Control agent having antibacterial activity against base rot fungus, which is soil-borne pathogenic fungus, and method for controlling soil-borne disease using the same

Abstract

To provide a microbial control agent exhibiting high control efficacy against soil-borne diseases caused by phytopathogenic filamentous fungi, being safe, free from phytotoxicity, and imposing a small environmental burden, and further to provide a method for effectively controlling soil-borne diseases through use of the microbial control agent.SOLUTION: Provided is a soil-borne disease control agent for phytopathogenic filamentous fungi, which includes, as an active ingredient, the isolates KUFF23 N1 and KUFF23 H1 alone or in combination with photosynthetic bacteria. In addition, provided is a method for controlling soil-borne diseases by applying the control agent to soil or the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a soil-borne disease control agent containing, as an active ingredient, a microorganism having antibacterial activity against root-rot pathogenic fungi that cause soil-borne diseases, and a method for preventing or controlling the onset of soil-borne diseases using the same. [Background technology]

[0002] Soil-borne diseases are agriculturally important contagious plant diseases that spread through soil. Among these, sweet potato root rot, caused by the fungus Diaporthe destruens, was first confirmed in Okinawa and Kagoshima in 2018 and has since spread throughout Japan, causing significant damage, including significant yield reductions. The main symptoms of sweet potato root rot are root rot, blackening of the stem, wilting, and death. These symptoms have caused significant damage in southern Kyushu, including a severe decline in sweet potato quality and reduced yields.

[0003] Conventional methods for controlling soil-borne diseases have primarily focused on chemical control, which involves soil disinfection using methyl bromide, a simple and versatile agent, as well as chloropicrin and dazomet. However, the use of methyl bromide was generally phased out in 2005 and completely phased out in 2013. New methods to replace methyl bromide were therefore explored, but chemical control methods using chloropicrin, dazomet, and basamide had many issues with efficacy and application compared to methyl bromide, making them inadequate alternatives to methyl bromide. In recent years, chemical pesticide control has been criticized for its significant environmental impact and safety for workers and food. Furthermore, chemical pesticide application to soil can significantly impact beneficial microflora in addition to the targeted pathogens. Therefore, there is a need for safe and sustainable alternatives to chemical control methods for soil-borne diseases.

[0004] In the circumstances described above, research has been underway into microbial materials that utilize microorganisms (antibacterial microorganisms) that have antibacterial properties against plant pathogens to suppress disease. Antibacterial microorganisms are microorganisms that suppress the growth and activity of specific microorganisms. In recent years, there has been a demand for a transition to ecosystem-based (or environmentally friendly) agriculture, such as pesticide-free farming, and it is no exaggeration to say that technology that uses antibacterial microorganisms as microbial pesticides to suppress the growth and activity of pathogens, thereby reducing plant disease, is a dream technology, and its development is eagerly awaited (Patent Document 1).

[0005] For example, Bacillus subtilis is known to have antibacterial properties against certain pathogenic bacteria that cause plant diseases, and is already registered as a pesticide in Japan as an agent for controlling gray mold in eggplants and tomatoes.

[0006] Furthermore, Patent Document 2 discloses a disease control technology for agricultural and horticultural plants, which contains a spore fraction prepared from a culture of bacteria belonging to the genus Bacillus, such as Bacillus subtilis, so that the fraction contains 50% or more spores by dry weight.

[0007] Furthermore, Patent Document 3 discloses a fungal disease control agent that is composed of an Amycolatopsis A1 strain of actinomycetes, is non-pathogenic to plants, is easily adaptable to the agricultural field, exhibits high plant adhesion, and exhibits excellent fungal disease control effects.

[0008] Furthermore, as shown in Patent Document 4, "Soil-borne disease control agent and soil-borne disease control method using same," researchers at Kagoshima University have discovered that when a photosynthetic bacterium of the genus Rhodopseudomonas is applied in combination with a soil bacterium of the genus Bacillus, the combined effect shows extremely high antibacterial properties and fertilizer effects against the plant pathogenic fungus (purple root rot pathogen) that causes a soil-borne disease (purple root rot of sweet potato), and this discovery has already been patented. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent Publication No. 2011-241178 [Patent Document 2] JP 8-175919 [Patent Document 3] Patent Publication No. 2006-290816 [Patent Document 4] Patent No. 6232602 Summary of the Invention [Problem to be solved by the invention]

[0010] The objective of this invention is to develop and provide a microbial control agent that is highly effective against soil-borne diseases caused by sweet potato root rot (Diaporthe destruens), that has a low environmental impact, is safe, and causes no chemical damage, and to develop a method for effectively controlling soil-borne diseases using this microbial control agent, thereby reducing labor and costs and ensuring a stable supply of safe agricultural products. [Means for solving the problem]

[0011] As mentioned above, the present inventors have already discovered that when a photosynthetic bacterium of the genus Rhodopseudomonas and a soil bacterium of the genus Bacillus are applied in combination to combat the pathogenic fungus of purple root rot, the combined effect shows extremely high antibacterial properties and fertilizer effects against plant pathogenic filamentous fungi that cause soil-borne diseases, and have obtained a patent for this discovery (Patent Document 4).

[0012] The present application provides the following specific means for stably maintaining the control effect by isolating two strains of bacteria that have a high control effect against sweet potato root rot bacteria even when used alone, and mixing them with an already patented photosynthetic bacterium.

