carriers, anti-aggregating agents, anti-degradation agents, expanders, dispersants, binding materials, disintegrants, and surfactants. Solid carriers can include diatomaceous earth, vermiculite, clay, talc, bentonite, perlite, charcoal, rice husks, bone meal, and calcium carbonate. Liquid carriers include water, alcohols, ketones (acetone, methyl ethyl ketone, cyclohexanone, etc.), aromatic hydrocarbons (e.g., benzene, toluene, xylene, ethylbenzene, methylnaphthalene), aliphatic hydrocarbons (n-hexane, kerosene, etc.), esters, nitriles, ethers, amides, and halogenated hydrocarbons. Gaseous carriers include LPG, air, nitrogen, carbon dioxide, and dimethyl ether.

[0078] As surfactants or dispersants, alkyl sulfates, alkyl (aryl) sulfonates, polyoxyalkyl (aryl) ethers, polyol esters, lignin sulfonates, etc., can be used.

[0079] As long as the control agent does not lose its ability to control plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis, it can contain microorganisms other than bacteria a to d, chemical control substances, etc.

[0080] The control agents involved in this invention have a low environmental impact and low toxicity to humans. It is believed that the control agents involved in this invention are independent of crop variety and have direct antibacterial effects against plant pathogenic microorganisms. Based on the control agents involved in this invention, stable agricultural production can be achieved.

[0081] One embodiment of the present invention is the application of a bacterial preparation selected from at least one of the group consisting of bacteria a to d in the preparation of an agent for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis.

[0082] One embodiment of the present invention relates to a control method including the step of applying the aforementioned control agent. According to the control method of the present invention, plant diseases caused by *Ralstonia solanacearum* or *Ralstonia pseudosporidis* can be controlled.

[0083] As an example of the process of applying a control agent, the process of applying a liquid control agent to plants can be cited. This process includes spraying or soaking the plant seeds with the control agent, and applying the control agent to the seeds. It also includes soaking seedlings before planting in the control agent, soaking the roots of seedlings in the control agent, and spraying the control agent onto the roots, leaves, and stems.

[0084] Other examples of the application of control agents include the process of applying liquid control agents to nutrient solutions or soil. The process of applying liquid control agents to nutrient solutions includes adding the control agent to the nutrient solution or diluting the control agent with the nutrient solution. The process of applying liquid control agents to soil includes spraying, spreading, or drenching the soil with the control agent. The application of control agents to the soil can be carried out before planting the plants or before sowing the seeds, or it can be carried out after planting or sowing.

[0085] Other examples of the process of applying control agents include the process of applying powdered or granular control agents to plants. The process of applying powdered or granular control agents to plants includes processes of applying the powdered or granular control agent to the seed surface, and processes of mixing the powdered or granular control agent with the seeds and then sowing them, etc.

[0086] Other examples of the process of applying control agents include the process of applying powdered or granular control agents to nutrient solutions or soil. The process of applying powdered or granular control agents to nutrient solutions includes the process of dispersing or dispersing the control agent in the nutrient solution. The process of applying powdered or granular control agents to soil includes the process of mixing the powdered or granular control agent into the soil, the process of dispersing it on the soil, etc.

[0087] Solid or liquid control agents can be applied by suspending or diluting them in water or nutrient solution. When applying control agents to nutrient solution, the control agent preferably contains bacterial cells and / or bacterial cultures selected from at least one of the group consisting of bacteria a to d. When applying control agents to soil, the control agent preferably contains bacterial cells and / or bacterial cultures selected from at least one of the group consisting of bacteria a to d, more preferably containing live bacteria selected from at least one of the group consisting of bacteria a to d. The application of control agents can be performed multiple times or in combination of the above-described steps.

[0088] The application of control agents can be carried out under conditions that do not diminish their control effectiveness. For example, it can be done at temperatures of 20–40°C or 25–30°C. Additionally, it can be done at pH levels of 3.0–8.0 or 4.0–6.0.

[0089] There is no particular limit to the total concentration of bacteria a to d in the control agent; it can be 1 × 10⁻⁶.3 ~1×10 12 cfu (colony forming units) / g, or 1×10 4 ~1×10 12 cfu / g, or 1×10 5 ~1×10 12 The concentration of the bacterial culture or extract in the control agent is not particularly limited and can be 0.1–100,000 ppm, 1–100,000 ppm, or 10–100,000 ppm. The control agent can be prepared by mixing 0.01–100 mL of the following solution with water per 50 g of soil: the solution is prepared to the above concentration by culturing the supernatant (bacterial culture) of at least one selected from the group consisting of bacteria a–d. The control agent can also be prepared by mixing the supernatant of the culture of at least one selected from the group consisting of bacteria a–d with nutrient solution at a ratio of 1 / 10000–1 / 10. The control agent should be applied in an amount suitable for meteorological conditions, formulation form, application time, application method, application location, and target plant.

[0090] In this specification, "plant" refers to plants that produce diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis. Such plants include, for example, agricultural and horticultural plants, specifically plants of the Solanaceae, Cucurbitaceae, Brassicaceae, Zingiberaceae, Rosaceae, Asteraceae, Leguminosae, Musaceae, Lamiaceae, Myrtaceae, Pedaliaceae, Moraceae, Strelitziaceae, Euphorbiaceae, Plumbaginaceae, and Gentianaceae families. Examples of plants in the Solanaceae family include tomatoes, eggplants, green bell peppers, sweet green bell peppers, chili peppers, red chili peppers, potatoes, and tobacco. Examples of plants in the Cucurbitaceae family include cucumbers, pumpkins, bitter melons, watermelons, cantaloupes, and zucchini. Examples of plants in the Cruciferae family include radishes, turnips, rapeseed, bok choy, komatsuna, cauliflower, broccoli, and cabbage. Examples of plants in the Zingiberaceae family include ginger and ginger root. Examples of plants in the Rosaceae family include strawberries. Examples of plants in the Asteraceae family include garland chrysanthemum and lettuce. Examples of plants in the Leguminosae family include peanuts, kidney beans, broad beans, peas, and soybeans. Examples of plants in the Musaceae family include bananas and plantains. Examples of plants in the Lamiaceae family include perilla and basil. Examples of plants in the Myrtaceae family include cloves. Examples of plants in the Pedaliaceae family include sesame. Examples of plants in the Moraceae family include mulberry trees. Examples of plants in the Strelitzia family include bird of paradise. Examples of plants in the Euphorbiaceae family include cassava. Examples of plants in the Plumbaginaceae family include limonite. Examples of plants in the Gentianaceae family include lisianthus. These specific examples mainly focus on agricultural crops, but plants can also be flowering plants.

[0091] One embodiment of the present invention is the application of the above-mentioned control agent for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis. Another embodiment of the present invention is the application of a bacterial preparation selected from at least one of the group consisting of bacteria a to d for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosporidis.

