Predatory bacterium or variant thereof, and method for controlling soilborne plant disease

Abstract

Provided are a method for controlling a soilborne disease in which a load on an environment and a human body is reduced, and a novel predatory bacterium therefor.　Provided are: a predatory bacterium or a variant thereof selected from the group consisting of bacteria of the genus Bacteriovorax and bacteria of the genus Bdellovibrio which selectively prey on a pathogenic bacterium belonging to the family Burkholderiaceae, the family Pectobacteriaceae, or the family Lysobacteraceae; a method for controlling a soilborne plant disease caused by the pathogenic bacterium, the method characterized in that the predatory bacterium or variant thereof is applied to a seed, root, or underground stem of a plant, rhizosphere soil, or soil; and a composition.

Description

Predatory bacteria or mutants thereof, and method for controlling soil-borne plant diseases

[0001] The present invention relates to a predatory bacterium or a mutant thereof that selectively preys on specific pathogenic bacteria, a method for controlling soil-borne plant diseases caused by specific pathogenic bacteria, and a method for promoting plant growth.

[0002] In recent years, it has been estimated that crop yield losses due to plant diseases are about 40%, and among plant diseases, soil-borne plant diseases are considered to be the main cause of yield loss.

[0003] In particular, diseases caused by soil-borne pathogenic bacteria belonging to the Burkholderiaceae, Pectobacterioceae, or Rhizobacterioceae families are known to be difficult to control and cause significant agricultural damage. For example, pathogenic bacteria that cause bacterial wilt (e.g., Ralstonia solanacearum) infect more than 200 species of plants, including those of the Solanaceae family, causing severe damage to agricultural products.

[0004] Until now, disease control has mainly been achieved by spraying large amounts of chemical pesticides, but there is a strong demand both in Japan and overseas to reduce the use of chemical pesticides due to concerns about their impact on ecosystems and the emergence of drug-resistant microorganisms. Therefore, research and development is being conducted focusing on bacterivorous bacteria (meaning "Bdellovibrio and similar microorganisms," also written as "BALOs") as a disease control technology to replace chemical pesticides.

[0005] Examples of BALOs include Bdellovibrio bacteriovorus strain HD100, Bdellovibrio reynosensis strain LBG001, Pseudobdellovibrio exovorus strain JSS, Bdellovibrio sp. strain NC01, Bacteriovorax stolpii strain DSM12778, Halobacteriovorax marinus strain SJ, Peredibacter starrii strain A3.12, and Micavibrio aeruginosavorus. aeruginosavorus ARL-13 strain.

[0006] Although many reports have shown that BALOs primarily prey on Gram-negative bacteria, particularly those belonging to the Pseudomonadota phylum, it is known that different predatory bacteria prey on different bacteria. For example, it has been reported that the Bdellovibrio bacteriovorus HD100 strain preys on Acinetobacter 0036, Aeromonas 0031, Raoultella 0037, Pseudomonas NC02, Serratia 0043, and Escherichia ML35, all of which belong to the Pseudomonadota phylum. On the other hand, it is known that Bdellovibrio sp. strain NC01 (hereinafter simply referred to as "strain NC01") can prey on Aeromonas genus strain 0031, Pseudomonas genus strain NC02, Serratia genus strain 0043, and Escherichia genus strain ML35, but cannot prey on Acinetobacter genus strain 0036 or Raoultella genus strain 0037 (Non-Patent Documents 1 and 2).

[0007] As examples of the control effects of BALOs against soil-borne pathogenic bacteria, there are reports on the protective effect of Bdellovibrio bacteriovorus strain HD100 against potato black leg disease (Pectobacterium carotovorum subsp. brasiliense) (Non-Patent Document 3) and on the protective effect of Bdellovibrio strain SOIR-1 against potato diseases caused by onion bulb rot (Pantoea sp.) or Xanthomonas campestris (Non-Patent Document 4). However, in both cases, the control effect was evaluated using potato or onion slices as a simple test.

[0008] It has also been reported that a bacterivorous bacterial strain belonging to the genus Bdellovibrio was isolated by the double-plate method (agar medium at pH 7.4) using Escherichia coli as bait cells, and its predatory activity against plant pathogenic bacteria such as Ralstonia solanacearum was evaluated in a liquid medium (pH 7.4) (Non-Patent Document 5).

[0009] However, there have been no reports to date that have confirmed disease suppression by BALOs in soil environments. The reason for the lack of reports on the disease suppression effect of BALOs in soil environments is thought to be that no BALOs have been found to date that exhibit high predatory activity in soil environments.

[0010] Williams, LE, Cullen, N. et al. (2019), Microbiology, 165(12), 1315-1330Mulvey, K., Brosnan, K. et al. (2023), Applied and Environmental Microbiology, 89(1), e01776-22Youdkes, D., Helman, Y. et al. (2020), Applied and environmental microbiology 86(6), e02543-19Odooli, S., Roghanian, R. et al. (2021). International Microbiology, 24(3), 399-413Tajabadi H., Moeinzadeh F., et al. (2022), Journal of Plant Pathology, 104, pages 631-640 (2022)

[0011] Therefore, there has been a demand for a method for controlling soil-borne pathogenic bacteria that reduces the burden on the environment and the human body, and for the search for novel predatory bacteria for this purpose.

[0012] As a result of extensive research to achieve the above object, the present inventors have discovered bacteria from BALOs that selectively prey on pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family in soil environments, thereby completing the present invention. Specifically, the present inventors have discovered the Bacteriovorax genus strain MRP-4 (hereinafter sometimes simply referred to as "strain MRP-4") and the Bdellovibrio genus strain MRP-1 (hereinafter sometimes simply referred to as "strain MRP-1") as such predatory bacteria. Furthermore, the present inventors have found that these predatory bacteria can effectively control soil-borne plant diseases caused by pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family, thereby completing the present invention. Furthermore, the present inventors have found that these predatory bacteria can promote plant growth, thereby completing the present invention.

