Microorganisms and microbial preparations

Abstract

The present invention provides novel microorganisms and microbial formulations utilizing such microorganisms, which can be used for controlling plant diseases and aquatic animal diseases. [Solution] A microorganism belonging to the genus Bacillus, deposited under accession number NITE P-04187. A microbial preparation containing the said microorganism or its culture.

Description

Technical Field

[0001] The present invention relates to microorganisms and microbial preparations.

Background Art

[0002] Filamentous fungi are commonly called molds and are present around us. They are microorganisms that can easily grow even under low-nutrient conditions. While they have been used since ancient times as antibiotics for pharmaceuticals and fermentation raw materials for foods, they have also been regarded as harmful. For example, they cause food spoilage, crop diseases in the agricultural field, and fish diseases in the fish farming field.

[0003] To solve these problems, chemical agents for suppressing filamentous fungi have been used. However, as we move towards sustainable production, there is a need for alternatives to chemical agents. Since microorganisms originally exist in the environment, they are attracting attention as an alternative to chemical agents in realizing a society-friendly and environmentally friendly recycling society. Therefore, in recent years, methods for controlling diseases of crops, fish, etc. using microorganisms have been proposed (for example, Patent Documents 1, 2, etc.).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] Under such circumstances, an object of the present invention is to provide a novel microorganism that can be used for controlling plant diseases and controlling aquatic animal diseases. Another object is to provide a microbial preparation using the microorganism.

Means for Solving the Problems

[0006] The inventors of this invention have conducted extensive research to solve the above problems and have found that the following invention is suitable for the above purpose, leading to the present invention. That is, the present invention relates to the following invention.

[0007] <1> A microorganism belonging to the genus Bacillus, deposited under accession number NITE P-04187. <2> The aforementioned <1> A microbial preparation containing the microorganisms described herein, or their cultures. <3> The above-mentioned product is used for one or more applications selected from the group consisting of plant disease control, aquatic animal disease control, organic matter decomposition, and water purification. <2> The microbial preparations described above. [Effects of the Invention]

[0008] The present invention provides a novel microorganism that can be used for controlling plant diseases and aquatic animal diseases. Furthermore, it provides a microbial preparation utilizing the said microorganism. [Brief explanation of the drawing]

[0009] [Figure 1] This is a photograph showing an antagonist test against water mold using the microorganism of the present invention. [Figure 2] This is a photograph showing an antagonist test against Aeromonas bacteria using the microorganism of the present invention. [Figure 3] This is a photograph showing an antagonist test against Columnaris bacteria using the microorganism of the present invention. [Figure 4] This figure shows the results of the salt resistance test. [Figure 5] This figure shows the number of fungal infections relative to the number of days elapsed in a water mold disease suppression test using medaka eggs. [Figure 6] This figure shows the number of hatches relative to the number of days elapsed in a water mold disease suppression experiment using medaka eggs. [Modes for carrying out the invention]

[0010] Hereinafter, embodiments of the present invention will be described in detail. However, the description of the constituent elements described below is an example (representative example) of an embodiment of the present invention, and the present invention is not limited to the following content unless the gist thereof is changed. In the present specification, when the expression "~" is used, it shall be used as an expression including the numerical values or physical property values before and after it.

[0011] <Microorganism of the present invention> The present invention relates to a microorganism belonging to the genus Bacillus (hereinafter sometimes referred to as "the microorganism of the present invention") deposited under the accession number NITE P-04187.