[0092] [Disinfectants and disinfection methods for plants, nutrient solutions, soil, nutrient solution cultivation materials, or soil cultivation materials contaminated with Ralstonia solanacearum or Ralstonia pseudosporidis]

[0093] One embodiment of the present invention relates to a disinfectant comprising a bacterial cell preparation of any one of bacteria a to c. Another embodiment of the present invention relates to a disinfectant comprising a bacterial cell preparation of bacteria d. The disinfectant may comprise two or more bacterial cell preparations selected from the group consisting of bacteria a to d. The disinfectant of the present invention can disinfect plants, nutrient solutions, soil, nutrient solution cultivation materials, or soil cultivation materials contaminated with *Ralstonia solanacearum* or *Ralstonia solanacearum*. If plants are cultivated using disinfected plants, nutrient solutions, soil, nutrient solution cultivation materials, or soil cultivation materials, the occurrence of plant diseases caused by *Ralstonia solanacearum* or *Ralstonia solanacearum* can be inhibited. The disinfectant of the present invention also has the effect of acting as a fungicide against *Ralstonia solanacearum* or *Ralstonia solanacearum*.

[0094] The effectiveness of a disinfectant can be evaluated by the following method, in which Ralstonia solanacearum or Ralstonia pseudosorcinia solanacearum is cultured in the presence of the disinfectant involved in this invention, and the effectiveness of the disinfectant is verified to be whether the Ralstonia solanacearum or Ralstonia pseudosorcinia solanacearum is killed, degraded or its proliferation is inhibited.

[0095] The disinfectant can be the same as the aforementioned preventative agents. The disinfectant may contain substances used to disinfect other pathogenic microorganisms.

[0096] Plants consist of seeds, seedlings, roots, tubers, bulbs, rhizomes, and stems and leaves (including flowers). From the perspective of preventing infection by pathogens, disinfection of plant seedlings, roots, etc., is particularly useful.

[0097] Nutrient solution cultivation is a cultivation method that does not use soil, providing the nutrients and water needed for plant growth as liquid fertilizer (nutrient solution). Representative examples of nutrient solution cultivation include hydroponics (rooting in nutrient solution), solid culture medium cultivation (growing crops on a solid medium that replaces soil), and spray cultivation (spraying nutrient solution into the roots). Nutrient solutions contain nitrogen (N), phosphate (P2O5), potassium (K2O), etc. Materials used in nutrient solution cultivation include cultivation beds, cultivation boards, planting panels, seedling pots, solid culture media (asbestos, etc.), nutrient solution tanks, nutrient solution pipes, irrigation pipes, and small agricultural tools (pruning shears, thermometers, hygrometers, etc.).

[0098] Soil cultivation is a cultivation method that uses soil. Soil includes soil for sowing, soil for seedling raising, sand, pumice, farm soil, etc. Materials used in soil cultivation include flower pots, seedling trays, cultivation boards, cultivation containers, spreaders, irrigation pipes, and small farm tools (pruning shears, thermometers, hygrometers, etc.).

[0099] One embodiment of the present invention is the application of a bacterial preparation selected from at least one of the group consisting of bacteria a to d in the preparation of disinfectants or bactericides for plants, nutrient solutions, soil, nutrient solution cultivation materials or soil cultivation materials contaminated by Ralstonia solanacearum or Ralstonia pseudosporidis.

[0100] One embodiment of the present invention relates to a disinfection method that includes the step of using the aforementioned disinfectant. According to the disinfection method of the present invention, plants, nutrient solutions, soil, nutrient solution cultivation materials, and soil cultivation materials contaminated with Ralstonia solanacearum or Ralstonia pseudosporidis can be disinfected.

[0101] Disinfectants can be used on plants, nutrient solutions, soil, hydroponic materials, or soil-grown materials that are contaminated with or potentially contaminated by *Ralstonia solanacearum* or *Ralstonia pseudosporidis*. In methods for disinfecting soil, nutrient solutions, and plants, the step of using a disinfectant can be performed in the same manner as applying the aforementioned control agents. In methods for disinfecting hydroponic or soil-grown materials, the step of using a disinfectant includes, for example, spraying, distributing, or immersing the hydroponic or soil-grown materials in a disinfectant solution, either by spraying, distributing, or immersing them in the disinfectant. The step of using a disinfectant can be performed simultaneously with known methods for disinfecting plants, nutrient solutions, soil, hydroponic materials, or soil-grown materials, such as soil fumigation.

[0102] There is no specific limit to the total concentration of bacteria a to d in the disinfectant; it can be 1 × 10⁻⁶. 3 ~1×10 12 cfu / g, or 1×10 4 ~1×10 12 cfu / g, or 1×10 5 ~1×10 12 CFU / g. There are no particular limitations on the concentration of bacterial culture or extract in the disinfectant; it can be 0.1–100,000 ppm, 1–100,000 ppm, or 10–100,000 ppm. The amount of the control agent should be appropriate for the weather conditions, formulation, application period, method of application, application location, and target plant.

[0103] One embodiment of the present invention is the application of the aforementioned disinfectant for disinfecting or sterilizing plants, nutrient solutions, soil, nutrient solution cultivation materials, or soil cultivation materials contaminated with *Ralstonia solanacearum* or *Ralstonia solanacearum*. Another embodiment of the present invention is the application of a bacterial preparation selected from at least one of the group consisting of bacteria a to d for disinfecting or sterilizing plants, nutrient solutions, soil, nutrient solution cultivation materials, or soil cultivation materials contaminated with *Ralstonia solanacearum* or *Ralstonia solanacearum*.

[0104] [Inhibitors and methods for inhibiting the proliferation of Ralstonia solanacearum or Ralstonia pseudosporidis]

[0105] One embodiment of the present invention relates to a proliferation inhibitor comprising a cell preparation of any one of bacteria a to c. Another embodiment of the present invention relates to a proliferation inhibitor comprising a cell preparation of bacteria d. The proliferation inhibitor may comprise two or more cell preparations selected from the group consisting of bacteria a to d. The proliferation inhibitor of the present invention is capable of inhibiting the proliferation of *Ralstonia solanacearum* or *Ralstonia solanacearum*. Inhibiting the proliferation of *Ralstonia solanacearum* or *Ralstonia solanacearum* inhibits the occurrence of plant diseases caused by these bacteria.

[0106] Growth inhibitors can be used, similar to the control agents mentioned above. Growth inhibitors may contain substances that inhibit the growth of other pathogenic microorganisms.

[0107] The effect of proliferation inhibitors can be evaluated by culturing Ralstonia solanacearum or Ralstonia pseudosporidis in the presence of a proliferation inhibitor to verify whether the proliferation of Ralstonia solanacearum or Ralstonia pseudosporidis is inhibited.

[0108] One embodiment of the present invention is the application of a bacterial cell preparation selected from at least one of the group consisting of bacteria a to d in the preparation of a proliferation inhibitor of Ralstonia solanacearum or Ralstonia pseudosporidis.