[0013] That is, the present invention provides the following: (1) A predatory bacterium or a mutant thereof selected from the group consisting of bacteria of the genus Bacteriovorax and bacteria of the genus Bdellovibrio, which selectively preys on pathogenic bacteria belonging to the family Burkholderiaceae, Pectobacteriaceae, or Lysobacteraceae. (2) The predatory bacterium or a mutant thereof according to (1), wherein the predatory bacterium or a mutant thereof has a base sequence identity of more than 97.7% with the 16S rRNA base sequence (SEQ ID NO: 6) of Bacteriovorax stolpii strain DSM12778 (accession number AJ288899) and a base sequence identity of more than 75.1% with the base sequence of the entire genome of the DSM12778 strain, or a base sequence identity of 100% with the 16S rRNA base sequence (SEQ ID NO: 2) of Bdellovibrio sp. strain NC01 (accession number CP030034) and a base sequence identity of more than 96.2% with the base sequence of the entire genome of the NC01 strain. (3) The predatory bacterium or mutant thereof according to (1) or (2), wherein the predatory bacterium or mutant thereof is the Bacteriovorax genus MRP-4 strain (accession number NITE BP-03910), the Bdellovibrio genus MRP-1 strain (accession number NITE BP-03909), or a mutant thereof. (4) The predatory bacterium or mutant thereof according to any one of (1) to (3), wherein the predatory bacterium or mutant thereof is the Bacteriovorax genus MRP-4 strain or a mutant thereof.(5) The predatory bacterium or mutant thereof according to any one of (1) to (4), wherein the pathogenic bacterium belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family is selected from the group consisting of bacteria of the genus Ralstonia of the Burkholderiaceae family, bacteria of the genus Burkholderia of the Burkholderiaceae family, bacteria of the genus Pectobacterium of the Pectobacteriaceae family, bacteria of the genus Dickeya of the Pectobacteriaceae family, and bacteria of the genus Xanthomonas of the Rhizobacteraceae family. (6) The pathogenic bacterium belonging to the family Burkholderiaceae, Pectobacteraceae, or Rhizobacteraceae is selected from the group consisting of Burkholderia plantarii, Burkholderia glumae, Burkholderia cepacia, and Burkholderia gladioli of the family Burkholderiaceae; Ralstonia pseudosolanacearum, Ralstonia solanacearum, Ralstonia syzygii subsp. syzygii, and Ralstonia syzygii subsp. indonesiensis of the family Burkholderiaceae. indonesiensis, Ralstonia syzygii subsp. celebesensis; Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum, Pectobacterium carotovorum subsp. brasilens from the Pectobacteriaceae family.brasiliense, Pectobacterium atrosepticum, Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium parmentieri, Dickeya dadantii, Dickeya solani, Dickeya dianthicola, Dickeya chrysanthemi, Dickeya oryzae, Dickeya zeae; or Xanthomonas campestris from the family Rhizobacteraceae. The predatory bacterium or its mutant according to any one of (1) to (5), wherein the predatory bacterium is Pseudomonas campestris. (7) A method for controlling soil-borne plant diseases caused by pathogenic bacteria belonging to the family Burkholderiaceae, Pectobacteriaceae, or Lysobacteraceae, characterized by applying a predatory bacterium selected from the group consisting of bacteria of the genus Bacteriovorax and bacteria of the genus Bdellovibrio, or a mutant thereof, to plant seeds, roots, rhizomes, rhizosphere soil, or soil. (8) A method for promoting plant growth, characterized by applying a predatory bacterium selected from the group consisting of bacteria of the genus Bacteriovorax and bacteria of the genus Bdellovibrio, or a mutant thereof, to plant seeds, roots, rhizomes, rhizosphere soil, or soil. (9) The predatory bacterium or mutant thereof is 16S of Bacteriovorax stolpii strain DSM12778 (accession number AJ288899)The method according to (7) or (8), wherein the 16S rRNA base sequence (SEQ ID NO: 6) of the Bdellovibrio sp. strain NC01 (accession number CP030034) has a base sequence identity of more than 97.7% and a base sequence identity of more than 75.1% with the base sequence of the entire genome of the DSM12778 strain, or the 16S rRNA base sequence (SEQ ID NO: 2) of the Bdellovibrio sp. strain NC01 (accession number CP030034) has a base sequence identity of 100% and a base sequence identity of more than 96.2% with the base sequence of the entire genome of the NC01 strain. (10) The method according to any one of (7) to (9), wherein the predatory bacterium or its mutant is the Bacteriovorax genus MRP-4 strain (accession number NITE BP-03910), the Bdellovibrio genus MRP-1 strain (accession number NITE BP-03909), or a mutant thereof. (11) The method according to any one of (7) to (10), wherein the predatory bacterium or its mutant is the Bacteriovorax genus MRP-4 strain or a mutant thereof. (12) The method according to exactly any one of (7) and (9) to (11), wherein the pathogenic bacterium belonging to the Burkholderiaceae family, Pectobacterialceae family, or Rhizobacteraceae family is selected from the group consisting of bacteria of the genus Ralstonia of the Burkholderiaceae family, bacteria of the genus Burkholderia of the Burkholderiaceae family, bacteria of the genus Pectobacterium of the Pectobacterialceae family, bacteria of the genus Dickeya of the Pectobacterialceae family, and bacteria of the genus Xanthomonas of the Rhizobacteraceae family. (13) The pathogenic bacterium belonging to the family Burkholderiaceae, Pectobacterialceae, or Rhizobacteraceae is selected from the group consisting of Burkholderia plantarii, Burkholderia glumae, Burkholderia cepacia, and Burkholderia gladioli; Ralstonia pseudosolanancellum;pseudosolanacearum), Ralstonia solanacearum, Ralstonia syzygii subsp. syzygii, Ralstonia syzygii subsp. indonesiensis, Ralstonia syzygii subsp. celebesensis; Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum, Pectobacterium carotovorum subsp. brasilens from the Pectobacteriumaceae family. brasiliense, Pectobacterium atrosepticum, Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium parmentieri, Dickeya dadantii, Dickeya solani, Dickeya dianthicola, Dickeya chrysanthemi, Dickeya oryzae, Dickeya zeae; or Xanthomonas campestris from the family Rhizobacteraceae. The method according to any one of (7) and (9) to (12), wherein the strain is Lactobacillus campestris.(14) The method according to any one of (7) and (9) to (13), wherein the soil-borne plant disease caused by the pathogenic bacterium is selected from the group consisting of bacterial wilt, rice bacterial grain rot, rice seedling blight, onion rot, brown rot of leeks, vegetable soft rot, potato black leg, soft rot of pineapple and banana, sudden withering of fruit trees, and black rot. (15) The method according to any one of (7) and (9) to (14), wherein the soil-borne plant disease caused by the pathogenic bacterium is bacterial wilt. (16) A composition for controlling soil-borne plant diseases caused by pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacterioceae family, or Rhizobacteraceae family, or for promoting plant growth, comprising the predatory bacterium according to any one of (1) to (6) or a mutant thereof. (17) The composition described in (16), wherein the pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family are selected from the group consisting of bacteria of the genus Ralstonia of the Burkholderiaceae family, bacteria of the genus Burkholderia of the Burkholderiaceae family, bacteria of the genus Pectobacterium of the Pectobacteriaceae family, bacteria of the genus Dickeya of the Pectobacteriaceae family, or bacteria of the genus Xanthomonas of the Rhizobacteraceae family.

[0014] According to the present invention, a predatory bacterium or a mutant thereof selected from the group consisting of bacteria of the genus Bacteriovorax and bacteria of the genus Bdellovibrio can effectively control soil-borne plant diseases caused by pathogenic bacteria belonging to the family Burkholderiaceae, Pectobacteriaceae, or Rhizobacteraceae, regardless of the pH at the time of application of the predatory bacterium. The predatory bacterium or a mutant thereof has an excellent control effect against soil-borne plant diseases even in actual field soil. As a result, plant growth can be promoted.

[0015] FIG. 1 is a graph showing that strains MRP-1 and MRP-4 have disease-suppressing effects against tomato bacterial wilt in an artificial soil (vermiculite) environment. The AIG-1 strain of Ralstonia pseudosolanacearum, a pathogen that causes tomato bacterial wilt, was inoculated into artificial soil (15 plants per treatment, replicated three times). The average diseased plant rates were compared. Compared to the controls (no application of strain MRP-1 or MRP-4), strain MRP-1 exhibited a high control effect at pH 6.0, while strain MRP-4 exhibited excellent control effects at pH 7.0 and pH 6.0. This graph shows that strain MRP-4 not only controls tomato bacterial wilt in an artificial soil environment, but also promotes tomato growth. A comparison of the average height above ground of tomato seedlings in the control and treatment plots (inoculated with MRP-4 strain) in Figure 1 showed that MRP-4 strain promoted tomato growth at pH 6.0 compared with the control. Figure 3 shows the time course of the density of R. pseudosolanacearum AIG-1 strain, a pathogen that causes bacterial wilt of tomato, in an artificial soil environment when MRP-1 and MRP-4 strains were applied. A comparison of the density of AIG-1 strain in the control and each treatment plot based on the average values ​​of three replicates showed that the MRP-1 strain application plot showed a gradual decrease in the density of AIG-1 strain at both pH 7.0 and 6.0, while the MRP-4 strain application plot showed a rapid decrease in the density of AIG-1 strain at both pH 7.0 and 6.0. 4A and 4B are graphs showing that the MRP-4 strain has a disease control effect against tomato bacterial wilt in disease-affected soil. Different soils were inoculated with the R. pseudosolanacearum AIG-1 strain, a pathogen that causes tomato bacterial wilt, and tomatoes were continuously cultivated in these soils to produce soils that were susceptible to tomato bacterial wilt. The average percentage of infected plants was compared using these soils. The MRP-4 strain demonstrated significantly superior disease control effects in soils A and B compared with the controls.5A and 5B show the density of R. pseudosolanacearum AIG-1, a pathogen that causes bacterial wilt of tomato, in soil continuously cultivated with MRP-4. The density of AIG-1 in each treatment plot was compared based on the average value of three replicate tests. The results showed that the density of AIG-1 in both soil A and soil B in the MRP-4 application plot was reduced to about one-tenth of the original density.