[0012] The inventor of the present invention screened about 200 soil samples, selected microorganisms that form heat- and drought-resistant spores, examined the antagonistic effect against the pathogen causing fish mycosis, and obtained the MK55 strain as a microbial strain having a growth inhibitory effect against this pathogen. The MK55 strain was confirmed to have an antagonistic effect against Aspergillus and Bacillus anthracis. Further, as a result of the nucleotide sequence analysis of 16S rDNA (16S rRNA gene), the MK55 strain contained the nucleotide sequence shown in SEQ ID NO: 1 as the 16S rRNA gene. The MK55 strain has been deposited with the depository institution as follows. · Depository institution: National Institute of Technology and Evaluation, Patent Microorganisms Depositary (2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture) · Identification label: MK55 · Accession number: NITE AP-04187 · Date of receipt: October 25, 2024 · Deposit number: NITE P-04187

[0013] The microorganism of the present invention has the following properties. These properties can be evaluated according to the methods described later in the examples. (1) It has a growth inhibitory effect against pathogenic filamentous fungi of plants. (2) It has a growth inhibitory effect against pathogens of aquatic animals. (3) It can grow under the condition of a salt concentration of 5%. (4) It has the ability to decompose organic matter. (5) It has the ability to nitrify.

[0014] Since the microorganism of the present invention has an inhibitory effect on the growth of pathogenic filamentous fungi of plants, as described below, it can be used in microbial preparations for controlling plant diseases. For example, the microorganism of the present invention has an antagonistic effect on powdery mildew fungi and anthrax bacteria and can suppress their growth.

[0015] Since the microorganism of the present invention has an inhibitory effect on the growth of pathogenic bacteria of aquatic animals, as described below, it can be used in microbial preparations for controlling diseases of aquatic animals. For example, the microorganism of the present invention has an antagonistic effect on the pathogenic bacteria causing saprolegniasis, Aeromonas bacteria, and Columnaris bacteria and can suppress their growth.

[0016] Examples of the pathogenic bacteria causing saprolegniasis in fish include molds of the genus Saprolegnia in the order Saprolegniales, family Saprolegniaceae, class Oomycetes, subphylum Mastigomycota. Representative species include Saprolegnia diclina, Saprolegnia parasitica, Saprolegnia ferax, etc.

[0017] In addition, the microorganism of the present invention is excellent in salt tolerance and can grow under the condition of a sodium chloride concentration of 3% in seawater. Therefore, it can be used for both freshwater and seawater applications.

[0018] The microorganism of the present invention has the ability to decompose organic matter and can decompose any of the four major organic substances, namely, protein, fiber, starch, and oil. Therefore, by applying it to water containing organic components, it can decompose the organic substances that cause water pollution and the like and purify the water quality. Also, in agriculture, by decomposing organic matter, it can be made into a nutrient source that is easily taken up by crops.

[0019] The microorganisms of the present invention possess nitrifying ability and can be used for ammonia removal and nitrite removal treatments. Nitrification ability is an important factor when used in agriculture and aquaculture.

[0020] The microorganisms of the present invention have a biosafety level (BSL) of 1 and can be used safely.

[0021] The microorganisms of the present invention can be cultured by known methods. For example, the microorganisms of the present invention can be added to known culture media such as liquid media or agar media and cultured at 20-37°C under aerobic conditions. The culture can be used as is, or diluted, concentrated, or dried as appropriate, in the microbial formulations of the present invention described later.

[0022] <Microbial preparation of the present invention> This invention relates to a microbial preparation containing the microorganism of the present invention or a culture thereof. Because the microbial preparation of the present invention possesses the properties of the microorganism of the present invention, it can be used for applications such as a plant disease control agent, an aquatic animal disease control agent, and a decomposition agent for organic pollutants.

[0023] The dosage form of the microbial formulation of the present invention is not particularly limited and can be appropriately designed according to the purpose. It may be in liquid form, semi-solid form such as gel or paste, or solid form such as powder, granules, tablets, or capsules. The formulation method is not particularly limited as long as it does not impair the desired properties and can be formulated by known methods. Depending on the dosage form, it may contain additives such as preservatives, surfactants, dispersants, carriers, and binders. Furthermore, it may contain microorganisms other than the microorganism of the present invention, as long as it does not impair the desired properties.