[0013] (1) A soil-borne disease control agent for root rot (Diaporthe destruens) containing a type of lactic acid bacterium (Lactobacillus), designated KUFF23N1 in this case, as an active ingredient.

[0014] (2) The soil-borne disease control agent according to (1), wherein the lactic acid bacterium (Lactobacillus) is a bacterium designated as KUFF23N1 under accession number P-04060.

[0015] (3) A soil-borne disease control agent for root rot fungus (Diaporthe destruens) containing as an active ingredient a bacterium called KUFF23H1 in this case, which is a type of Microvirgula aerodenitrificans.

[0016] (4) The soil-borne disease control agent according to (3), wherein the species of Microvirgula aerodenitrificans is a bacterium designated as KUFF23H1 under accession number P-04061.

[0017] (5) A soil-borne disease control agent for plant pathogenic filamentous fungi, containing KUFF23N1, KUFF23H1 and photosynthetic bacteria as active ingredients.

[0018] (6) The soil-borne disease control agent according to (5), wherein the photosynthetic bacterium is a bacterium of the genus Rhodopseudomonas.

[0019] (7) A soil-borne disease control agent according to (6), wherein the mixing ratio of KUFF23N1, KUFF23H1 and photosynthetic bacteria in the soil-borne disease control agent is not particularly limited as long as the above three types of bacteria are included, but preferably the ratio of photosynthetic bacteria:KUFF23N1:KUFF23H1 is 1:1 to 1000:1 to 1000.

[0020] (8) The soil-borne disease control agent according to (7), which is a type of lactic acid bacterium (Lactobacillus) for use as an active ingredient in a soil-borne disease control agent against root rot bacteria (Diaporthe destruens).

[0021] (9) The soil-borne disease control agent according to (7), which is a bacterium of the type Microvirgula aerodenitrificans, for use as an active ingredient in a soil-borne disease control agent against root rot fungi (Diaporthe destruens).

[0022] (10) The soil-borne disease control agent according to any one of (1) to (9), wherein the plant pathogenic filamentous fungus is selected from the group consisting of root rot fungus (Diaporthe destruens), Helicobasidium fungus, Rosellinia fungus, Fusarium fungus, Pythium fungus, Pyrenochaeta fungus, Monosporascus fungus, Verticillium fungus, Rhizoctonia fungus, Plasmodiophora fungus, Phitophthora fungus, and Sclerotium fungus.

[0023] (11) The soil-borne disease control agent according to any one of (1) to (9), wherein the soil-borne disease is selected from the group consisting of root rot, root rot, clubroot, damping-off, wilt, root rot, southern blight, and phytophthora blight.

[0024] (12) A method for controlling a soil-borne disease, which comprises applying the soil-borne disease control agent according to any one of (1) to (9) to soil or a medium.

[0025] (13) The method for controlling a soil-borne disease according to (12), wherein the soil is soil for planting a target plant.

[0026] (14) The method for controlling a soil-borne disease according to (13), wherein the method is applied before planting of a target plant. [Effects of the Invention]

[0027] According to the present invention, KUFF23N1 and KUFF23H1 can be used alone, or a mixture of KUFF23N1 and KUFF23H1 with photosynthetic bacteria can efficiently suppress the growth and activity of plant pathogenic filamentous fungi.

[0028] The soil-borne disease control agent of the present invention can provide a microbial control agent that is not harmful to plants, has a high control effect against soil-borne diseases caused by plant pathogenic filamentous fungi, is environmentally friendly, is safe, and does not cause phytotoxicity.

[0029] According to the method for controlling soil-borne diseases of the present invention, soil-borne diseases can be effectively controlled using the agent for controlling soil-borne diseases of the present invention, thereby reducing labor and costs and enabling the stable provision of safe agricultural products. [Brief explanation of the drawings]

[0030] [Figure 1] A microscopic photograph of KUFF23N1 (lactic acid bacteria: a type of Lactobacillus) is shown. [Figure 2] A photograph of a plate culture of KUFF23N1 (lactic acid bacteria: a type of Lactobacillus) is shown. [Figure 3] The base sequence of 16S rDNA of KUFF23N1 (lactic acid bacteria: a type of Lactobacillus) is shown below. [Figure 4] This shows the test of KUFF23N1 (lactic acid bacteria: a type of Lactobacillus) using a lactic acid bacteria detection kit. [Figure 5] A micrograph of KUFF23H1 (a species of Microburgula aerodinitrificans) is shown. [Figure 6]A photograph of a plate culture of KUFF23H1 (a species of Microbergia aerodinitrificans) is shown. [Figure 7] The base sequence of 16S rDNA of KUFF23H1 (a species of Microburgula aerodinitrificans) is shown. [Figure 8] The growth characteristics of each medium for the target root rot fungi are shown. [Figure 9] This shows the antibacterial properties of the drug against root rot bacteria. [Figure 10] It exhibits antibacterial properties such as being a sole control agent against base rot fungi. [Figure 11] It exhibits antibacterial properties such as an inhibitor of base rot fungi. DETAILED DESCRIPTION OF THE INVENTION

[0031] 1. Soil-borne disease control agents 1-1. Overview and definition A first aspect of the present invention is a soil-borne disease control agent for plant pathogenic filamentous fungi. The soil-borne disease control agent of this aspect uses a specific bacterium as an active ingredient and can suppress the growth and activity of the target plant pathogenic filamentous fungi through the occupancy and antibacterial effects of the bacterium. Therefore, by applying the agent to a desired plant, it is possible to reduce diseases caused by plant pathogenic filamentous fungi on the plant.