[0016] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be practiced with appropriate modifications.

[0017] [First Embodiment] The first embodiment of the present invention is a predatory bacterium or a mutant thereof selected from the group consisting of Bacteriovorax bacteria and Bdellovibrio bacteria, which selectively prey on pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family. In the present invention, "pathogenic bacteria" refers to microorganisms that are preyed upon by predatory bacteria and that belong to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family, which cause soil-borne plant diseases and are decomposed or lysed by predatory bacteria. Pathogenic bacteria belonging to these families may also be referred to simply as "pathogenic bacteria." In the present invention, "predatory bacteria" refers to bacteria that have the ability to selectively decompose or lyse the pathogenic bacteria. For example, bacteria that invade the microorganism, kill the microorganism, and absorb its cellular components as nutrients may be mentioned. The cells of predatory bacteria are smaller than those of ordinary bacteria (width: 0.2-0.5 μm, length: 0.5-1.6 μm), and can be distinguished using a filter with a pore size of 0.45 μm.

[0018] In the present invention, the term "selective" refers to a condition in which the ability of the "predatory bacteria" of the present invention to prey on the above-mentioned pathogenic bacteria is higher than the ability to prey on bacteria other than the pathogenic bacteria. Examples of bacteria other than bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family include bacteria of the Alphaproteobacteria class of the Pseudomonadota phylum, the Pseudomonadales order of the Gammaproteobacteria class, the Actinobacteriota phylum, and the Bacteroidota phylum, as described in Example 5 below.

[0019] Specifically, the predatory bacteria of the present invention are the MRP-4 strain belonging to the genus Bubacteriovorax (hereinafter sometimes simply referred to as "Bdellovibrio MRP-4 strain" or "MRP-4 strain") and the MRP-1 strain belonging to the genus Bdellovibrio (hereinafter sometimes simply referred to as "Bdellovibrio MRP-1 strain" or "MRP-1 strain"), which prey on the bacterial wilt bacterium (Ralstonia pseudosolanacearum), isolated from soil. Hereinafter, the term "predatory bacteria" in the present invention may be used to mean predatory bacteria or mutants thereof.

[0020] The predatory bacteria of the present invention can be isolated by conventional methods (see, for example, Jurkevitch, E. (2012). Current protocols in microbiology, 26(1), 7B-1) or modified methods thereof. Specifically, soil is collected, suspended in a washing solution, and centrifuged to obtain a supernatant. This supernatant is further centrifuged to obtain a precipitate, and a suspension of the precipitate is used to detect plaques (bacteriolytic plaques) by the double plate method.

[0021] Regarding the phylogenetic properties of the Bdellovibrio MRP-4 strain of the present invention, identity analysis of the nucleotide sequence of the 16S rRNA gene was performed, and it was found that the known bacterial species most closely related to the Bacteriovorax MRP-4 strain is the Bacteriovorax stolpii DSM12778 strain (accession number AJ288899), and that the nucleotide sequence of the 16S rRNA gene of the DSM12778 strain (SEQ ID NO: 6) has approximately 97.7% nucleotide sequence identity to the nucleotide sequence of the 16S rRNA gene of the MRP-4 strain (SEQ ID NO: 5). Furthermore, the "average nucleotide identity" (hereinafter referred to as "ANI") between the genome sequence of the MRP-4 strain (DDBJ / EMBL / GenBank accession number AP031397) and the genome sequence of the DSM12778 strain was calculated using phylogenetic analysis software (EZBioCould Microbiome Taxonomic Profiling), revealing a low identity of approximately 75.1%. The second and third most closely related known microorganisms to the MRP-4 strain are Bacteriovorax sp. Gunpowder strain (accession number AF084853) and Bacteriovorax sp. FukuN9 strain (accession number AJ290009), respectively, and the 16S rRNA gene sequence identities were approximately 97.0% and approximately 96.7%, respectively.

[0022] On the other hand, the known bacterial species most closely related to the MRP-1 strain is the Bdellovibrio sp. strain NC01 (accession number CP030034), and it was found that the nucleotide sequence of the 16S rRNA gene of the NC01 strain (SEQ ID NO: 2) has 100% nucleotide sequence identity with the nucleotide sequence of the 16S rRNA gene of the MRP-1 strain (SEQ ID NO: 1). Furthermore, the ANI between the nucleotide sequence of the genome of the MRP-1 strain (DDBJ / EMBL / GenBank accession number AP031396) and the nucleotide sequence of the genome of the NC01 strain was approximately 96.2%, making it clear that the MRP-1 strain is a different strain from the NC01 strain. The second and third most closely related known microorganisms to strain MRP-1 are Bdellovibrio sp. strain EC13 (accession number LUKD01000010) and Bdellovibrio sp. strain BER2 (accession number LUKF01000014), respectively, and the nucleotide sequence identity of the 16S rRNA gene between these strains was approximately 97.6%. Therefore, the above two strains of the present invention were identified as novel strains distinct from known bacterial species.

[0023] In the present invention, "sequence identity" refers to a value that can be calculated by appropriately aligning at least two sequences to be compared, determining the identical bases present in each sequence, determining the number of matching sites, dividing the number of matching sites by the total number of bases in the sequence region to be compared, and multiplying the resulting value by 100. Specifically, such sequence identity can be calculated, for example, using phylogenetic analysis software (EZBioCould Microbiome Taxonomic Profiling). Furthermore, "average nucleotide identity" (ANI) can be obtained by cutting the entire genome sequence of the analyzed strain into approximately 1,000 bases and calculating the average sequence similarity between the genome sequence of each fragment and the genome sequence of the comparison strain (e.g., Richter and Rossello-Mora, Proc Natl Acad Sci USA. 2009 Nov 10;106(45):19126-31. doi: 10.1073 / pnas.0906412106).

[0024] The inventors applied to deposit the above two strains with the Patent Microorganism Depositary, Biotechnology Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture), and the above two strains were received on June 13, 2023 under the following accession numbers (notification date: November 28, 2024), and are currently preserved at the institution. (1) MRP-1 strain (accession number NITE BP-03909) (2) MRP-4 strain (accession number NITE BP-03910)

[0025] The predatory bacteria of the present invention may be viable bacteria, and a culture solution containing the viable bacteria may also be preferably used. The viable bacteria may be in a form that can survive and be active in the seeds, roots, rhizomes, rhizosphere soil, or soil of the plant to which the bacteria are applied.

[0026] In the present invention, the term "predatory bacterium mutant" includes any mutant derived from the predatory bacterium of the present invention, as long as it has the effect of selectively preying on pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family. The mutation may be a substitution, deletion, insertion, and / or addition of bases in the entire genome sequence of the predatory bacterium of the present invention. Examples of mutants include strains generated by spontaneous mutations that occur in nature, including the cultivation and subculture of the predatory bacterium; strains created by artificial mutations induced by exposure to radiation, ultraviolet rays, X-rays, chemicals, etc.; strains created by genetic recombination; and strains created by genome editing.

[0027] The 16S rRNA base sequence of the mutant MRP-4 strain of the present invention preferably exhibits at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 97.7%, more preferably at least 97.8%, more preferably at least 97.9%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, and even more preferably at least 99.3% identity to the 16S rRNA base sequence of the MRP-4 strain. Further, the base sequence of the entire genome of the mutant MRP-4 strain of the present invention is preferably at least 80%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.9%, more preferably at least 99.91%, more preferably at least 99.92%, more preferably at least 99.93%, more preferably at least 99.94%, at least 99.95%, more preferably at least 99.96%, more preferably at least 99.97%, more preferably at least 99.98%, and even more preferably at least 99.99% identity.