[0024] The microbial preparation of the present invention may be used as is without dilution, or it may be used after dilution with water or the like. The amount of the microorganism of the present invention in the microbial preparation of the present invention can be appropriately designed according to the purpose of use and method of use, and is not particularly limited as long as it has the desired properties, but for example, if the bacterial concentration is 1 × 10 4 ~1 × 1011 This can be expressed as CFU / mL.

[0025] (Microbial preparations for controlling plant diseases) The microbial formulation of the present invention can be used to control plant diseases. By applying the microbial formulation of the present invention to plants such as crops, the growth of pathogenic filamentous fungi can be suppressed due to the growth-inhibiting effect of the microorganisms of the present invention, thereby suppressing plant diseases. Furthermore, due to the organic matter decomposition ability of the microorganisms of the present invention, organic matter that serves as a nutrient source is broken down, making it easier for crops to absorb nutrients and promoting growth.

[0026] The plants to which the microbial preparation of the present invention is applied are not particularly limited, but can include, for example, vegetables, fruit trees, and grains. Specifically, examples include cucurbitaceous plants such as cucumbers, white melons, watermelons, melons, pumpkins, zucchini, bitter melons, winter melons, and bitter gourds; nightshadeaceous plants such as eggplants, tomatoes, bell peppers, paprika, chili peppers, shishito peppers, and potatoes; legumes such as green beans, peas, soybeans, broad beans, peanuts, mung beans, and adzuki beans; roseaceous plants such as strawberries; and fruit trees such as grapes.

[0027] Furthermore, the present invention is not limited to plants grown in soil. Since the microbial preparation of the present invention can decompose organic matter in water and maintain water quality, it may also be applied to hydroponically grown plants.

[0028] The method of applying the microbial preparation of the present invention to plants is not particularly limited, and known methods can be appropriately selected depending on the dosage form, etc. For example, methods include applying it to the whole or a part of the plant (leaves, stems, buds, flowers, fruits, seeds, roots, branches, etc.) by coating or spraying, or mixing it into the soil. The amount to be applied is appropriately determined depending on the dosage form, the type and condition of the target plant, etc. For example, the microbial preparation of the present invention can be appropriately diluted with water, etc., and applied at a rate of 200 to 700 L per 10a, or at a rate of 5 to 20 mL per plant.

[0029] (Microbial preparations for controlling diseases in aquatic animals) The microbial formulation of the present invention can be used to control diseases in aquatic animals. Due to the inhibitory effect of the microorganisms of the present invention on the growth of aquatic animal pathogens, the growth of aquatic animal pathogens in water is suppressed, thereby suppressing diseases in aquatic animals. Furthermore, because the microorganisms of the present invention can decompose organic matter, water quality can be maintained and improved, thus suppressing diseases. Moreover, because the microorganisms of the present invention have excellent salt tolerance, the microbial formulation of the present invention can be used as a microbial control agent for both freshwater and saltwater aquatic animals.

[0030] Aquatic animals to which the microbial formulation of the present invention is applied include fish, crustaceans, and shellfish. The aquatic animals are not particularly limited, but examples of fish include freshwater fish such as goldfish, medaka, and tilapia; and saltwater fish such as yellowtail and amberjack; and examples of crustaceans include shrimp.

[0031] The microbial formulation of the present invention may be applied to either ornamental aquatic organisms or aquatic organisms for aquaculture, and may be applied at any growth stage, such as eggs / fry, juvenile fish, young fish, adult fish, juvenile, adult, juvenile shellfish, and adult shellfish.

[0032] For example, by applying it to juvenile and adult fish, diseases in juvenile and adult fish can be suppressed. Furthermore, by applying the microbial formulation of the present invention to fish eggs, the growth of mold on fish eggs can be suppressed, thereby increasing the hatching rate.