[0032] As used herein, the term "agent for controlling soil-borne diseases" refers to a microbial pesticide intended to prevent and control the onset of soil-borne diseases.

[0033] "Microbial pesticides" refer to pesticides that use live microorganisms as active ingredients and utilize the properties and activity of those microorganisms to inhibit the growth, activity, growth inhibition, or death of target organisms that primarily cause plant diseases. As used herein, "microorganisms" generally refer to tiny organisms that are difficult to see with the naked eye, such as bacteria (eubacteria) and fungi. Fungi are a group of eukaryotic organisms belonging to the kingdom Fungi, and are also called fungi or filamentous fungi. Fungi include unicellular eukaryotic microorganisms such as yeast, and multicellular eukaryotic microorganisms such as filamentous fungi (including molds) or mushrooms that are relatively difficult to see with the naked eye.

[0034] As used herein, the term "soil-borne disease" refers to a disease that develops in plants due to infection with a pathogenic microorganism transmitted via soil or planting medium (including liquid medium). Examples include root rot, root rot (including purple root rot and white root rot), clubroot, seedling damping-off (including rice seedling damping-off), wilt (including tomato wilt and eggplant verticillate wilt), root rot, southern blight, and phytosporum flavum. Root rot, particularly purple root rot, is a preferred target for prevention and control by the soil-borne disease control agent of this embodiment.

[0035] As used herein, the term "plant-pathogenic filamentous fungus" refers to a microorganism that is the target of the soil-borne disease control agent of this embodiment, and is a filamentous fungus that infects living plants and causes soil-borne diseases. Examples of plant pathogenic filamentous fungi of this embodiment include root rot fungi (Diaporthe destruens), Helicobasidium, Rosellinia, Fusarium, Pythium, Pyrenochaeta, Monosporascus, Verticillium, Rhizoctonia, Plasmodiophora, Phitophthora, and Sclerotium. Among these, Helicobasidium can be a suitable target for the soil-borne disease control agent of this embodiment. Helicobasidium fungi are known to form abscissive, felt-like fruiting bodies at the base of host plants, and to produce curved, cylindrical basidia and colorless, ovoid to elliptical basidiospores with protruding bases on the surface of the fruiting bodies. Currently, H. mompa, H. brebissonii (H. purpureum), and H. longisporum have been identified as taxonomically effective species, and all of these can be targets of the soil-borne disease control agent of this embodiment, but root rot fungi are particularly suitable.

[0036] 1-2.Configuration 1-2-1. Composition The soil-borne disease control agent of this embodiment contains, as active ingredients, essential acid bacteria: KUFF23N1 (Accession Number: P-04060), a type of Lactobacillus; and KUFF23H1 (Accession Number: P-04061), a type of Microvirgula aerodenitrificans; and contains the photosynthetic bacterium Rhodopseudomonas strain A (Accession Number: FERM P-19431, Patent No. 6232602) as a carrier.

[0037] A. Active ingredient The soil-borne disease control agent of this embodiment contains active ingredients from at least two different types of bacteria, namely, lactic acid bacteria: KUFF23N1 (Accession Number: P-04060), a type of Lactobacillus, and KUFF23H1 (Accession Number: P-04061), a type of Microvirgula aerodenitrificans, either alone or in combination with KUFF23N1, KUFF23H1, and photosynthetic bacteria. Each type of bacteria will be described in detail below.

[0038] (1) KUFF23N1 (Lactic acid bacteria: a type of Lactobacillus) The properties of KUFF23N1 (accession number: P-04060) are that the cell morphology is rod-shaped, the colonies are cream-colored, the characteristics are rod-shaped, non-motile, Gram-positive, catalase- and oxidase-negative, facultative anaerobic, and positive in lactic acid bacteria detection medium (manufactured by Prima Meat Packers). KUFF23N1 (accession number: P-04060) was received and entrusted to the National Institute of Technology and Evaluation on January 12, 2024.

[0039] The culture medium was NB medium (0.5% (w / v) Pepton Casin (Nacalai Tesque special grade microbial culture reagent), 0.3% (w / v) Bact Yeast Extract (BD Biosciences), tap water (pH 7.0-7.2)), the isolation location was sweet potato field soil in Osaki Town, Kagoshima Prefecture, the culture temperature was 25°C, the culture method was static culture, the storage method was 15% (V / V) glycerol, the storage temperature was -80°C, the condensate was sterilized saline, and the sterilization method was 121°C for 15 minutes.

[0040] Figure 1 shows a micrograph of KUFF23N1 (a type of lactic acid bacteria: Lactobacillus) (Olympus optical microscope, 1000x magnification (cell density 1.2 × 10 12 cells / ml), and Figure 2 shows a photograph of the colony on a plate culture of KUFF23N1 (a type of lactic acid bacteria: Lactobacillus).