[0028] The 16S rRNA base sequence of the mutant MRP-1 strain of the present invention preferably has at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, and even more preferably at least 99.3% base sequence identity to the 16S rRNA base sequence of the MRP-1 strain. Further, the base sequence of the entire genome of the MRP-1 strain mutant of the present invention is preferably at least 80%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.9%, more preferably at least 99.91%, more preferably at least 99.92%, more preferably at least 99.93%, more preferably at least 99.94%, at least 99.95%, more preferably at least 99.96%, more preferably at least 99.97%, more preferably at least 99.98%, and even more preferably at least 99.99% identity.

[0029] In the mutants of the MRP-4 or MRP-1 strain of the present invention, the number of substituted, deleted, inserted, and / or added bases is not particularly limited as long as the predatory activity of the protein encoded by the base sequence against a specific pathogenic bacterium is not lost, and may be, for example, 1 to about 8,000, preferably 1 to about 1,000, more preferably 1 to about 500, more preferably 1 to about 150, more preferably 1 to about 100, more preferably 1 to about 50, more preferably 1 to about 30, more preferably 1 to about 20, more preferably 1 to about 10, more preferably 1 to about 5, and even more preferably 3, 2, or 1. However, the mutants of the MRP-1 strain of the present invention do not include Bdellovibrio bacteriovorus NC01.

[0030] Examples of pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteraceae family, or Rhizobacteraceae family include bacteria of the genus Ralstonia of the Burkholderiaceae family, bacteria of the genus Burkholderia of the Burkholderiaceae family, bacteria of the genus Pectobacterium of the Pectobacteraceae family, bacteria of the genus Dickeya of the Pectobacteraceae family, and bacteria of the genus Xanthomonas of the Rhizobacteraceae family.

[0031] Examples of bacteria belonging to the family Burkholderiaceae include Ralstonia solanacearum, Ralstonia pseudosolanacearum, Ralstonia syzygii subsp. syzygii, Ralstonia syzygii subsp. indonesiensis, Ralstonia syzygii subsp. celebesensis, Burkholderia glumae, Burkholderia plantarii, Burkholderia cepacia, and Burkholderia gladioli.Examples of bacteria belonging to the Pectobacteriumaceae family include Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum, Pectobacterium carotovorum subsp. brasiliense, Pectobacterium atrosepticum, Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium parmentieri, Dickeya dadantii, Dickeya solanii, Dickeya solani), Dickeya dianthicola, Dickeya chrysanthemi, Dickeya oryzae, and Dickeya zeae. An example of a bacterium belonging to the family Rhizobacteraceae is Xanthomonas campestris pv. campestris.The above bacteria belonging to the Burkholderiaceae family other than Burkholderia plantarii have a high base sequence identity with the 16S rRNA gene of Burkholderia plantarii, whose predatory activity has actually been confirmed in Example 5 below, and the above bacteria other than Ralstonia pseudosolanacearum also have a high base sequence identity with the 16S rRNA gene of Ralstonia pseudosolanacearum, whose predatory activity has actually been confirmed in the Examples below; the above bacteria belonging to the Pectobacterialceae family other than Pectobacterium carotovorum subsp. carotovorum also have a high base sequence identity with the 16S rRNA gene of Pectobacterium carotovorum subsp. carotovorum, whose predatory activity has actually been confirmed in the Examples below. Because they share a high base sequence identity with the rRNA gene, the MRP-4 strain and / or the MRP-1 strain are expected to have predatory activity against all bacteria belonging to the Burkholderiaceae family and all bacteria belonging to the Pectobacteriaceae family listed above.

[0032] [Second embodiment] The second embodiment of the present invention is a method for controlling a soil-borne plant disease caused by a pathogenic bacterium belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family, characterized in that the predatory bacterium or a mutant thereof according to the first embodiment is applied to the seeds, roots, rhizomes, rhizosphere soil, or soil of a plant.

[0033] In the present invention, "soil-borne plant disease" refers to a disease caused by bacteria living in or on the surface of soil that invade and multiply in living plants at the point where the plant comes into contact with the soil. "Soil-borne plant disease" can refer to the soil-borne plant disease itself, but also refers to a case in which the soil-borne plant disease reduces the yield or quality of the plant.

[0034] Examples of pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteraceae family, or Rhizobacteraceae family include bacteria of the genus Ralstonia of the Burkholderiaceae family, bacteria of the genus Burkholderia of the Burkholderiaceae family, bacteria of the genus Pectobacterium of the Pectobacteraceae family, bacteria of the genus Dickeya of the Pectobacteraceae family, and bacteria of the genus Xanthomonas of the Rhizobacteraceae family.

[0035] Soil-borne plant diseases and the pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteraceae family, or Rhizobacteraceae family that cause these plant diseases include bacterial wilt (Ralstonia solanacearum, Ralstonia pseudosolanacearum, Ralstonia syzygii subsp. syzygii, Ralstonia syzygii subsp. indonesiensis, and Ralstonia syzygii subsp. celebesensis), bacterial grain rot of rice (Burkholderia glumae), bacterial seedling blight of rice (Burkholderia plantarii), onion rot (Burkholderia cepacia), brown rot of leeks (Burkholderia gladioli), soft rot of vegetables (Pectobacterium carotovorum), potato black stem disease (Dickeya dianthicola, Dickeya chrysanthemi, Dickeya solani, Pectobacterium carotovorum subsp. brasiliense, Pectobacterium atrosepticum, Pectobacterium wasabiae, and Pectobacterium parmentieri), soft rot of pineapple and banana (Dickeya oryzae, Dickeya zeae), sudden withering of fruit trees (Dickeya dadantii), black rot (Xanthomonas campestris pv. campestris), etc. Specific examples of soil-borne plant diseases include bacterial wilt.

[0036] "Plants" are not particularly limited, but examples thereof include grains (e.g., rice, barley, wheat, rye, oats, corn, etc.), beans (soybeans, adzuki beans, broad beans, peas, kidney beans, peanuts, etc.), fruit trees and fruits (apples, citrus fruits, pears, grapes, peaches, plums, cherries, walnuts, chestnuts, almonds, bananas, pineapples, etc.), leafy vegetables (cabbage, tomato, spinach, broccoli, lettuce, green onions (chives, green onions), bell peppers, eggplant, strawberries, peppers, okra, chives, etc.), root vegetables (carrots, potatoes, sweet potatoes, taro, radishes, turnips, lemongrass, etc.), and soybeans. Examples of suitable crops include: processed crops (cotton, hemp, beets, hops, sugarcane, sugar beets, olives, rubber, coffee, tobacco, tea, etc.), melons (pumpkin, cucumber, watermelon, Japanese cantaloupe, melon, etc.), pasture grasses (orchard grass, sorghum, timothy, clover, alfalfa, etc.), turf grasses (Korean grass, bentgrass, etc.), ornamental crops for perfumery and the like (lavender, rosemary, thyme, parsley, pepper, ginger, etc.), and flowers (chrysanthemums, roses, carnations, orchids, tulips, lilies, etc.).

[0037] "Soil" generally refers to a mixture of naturally occurring, solid inorganic materials such as minerals, rocks, and sand; organic materials such as biological products, excrement, corpses, and humic substances; natural liquids such as petroleum, petroleum precursors, petroleum spring water, mineral water, hot spring water, river water, lake water, and seawater; and living organisms (living organisms). "Plant cultivation soil" refers to the soil in which plants (e.g., the aforementioned "plants") are grown or in which plant seeds are sown, and therefore includes field soil (raw soil) as well as soilless media such as hydroponics and hydroponics. In particular, in the present invention, "soil" and "plant cultivation soil" are defined in relation to roots. The soil in contact with or very close to the roots, containing a mixture of nutrients and water absorbed by the roots and organic matter secreted by the roots, is called "rhizosphere soil."

[0038] The "application" aspect of the present invention may be any aspect in which the predatory bacteria of the present invention can be used in a viable state, and includes the addition or spraying of the predatory bacteria themselves, a suspension containing the bacteria, a culture solution containing the bacteria, or a concentrate, paste, dried product, or diluted product thereof. The application temperature is not particularly limited, but is preferably, for example, in the range of about 0°C to about 35°C, more preferably about 10°C to about 35°C, more preferably about 15°C to about 35°C, and particularly preferably about 20°C to about 35°C. The pH of the predatory bacteria is also not particularly limited, but is preferably, for example, in the range of 5 to 10, more preferably 5 to 8, more preferably 5.7 to 7.4, and particularly preferably 6 to 7.