[0109] One embodiment of the present invention relates to a proliferation inhibition method including the step of using the above-mentioned proliferation inhibitor. According to the proliferation inhibition method of the present invention, the proliferation of *Ralstonia solanacearum* or *Ralstonia pseudosporidis* can be inhibited, and the occurrence of plant diseases caused by *Ralstonia solanacearum* or *Ralstonia pseudosporidis* can be inhibited.

[0110] There are no particular limitations on the procedure for using the proliferation inhibitor; it can be applied in a manner that brings the proliferation inhibitor into contact with or near Ralstonia solanacearum or Ralstonia pseudosporidis. The proliferation inhibitor can be applied using the same method as the control agents described above.

[0111] The total concentration of bacteria a to d in the proliferation inhibitor is not specifically limited and can be 1 × 10⁻⁶. 3 ~1×10 12 cfu / g, or 1×10 4 ~1×10 12 cfu / g, or 1×10 5 ~1×10 12cfu / g. The concentration of bacterial culture or extract in the proliferation inhibitor is not particularly limited and can be 0.1–100,000 ppm, 1–100,000 ppm, or 10–100,000 ppm.

[0112] One embodiment of the present invention is the application of the aforementioned proliferation inhibitor for inhibiting the proliferation of *Ralstonia solanacearum* or *Ralstonia solanacearum*. Another embodiment of the present invention is the application of a bacterial cell preparation selected from at least one of the group consisting of bacteria a to d for inhibiting the proliferation of *Ralstonia solanacearum* or *Ralstonia solanacearum*.

[0113] Plant cultivation methods

[0114] One embodiment of the present invention relates to a plant cultivation method comprising the step of cultivating plants in a nutrient solution or soil containing a bacterial cell preparation of at least one bacterium selected from the group consisting of bacteria a to c. According to the plant cultivation method of the present invention, plant diseases caused by *Ralstonia solanacearum* or *Ralstonia pseudosporidis* can be suppressed.

[0115] The method for containing a bacterial preparation of at least one bacterium selected from the group consisting of bacteria a to c in a nutrient solution or soil is not particularly limited, and the same method as the application method of the control agent described above can be used. A bacterial preparation of at least one bacterium selected from the group consisting of bacteria a to c can be contained in a nutrient solution or soil as the control agent described above.

[0116] Plant cultivation methods include hydroponics or soil cultivation. These methods may include, for example, the steps of preparing nutrient solutions or soil, sowing seeds, irradiating with light, thinning seedlings, and allowing plants to grow. Nutrient solutions or soil containing a bacterial cell preparation of at least one bacterium selected from the group consisting of bacteria a to c may be used in at least some of these steps, or in all steps.

[0117] One embodiment of the present invention is the application of the above-mentioned control agent in a method of cultivating plants in nutrient solution or soil. Another embodiment of the present invention is the application of a bacterial preparation selected from at least one of the group consisting of bacteria a to d in a method of cultivating plants in nutrient solution or soil. Example

[0118] The present invention will be described in more detail below with reference to specific embodiments, but the present invention is not limited to these embodiments.

[0119] [Experiment 1: Isolation of New Microorganisms]

[0120] The isolated source (Quercus acutissima) was ground together with sterile water. The ground liquid was appropriately diluted and added to 1 / 2 MRS liquid medium for enrichment culture. The enriched culture was spread on MRS agar medium containing calcium carbonate, and the microorganisms that formed halos were isolated. Hereinafter, this isolate will be referred to as isolate A. When the culture medium of isolate A was suspended in hydrogen peroxide, no bubbles were produced. Since isolate A does not have catalase activity, it was confirmed to be a lactic acid bacterium.

[0121] [Experiment 2: Identification of Isolator A]

[0122] Isolator A was identified through 16S rRNA gene analysis, morphological observation, and physiological and biochemical trait tests.

[0123] (1) 16S rRNA gene analysis

[0124] Genomic DNA was extracted from isolate A. Using this genomic DNA as a template, PCR amplification of the 16S rRNA gene was performed using forward primer 9F and reverse primer 1510R (Yoshikata Nakagawa: Gene Analysis Method for Determining the Base Sequence of the 16S rRNA Gene, edited by the Japanese Society for Actinomycetes, Classification and Identification of Actinomycetes, 88-117pp. Japan Society Affairs Center, 2001). PCR amplification was performed using Tks Gflex DNA polymerase (manufactured by Takara Bio), and the amplified products were purified.

[0125] The purified PCR amplification products were used for cyclic sequencing. Cyclic sequencing was performed using the BigDye terminator v3.1 cyclic sequencing kit. The resulting reaction solution was purified, and DNA sequencing analysis (3130×1 DNA analyzer) was performed to determine the nucleotide sequence (accession number 1) of the 16S rRNA gene from the template DNA extracted from isolate A. Primers used for sequencing analysis included 9F, 515F, 1099F, 536R, 926R, and 1510R (Yoshikata Nakagawa: Gene Analysis Method for Determining the Base Sequence of the 16S rRNA Gene, edited by the Japanese Society for Actinomycetes, Classification and Identification of Actinomycetes, 88-117pp. Japan Society Affairs Center, 2001).

[0126] Using the microbial identification system "ENKI" (manufactured by TechnoSuruga Laboratory), a BLAST homology search was performed on the 16S rRNA gene sequence of isolate A in the microbial identification database DB-BA15.0 (manufactured by TechnoSuruga Laboratory) and the international base sequence databases (DDBJ / ENA(EMBL) / GenBank). The sequence identity with respect to the 16S rRNA gene of Lactobacillus mali (NBRC 102159) was 99.87%, with respect to the 16S rRNA gene of Lactobacillus cacaonum (LMG24285) was 98.72%, and with respect to the 16S rRNA gene of Lactobacillus aquatius (IMCC1736) was 97.58%. However, there is no microorganism with a 16S rRNA gene that has a completely identical base sequence to that of isolate A.

[0127] (2) Morphological observation and physiological and biochemical trait tests

[0128] Bacterial isolate A was plated on MRS agar medium and cultured aerobically at 30°C for 48 hours. Cell morphology, Gram staining, motility, and colony morphology were observed using the following methods. The physiological and biochemical characteristics of the bacteria were examined using the API50CHB kit (bioMerieux, France).