[0041] Figure 3 shows the base sequence of the 16S rDNA of KUFF23N1 (a type of lactobacillus). The sequence results show that the bacterium is 93% similar to Bacillus toyonensis H41, but its cell morphology and physiological conditions are characteristic of Lactobacillus. The DDBJ accession number is MH681471. Furthermore, it tested positive using a lactic acid bacteria detection kit (manufactured by Prima Ham). Therefore, KUFF23N1 is considered to be a type of Lactobacillus.

[0042] FIG. 4 shows the results of a test using a lactic acid bacteria detection kit (manufactured by Prima Meat Packers) for KUFF23N1 (lactic acid bacteria: a type of Lactobacillus), with N1 indicating positive and the others indicating negative.

[0043] (2) KUFF23H1 (Microvirgula aerodenitrificans: a species of Microvirgula aerodenitrificans) KUFF23H1 (accession number: P-04061) has the characteristics of a bacillus cell morphology, cream-colored colonies, characteristically curved rods (vibrios), motile, Gram-negative, catalase- and oxidase-positive, aerobic denitrifying bacteria. KUFF23H1 (accession number: P-04061) was received and entrusted to the National Institute of Technology and Evaluation on January 12, 2024.

[0044] The culture medium was NB medium (0.5% (w / v) Pepton Casin (Nacalai Tesque special grade microbial culture reagent), 0.3% (w / v) Bact Yeast Extract (BD Biosciences), tap water (pH 7.0-7.2)), the isolation location was sweet potato field soil in Osaki Town, Kagoshima Prefecture, the culture temperature was 25°C, the culture method was aerobic conditions (shaking culture), the storage method was 15% (V / V) glycerol, the storage temperature was -80°C, and the sterilization method was 121°C for 15 minutes.

[0045] Figure 5 shows a micrograph of KUFF23H1 (a species of Microburgula aerodinitrificans) (Olympus optical microscope, 1000x magnification (cell density 3.2 × 10 13 cells / ml), and FIG. 6 shows a photograph of a plate culture of KUFF23H1 (a species of Microbergia aerodinitrificans), which is a colony photograph.

[0046] Figure 7 shows the base sequence of the 16S rDNA of KUFF23H1 (a species of Microvirgula aerodenitrificans). The sequence results indicate that the bacterium is 99% similar to KUFF23H1 (Microvirgula aerodenitrificans), and the cell morphology and physiological conditions are also identical. The DDBJ accession number is CP028519. Based on the above, KUFF23H1 is considered to be a species of Microvirgula aerodenitrificans.

[0047] KUFF23H1 (Microvirgula aerodenitrificans) was first isolated from activated sludge by Patureau et al. in 1998. This microorganism is a curved, rod-shaped (vibrio), motile, Gram-negative, catalase- and oxidase-positive, aerobically denitrifying bacterium. It is also mesophilic and neutrophilic, with maximum growth occurring at 35°C and pH 7. This organism also exhibits growth at temperatures ranging from 15°C to 45°C and pH below 6, with cell size varying within the growth phase. At the beginning of the growth phase, cells are slender, but by the late stationary phase, they enlarge and form associations of four to five cells. Based on these phenotypic characteristics, M. aerodenitrificans is most closely related to Cornarnonas testosteroni and Pseudomonas acaligenes. M. aerodenitrificans has a typical Gram-negative cell wall structure, containing two membrane layers, an outer and an inner membrane, and a wavy outer membrane. Colonies isolated after a 24-hour incubation period are shown to be round, cream-colored, and 1 ± 2 mm in diameter. In young cultures, cells occur singly or in pairs. Under negative-stain electron microscopy, thin sections of M. aerodenitrificans show bipolar tufts of flagella.

[0048] (3) Photosynthetic bacteria: PSB for short Photosynthetic bacteria are bacteria that perform photosynthesis and include purple sulfur bacteria, purple non-sulfur bacteria, green sulfur bacteria, and green non-sulfur bacteria. Although photosynthetic bacteria are not essential active ingredients in the soil-borne disease control agent of the present invention, they possess the property of maintaining soil-borne disease control efficacy when mixed with the above two strains. These bacteria belong to the genus Rhodopseudomonas. The present bacteria are Rhodopseudomonas strains A and B (Accession Nos. FERM P-19431 and FERM P-19432, Patent No. 6232602), which are closely related to Rhodopseudomonas faecalis. Their bacteriologic properties include alkali tolerance and the presence of bacterial chlorophyll a, neurosporene, and lyocopene. As used herein, "alkali tolerance" primarily refers to the property of having a maximum growth rate at a pH of 8 to 10. Rhodopseudomonas strains A and B are described in detail in Japanese Patent No. 3699987 and are currently deposited at the National Institute of Technology and Evaluation (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan). Two or more species of photosynthetic bacteria, for example, Rhodopseudomonas strain A and Rhodopseudomonas strain B, may be contained as active ingredients.

[0049] (4) Mixing ratio, etc. As described above, the soil-borne disease control agent of this embodiment contains at least KUFF23N1 (lactic acid bacteria: a species of Lactobacillus) and KUFF23H1 (a species of Microburgula aerodinitrificans), which have control effects on their own, but by mixing them with photosynthetic bacteria (Rhodopseudomonas strain A (Accession No. FERM P-19431, Japanese Patent No. 6232602)), the control effect is maintained for a longer period of time and the bacteria are contained in a viable state as an active ingredient. In this case, the mixing ratio of the KUFF23N1 and KUFF23H1 photosynthetic bacteria in the soil-borne disease control agent is not particularly limited as long as the agent contains the above three species of bacteria, but preferably the ratio of photosynthetic bacteria:KUFF23N1:KUFF23H1 is 1:1 to 1000:1 to 1000.