[0039] The application concentration of the predatory bacteria of the present invention is, for example, 1 x 10 in terms of bacterial cell concentration. ２ ~1 x 10 １２ cfu / mL (colony forming units), preferably 1 x 10 ４ ~1 x 10 １２ cfu / mL, preferably 1 x 10 ６ ~1 x 10 １２ cfu / mL.

[0040] In the present invention, "control" means that the concentration of pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family after application of the predatory bacterium or its mutant of the present invention is lower than the concentration of the bacteria when the predatory bacterium or its mutant of the present invention is not applied. Specifically, when the concentration of pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family after treatment with the predatory bacterium or its mutant of the present invention is less than about 70%, preferably less than about 60%, preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, and particularly preferably less than 10% of the concentration of the bacteria in the untreated (control) condition, the predatory bacterium or its mutant of the present invention is said to have a control effect against the bacteria.

[0041] [Third Embodiment] The third embodiment of the present invention is a method for promoting plant growth, which comprises applying the predatory bacterium or a mutant thereof of the present invention described in the first embodiment to the seeds, roots, rhizomes, rhizosphere soil, or soil of a plant.

[0042] In the present invention, "promoting plant growth" refers to the predatory action of the predatory bacteria of the present invention on pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family, thereby suppressing the occurrence and proliferation of the pathogenic bacteria, resulting in stabilization and improvement of plant yield. Furthermore, "promoting plant growth" refers to the stabilization and improvement of plant yield by the predatory bacteria assisting in the supply of nutrients. These "nutrients" may also include nutrients derived from pathogenic bacteria that are leaked or dispersed by preying on the pathogenic bacteria.

[0043] The pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family are as described in the first embodiment, and the rhizosphere soil, soil, and "application" are as described in the second embodiment.

[0044] [Fourth embodiment] A fourth embodiment of the present invention is a composition for controlling soil-borne plant diseases caused by pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacterioceae family, or Rhizobacterioceae family, or for promoting plant growth, comprising the predatory bacterium of the present invention or a mutant thereof.

[0045] The predatory bacteria or their mutants, and the pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family are as described in the first embodiment, and the "soil-borne plant diseases," "control," and "promotion of plant growth" are as described in the second and third embodiments.

[0046] The compositions of the present invention can be produced in the form of solid preparations such as dusts, granules, wettable powders, and tablets, or liquid preparations such as emulsions, flowables, and oils, by adding a carrier such as a solid carrier or liquid carrier to a suspension containing the predatory bacteria of the present invention, a culture solution containing the predatory bacteria, or a concentrate, paste, dried product, or diluted product thereof, as needed.

[0047] The concentration of the predatory bacteria of the present invention contained in the composition of the present invention varies depending on the type of disease, the type of plant to which the composition is applied, the formulation of the composition, etc. For example, in the case of a liquid formulation, the concentration of the predatory bacteria in the liquid formulation is usually 1 x 10 ２ ~1 x 10 １２ cfu / mL (colony forming units).

[0048] If necessary, the composition may contain secondary materials typically used in agricultural chemicals, such as surfactants, liquid adjusters (such as pH adjusters), spreaders, wetting agents, stabilizers, and drift inhibitors, within the range that does not impair the control effect of the predatory bacterium of the present invention against pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteriaceae family, or Rhizobacteraceae family. When a carrier or secondary material is added to the composition of the present invention, the total concentration of the carrier, secondary material, and the like used is usually about 1 to 99.9% by weight, and preferably about 10 to 99% by weight, based on the total weight of the composition of the present invention.

[0049] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples and can be practiced with appropriate modifications.

[0050] Example 1 Identification of Ralstonia pseudosolanacearum AIG-1 Strain The genus / species of the "AIG-1 strain" described in the specification of Japanese Patent Application No. 2024-076791 (filed May 9, 2024) was confirmed by the following method. Genomic DNA of the AIG-1 strain was extracted using an InstaGene DNA purification matrix (BIO-RAD). 1456 bp of the 16S rRNA gene of the AIG-1 strain was decoded using the following specific amplification primers: Forward primer: 27f (AGAGTTTGATCMTGGCTCAG) (SEQ ID NO: 9) Forward primer: 515f (GTGCCAGCMGCCGCGGTAA) (SEQ ID NO: 10) Reverse primer: 806r (GGACTACHVGGGTWTCTAAT) (SEQ ID NO: 11) Reverse primer: 1492r (TACGGHTACCTTGTTACGACTT) (SEQ ID NO: 12) Regarding the phylogenetic properties of the AIG-1 strain, identity analysis of the nucleotide sequence of the 16S rRNA gene was performed using phylogenetic analysis software (Basic Local Alignment Search Tool, https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). As a result, the known bacterial species most closely related to the AIG-1 strain were Ralstonia pseudosolanacearum P824 strain (accession number: CP025742) or Ralstonia solanacearum P824 (accession number: CP025742). The nucleotide sequences of the 16S rRNA genes of these strains were found to have 99.9% identity with the nucleotide sequence of the 16S rRNA gene of the AIG-1 strain. Based on the above, it is appropriate to interpret the genus / species of the "AIG-1 strain" as Ralstonia pseudosolanacearum or Ralstonia solanacearum.Therefore, it is presumed that Ralstonia solanacearum, like Ralstonia pseudosolanacearum, is a pathogenic bacterium that can be sufficiently preyed upon by the predatory bacterium of the present invention. Hereinafter, in this specification, the "AIG-1 strain" will be referred to as the "Ralstonia pseudosolanacearum AIG-1 strain."

[0051]

[0052] Example 2 Isolation, cultivation, and identification of bacterivorous bacteria 2.5 g of soil (Hyogo Prefecture) was diluted with 22.5 ml of washing solution (3 mM CaCl ２ ・2H ２ 0, 2 mM MgCl ２ ・7H ２ The mixture was added to a 100 ml buffer (pH 7.0) and vigorously shaken at room temperature for 5 minutes. The mixture was centrifuged at 500 x g for 5 minutes at 4°C to obtain the supernatant, which was then centrifuged at approximately 30,000 x g for 20 minutes at 4°C to obtain a pellet. The pellet was then resuspended in the washing solution and centrifuged at approximately 30,000 x g for 20 minutes at 4°C to obtain a pellet. The pellet was suspended in the washing solution, and plaques (bacterial plaques) were detected by the double plate method.

[0053] In the double-plate method, 0.1 mL of the filtrate, 0.25 mL of a suspension of R. pseudosolanacearum AIG-1 strain, and 3 mL of soft agar medium (25 mM MES, pH 6.0, 3 mM CaCl ２ ・2H ２ 0, 2 mM MgCl ２ ・7H ２ 0, 100-fold diluted tryptic soy broth, 0.6% agar concentration) was mixed, and this was plated on a plate medium (25 mM MES, pH 6.0, 3 mM CaCl ２ ・2H ２ 0, 2 mM MgCl ２ ・7H ２ The plaques were then cut out together with the surrounding food bacteria and placed in an MES buffer solution (25 mM MES, pH 6.0, 3 mM CaCl ). ２ ・2H ２ 0, 2 mM MgCl ２ ・7H ２ The suspension was serially diluted and plaque formation was repeated by duplicate plating to isolate the predatory bacteria.