[0129] Colony observation was performed using a stereomicroscope SMZ800N (Nikon), and morphological observation was performed using an optical microscope BX50F4 (Olympus). Tests were conducted on catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F) based on methods described in Barrow & Feltham's (Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd ed. Cambridge: Cambridge University Press; 1993). Gram staining was performed using Favor G "NISSUI" (Nissui Pharmaceutical Co., Ltd.). Figure 1 As shown in (A), isolate A formed circular colonies. Figure 1(B) shows that isolate A is Gram-positive. The results of physiological and biochemical phenotypic tests and fermentation tests for isolate A are shown in Tables 1 and 2. Isolate A is a motile Gram-positive bacillus that ferments glucose, and is negative for catalase and oxidase reactions. These traits are consistent with those of the *Lactobacillus* genus as determined by partial 16S rDNA sequence analysis. Fermentation tests using the API CHL50 kit showed that isolate A ferments fructose, mannose, mannitol, and salicin, but not galactose or lactose. Furthermore, growth at 15°C did not show arginine dihydrogenase activity. These traits are almost identical to those of Lactobacillus mali. The results of partial 16S rDNA sequence analysis suggest that the two are closely related, but differ in their fermentation of mannitol (Hammes WP and Hertel C. Lactobacillus. In: Whitman WB, Rainey F, Kampfer P, Trujillo M, Chun J et al. (editors). Bergey's Manual of Systematics of Archaea and Bacteria. Chichester: John Wiley & Sons; 2015. doi: 10.1002 / 9781118960608.gbm00604). As mentioned above, isolate A belongs to the genus *Lactobacillus* and is most closely related to *Lactobacillus mali* among known species. However, 16S rDNA sequence analysis and physiological and biochemical phenotypic tests indicate that isolate A differs slightly from *Lactobacillus mali*. Therefore, it is considered a new isolate closely related to *Lactobacillus mali*. Isolate A is named *Lactobacillus sp.* SC-2001 and deposited as NITE BP-03197.

[0130] [Table 1]

[0131]

[0132] +: Positive, -: Negative

[0133] [Table 2]

[0134] project matrix components Test results project matrix components Test results 0 Comparison - 25 Aesculin + 1 Glycerol - 26 Salicylic acid + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 maltose - 4 L-arabinose - 29 lactose - 5 Ribose - 30 Melibi - 6 D-xylose - 31 sucrose + 7 L-xylose - 32 Trehalose + 8 Calendula alcohol - 33 Inulin - 9 β-methyl-D-xylose - 34 Ginsenosides - 10 Galactose - 35 Raffinose - 11 glucose + 36 starch - 12 fructose + 37 Glycogen - 13 Mannose + 38 Xylitol - 14 sorbose - 39 Gentian disaccharide - 15 Rhamnose - 40 D-Melalose - 16 Euonymus alcohol - 41 D-Lysulose - 17 Inositol - 42 D-Tagose + 18 Mannitol + 43 D-fucose - 19 Sorbitol - 44 L-fucose - 20 α-Methyl-D-mannoside + 45 D-Arabatool - 21 α-Methyl-D-glucoside - 46 L-Arabatool - 22 N-acetylglucosamine + 47 gluconate - 23 amygdalin + 48 2-Ketogluconic acid - 24 Arbutin + 49 5-Ketogluconic acid -

[0135] +: Positive, -: Negative

[0136] [Experiment 3: Isolation and Identification of Isolator B]

[0137] Salted cuttlefish was used as the isolation source, and isolate B was isolated using the same method as in Experiment 1. Identification of isolate B was performed using the same method as in Experiment 2.

[0138] The 16S rRNA gene sequence of isolate B showed 99.87% identity with the 16S rRNA gene of *Lactobacillus pentosus* (JCM1558), 99.87% identity with the 16S rRNA gene of *Lactobacillus plantarum subsp. plantarum* (JCM1149), and 99.73% identity with the 16S rRNA gene of *Lactobacillus paraplantarum* (DSM10667). However, no microorganism possessed a 16S rRNA gene with a completely identical 16S rRNA gene sequence to isolate B.

[0139] like Figure 2 As shown in (A), isolate B formed circular colonies. Figure 2 As shown in (B), isolate B was Gram-positive. The results of physiological and biochemical phenotypic tests and fermentation tests for isolate B are shown in Tables 3 and 4. Isolate B is a non-motile Gram-positive bacillus that does not form spores, and is negative for catalase and oxidase reactions, fermenting glucose. These characteristics are consistent with those of *Lactobacillus* species indicated by partial 16S rDNA sequence analysis. Fermentation tests using the API kit showed that isolate B fermented galactose, fructose, and menotriose, but not glycerol or D-xylose. Furthermore, it did not exhibit arginine dihydrogenase activity and grew at 15°C. These characteristics are consistent with those of *Lactobacillus pentosus* and *Lactobacillus plantarum*, which were identified through partial 16S rDNA sequence analysis, but differ from *Lactobacillus pentosus* at the point where fermentation does not show glycerol and D-xylose. Therefore, isolate B is a new isolate belonging to *Lactobacillus plantarum*. Isolate B is deposited internationally as NITE BP-03198.

[0140] [Table 3]

[0141]

[0142] +: Positive, -: Negative

[0143] [Table 4]

[0144] project matrix components Test results project matrix components Test results 0 Comparison - 25 Aesculin + 1 Glycerol - 26 Salicylic acid + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 maltose + 4 L-arabinose + 29 lactose + 5 Ribose + 30 Melibi + 6 D-xylose - 31 sucrose + 7 L-xylose - 32 Trehalose + 8 Calendula alcohol - 33 Inulin - 9 β-methyl-D-xylose - 34 Ginsenosides + 10 Galactose + 35 Raffinose + 11 glucose + 36 starch - 12 fructose + 37 Glycogen - 13 Mannose + 38 Xylitol - 14 sorbose - 39 Gentian disaccharide + 15 Rhamnose - 40 D-Melalose + 16 Euonymus alcohol - 41 D-Lysulose - 17 Inositol - 42 D-Tagose - 18 Mannitol + 43 D-fucose - 19 Sorbitol + 44 L-fucose - 20 α-Methyl-D-mannoside + 45 D-Arabatool - 21 α-Methyl-D-glucoside - 46 L-Arabatool - 22 N-acetylglucosamine + 47 gluconate + 23 amygdalin + 48 2-Ketogluconic acid - 24 Arbutin + 49 5-Ketogluconic acid -

[0145] +: Positive, -: Negative

[0146] [Experiment 4: Isolation and Identification of Isolator C]

[0147] Wax apples were used as the source of isolation, and isolate C was isolated using the same method as in Experiment 1. Identification of isolate C was performed using the same method as in Experiment 2.

[0148] The 16S rRNA gene sequence of isolate C showed 100.0% identity with the 16S rRNA gene of *Lactobacillus pentosus* (JCM1558), 100.0% identity with the 16S rRNA gene of *Lactobacillus plantarum subsp. plantarum* (JCM1149), and 99.80% identity with the 16S rRNA gene of *Lactobacillus paraplantarum* (DSM10667).

[0149] like Figure 3 As shown in (A), isolate C formed circular colonies. Figure 3As shown in (B), isolate C was Gram-positive. The results of physiological and biochemical phenotypic and fermentation tests for isolate C are shown in Tables 5 and 6. Isolate C was a non-motile Gram-positive bacillus that did not form spores, showed negative catalase and oxidase reactions, and fermented glucose. These characteristics are consistent with those of *Lactobacillus* species indicated by partial 16S rDNA sequence analysis. Fermentation tests using the API kit showed that isolate C fermented galactose, fructose, α-methyl-D-mannoside, and menotriose, but not glycerol or D-xylose. Furthermore, it did not show arginine dihydrogenase activity and grew at 15°C. These characteristics are consistent with those of *Lactobacillus pentosus* and *Lactobacillus plantarum*, which were identified through partial 16S rDNA sequence analysis, but differ from *Lactobacillus pentosus* at the point where fermentation does not show glycerol and D-xylose. Therefore, isolate C is a new isolate belonging to *Lactobacillus plantarum*. Isolate C is deposited internationally as NITEBP-03199.