[0050] The KUFF23N1, KUFF23H1, and photosynthetic bacteria may be in a mixed state when the soil-borne disease control agent is applied; however, the two bacteria do not necessarily have to be in a mixed state before application, for example, during storage. Furthermore, as used herein, "viable state" refers to a state in which the bacteria are capable of metabolism and / or division and proliferation. Therefore, even if they are frozen, they are considered to be viable. However, the soil-borne disease control agent may contain dead KUFF23N1, KUFF23H1, and photosynthetic bacteria.

[0051] B. Carrier The soil-borne disease control agent of this embodiment can contain the active ingredients KUFF23N1 and KUFF23H1 and a carrier acceptable for agricultural chemical formulations to the extent that it does not inhibit or suppress the survival and physiological activity of photosynthetic bacteria.

[0052] The term "pesticidally acceptable carrier" refers to a substance that facilitates application of the soil-borne disease control agent, maintains the survival of KUFF23N1 and KUFF23H1 and photosynthetic bacteria, and maintains antibacterial or inhibitory activity against plant pathogenic filamentous fungi, and / or controls the rate of action of the soil-borne disease control agent, and has no or little harmful effect on the environment, such as soil and water quality, and / or has no or little harmful effect on animals, particularly humans. Examples of carriers in the soil-borne disease control agent of this embodiment include excipients. Excipients include pulverized natural minerals, pulverized synthetic minerals, emulsifiers, dispersants, surfactants, etc.

[0053] Ground natural minerals include, for example, kaolin, clay, talc, and chalk, and ground synthetic minerals include, for example, highly dispersed silica and silicates. Emulsifiers include nonionic and anionic emulsifiers (e.g., polyoxyethylene fatty alcohol ethers, alkyl and aryl sulfonates).

[0054] Examples of dispersants include lignosulfite waste liquor and methyl cellulose. Examples of surfactants include alkali metal salts, alkaline earth metal salts, and ammonium salts of lignosulfonic acid, naphthalenesulfonic acid, phenolsulfonic acid, and dibutylnaphthalenesulfonic acid, alkylarylsulfonates, alkyl sulfates, alkylsulfonates, fatty alcohol sulfates, fatty acids, and sulfated fatty alcohol glycol ethers, as well as condensates of sulfonated naphthalene and naphthalene derivatives with formaldehyde, condensates of naphthalene or naphthalenesulfonic acid with phenol and formaldehyde, and polyoxyethylene octylphenyl ether. ether, ethoxylated isooctylphenol, octylphenol, nonylphenol, alkylphenyl polyglycol ether, tributylphenyl polyglycol ether, tristearylphenyl polyglycol ether, alkylaryl polyether alcohol, alcohol and fatty alcohol / ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkyl ether, ethoxylated polyoxypropylene, lauryl alcohol polyglycol ether acetal, sorbitol esters, lignosulfite waste liquor, and methylcellulose.

[0055] The soil-borne disease control agent of the present invention can contain one or more pesticide-acceptable carriers, and can also contain other active ingredients with pharmacological actions, such as herbicides, fungicides, insecticides, and fertilizers (e.g., urea, ammonium nitrate, and superphosphate), as long as they do not affect the bacteria of the active ingredient.

[0056] 1-2-2. Dosage form The formulation of the soil-borne disease control agent of the present invention is not particularly limited as long as it can maintain the active ingredients KUFF23N1 and KUFF23H1 and photosynthetic bacteria in a viable state. For example, the agent can be in a liquid state in which each bacterium is suspended in an appropriate solution, a solid state, or a combination thereof. In the liquid state, the photosynthetic bacteria and / or the KUFF23N1 and KUFF23H1 bacteria can be suspended in an appropriate solution. Examples of suitable solutions include water (including sterilized water, deionized water, and ultrapure water), physiological saline, buffers (including phosphate buffer and carbonate buffer), and culture media for the bacteria. In the solid state, examples include granules, powders, and semi-solid forms such as gels. Specific examples of these include liquid formulations, dusts, granules, and seedling soil.

[0057] 1-2-3.Content The content of KUFF23N1, KUFF23H1, and photosynthetic bacteria as active ingredients per predetermined amount of the soil-borne disease control agent of the present invention varies depending on various conditions, such as the type and combination of bacteria, the type of plant to be treated, the formulation, and the application method. Generally, when applying the soil-borne disease control agent of the present invention, it is preferable that KUFF23N1, KUFF23H1, and photosynthetic bacteria are contained in an amount sufficient to exert an antibacterial or inhibitory effect against plant pathogenic filamentous fungi. This content may be determined, taking into consideration various conditions within the scope of common general technical knowledge in the field, so that KUFF23N1, KUFF23H1, and photosynthetic bacteria are present in the desired amount per predetermined volume of soil after application. For example, the content of KUFF23N1, KUFF23H1, and photosynthetic bacteria in the soil-borne disease control agent of this embodiment may be determined as a concentration of both bacteria of 10 6 ~10 11 In this case, the compound may be diluted 10 to 1000 times with water, physiological saline, buffer, or the like before application, if necessary.