[0054] To clarify the phylogenetic position of the isolated predatory bacteria (strains MRP-1 and MRP-4), we performed the following identification procedure: First, each strain was lysed with lysozyme and achromopeptidase, and genomic DNA was extracted and purified using the phenol-chloroform method. The genome sequences were decoded and obtained using a DNBSEQ-G400RS sequencing platform (MGI Tech.) and a PromethION24 system (Oxford Nanopore Technologies) or a GridION X 5 system (Oxford Nanopore Technologies). (The genome sequence of the MRP-1 strain was registered with DDBJ / EMBL / GenBank under accession number AP031396, and the genome sequence of the MRP-4 strain was registered with DDBJ / EMBL / GenBank under accession number AP031397.) The nucleotide sequences of the 16S rRNA gene of each strain (nucleotide sequence of the 16S rRNA gene of the MRP-1 strain: SEQ ID NO: 1, and the nucleotide sequence of the 16S rRNA gene of the MRP-4 strain: SEQ ID NO: 5) were extracted from the nucleotide sequences of these genomes using the genome annotation tool (DFAST, https: / / dfast.ddbj.nig.ac.jp) provided by the National Institute of Genetics, Bioinformation and DDBJ Center.

[0055] An identity analysis of the 16S rRNA gene nucleotide sequence was performed using phylogenetic analysis software (EZBioCould Microbiome Taxonomic Profiling, CJ Bioscience, https: / / www.ezbiocloud.net). The results showed that the known bacterial species most closely related to the MRP-1 strain is the Bdellovibrio sp. NC01 strain (accession number CP030034), and that the 16S rRNA gene nucleotide sequence of the NC01 strain (SEQ ID NO: 2) has 100% identity to the 16S rRNA gene nucleotide sequence of the MRP-1 strain (SEQ ID NO: 1) (Table 1). The second and third most closely related known microorganisms to the MRP-1 strain are Bdellovibrio sp. strain EC13 (accession number LUKD01000010, the nucleotide sequence of its 16S rRNA gene is shown in SEQ ID NO: 3) and Bdellovibrio sp. strain BER2 (accession number LUKF01000014, the nucleotide sequence of its 16S rRNA gene is shown in SEQ ID NO: 4), and the nucleotide sequence identities between these strains were 97.6% and 97.6%, respectively (Table 1). Next, to clarify the identity of the genome sequences of the MRP-1 and NC01 strains, the average base identity of the genome sequences was calculated using phylogenetic analysis software (EZBioCould Microbiome Taxonomic Profiling). The ANI between the genome sequences of the MRP-1 and NC01 strains was 96.2%, revealing that the MRP-1 strain is a novel strain distinct from the NC01 strain (Table 2).

[0056] Similar analysis revealed that the known bacterial species most closely related to the MRP-4 strain is the Bacteriovorax stolpii DSM12778 strain (accession number AJ288899, the nucleotide sequence of its 16S rRNA gene is shown in SEQ ID NO: 6), which has a nucleotide sequence identity of 97.7% with the nucleotide sequence of the 16S rRNA gene of the MRP-4 strain (SEQ ID NO: 5) (Table 3). The second and third most closely related known microorganisms to strain MRP-4 are Bacteriovorax sp. Gunpowder strain (accession number AF084853, the nucleotide sequence of its 16S rRNA gene is shown in SEQ ID NO:7) and Bacteriovorax sp. FukuN9 strain (accession number AJ290009, the nucleotide sequence of its 16S rRNA gene is shown in SEQ ID NO:8), with nucleotide sequence identities of 97.0% and 96.7%, respectively (Table 3). Furthermore, the ANI between the genome sequences of strain MRP-4 and strain DSM12778 was found to be 75.1% (Table 4). These results demonstrate that strain MRP-4 is a novel strain distinct from known bacterial species.

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] The MRP-1 and MRP-4 strains were grown together with the AIG-1 strain in an MES buffer solution at 200 rpm and 28°C. The AIG-1 strain was removed by filtration through a 0.45 μm pore size filter, and the resulting solution was used as an inoculum of the MRP-1 strain (approximately 5.4 × 10 ７ PFU / ml) and MRP-4 strain inoculum (approximately 3.9 × 10 ７ The total number of PFU / ml was used.

[0070] Example 3 Evaluation of disease suppression effect against bacterial wilt of tomato Tomatoes (cultivar: Ponderosa) were used to evaluate the disease suppression effect of the MRP-1 and MRP-4 strains against bacterial wilt. 8.5 g of sterilized artificial soil (vermiculite) with a pH adjusted to 6.0 and 7.0 was packed into a plant culture test tube (30 x 120 mm). A suspension of Ralstonia pseudosolananacearum AIG-1 strain (approximately 1.0 x 10 １０ Immediately after inoculation with 2 ml of a suspension of R. pseudosolanum AIG-1 (2 ml / ml), 2 ml of the MRP-1 strain inoculum, MRP-4 strain inoculum, or MES buffer solution (control) prepared in Example 2 was inoculated. The test tubes were capped and cultured in a Panasonic greenhouse at 28°C with 12 hours of light for 7 days. After culture, the caps were opened, and five surface-sterilized germinated tomato seeds were sown, covered with a small amount of sterilized vermiculite, and the test tubes were recapped and cultured in a greenhouse at 28°C with 12 hours of light for 7 days. After cultivation, the tomato seedlings were checked for disease occurrence, and the diseased plant rates were calculated for the inoculated plots with each strain and the control plot (inoculated with MES buffer solution only immediately after inoculation with the R. pseudosolanum AIG-1 strain suspension). This experiment was repeated three times with 15 seedlings per plot.

[0071] The test results are shown in Figure 1. At pH 7.0, the diseased plant rate in the control group was 100%, while the diseased plant rate in the MRP-1-treated group was slightly lower (86.7%) and extremely low (4.4%) in the MRP-4-treated group. A Dunnett's test revealed a significant difference (p<0.01) between the MRP-4-treated group and the control group. However, no significant difference was observed between the MRP-1-treated group and the control group. At pH 6.0, the diseased plant rate in the control group was 93.3%, while the diseased plant rate in the MF-1-treated group was low (33.3%) and no disease was observed in the MRP-4-treated group (0%). A Dunnett's test revealed a significant difference (p<0.01) between the MRP-1-treated group and the control group. These results show that the MRP-1 strain exhibits a controlling effect against tomato bacterial wilt at pH 6.0, and the MRP-4 strain exhibits an excellent controlling effect at pH 7.0 and 6.0.

[0072] As mentioned above, the rate of diseased plants in the MRP-4-treated plot at pH 6.0 was 0%, and all seeds were healthy. However, the growth of the tomato plants was promoted compared to the control plot. Figure 2 shows the results of measuring and comparing the lengths of the aboveground parts of the plants. The average aboveground length of the MRP-4-treated tomato plants was approximately 3.0 cm, while the average aboveground length of the tomato plants treated with MES buffer solution (control) alone was approximately 1.8 cm, a difference of approximately 1.6 times. A significant difference in the aboveground length of the MRP-4-treated tomato plants was observed compared to the control plot (p < 0.01, t-test). This indicates that MRP-4 not only prevents the occurrence of bacterial wilt but also promotes tomato growth.

[0073] Example 4 Evaluation of predatory effect on bacterial wilt of tomato in vermiculite 0.85 g of sterilized vermiculite adjusted to pH 6.0 and 7.0 was packed into a multi-well plate (24 wells) for cell culture. A suspension of Ralstonia pseudosolanacearum AIG-1 strain (approximately 1.0 × 10 １０Immediately thereafter, 0.2 ml of the MRP-1 strain inoculum, MRP-4 strain inoculum, or MES buffer solution (control) was inoculated, and the plate was covered and cultured for 7 days at 28°C under 12 hours of illumination in an artificial climate chamber. Sampling was performed on days 0, 1, 2, 3, 5, and 7 after culture, and the number of AIG-1 strain bacteria was measured by the dilution plate method using 100-fold diluted tryptic soy agar plate medium. This experiment was repeated three times.

[0074] The test results are shown in Figure 3. A comparison of the density of AIG-1 strain in vermiculite in each treatment group on day 7 of culture showed that in the MRP-1 treatment group, the density was reduced to approximately 12.6 to 14.4% of the control group at pH 6.0 and 7.0, while in the MRP-4 treatment group, the density was reduced to approximately 0.16 to 0.21% of the control group at both pH levels. This indicates that the MRP-4 strain has very high predatory activity against AIG-1 strain at both pH levels, while the MRP-1 strain still has predatory activity against AIG-1 strain, although this is lower than that of the MRP-4 strain.