[0150] [Table 5]

[0151]

[0152] +: Positive, -: Negative

[0153] [Table 6]

[0154] project matrix components Test results project matrix components Test results 0 Comparison - 25 Aesculin + 1 Glycerol - 26 Salicylic acid + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 maltose + 4 L-arabinose + 29 lactose + 5 Ribose + 30 Melibi + 6 D-xylose - 31 sucrose + 7 L-xylose - 32 Trehalose + 8 Calendula alcohol - 33 Inulin - 9 β-methyl-D-xylose - 34 Ginsenosides + 10 Galactose + 35 Raffinose + 11 glucose + 36 starch - 12 fructose + 37 Glycogen - 13 Mannose + 38 Xylitol - 14 sorbose - 39 Gentian disaccharide + 15 Rhamnose - 40 D-Melalose + 16 Euonymus alcohol - 41 D-Lysulose - 17 Inositol - 42 D-Tagose - 18 Mannitol + 43 D-fucose - 19 Sorbitol + 44 L-fucose - 20 α-Methyl-D-mannoside + 45 D-Arabatool - 21 α-Methyl-D-glucoside + 46 L-Arabatool - 22 N-acetylglucosamine + 47 gluconate + 23 amygdalin + 48 2-Ketogluconic acid - 24 Arbutin + 49 5-Ketogluconic acid -

[0155] +: Positive, -: Negative

[0156] [Experiment 5: Isolation and Identification of Isolator D]

[0157] Pandanus odoratissimus was used as the source of isolation, and isolate D was isolated using the same method as in Experiment 1. Identification of isolate D was performed using the same method as in Experiment 2.

[0158] The 16S rRNA gene sequence of isolate D showed 99.87% identity with the 16S rRNA gene of *Lactobacillus pentosus* (JCM1558), 99.87% identity with the 16S rRNA gene of *Lactobacillus plantarum subsp. plantarum* (JCM1149), and 99.66% identity with the 16S rRNA gene of *Lactobacillus paraplantarum* (DSM10667). However, no microorganism possessed a 16S rRNA gene with a completely identical 16S rRNA gene sequence to that of isolate D.

[0159] like Figure 4 As shown in (A), isolate D formed circular colonies. Figure 4 As shown in (B), isolate D was Gram-positive. The results of physiological and biochemical phenotypic and fermentation tests for isolate D are shown in Tables 7 and 8. Isolate D was a non-motile Gram-positive bacillus that did not form spores, showed negative catalase and oxidase reactions, and fermented glucose. These characteristics are consistent with those of *Lactobacillus* species indicated by partial 16S rDNA sequence analysis. Fermentation tests using the API kit showed that isolate D fermented galactose, fructose, α-methyl-D-mannoside, and menotriose, but not glycerol or D-xylose. Furthermore, it did not show arginine dihydrogenase activity and grew at 15°C. These characteristics are consistent with those of *Lactobacillus pentosus* and *Lactobacillus plantarum*, which are closely related to *Lactobacillus pentosus* and *Lactobacillus plantarum*, as shown by partial 16S rDNA sequence analysis. However, they differ from *Lactobacillus pentosus* at the point where fermentation does not show glycerol and D-xylose. Therefore, isolate D is a new isolate belonging to *Lactobacillus plantarum*. Isolate D is deposited internationally as NITEBP-03200.

[0160] [Table 7]

[0161]

[0162] +: Positive, -: Negative

[0163] [Table 8]

[0164] project matrix components Test results project matrix components Test results 0 Comparison - 25 Aesculin + 1 Glycerol - 26 Salicylic acid + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 maltose + 4 L-arabinose - 29 lactose + 5 Ribose + 30 Melibi + 6 D-xylose - 31 sucrose + 7 L-xylose - 32 Trehalose + 8 Calendula alcohol - 33 Inulin - 9 β-methyl-D-xylose - 34 Ginsenosides + 10 Galactose + 35 Raffinose + 11 glucose + 36 starch - 12 fructose + 37 Glycogen - 13 Mannose + 38 Xylitol - 14 sorbose - 39 Gentian disaccharide + 15 Rhamnose - 40 D-Melalose + 16 Euonymus alcohol - 41 D-Lysulose - 17 Inositol - 42 D-Tagose - 18 Mannitol + 43 D-fucose - 19 Sorbitol + 44 L-fucose - 20 α-Methyl-D-mannoside + 45 D-Arabatool - 21 α-Methyl-D-glucoside - 46 L-Arabatool - 22 N-acetylglucosamine + 47 gluconate + 23 amygdalin + 48 2-Ketogluconic acid - 24 Arbutin + 49 5-Ketogluconic acid -

[0165] +: Positive, -: Negative

[0166] [Experiment 6: Isolation and Identification of Isolator E]

[0167] Ficus variegata was used as the isolation source, and isolate E was isolated using the same method as in Experiment 1. Identification of isolate E was performed using the same method as in Experiment 2.

[0168] The 16S rRNA gene sequence of isolate E showed 99.93% identity with the 16S rRNA gene of *Lactobacillus pentosus* (JCM1558), 99.93% identity with the 16S rRNA gene of *Lactobacillus plantarum subsp. plantarum* (JCM1149), and 99.73% identity with the 16S rRNA gene of *Lactobacillus paraplantarum* (DSM10667). However, no microorganism possessed a 16S rRNA gene with a completely identical 16S rRNA gene sequence to isolate E.

[0169] like Figure 5 As shown in (A), isolate E formed circular colonies. Figure 5As shown in (B), isolate E was Gram-positive. The results of physiological and biochemical phenotypic tests and fermentation tests for isolate E are shown in Tables 9 and 10. Isolate E was a non-motile Gram-positive bacillus that did not form spores, showed negative catalase and oxidase reactions, and fermented glucose. These characteristics are consistent with those of *Lactobacillus* species indicated by partial 16S rDNA sequence analysis. Fermentation tests using the API kit showed that isolate E fermented galactose, fructose, and menotriose, but not glycerol or D-xylose. Furthermore, no arginine dihydrogenase activity was observed, and it grew at 15°C. These characteristics are consistent with those of *Lactobacillus pentosus* and *Lactobacillus plantarum*, which were identified through partial 16S rDNA sequence analysis. However, they differ from *Lactobacillus pentosus* at the point where fermentation does not show glycerol and D-xylose. Therefore, isolate E is a novel isolate belonging to *Lactobacillus plantarum*. Isolate E is deposited internationally as NITE BP-03201.