[0058] 1-3.Effects The soil-borne disease control agent of this embodiment contains photosynthetic bacteria (particularly bacteria of the genus Rhodopseudomonas) and bacteria of the genera KUFF23N1 and KUFF23H1 as active ingredients, and can efficiently suppress the growth and activity of plant pathogenic filamentous fungi.

[0059] The soil-borne disease control agent of the present invention is non-toxic and non-pathogenic to plants and has a high control effect against soil-borne diseases caused by plant pathogenic filamentous fungi. Furthermore, since it is a microbial pesticide containing naturally occurring bacteria as an active ingredient, it is highly safe and has a smaller impact on the environment than chemical pesticides. Even when applied to soil, it does not pose a risk to humans or animals, or pose problems such as environmental pollution or residues in crops. Therefore, it is possible to provide consumers with safe, phytotoxic crops. Furthermore, compared to chemical pesticides, the incidence of emergence of resistant strains of the target plant pathogenic filamentous fungi is extremely low.

[0060] The soil-borne disease control agent of the present invention also has a plant activation effect. Furthermore, the soil-borne disease control agent of the present invention improves microbial activity (FDA hydrolysis activity) in the soil and increases the microbial density, which is thought to activate beneficial soil microorganisms that naturally inhabit the soil, and the resulting microbial community (bacterial flora) acts suppressively against plant pathogenic filamentous fungi, creating an environment that suppresses the onset of soil-borne diseases in plants.

[0061] 2. Soil-borne disease control methods 2-1. Overview and definition A second aspect of the present invention is a method for controlling soil-borne diseases, which comprises applying the soil-borne disease control agent according to the first aspect to soil or a medium to prevent or control the occurrence of soil-borne diseases in target plants.

[0062] As used herein, the term "target plant" refers to a plant to which a soil-borne disease control agent is applied. The target plant may be of any type, as long as it is a plant that can develop a soil-borne disease due to infection with a plant pathogenic fungus. The target plant may be either an angiosperm or a gymnosperm. Angiosperms include dicotyledonous plants and monocotyledonous plants. Examples of monocotyledonous plants include plants of the Poaceae family. Examples of dicotyledonous plants include plants of the Convolvulaceae family, Rosaceae family, Apiaceae family, Solanaceae family, Liliaceae family, Fabaceae family, Cucurbitaceae family, and Brassicaceae family.

[0063] Specific target plants may vary depending on the type of plant pathogenic fungus that causes the soil-borne disease. For example, if the plant pathogenic fungus causes root rot or root rot, examples of target plants include sweet potato (Ipomoea batatas), carrot (Daucus carota subsp. sativus), potato (Solanum tuberosum), asparagus (Asparagus spp.), apple (Malus pumila), and pear (Pyrus pyrifolia var. culta, P. bretschneideri, P. communis).

[0064] 2-2. Method 2-2-1. Application process The soil-borne disease control method of this embodiment includes an application step. In this specification, the term "application step" refers to a step of applying the soil-borne disease control agent of the first embodiment to soil or a culture medium. The "soil" used here is not particularly limited as long as it is soil suitable for the growth of the target plant. Typically, soil for cultivating the target plant containing appropriate nutrients (nitrogen, phosphorus, potassium, etc.) and having an appropriate pH value is used. The location of the soil is not important. It may be either field soil or nursery soil before sowing or planting of seedlings. Furthermore, the term "culture medium" refers to an artificially prepared culture medium for cultivating the target plant. It may be a solid medium such as an agar medium, or a liquid medium. Examples of culture medium include isolation beds, root-zone restricted pots, and seedbeds. The composition of the culture medium may be any known medium composition in the art. It can be appropriately selected depending on the type of crop to be used.

[0065] The method of application of the soil-borne disease control agent can be appropriately selected depending on the type of soil-borne disease, the type of crop to be applied, etc. For example, the soil-borne disease control agent of the present invention may be applied by mixing, spraying, irrigation, or the like to the planting soil of a target plant, preferably to a portion corresponding to the root zone of the target plant, such as a furrow portion or a portion where the incidence of soil-borne disease is high.

[0066] If necessary, manure and the like may be used in combination with a soil-borne disease control agent. If there are fertilizer components to be supplemented, they can also be used in combination with chemical fertilizers of various components, soil conditioners, organic fertilizers, and the like.

[0067] 2-2-2. Application period In the soil-borne disease control method of this embodiment, the application time of the soil-borne disease control agent of the first embodiment is not limited, but is preferably before planting of the target crop, for example, before sowing or transplanting. By applying the soil-borne disease control agent before planting, the active ingredients KUFF23N1 and KUFF23H1 and photosynthetic bacteria proliferate in the soil, preventing the invasion of plant pathogenic fungi through their occupancy action and eliminating existing plant pathogenic fungi through their antibacterial action, thereby reducing the infection rate of the target crop with plant pathogenic fungi and efficiently preventing or controlling the onset of soil-borne diseases. The target plant is planted in the applied soil 2 days to 3 weeks, preferably 10 days to 2 weeks, after application of the soil-borne disease control agent.