[0033] The method of applying the microbial preparation of the present invention to aquatic organisms is not particularly limited, and known methods can be appropriately selected depending on the dosage form, etc. For example, methods include adding the microbial preparation of the present invention to a tank or pond where aquatic organisms are raised or cultivated, mixing the microbial preparation of the present invention with a certain amount of water and adding this mixture to the tank or pond, or supplying the microbial preparation of the present invention to the tank or pond using a pump or the like. The amount used can be appropriately determined depending on the dosage form, the size of the tank, the type and number of aquatic organisms, etc. For example, the final concentration (for example, the bacterial concentration in the tank after adding the microbial preparation of the present invention when adding it to a tank) may be 1 × 10⁻⁶. 1 ~1 × 105 It can be used to achieve a CFU / mL concentration.

[0034] (Microbial preparations for organic matter decomposition, microbial preparations for water purification) The microbial formulation of the present invention can be used as a microbial formulation for the decomposition of organic matter. For example, since it can decompose organic matter in water, it can be used as a microbial formulation for water purification. By applying the microbial formulation of the present invention to water, water pollutants such as organic matter and ammonia nitrogen are decomposed and removed, thereby purifying the water.

[0035] Furthermore, because the microbial formulation of the present invention can decompose the four major organic substances, it can be used for applications such as promoting the composting of organic waste and livestock manure, wastewater treatment in factories such as food processing plants, wastewater treatment for livestock farmers, and decomposition of food waste. For example, in wastewater treatment, a phenomenon called "bulking" caused by filamentous fungi is a major problem. The microbial formulation of the present invention can also be applied to suppress bulking. [Examples]

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples unless its essence is changed.

[0037] 1. Screening Microorganisms that suppress filamentous fungi were screened from soil. First, bacteria that show antagonistic properties against Saprolegnia (water mold) were screened from approximately 200 soil samples. While there are as many as 10 billion microorganisms in soil, to commercialize these microorganisms, it is necessary to use those with high shelf life. Therefore, we first selected microorganisms that form spores that are resistant to heat and drought. Microorganisms that do not form spores were sterilized by dissolving 1 mL of physiological saline and approximately 10 mg of soil in a microcentrifuge tube and heating at 65°C for 30 minutes (soil solution). 1 mL each of No. 802 liquid medium (polypeptone: 10.0 g, yeast extract: 2.0 g, magnesium sulfate heptahydrate: 1.0 g, distilled water: 1 L (pH 7.0)) was packed into a 24-well plate, 10 μL of the soil solution was added, and the mixture was incubated at 30°C for 24 hours (soil culture solution).

[0038] Paper discs with a diameter of 8 mm were arranged on a YM / 2 agar medium (yeast extract: 1.5 g, malt extract: 1.5 g, polypeptone: 2.5 g, glucose: 5.0 g, agar: 15.0 g, distilled water: 1 L (pH 7.0)). 30 μL of a solution of water mold (NBRC32710 Saprolegnia diclina) was impregnated into the center of the paper disc, and 30 μL each of soil culture solution was impregnated into the other paper discs. The mixture was then incubated at 25°C for 5 days. Filamentous fungi spread by moving horizontally across the agar plate, but a zone of inhibition is formed around the paper disc containing antagonistic bacteria. Therefore, the presence or absence of a zone of inhibition was used as the selection criterion.

[0039] We isolated multiple microorganisms from the soil culture solution that formed a zone of inhibition. Specifically, soil culture solution diluted appropriately was spread onto No. 802 agar medium (No. 802 liquid medium + agar) and incubated at 30°C for 1-2 days. Next, each colony that had grown was picked and inoculated into a test tube containing 1 mL of No. 802 liquid medium, and incubated at 30°C for 1 day to obtain culture solution (A). Paper discs with a diameter of 8 mm were arranged on YM / 2 agar medium, and 30 μL of a solution of water mold (NBRC32710 Saprolegnia diclina) was impregnated into the center, while 30 μL of culture solution (A) was impregnated into the other paper discs. After incubation at 25°C for 5 days, the presence or absence of inhibition zones was evaluated.

[0040] As described above, by repeatedly performing antagonism tests and isolation procedures, three strains with high antagonism ability were obtained. Of these three strains, the one that could grow in a culture medium containing 3% sodium chloride was selected and designated as strain MK55.