[0170] [Table 9]

[0171]

[0172] +: Positive, -: Negative

[0173] [Table 10]

[0174] project matrix components Test results project matrix components Test results 0 Comparison - 25 Aesculin + 1 Glycerol - 26 Salicylic acid + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 maltose + 4 L-arabinose + 29 lactose + 5 Ribose + 30 Melibi + 6 D-xylose - 31 sucrose + 7 L-xylose - 32 Trehalose + 8 Calendula alcohol - 33 Inulin - 9 β-methyl-D-xylose - 34 Ginsenosides + 10 Galactose + 35 Raffinose + 11 glucose + 36 starch - 12 fructose + 37 Glycogen - 13 Mannose + 38 Xylitol - 14 sorbose - 39 Gentian disaccharide + 15 Rhamnose - 40 D-Melalose + 16 Euonymus alcohol - 41 D-Lysulose - 17 Inositol - 42 D-Tagose - 18 Mannitol + 43 D-fucose - 19 Sorbitol + 44 L-fucose - 20 α-Methyl-D-mannoside - 45 D-Arabatool - 21 α-Methyl-D-glucoside - 46 L-Arabatool - 22 N-acetylglucosamine + 47 gluconate + 23 amygdalin + 48 2-Ketogluconic acid - 24 Arbutin + 49 5-Ketogluconic acid -

[0175] +: Positive, -: Negative

[0176] [Experiment 7: Isolation and Identification of Isolator F]

[0177] Pine cones were used as the source of isolation, and isolate F was isolated using the same method as in Experiment 1. Identification of isolate F was performed using the same method as in Experiment 2.

[0178] The 16S rRNA gene sequence of isolate F showed 99.93% identity with the 16S rRNA gene of *Lactobacillus pentosus* (JCM1558), 99.93% identity with the 16S rRNA gene of *Lactobacillus plantarum subsp. plantarum* (JCM1149), and 99.73% identity with the 16S rRNA gene of *Lactobacillus paraplantarum* (DSM10667). However, no microorganism possessed a 16S rRNA gene with a completely identical 16S rRNA gene sequence to isolate F.

[0179] like Figure 6 As shown in (A), isolate F formed circular colonies. Figure 6 As shown in (B), isolate F was Gram-positive. The results of physiological and biochemical phenotypic and fermentation tests for isolate F are shown in Tables 11 and 12. Isolate F was a non-motile Gram-positive bacillus that did not form spores, showed negative catalase and oxidase reactions, and fermented glucose. These characteristics are consistent with those of *Lactobacillus* species indicated by partial 16S rDNA sequence analysis. Fermentation tests using the API kit showed that isolate F fermented galactose, fructose, α-methyl-D-mannoside, and menotriose, but not glycerol or D-xylose. Furthermore, it did not show arginine dihydrogenase activity and grew at 15°C. These characteristics are consistent with those of *Lactobacillus pentosus* and *Lactobacillus plantarum*, which were identified through partial 16S rDNA sequence analysis, but differ from *Lactobacillus pentosus* at the point where fermentation does not show glycerol and D-xylose. Therefore, isolate F is a new isolate belonging to *Lactobacillus plantarum*. Isolate F is deposited internationally as NITEBP-03202.

[0180] [Table 11]

[0181]

[0182] +: Positive, -: Negative

[0183] [Table 12]

[0184] project matrix components Test results project matrix components Test results 0 Comparison - 25 Aesculin + 1 Glycerol - 26 Salicylic acid + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 maltose + 4 L-arabinose + 29 lactose + 5 Ribose + 30 Melibi + 6 D-xylose - 31 sucrose + 7 L-xylose - 32 Trehalose + 8 Calendula alcohol - 33 Inulin - 9 β-methyl-D-xylose - 34 Ginsenosides + 10 Galactose + 35 Raffinose + 11 glucose + 36 starch - 12 fructose + 37 Glycogen - 13 Mannose + 38 Xylitol - 14 sorbose - 39 Gentian disaccharide + 15 Rhamnose - 40 D-Melalose + 16 Euonymus alcohol - 41 D-Lysulose - 17 Inositol - 42 D-Tagose - 18 Mannitol + 43 D-fucose - 19 Sorbitol + 44 L-fucose - 20 α-Methyl-D-mannoside + 45 D-Arabatool - 21 α-Methyl-D-glucoside + 46 L-Arabatool - 22 N-acetylglucosamine + 47 gluconate + 23 amygdalin + 48 2-Ketogluconic acid - 24 Arbutin + 49 5-Ketogluconic acid -

[0185] +: Positive, -: Negative

[0186] [Experiment 8: Evaluation of Antibacterial Activity of Bacterial Cell Cultures]

[0187] Verify whether bacterial cultures of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 possess antibacterial activity. Based on... Figure 7 The steps of Experiment 8 are explained. *Ralstonia solanacearum* strains 1-6, as shown in Table 13, were used in the experiment. The turbidity of the pre-culture medium for each *Ralstonia solanacearum* strain was measured at a wavelength of 600 nm. *Ralstonia solanacearum* strain 1 was diluted in soft agar medium 11 with a concentration of 0.1 OD units for each strain and 0.4 OD units for the others. Furthermore, the bacterial count in 1 mL of culture medium with an OD600 of 1 was defined as 1 OD unit. Soft agar medium 11 containing each *Ralstonia solanacearum* strain was layered on agar medium 12 and allowed to dry to prepare the growth medium for each strain. NITEBP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, and NITE BP-03202 were cultured in MRS liquid medium at 30°C. The bacterial cells were separated by centrifugation, and the culture supernatant (bacterial culture) was prepared as sample 13. 10 μL of sample 13 was added dropwise to the growth medium of each bacterial wilt pathogen and incubated at 30°C under aerobic conditions for 20 hours. Growth inhibition circles 14 were formed when the bacterial culture supernatant exhibited antibacterial activity against bacterial wilt pathogens. As a control experiment, tetracycline (50 μg / mL) with antibacterial activity was used.

[0188] [Table 13]

[0189]

[0190] The results of Experiment 8 are shown below. Figure 8 In the study, the culture supernatants of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 all formed growth inhibition circles on the growth media of Ralstonia solanacearum 1–6. Cell cultures of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 all showed inhibition of the proliferation of Ralstonia solanacearum or Ralstonia pseudosporidis.

[0191] [Experiment 9: Evaluation of antibacterial activity of bacterial cells and mixtures of bacterial cell cultures]

[0192] To verify whether the bacterial cells and cultures of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 possess antibacterial activity. As sample 13, a mixture of bacterial cells and culture supernatant was used; otherwise, Experiment 9 was identical to Experiment 8. The experimental procedures are shown below. Figure 9 First, the growth medium for each bacterial wilt pathogen was prepared using the same method as in Experiment 8. NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, and NITE BP-03202 were cultured in MRS liquid medium at 30°C. The mixture of bacterial cells and culture supernatant (bacterial culture) was prepared as sample 13. A filter 15 containing 60 μL of sample 13 was placed in the growth medium of each bacterial wilt pathogen and cultured at 30°C under aerobic conditions for 20 hours. After culture, the formation of growth inhibition circles was confirmed.