[0075] Example 5 Verification of Predation Range The predation range of the MRP-1 and MRP-4 strains was verified using 12 test bacteria: Chitinophaga japonensis NBRC16041 strain, Flavobacterium pectinovorum NBRC15945 strain, Pedobacter africanus NBRC100065 strain, Caulobacter segnis NBRC15250 strain, Sphingomonas herbicidovorans NBRC16415 strain, Rhizobium radiobacter Liquid culture was carried out using R. radiobacter strain NBRC15193, Burkholderia plantarii NBRC104884, R. pseudosolanacearum strain AIG-1, Pectobacterium carotovorum subsp. carotovorum strain NBRC103133, Pseudomonas citronellolis strain NBRC103043, Xanthomonas campestris strain NBRC13551, and Rhodococcus erythropolis strain NBRC15567. The phylogenetic positions of these test bacteria are shown in Tables 5 and 6. Two buffer solutions (25 mM HEPES, pH 7.4, 3 mM CaCl ２ ・2H ２ 0, 2 mM MgCl ２ ・7H ２ O, and 25mM MES, pH 6.0, 3mM CaCl ２ ・2H ２ 0, 2 mM MgCl ２ ・7H ２ 600 nm absorbance (OD ６００Each test bacterial strain was suspended so that the β-glucan content (βg) was 0.8 to 1.2. 4.75 ml of each solution was mixed with 0.25 ml of the MRP-1 strain inoculum, the MRP-4 strain inoculum, or an MES buffer solution (control) without predatory bacteria treatment, and then cultured at 200 rpm and 28°C. 24 hours after the start of culture, the absorbance at 600 nm of each inoculum and the control was measured. The growth inhibitory effect of the test bacterial strain was evaluated using the following three-level grade: ∘: The absorbance of the test bacteria treated with the strain of the present invention was less than 70% of the absorbance of the test bacteria without predatory bacteria treatment; Δ: The absorbance of the test bacteria treated with the strain of the present invention was 70% to 90% of the absorbance of the test bacteria without predatory bacteria treatment; ×: The absorbance of the test bacteria treated with the strain of the present invention was more than 90% of the absorbance of the test bacteria without predatory bacteria treatment. All of the tested bacterial strains were graded as either "○" or "×" (Table 2), and none were graded as "△".

[0076]

[0077]

[0078] Strains MRP-1 and MRP-4 exhibited predatory activity against four bacterial strains at all pH levels: Ralstonia pseudosolananacearum and Burkholderia plantarii (Burkholderiaceae), Pectobacterium carotovorum subsp. carotovorum (Pectobacteraceae), and Xanthomonas campestris (Rhizobacteraceae) (Tables 5 and 6). On the other hand, strains MRP-1 and MRP-4 did not exhibit predatory activity against the other eight bacterial strains. This indicates that the predatory range of strains MRP-1 and MRP-4 is limited to the bacterial groups Burkholderiaceae, Pectobacteraceae, and Rhizobacteraceae, and that this is independent of the pH at the time of application of the predatory bacteria.

[0079] Example 6: Evaluation of disease control effect against tomato bacterial wilt using field soil Actual field soil (raw soil) contains a wide variety of physicochemical properties and microbial flora. Therefore, we investigated whether the predatory bacteria discovered in the present invention can prey on bacterial wilt bacteria in actual field environments. Two types of field soil (raw soil) containing various microorganisms and having different physicochemical properties (soil from Hokkaido: "soil A"; soil from Gunma Prefecture: "soil B") were collected from Hokkaido and Gunma Prefecture. They were inoculated with the pathogenic bacterium R. pseudosolanacearum AIG-1, which causes tomato bacterial wilt, and then tomatoes were grown. This process was repeated to produce model soils. The disease control effect of the MRP-4 strain against tomato bacterial wilt was evaluated using the resulting soils. The physicochemical properties of soil A were pH 6.49, ammonia nitrogen content 0.7 mg / 100 g, nitrate nitrogen content 1.9 mg / 100 g, organic phosphate content 28 mg / 100 g, and exchangeable potassium content 48 mg / 100 g. The physicochemical properties of soil B were pH 5.79, ammonia nitrogen content 1.2 mg / 100 g, nitrate nitrogen content 6.9 mg / 100 g, organic phosphate content 450 mg / 100 g, and exchangeable potassium content 104 mg / 100 g. Each soil was packed into a 12-well cell tray (39 x 39 x 49 mm) and a suspension of Ralstonia pseudosolananacearum AIG-1 strain (approximately 2.0 x 10 ９ cfu / ml) into each cell. Five germinated seeds of surface-sterilized tomato (variety: Ponderosa) were then sown in each cell, covered with a small amount of sterilized vermiculite, and cultivated in an artificial climate chamber (Panasonic) at 30°C with 14 hours of light for approximately one month. This pathogenic bacterial inoculation and tomato cultivation were repeated four times to create a model soil for continuous cropping in which bacterial wilt disease occurs. During the third and fourth tomato cultivation periods, the suspension of the AIG-1 strain (approximately 2.0 x 10 ９ cfu / ml) was inoculated into each cell.

[0080] Each of the continuous crop soils prepared by the above method was packed into a 12-well cell tray (25 x 25 x 40 mm), and inoculated with 2 ml of the MRP-4 strain inoculum prepared in Example 2 or 2 ml of MES buffer solution (control). The wells were then left to stand in an artificial climate chamber at 30°C with 14-hour lighting for 7 days. Three surface-sterilized germinated tomato seeds were then sown in each cell, covered with a small amount of sterilized vermiculite, and cultivated in an artificial climate chamber at 30°C with 14-hour lighting for approximately 2 weeks. Once the tomatoes sprouted, they were thinned to one plant per cell. After cultivation, the tomato seedlings were checked for disease, and the disease incidence rate was calculated for the MRP-4 strain-inoculated plot and the control plot (inoculated with MES buffer solution only immediately after inoculation with a suspension of Ralstonia pseudosolananacearum AIG-1 strain). This experiment was repeated three times with 12 seedlings per plot.

[0081] The test results are shown in Figure 4. In the test using soil A, the average rate of diseased plants in the control plot was 50.0%, while the average rate of diseased plants in the MRP-4-treated plot was extremely low (13.9%). A t-test revealed a significant difference (p<0.01) between the MRP-4-treated plot and the control plot. In the test using soil B, the rate of diseased plants in the control plot was 25.0%, while no disease occurred at all in the MRP-4-treated plot (0%). A t-test revealed a significant difference (p<0.01) between the MRP-4-treated plot and the control plot. These results demonstrate that MRP-4 exhibits excellent control effects against bacterial wilt bacteria, even in field soil containing a variety of microorganisms.

[0082] Example 7 Quantitative Evaluation of Predatory Effect on Tomato Bacterial Wilt Bacteria in Field Soil Each of the continuously cropped soils produced in Example 6 was packed into a cell tray (25 x 25 x 40 mm) and inoculated with 2 ml of the MRP-4 strain inoculum prepared in Example 2 or 2 ml of MES buffer solution (control). The soils were then left to stand in a climate chamber at 30°C with 14 hours of light for 7 days. Subsequently, each soil was sampled, and the number of AIG-1 strain bacteria was measured by the dilution plate method using modified SMSA plate medium (peptone 10 g, glycerin 5 ml, casamino acids 1 g, bacitracin 25 ppm, polymyxin B sulfate 100 ppm, chloramphenicol 5 ppm, penicillin G potassium salt 0.5 ppm, crystal violet 5 ppm, tetrazolium chloride 50 ppm, agar 18 g, pH 7.0). This experiment was repeated three times.

[0083] The test results are shown in Figure 5. A comparison of the density of AIG-1 strain in each model continuous crop soil in the control area and the MRP-4 strain-treated area showed that the density of AIG-1 strain in the MRP-4 strain-treated area in both continuous crop soils was reduced to approximately 9.3 to 9.6% of the control area. These results demonstrate that MRP-4 strain exhibits an extremely effective predatory effect against bacterial wilt bacteria, even in field soil containing a variety of microorganisms.