[0041] 2. Antagonism test against three types of filamentous fungi Water mold, powdery mildew infecting the leaves and fruits of cultivated strawberries, and anthrax fungi were isolated and used in the tests.

[0042] (Water mold) The MK55 strain was inoculated into liquid medium No. 802 and cultured at 30°C for 1 day to prepare the culture medium. Pre-cultured water mold was diluted as needed and spread over the entire YM / 2 agar plate. Then, an 8 mm diameter paper disc was placed in the center of the YM / 2 agar plate and impregnated with 30 μL of the MK55 strain culture medium. After culturing at 25°C for 5 days, the zone of inhibition formed around the paper disc was observed (Figure 1). The distance between the pathogen and the antagonistic bacteria (distance between colonies) was also measured.

[0043] Instead of MK55, we used three strains registered as antagonistic in NBRC and a reference strain of Bacillus subtilis, and conducted similar tests for comparison.

[0044] (Powdery mildew, anthrax) The same tests as those for the antagonism test against water mold described above were performed, except that powdery mildew or anthrax fungus were used instead of water mold.

[0045] Table 1 shows the distance between colonies [mm]. A larger distance between colonies was considered to indicate higher antagonistic ability. The results of the test showed that the control strain showed slight antagonistic ability, but it was not as effective as the MK55 strain.

[0046] [Table 1]

[0047] 3. Antagonism against bacteria The tests primarily used Aeromonas bacteria (NBRC3820 Aeromonas hydrophila, NBRC13784 Aeromonas salmonicida) and Columnaris bacteria (NBRC100251 Flavobacterium columnare), which commonly infect fish.

[0048] (Culture conditions) Culture was performed under the following conditions: NBRC3820 was cultured in No. 802 medium (liquid or agar medium) at 30°C, NBRC13784 in No. 802 medium (liquid or agar medium) at 24°C, and NBRC100251 in No. 334 medium (liquid or agar medium) at 20°C. The No. 334 culture medium consists of: polypeptone: 2.0g, meat extract: 0.5g, yeast extract: 0.5g, sodium acetate: 0.2g, distilled water: 1L (pH 7.2-7.4), and agar (if using an agar medium).

[0049] (test) Aeromonas and Columnaris bacteria, cultured in the liquid medium and at the above-mentioned culture temperature, were diluted as needed and spread over the entire surface of each agar plate. Then, an 8 mm diameter paper disc was placed in the center of each agar plate and impregnated with 30 μL of the culture solution of strain MK55 (the same as in "2. Antagonism Test against Three Filamentous Fungi"). The cultures were then incubated under each culture condition, and the zone of inhibition formed around the paper disc was observed. The test results showed that the MK55 strain exhibits antagonistic activity against bacteria such as Aeromonas and Columnaris (Figures 2 and 3).

[0050] 4. Salt resistance test Since a high-salinity environment is expected to be a condition that suppresses filamentous fungi, we conducted tests to determine how high a salinity concentration the fungi could tolerate. First, the MK55 strain was cultured in liquid medium No. 802 to obtain a pre-culture solution. 5 mL of liquid medium with progressively different sodium chloride concentrations was placed in each test tube, and the MK55 strain pre-culture solution was inoculated at 0.1%. The culture was then incubated at 30°C with shaking at 150 rpm. The OD (Oxygen Demand) was monitored over time using an OD monitor. 600 The growth of the bacterial strain was monitored by recording the data. The results are shown in Figure 4. As shown in Figure 4, the microbial strain did not grow at 6%, but it was found to grow up to 5%. Since the standard value for microbial salt tolerance is 2%, it was found that the MK55 strain has high salt tolerance.

[0051] 5. Organic matter decomposition test Organic matter is an essential source of nutrients for animals, but excess organic matter contributes to environmental pollution, such as water pollution. In agriculture, however, the decomposition of organic matter makes it a more readily absorbable source of nutrients for crops. The microorganisms shown in Table 2 were used as test subjects, and their ability to decompose proteins, fibers, starches, and oils, which are representative organic substances, was tested.