[0193] The results of Experiment 9 are shown below. Figure 10 In the study, mixtures of cell and culture supernatant of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 all formed growth inhibition circles on growth media of Ralstonia solanacearum 1–6. Mixtures of cell and cell cultures of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 all showed inhibitory effects on the proliferation of Ralstonia solanacearum or Ralstonia pseudosporidis.

[0194] [Experiment 10: Long-term antibacterial activity evaluation test of bacterial cells and mixtures of bacterial cell cultures]

[0195] To verify whether the bacterial cells and cultures of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 possess long-term antibacterial activity. As for *Ralstonia solanacearum*, *Ralstonia solanacearum* 5 as described in Table 13 was used, with the incubation period starting from filter 15 in the *Ralstonia solanacearum* growth medium extended to 3 days, 7 days, or 15 days. Otherwise, Experiment 10 was performed using the same method as Experiment 9. As controls, lactic acid bacteria medium, no treatment, or tetracycline (50 μg / mL) were used.

[0196] The results of Experiment 10 are shown below. Figure 11 Even after 15 days of filtering and allowing the bacterial cells and culture supernatants of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 to stand in the growth medium of Ralstonia solanacearum, growth inhibition circles were formed. This indicates that mixtures of bacterial cells and cultures of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 can inhibit the proliferation of Ralstonia solanacearum for a long period.

[0197] [Experiment 11: Evaluation of antibacterial activity against Ralstonia solanacearum in nutrient solution]

[0198] To verify whether bacterial cultures of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 can inhibit the proliferation of Ralstonia solanacearum in nutrient solution. Figure 12The experimental procedure is as follows: NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, and NITE BP-03202 were cultured in MRS liquid medium at 30°C. The bacterial cells were separated by centrifugation, and the supernatant (bacterial culture) was prepared as sample 23. As nutrient solution 22, OAT Agrio's OAT A formulation (containing 1.5 g / L OAT House 1 and 1.0 g / L OAT House 2 aqueous solution) was used. Ralstonia solanacearum 21 was suspended in nutrient solution 22 at a concentration of 0.01 OD units to prepare nutrient solution 22 contaminated with Ralstonia solanacearum 21. 10 μL of sample 23 was added dropwise to 90 μL of nutrient solution 22, stirred, and cultured at 30°C for 5 hours. The culture medium was diluted 100-fold and spread onto agar medium 24 for detecting Ralstonia solanacearum, and further incubated at 30°C for 48 hours. The number of colonies 25 on the incubated agar medium 24 was counted. Ralstonia solanacearum 5, listed in Table 13, was used as the pathogen. As a control, lactic acid bacteria medium or tetracycline (50 μg / mL) was used instead of the sample.

[0199] The results of Experiment 11 are shown below Figure 13 In nutrient solutions containing culture supernatants of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202, the colony formation of *Ralstonia solanacearum* was inhibited. This indicates that bacterial cultures containing NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 can kill *Ralstonia solanacearum* in the nutrient solution or inhibit its proliferation.

[0200] [Experiment 12: Evaluation of antibacterial activity against *Ralstonia solanacearum* in soil]

[0201] To verify whether the bacterial cells of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202 can inhibit the proliferation of Ralstonia solanacearum in soil. 2.5 mL of Ralstonia solanacearum 5 bacterial suspension, adjusted to 0.06 OD units with distilled water (manufactured by Otsuka Pharmaceutical Co., Ltd.), was added to 50 g of soil (Akagi horticulture, herbaceous flower, and wild vegetable potting soil) to prepare contaminated soil. Mix 1g of reducing agent (molasses and other organic matter, manufactured by Sunpillars, Omalass 95) with the bacterial cells of NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201, or NITE BP-03202) and soil, and incubate at 30°C under anaerobic conditions for 14 days. For the bacterial cells, adjust the product of each bacterium cultured at 30°C on MRS liquid medium to 0.5 OD units with distilled water (manufactured by Otsuka Pharmaceutical Factory), and mix 2.5mL of bacterial cells into every 50g of soil. The reducing agent is used as a soil fumigant. Microbial DNA in the soil was purified using a soil DNA extraction kit (ISOIL for Beads Beating Kit, NIPPON GENE). PCR amplification was performed using a common primer set for *Ralstonia solanacearum* (759: GTCGCCGTCAACTCAACTTTCC (SEQ ID NO. 2), 760: GTCGCCGTCAGCAATGCGGAATCG (SEQ ID NO. 3)) and a PCR primer set specific to *Ralstonia solanacearum* 5 (Nmult21: 1F CGTTGATGAGGCGCGCAATTT (SEQ ID NO. 4), Nmult21: RRTTCGCTTGACCCTATAACGAGT (SEQ ID NO. 5)). PCR amplification was performed using KOD-Plus-Ver. 2 DNA polymerase (Toyobo Co., Ltd.), and the amplified products were purified. The purified PCR amplification products were electrophoresed with 4% E-Gel (Thermo Fisher Scientific), and the bands of 144 base pairs amplified using PCR primers specific to Ralstonia solanacearum 5 were observed using an image analyzer LAS-3000 (FUJIFILM).

[0202] In soil containing a mixture of NITEBP-03197, NITEBP-03198, NITEBP-03199, NITEBP-03200, NITEBP-03201, or NITEBP-03202, the band of 144 base pairs specific to Ralstonia solanacearum 5 was reduced. The presence of NITEBP-03197, NITEBP-03198, NITEBP-03199, NITEBP-03200, NITEBP-03201, or NITEBP-03202 cells can kill or inhibit the proliferation of Ralstonia solanacearum in the soil.

[0203] [Experiment 13: Effect of nutrient solution culture on the control of bacterial wilt]

[0204] This study aimed to verify whether adding cultures of NITE BP-03199, NITE BP-03200, or NITE BP-03201 to the nutrient solution could control bacterial wilt in hydroponic cultivation. First, using an LED planter (LED PlanterGreenteria, DeAGOSTINI), tomatoes (variety: Lirong) were cultivated to the 4-5 leaf stage in nutrient solution with OAT A formulation, under conditions of 25°C, 20-30% humidity, and a photoperiod of 16 hours light / 8 hours dark. Eight tomato plants were used in each of the experimental and control areas.

[0205] In the experimental area, before inoculation with *Ralstonia solanacearum*, a culture supernatant prepared from NITE BP-03201 medium with an OD600 value of 10 was added to the nutrient solution at a volume of 1 / 100. In the control area, before inoculation with *Ralstonia solanacearum*, MRS medium was added to the nutrient solution at a volume of 1 / 100. Then, a bacterial suspension of *Ralstonia solanacearum* 5 was added at a concentration of 2 × 10⁻⁶. 7 After adding the fungus to the nutrient solution at a rate of CFU / mL, the tip of the tomato root was cut off, and the fungus was then inoculated with bacterial wilt pathogen. During the 10–12 days from the inoculation date to the end of the experiment, the symptoms appearing on the plant were evaluated using the following five stages, according to the evaluation criteria described in Non-Patent Literature 1.