[0068] 2-2-3. Application amount In the soil-borne disease control method of this embodiment, the application amount of the soil-borne disease control agent of the first embodiment may be adjusted appropriately depending on the conditions, as it varies depending on the formulation, application method and timing, type of target plant, etc. For example, when the soil-borne disease control agent is a liquid formulation, it may be used at an application amount of 0.5 to 20 tonnes, preferably 1 to 10 tonnes, per 10 ares of soil.

[0069] 2-2-4. Number of applications In the soil-borne disease control method of this embodiment, there is no limit to the number of applications of the soil-borne disease control agent of the first embodiment. When applied before planting of the target plant, one application is generally sufficient. However, regardless of whether the application is before or after planting of the target plant, multiple applications increase the soil-borne disease control effect. Therefore, to form soil disease-suppressive soil, it is preferable to increase the number of applications of the soil-borne disease control agent. Furthermore, photosynthetic bacteria are known to be effective as organic fertilizers (Kitamura, Hiroshi, 1984, Photosynthetic Bacteria, 347-349). Therefore, combining the agent with conventional compost can further improve the soil microbial flora. As a result, the control effect of soil-borne diseases caused by plant pathogenic fungi is further improved. From this perspective, it is also preferable to apply the soil-borne disease control agent more frequently.

[0070] 2-3.Effects According to the method for controlling soil-borne diseases of this embodiment, soil-borne diseases can be effectively controlled by applying a soil-borne disease control agent to the soil in which a desired plant is planted, thereby reducing labor and costs and enabling safe agricultural products to be stably provided to consumers. [Example]

[0071] The present invention will be specifically explained below with reference to examples, but these examples are not intended to limit the scope of the present invention in any way.

[0072] <Antibacterial activity test of soil-borne disease control agents against plant pathogenic fungi> Materials and Methods 1. Microorganisms used to treat root rot The root rot fungus (Diaporthe destruens) was isolated from soil of a sweet potato field in Osaki Town, Kagoshima Prefecture, at 25°C using the dilution plate method, in which the fungus was spread onto the following medium.

[0073] [Potate Sucrose Agar (PSA) medium composition for filamentous fungi (Source: NITE)] This is the medium most commonly used for filamentous fungi, and in many cases, commercially available PDA medium can be substituted. In this example, 200 g of potato (preferably Danshaku), 20 g of sucrose, 20 g of agar, and 1 L of distilled water (pH 5.6) were used, and the procedure was as follows.

[0074] First, boil 200g of peeled and cut potatoes into 1cm cubes in 1L of boiling distilled water for 20 minutes, then filter the broth through muslin (canvas or triple-layered gauze will also work), measure up the filtered broth to 1L with distilled water, add sucrose and agar powder, stir and heat to dissolve, adjust the pH, and sterilize in an autoclave.

[0075] 2. Two microorganisms as disease control agents for root rot The culture method for the lactic acid bacteria KUFF23N1 (accession number: P-04060), a species of Lactobacillus, and the Microvirgula aerodenitrificans KUFF23H1 (accession number: P-04061), a species of Microvirgula aerodenitrificans, was as described above.

[0076] 3. Rhodopseudomonas bacteria, one of the active ingredients in soil-borne disease control agents The Rhodopseudomonas A strain described in Japanese Patent Publication No. 3699987 and designated by the accession number FERM P-19432 was used. Rhodopseudomonas strain A was cultured under the conditions described in Japanese Patent Publication No. 3699987, i.e., basal medium 1: 0.03% (w / v) KH2PO4, 0.03% (w / v) K2HPO4, 0.1% (w / v) NH4Cl, 0.02% (w / v) MgCl2·6H2O, 0.02% (w / v) NaCl, 0.005% (w / v) CaCl2·2H2O, 0.005% (w / v) yeast extract, 0.005% (w / v) Na2S2O3·5H2O, 0.01% (w / v) DL-Malic acid, 0.01% (w / v) sodium acetate, 0.1% growth factor solution (0.0001% (w / v) vitamin B2, 0.0001% (w / v) Pyridoxine Hydrochloride, 0.0003%(w / v) p-Aminobenzoic Acid, 0.0005%(w / v) D-Biotin), 0.1% Trace Element Solution(2%(w / v) EDTA-2Na, 2%(w / v) FeSO4·7H2O, 0.1%(w / v) H3BO3, Cultured at 25°C with 0.1% (w / v) ZnCl2, 0.1% (w / v) MnCl·4H2O) (pH 7.8).

[0077] 4. Antibacterial Test Method The root rot bacteria (Diaporthe destruens), KUFF23N1 and KUFF23H1, and the photosynthetic bacterium Rhodopseudomonas strain A (accession number FERM P-19431, Japanese Patent No. 6232602) were cultured under the above-mentioned conditions for two weeks before the antibacterial test.

[0078] Next, 10 μL of KUFF23N1, KUFF23H1, and photosynthetic bacteria cultures were mixed and inoculated onto a filter paper plate approximately 6 mm in diameter placed on the PSA medium in the dish. As a control, only one of the strains was inoculated onto a filter paper plate of the same size placed on the PSA medium. Agar pieces approximately 5 mm in diameter, obtained by hollowing out the mycelium of the purple root fungus, were placed on each paper plate and incubated in the dark at 25°C. After 5, 7, and 10 days of incubation, each dish was photographed to examine the growth of the root rot fungus mycelium.