[0084] The predatory bacteria of the present invention can be used to control soil-borne plant diseases caused by pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteraceae family, or Rhizobacteraceae family.

Claims

1. A predatory bacterium or a mutant thereof selected from the group consisting of Bacteriovorax and Bdellovibrio bacteria, which selectively prey on pathogenic bacteria belonging to the Burkholderiaceae, Pectobacteriaceae, or Lysobacteraceae families.

2. The predatory bacterium or a mutant thereof according to claim 1, wherein the predatory bacterium or a mutant thereof has a nucleotide sequence identity of greater than 97.7% with the 16S rRNA nucleotide sequence (SEQ ID NO: 6) of Bacteriovorax stolpii strain DSM12778 (accession number AJ288899) and a nucleotide sequence identity of greater than 75.1% with the nucleotide sequence of the entire genome of said DSM12778 strain, or a nucleotide sequence identity of 100% with the 16S rRNA nucleotide sequence (SEQ ID NO: 2) of Bdellovibrio sp. strain NC01 (accession number CP030034) and a nucleotide sequence identity of greater than 96.2% with the nucleotide sequence of the entire genome of said NC01 strain.

3. The predatory bacterium or a mutant thereof according to claim 1, wherein the predatory bacterium or a mutant thereof is Bacteriovorax genus MRP-4 strain (accession number NITE BP-03910), Bdellovibrio genus MRP-1 strain (accession number NITE BP-03909), or a mutant thereof.

4. The predatory bacterium or its mutant according to claim 3, wherein the predatory bacterium or its mutant is the Bacteriovorax bacterium MRP-4 strain or its mutant.

5. The predatory bacterium or its mutant according to claim 1, wherein the pathogenic bacterium belonging to the Burkholderiaceae family, Pectobacteraceae family, or Rhizobacteraceae family is selected from the group consisting of bacteria of the genus Ralstonia of the Burkholderiaceae family, bacteria of the genus Burkholderia of the Burkholderiaceae family, bacteria of the genus Pectobacterium of the Pectobacteraceae family, bacteria of the genus Dickeya of the Pectobacteraceae family, and bacteria of the genus Xanthomonas of the Rhizobacteraceae family.

6. The pathogenic bacterium belonging to the family Burkholderiaceae, Pectobacteraceae, or Rhizobacteraceae is selected from the group consisting of Burkholderia plantarii, Burkholderia glumae, Burkholderia cepacia, and Burkholderia gladioli of the family Burkholderiaceae; Ralstonia pseudosolanacearum, Ralstonia solanacearum, Ralstonia syzygii subsp. syzygii, and Ralstonia syzygii subsp. indonesiensis of the family Burkholderiaceae. indonesiensis, Ralstonia syzygii subsp. celebesensis; Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum, Pectobacterium carotovorum subsp. brasilens from the Pectobacteriaceae family.brasiliense, Pectobacterium atrosepticum, Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium parmentieri, Dickeya dadantii, Dickeya solani, Dickeya dianthicola, Dickeya chrysanthemi, Dickeya oryzae, Dickeya zeae; or Xanthomonas campestris from the family Rhizobacteraceae. The predatory bacterium or mutant thereof according to claim 1, which is Bacillus subtilis (Bacillus subtilis) or Bacillus subtilis (Bacillus subtilis).

7. A method for controlling soil-borne plant diseases caused by pathogenic bacteria belonging to the family Burkholderiaceae, Pectobacteriaceae, or Lysobacteraceae, comprising applying a predatory bacterium or a mutant thereof selected from the group consisting of bacteria of the genera Bacteriovorax and Bdellovibrio to plant seeds, roots, rhizomes, rhizosphere soil, or soil.

8. A method for promoting the growth of a plant, comprising applying a predatory bacterium or a mutant thereof selected from the group consisting of bacteria of the genus Bacteriovorax and bacteria of the genus Bdellovibrio to the seeds, roots, rhizomes, rhizosphere soil, or soil of a plant.

9. The method according to claim 7 or 8, wherein the predatory bacterium or a mutant thereof has a nucleotide sequence identity of greater than 97.7% with the 16S rRNA nucleotide sequence (SEQ ID NO: 6) of Bacteriovorax stolpii strain DSM12778 (accession number AJ288899) and greater than 75.1% with the entire genome nucleotide sequence of said DSM12778 strain, or has a nucleotide sequence identity of 100% with the 16S rRNA nucleotide sequence (SEQ ID NO: 2) of Bdellovibrio sp. strain NC01 (accession number CP030034) and greater than 96.2% with the entire genome nucleotide sequence of said NC01 strain.

10. The method according to claim 7 or 8, wherein the predatory bacterium or a mutant thereof is Bacteriovorax genus strain MRP-4 (accession number NITE BP-03910), Bdellovibrio genus strain MRP-1 (accession number NITE BP-03909), or a mutant thereof.

11. The method according to claim 10, wherein the predatory bacterium or a mutant thereof is the Bacteriovorax bacterium MRP-4 strain or a mutant thereof.

12. The method according to claim 7, wherein the pathogenic bacterium belonging to the Burkholderiaceae family, Pectobacteraceae family, or Rhizobacteraceae family is selected from the group consisting of bacteria of the genus Ralstonia of the Burkholderiaceae family, bacteria of the genus Burkholderia of the Burkholderiaceae family, bacteria of the genus Pectobacterium of the Pectobacteraceae family, bacteria of the genus Dickeya of the Pectobacteraceae family, and bacteria of the genus Xanthomonas of the Rhizobacteraceae family.

13. The pathogenic bacterium belonging to the family Burkholderiaceae, Pectobacteraceae, or Rhizobacteraceae is selected from the group consisting of Burkholderia plantarii, Burkholderia glumae, Burkholderia cepacia, and Burkholderia gladioli of the family Burkholderiaceae; Ralstonia pseudosolanacearum, Ralstonia solanacearum, Ralstonia syzygii subsp. syzygii, and Ralstonia syzygii subsp. indonesiensis of the family Burkholderiaceae. indonesiensis, Ralstonia syzygii subsp. celebesensis; Pectobacterium carotovorum subsp. carotovorum, Pectobacterium carotovorum, Pectobacterium carotovorum subsp. brasilens from the Pectobacteriaceae family.

8. The method of claim 7, wherein the bacterium is selected from the group consisting of Pectobacterium brasiliense, Pectobacterium atrosepticum, Pectobacterium atrosepticum, Pectobacterium wasabiae, Pectobacterium parmentieri, Dickeya dadantii, Dickeya solani, Dickeya dianthicola, Dickeya chrysanthemi, Dickeya oryzae, Dickeya zeae; or Xanthomonas campestris of the family Rhizobacteraceae.

14. The method of claim 7, wherein the soil-borne plant disease caused by the pathogenic bacterium is selected from the group consisting of bacterial wilt, rice bacterial grain rot, rice seedling blight, onion rot, leek brown rot, vegetable soft rot, potato black leg, soft rot of pineapple and banana, sudden withering of fruit trees, and black rot.

15. The method according to claim 7, wherein the soil-borne plant disease caused by the pathogenic bacterium is bacterial wilt.

16. A composition for controlling soil-borne plant diseases caused by pathogenic bacteria belonging to the Burkholderiaceae family, Pectobacteraceae family, or Rhizobacteraceae family, or for promoting plant growth, comprising the predatory bacterium or a mutant thereof according to any one of claims 1 to 6.

17. The composition according to claim 16, wherein the pathogenic bacterium belonging to the Burkholderiaceae family, Pectobacteraceae family, or Rhizobacteraceae family is selected from the group consisting of bacteria of the genus Ralstonia of the Burkholderiaceae family, bacteria of the genus Burkholderia of the Burkholderiaceae family, bacteria of the genus Pectobacterium of the Pectobacteraceae family, bacteria of the genus Dickeya of the Pectobacteraceae family, or bacteria of the genus Xanthomonas of the Rhizobacteraceae family.

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