[0206] 0: No symptom onset

[0207] 1:1 leaf withering

[0208] 2: Two or more leaves wither

[0209] 3: Except for the top leaves, the leaves wither.

[0210] 4: The whole body withers and dies.

[0211] Disease index = (0×N0+1×N1+2×N2+3×N3+4×N4) / 4×(N0+N1+N2+N3+N4)

[0212] NO to N4 represent the number of individuals for that value. The disease severity index indicates the degree of damage caused by the disease; a higher disease severity index means the disease progresses more rapidly.

[0213] Figure 14 The changes in the disease index over time after inoculation with *Ralstonia solanacearum* are shown. Severe symptoms appeared in the control area, while none were observed in the experimental area. Therefore, it can be concluded that the cell culture of NITE BP-03201 has a control effect on *Ralstonia solanacearum* in nutrient solution culture.

[0214] Using the same method, add 1 / 100 of the culture supernatant of NITE BP-03199 or NITE BP-03200 to the nutrient solution, and show the results of the test to see if it can prevent bacterial wilt. Figure 15 In the control area, severe symptoms appeared, while the disease index was significantly lower in the experimental area. Therefore, it can be concluded that the cell cultures of NITE BP-03199 or NITE BP-03200 also have a control effect on bacterial wilt in nutrient solution culture.

[0215] [Experiment 14: Effect of soil cultivation on the control of bacterial wilt]

[0216] This study aimed to verify whether the application of NITE BP-03200 bacterial culture could control bacterial wilt in soil cultivation. One week before planting, 50 mL of NITE BP-03200 culture supernatant was diluted 10, 50, or 100 times and applied as a drenching agent to each plant. Subsequently, tomato seedlings were inoculated with Bacterium wilt by planting them in a farm contaminated with Bacterium wilt. After planting in the farm, 500 mL of NITE BP-03200 culture supernatant was diluted 10, 50, or 100 times and applied as a drenching agent to each plant every week. The disease symptoms observed in the treated and untreated areas were compared to evaluate the control efficacy.

[0217] In the untreated area, wilt and death occurred; conversely, in the area treated with NITE BP-03200 culture supernatant, these symptoms were suppressed. Therefore, it can be concluded that NITE BP-03200 cell culture has a control effect against bacterial wilt in soil cultivation.

[0218] Symbol Explanation

[0219] 11: Soft agar medium; 12: Agar medium; 13: Sample; 14: Growth inhibition circle; 15: Filter; 21: Bacterial wilt pathogen; 22: Nutrient solution; 23: Sample; 24: Agar medium; 25: Colony. sequence list <110> Sumitomo Chemical Co., Ltd. <120> New microorganisms belonging to the genus *Lactobacillus*, and agents and methods for controlling plant diseases caused by *Ralstonia solanacearum* or *Ralstonia pseudosporidis*. <130> S44637WO01 <150> JP2020-111696 <151> 2020-06-29 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 1485 <212> DNA <213> Lactobacillus sp. SC-2001 (NITE BP-03197) <400> 1 gacgaacgct ggcggcgtgc ctaatacatg caagtcgaac gcaaaacttt caccgaatgc 60 ttgcattcac cggaagtttt gagtggcgaa cgggtgagta acacgtgggt aacctgccca 120 gaagaggggg ataacacttg gaaacaggtg ctaataccgc ataacaataa aaaccgcatg 180 gtttttattt aaaagatggt tttgctatca cttctggatg gacccgcggc gtattagcta 240 gttggtaagg taaaggctta ccaaggcaat gatacgtagc cgaactgaga ggttgatcgg 300 ccacattggg actgagacac ggcccaaact cctacggggag gcagcagtag ggaatcttcc 360 acaatggacg aaagtctgat ggagcaacgc cgcgtgagtg aagaaggttt tcggatcgta 420 aaactctgtt gttagagaag aacgtgtgtg aaggaactg ctcatgcagt gacggtatct 480 aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 540 600 660 gtggaactcc atgtgtagcg gtgaaatgcg tagatatg gaagaacacc agtggcgaaa 720 gcggctctct ggtctgtaac tgacgctgag gttcgaaagt gtgggtagca aacaggatta 780 gataccctgg tagtccacac cgtaaacgat gaatgctaag tgttggaggg tttccgccct 840 tcggtgctgc agctaacgca ttaagcattc cgcctgggga gtacgaccgc aaggttgaaa 900 ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 960 cgcgaagaac cttaccaggt cttgacatct tctgacaacc taagagatta ggtgttccct 1020 tcggggcag aatgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1080 ttaagtcccg caacgagcgc aacccttatt attagttgcc agcattaagt tgggcactct 1140 agtgagactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1200 ttatgacctg ggctacacac gtgctacaat ggacggtaca acgagtcgcg aaaccgcgag 1260 gtttagctaa tctcttaaag ccgttctcag ttcggattgt aggctgcaac tcgcctacat 1320 gaagtcggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1380 tgtacacacc gcccgtcaca ccatgagagt ttgtaacacc caaagccggt gaggtaacct 1440 ttatggaacc agccgtctaa ggtgggacag atgattgggg tgaag 1485 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 759 <400> 2 gtcgccgtca actcaacttt cc 22 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PCR primer 760 <400> 3 gtcgccgtca gcaatgcgga atcg 24 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR primers Nmult21:1F <400> 4 cgttgatgag gcgcgcaatt t 21 <210> 5 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> PCR primer Nmult21:RR <400> 5 ttcgcttgac cctataacga gt 22

Claims

1. A bacterium with accession numbers NITE BP-03197, NITE BP-03198, NITE BP-03199, NITE BP-03200, NITE BP-03201 or NITE BP-03202.

2. A bacterial cell or bacterial culture of the bacteria as described in claim 1, or an extract thereof.

3. An agent for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the bacterial cells or bacterial cultures of the bacteria as described in claim 1 or extracts thereof.

4. A method for controlling plant diseases caused by Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the step of applying the control agent as described in claim 3.

5. A disinfectant for plants, nutrient solutions, soil, nutrient solution cultivation materials or soil cultivation materials contaminated with Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising bacterial cells or bacterial cell cultures of the bacteria described in claim 1 or extracts thereof.

6. A method for disinfecting plants, nutrient solutions, soil, nutrient solution cultivation materials, or soil cultivation materials contaminated with Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the step of using the disinfectant described in claim 5.

7. A proliferation inhibitor of Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the bacterial cells or bacterial culture of the bacteria of claim 1 or extracts thereof.

8. A method for inhibiting the proliferation of Ralstonia solanacearum or Ralstonia pseudosolanacearum, comprising the step of using the proliferation inhibitor of claim 7.

9. A method for cultivating a plant, comprising the step of cultivating the plant in a nutrient solution or soil containing the bacterial cells or bacterial culture of the bacteria of claim 1 or extracts thereof.

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