[0079] (result) The results of the antibacterial test are shown in photographs in FIGS. 8 to 11, and the explanations are provided in Tables 1 to 3 below.

[0080] Figure 8 shows the growth characteristics of each medium (from left: KSC: Kogane Senkan (with bark), KSF: Kogane Senkan (without bark), SY: Shiroyutaka (with bark)) against the target root rot fungi, and shows that all are strongly affected by the root rot fungi. Note that the above-mentioned KSC was used in the antibacterial experiments from Figure 9 onwards.

[0081] FIG. 9 shows the antibacterial activity of the drugs (antibiotics) shown in Table 1 against root rot bacteria, demonstrating that these drugs have no antibacterial effect.

[0082] [Table 1]

[0083] Figure 10 shows the antibacterial properties of the individual control agents shown in Table 1 against the basal rot fungus, and it was confirmed that the photosynthetic bacteria (PSB) <KUFF23H1 <KUFF23N1 exhibit antibacterial effects in that order, with increasing strength.

[0084]

Table 2

[0085] Figure 11 shows the antibacterial properties of the control agents shown in Table 3 against the basal rot fungus, and it was confirmed that the control agent obtained by mixing photosynthetic bacteria with three types, namely KUFF23H1 and KUFF23N1, exhibits a stronger antibacterial effect than photosynthetic bacteria alone.

[0086]

Table 3

[0087] Summarizing the results of the antibacterial tests shown in Figures 8 to 11, we get the following.

[0088] 1. The basal rot fungus has different growth characteristics in the following three culture media components (KSC: kogane senkan (with skin), KSF: kogane senkan (without skin), SY: shiroyutaka (with skin)). The antibacterial experiment uses KSC: kogane senkan (with skin).

[0089] 2. Against the basal rot fungus, copper hydroxide, streptomycin, oxytetracycline, and erythromycin have no antibacterial effect.

[0090] 3. Against the basal rot fungus, individually, the photosynthetic bacteria (PSB) <KUFF23H1 <KUFF23N1 exhibit antibacterial effects in that order, with increasing strength.

[0091] 4. Against the basal rot fungus, compared to photosynthetic bacteria alone, mixing three types, namely photosynthetic bacteria (PSB), KUFF23H1, and KUFF23N1, further enhances the antibacterial effect.

[0092] These results revealed that by mixing three types of photosynthetic bacteria (PSB) with KUFF23H1 and KUFF23N1, the growth and activity of plant pathogenic filamentous fungi can be suppressed, and soil-borne diseases of target plants can be prevented or controlled.

Claims

1. A soil-borne disease control agent against root rot bacteria (Diaporthe destruens) containing a type of lactic acid bacterium (Lactobacillus) as the active ingredient.

2. The soil-borne disease control agent according to claim 1, wherein the lactic acid bacterium (Lactobacillus) is a bacterium with accession number P-04060 and DDBJ accession number MH681471.

3. A soil-borne disease control agent against root rot fungus (Diaporthe destruens) containing a species of Microvirgula aerodenitrificans as the active ingredient.

4. The soil-borne disease control agent according to claim 3, wherein the species of Microvirgula aerodenitrificans is a bacterium having accession number P-04061 and DDBJ ACCESSION number CP028519.

5. A soil-borne disease control agent for plant pathogenic filamentous fungi containing KUFF23N1, KUFF23H1 and the photosynthetic bacterium Rhodopseudomonas strain A (accession number FERM P-19431) as active ingredients.

6. The soil-borne disease control agent according to claim 5 , wherein the photosynthetic bacterium is a bacterium of the genus Rhodopseudomonas.

7. The soil-borne disease control agent according to claim 6, wherein the mixing ratio of KUFF23N1, KUFF23H1 and photosynthetic bacteria in the soil-borne disease control agent is not particularly limited as long as the above three types of bacteria are included, but preferably the ratio of photosynthetic bacteria:KUFF23N1:KUFF23H1 is 1:1 to 1000:1 to 1000.

8. The soil-borne disease control agent according to claim 7, which is a type of lactic acid bacterium (Lactobacillus) for use as an active ingredient in a soil-borne disease control agent against root rot fungus (Diaporthe destruens).

9. The soil-borne disease control agent according to claim 7, which is a bacterium of the type Microvirgula aerodenitrificans, for use as an active ingredient in a soil-borne disease control agent against root rot fungus (Diaporthe destruens).

10. The soil-borne disease control agent according to any one of claims 1 to 9, wherein the plant pathogenic filamentous fungus is selected from the group consisting of root rot fungus (Diaporthe destruens), Helicobasidium, Rosellinia, Fusarium, Pythium, Pyrenochaeta, Monosporascus, Verticillium, Rhizoctonia, Plasmodiophora, Phitophthora, and Sclerotium.

11. The soil-borne disease control agent according to any one of claims 1 to 9, wherein the soil-borne disease is selected from the group consisting of root rot, root rot, clubroot, damping-off, wilt, root rot, southern blight, and phytosophra blight.

12. A method for controlling soil-borne diseases, comprising applying the soil-borne disease control agent according to any one of claims 1 to 9 to soil or a medium.

13. The method for controlling a soil-borne disease according to claim 12, wherein the soil is soil for planting a target plant.

14. The method for controlling a soil-borne disease according to claim 13, which is applied before planting of a target plant.

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