[0052] 5-1. Protein The target microbial colonies were streaked onto agar medium for protein degradation testing (polypeptone: 0.25 g, yeast extract: 0.25 g, ferrous sulfate heptahydrate: 0.01 g, disodium hydrogen phosphate: 0.01 g, agar: 15 g, skim milk: 50 g, distilled water: 1 L (pH 6.8~7.2)) and incubated at 30°C for 3 days. If a clear zone formed around the colony, the test was considered positive for protein degradation.

[0053] 5-2. Fiber The target microbial colonies were streaked onto agar medium for fiber degradation testing (dipotassium hydrogen phosphate: 1.0 g, ammonium nitrate: 0.3 g, potassium chloride: 0.5 g, magnesium sulfate heptahydrate: 0.5 g, ferrous sulfate: 0.01 g, carboxymethylcellulose: 5.0 g, agar: 10.0 g (pH 6.8)) and incubated at 30°C for 3 days. 2 mL of 0.1% Congo Red solution was placed on the agar medium and allowed to stand for 15 minutes before being removed. Then, 2 mL of 6% sodium chloride solution was placed on top and allowed to stand for 15 minutes before being removed. If a clear zone formed around the colony, the test was considered positive for fiber degradation.

[0054] 5-3. Starch The target microbial colonies were streaked onto agar medium for starch degradation testing (soluble starch: 5.0 g, yeast extract: 0.5 g, agar: 15.0 g) and incubated at 30°C for 3 days. 2 mL of iodine tincture diluted 5-fold was placed on the agar medium, allowed to stand for 5 minutes, and then removed. If a clear zone formed around the colony, the test was considered positive for starch degradation.

[0055] 5-4.Oils and fats The target microorganisms were cultured at 30°C for 3 days in liquid culture medium No. 802 with 1% canola oil added. The collected culture supernatant was measured using Lipase Kit S (Sumitomo Bakelite Co., Ltd.), and those that tested positive for lipase activity were considered positive for lipid decomposition.

[0056] The organic matter decomposition test was evaluated relatively on a five-point scale, with "+++" representing the most advanced decomposition and "-" representing no decomposition. Table 2 shows the results of the organic matter test. As shown in Table 2, the MK55 strain was found to have the ability to decompose all of the representative organic substances: proteins, fibers, starches, and oils.

[0057] [Table 2]

[0058] 6. Nitrification capacity test Since nitrification ability is an important factor in both agricultural and aquaculture applications, we tested the nitrification ability to oxidize ammonia nitrogen to nitrate. In nitrification tests, nitrification ability can be confirmed by the disappearance of added ammonia nitrogen and the generation of nitrite and nitrate.

[0059] The microorganisms listed in Table 3 were cultured in liquid medium No. 802 to obtain pre-culture solutions. 20 mL of nitrification test medium was placed in a 200 mL flask with a claw, and 1% of each microorganism's pre-culture solution was inoculated. The cultures were then incubated at 25°C at 150 rpm for 3 days with shaking to obtain the culture solution. The supernatant was then centrifuged and filtered through a syringe filter with a pore size of 0.45 μm for analysis. Nitrification capacity was analyzed by measuring the decrease in ammonia nitrogen and the increase in nitrate nitrogen.

[0060] The composition of the culture medium for nitrification testing is as follows: Disodium hydrogen phosphate: 4.0g, Potassium dihydrogen phosphate: 1.5g, Magnesium sulfate heptahydrate: 0.2g, Sodium succinate: 8.5g, Casamino acid: 20mg, Ammonium sulfate: 70mg, Trace metals: 2ml, Distilled water: 1L (pH 7.0)

[0061] Furthermore, the composition of trace metals used in the culture medium for nitrification testing is as follows: EDTA-2Na: 50.0g, Zinc Sulfate Heptahydrate: 3.9g, Calcium Chloride: 5.5g, Manganese Chloride Tetrahydrate: 5.0g, Iron Sulfate Heptahydrate: 5.0g, Ammonium Molybdate: 1.1g, Copper Sulfate Pentahydrate: 1.6g, Cobalt Chloride Hexahydrate: 1.6g, Distilled Water: 1L

[0062] <Quantitative determination of ammonia nitrogen> (Creating a calibration curve) 0.3 mL of ammonia nitrogen standard solution, adjusted to the specified concentration, was taken. 0.6 mL of distilled water, 0.15 mL of phenol-nitroprusside sodium solution, and 0.15 mL of hypochlorous acid solution were added and the mixture was stirred. After standing at 25°C for 45 minutes, the absorbance was measured at 635 nm and a calibration curve was created. • Standard solution of ammonia nitrogen 0.382 g of special grade ammonium chloride, dried in a sulfuric acid desiccator for more than 4 hours, was dissolved in distilled water to make 1 L, and then diluted before use. • Sodium phenol-nitroprusside solution Phenol: 60g, Sodium nitroprusside: 0.2g, Buffer solution: 1L ·Buffer solution Trisodium phosphate: 30g, Trisodium citrate: 30g, EDTA-2Na: 3g, Distilled water: 1L • Hypochlorous acid solution Sodium hypochlorite solution (final concentration 0.1%), 40 g / L sodium hydroxide solution: 400 mL, distilled water: 1 L

[0063] (Quantitative) A 0.3 mL sample was taken from the appropriately diluted test sample. 0.6 mL of distilled water, 0.15 mL of phenol-nitroprusside sodium solution, and 0.15 mL of hypochlorous acid solution were added and the mixture was stirred. After standing at 25°C for 45 minutes, the absorbance was measured at 635 nm, and the concentration of ammonia nitrogen was determined based on the dilution ratio and calibration curve.

[0064] <Method for analyzing nitrate nitrogen> (Creating a calibration curve) In a microcentrifuge tube, 1 mL of nitrate nitrogen standard solution adjusted to a predetermined concentration, 200 μL of hydrochloric acid (1+16), and 20 μL of 0.5% aluminum chloride solution were added and stirred for 30 minutes. After removing the precipitate using a centrifuge for 3 minutes, the supernatant was collected and its absorbance at 220 nm was measured using a spectrophotometer to create a calibration curve. • Nitrate nitrogen standard solution: 0.7218g of potassium nitrate, dried at 105-110°C, was dissolved in water to make 1L (=100ppm), and then diluted before use.

[0065] (Quantitative) In a microcentrifuge tube, 1 mL of the appropriately diluted test sample, 200 μL of hydrochloric acid (1+16), and 20 μL of 0.5% aluminum chloride solution were added and stirred for 30 minutes. After removing the precipitate using a centrifuge for 3 minutes, the supernatant was collected and the absorbance at 220 nm was measured using a spectrophotometer. The concentration of nitrate nitrogen was determined based on the dilution ratio and calibration curve.

[0066] Table 3 shows the results of the nitrification test. In the nitrification reaction, there are bacteria that oxidize ammonia to nitrite and bacteria that oxidize nitrite to nitrate, but the nitrification test results showed that strain MK55 is a bacterium capable of oxidizing from ammonia nitrogen to nitrate nitrogen. Since nitrite is toxic to crops and fish, strain MK55 is suitable for use in agricultural and fish rearing environments.

[0067] [Table 3]

[0068] 7. Estimation of the taxonomic group of the MK55 strain Based on morphological observations and nucleotide sequence analysis of 16S rDNA (16S rRNA gene), the taxonomic group to which the sample (lower part of MK55) belongs was estimated.

[0069] (Culture conditions) • Culture medium: Difco® LB (Luria-Bertani) broth, Lennox (Becton Dickinson, USA) + agar ·Culture temperature: 30°C Other conditions: Aerobic culture

[0070] (Morphological observation) • Gram staining: Faber G "Nissui" (Nissui Pharmaceutical, Japan) • Optical microscope: BX50F4 (Olympus, Japan) • Stereomicroscope: SMZ(registered trademark)800N (Nikon, Japan)

[0071] (16S rDNA base sequence analysis) • DNA extraction: Achromopeptidase (registered trademark) (FUJIFILM Wako Pure Chemical, Japan) • PCR amplification: Tks Gflex(registered trademark) DNA Polymerase (Takara Bio, Japan) • Cycle Sequencing: BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) • Primer used PCR amplification: 9F, 1510R Sequence (approx. 1,500 bp): 9F, 515F, 1099F, 536R, 926R, 1510R • Sequencing: ABI PRISM 3500xL Genetic Analyzer System (Applied Biosystems) • Sequencing: ChromasPro 2.1 (Technelysium, AUS) ·BLAST homology search Analysis software: ENKI(registered trademark) v3.2 (TechnoSuruga Laboratory, Japan) Databases: DB-BA17.0 (TechnoSuruga Laboratory), International Nucleotide Sequence Database (DDBJ / ENA / GenBank) ·Simple molecular phylogenetic analysis: Phylogenetic tree estimation: Neighbor-joining method Base substitution model: Kimura-2-parameter Reliability evaluation of tree structure: Bootstrap method (1,000 repeats)

[0072] (Morphological observation results) The results are shown in Table 4.

[0073] [Table 4]

[0074] (Sequencing analysis of 16S rDNA (16S rRNA gene)) The extracted DNA from the MK55 strain was amplified by PCR. Subsequently, the nucleotide sequence of the 16S rDNA was determined by cycle sequencing. The nucleotide sequence of the 16S rDNA (16S rRNA gene) was as described in Sequence ID No. 1.

[0075] Based on the results of the above simplified morphological observation, physiological characteristic tests, and 16S rDNA sequencing analysis, strain MK55 was identified as a Bacillus sp. closely related to B. siamensis.

[0076] 8. Water mold disease suppression test using medaka eggs Water mold (NBRC32710 Saprolegnia diclina) was dissolved in physiological saline and counted on a hemocytometer, with a final concentration of 5 × 10⁻⁶. 3 The solution was diluted with physiological saline to achieve the desired result (water mold solution). In a sterile 24-well plate, 975 μL of water mold solution and one fresh medaka egg were placed in each of the 20 wells. Three identical sets were prepared as control, methylene blue, and MK55 strain test groups. A commercially available 8.2% methylene blue solution was diluted to 5 μL / mL (methylene blue dilution). MK55 strain cells were cultured in this solution, collected by centrifugation, rinsed with physiological saline, and then dissolved in distilled water to determine the number of viable cells (2 × 10⁶). 5 A solution adjusted to CFU / mL (MK55 strain dilution) was prepared. 25 μL of distilled water was added to the control group, 25 μL of diluted methylene blue was added to the methylene blue group, and 25 μL of MK55 strain was added to the MK55 strain group. The samples were left standing at 23°C, and the degree of contamination by water mold and the hatching rate were observed over time. Figure 5 shows the number of mold-contaminated eggs per day, and Figure 6 shows the number of hatched eggs per day. Table 5 shows the mold contamination rate after 8 days and the hatching rate after 10 days.

[0077] [Table 5] [Industrial applicability]

[0078] Recent climate change has raised concerns about future food shortages, necessitating increased efficiency in agriculture and aquaculture. The microbial formulations of the present invention can suppress plant and fish diseases, thereby improving the efficiency of agriculture and aquaculture. The microorganisms and microbial formulations of the present invention are applicable to agriculture and aquaculture and are industrially useful.

Claims

1. A microorganism belonging to the genus Bacillus, deposited under accession number NITE P-04187.

2. A microbial preparation comprising the microorganism described in claim 1, or a culture thereof.

3. A microbial preparation according to claim 2, used for one or more applications selected from the group consisting of plant disease control, aquatic animal disease control, organic matter decomposition, and water purification.

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