KR20220162897A - Bacillus marisflavi SN56 having activities of promoting plant growth and controlling plant disease, and uses thereof - Google Patents

Abstract

The purpose of the present invention is to provide bacillus marisflavi SN56 strain having activities of promoting plant growth and controlling plant disease, and the use thereof. Specifically, the bacillus marisflavi SN56 strain (KFCC11878P) has phosphate solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixation ability, IAA production ability, ACC deaminase activity, siderophore production ability, extracellular enzyme activity and antifungal activity against various plant pathogenic fungi, such that a composition containing the strain, culture thereof, or extract thereof as an active ingredient can be usefully used to promote the plant growth and control the plant diseases.

Description

Bacillus marisflavi SN56 strain having plant growth promotion and plant disease control activity and uses thereof {Bacillus marisflavi SN56 having activities of promoting plant growth and controlling plant disease, and uses thereof}

It relates to a Bacillus marisflavi SN56 strain having plant growth promoting and plant disease control activities and uses thereof.

It is important to find a microbial agent that will activate crop cultivation while protecting the soil and environment by reducing the use of chemical and inorganic fertilizers currently used in agriculture. Useful microorganisms existing in the rhizosphere have been attracting attention as substitutes for chemical fertilizers, which are the main cause of environmental pollution, and Pseudomonas, Burkholderia, Bacillus, Enterobacter, Arthrobacter, and Acinetobacter genera have been studied as plant growth promoting microorganisms. Plant roots release low-molecular-weight carbon sources that promote the growth of useful microorganisms in the soil rhizosphere, and useful microorganisms in the soil rhizosphere provide nutrients through nitrogen fixation, mineral fixation and solubilization, or indole-3-acetic acid, a growth hormone. It promotes plant growth through acid (IAA) and siderophore generation, antibiotics, and extracellular enzymes, and helps plant growth by suppressing the growth of plant pathogenic fungi.

Plant growth promoting rhizobacteria (PGPR) are soil microorganisms that exist in the rhizosphere of plants and promote plant growth, directly or indirectly helping plants grow and develop.

Phosphoric acid is an essential nutrient that plays an important role in all major metabolic processes, such as photosynthesis, energy transmission, signal transduction, and biosynthesis of macromolecules, and PGPR strains can directly help plant growth by solubilizing phosphoric acid.

In addition, nitrogen in the atmosphere exists in a form that cannot be used by living things, but the PGPR strain converts it into a form of nitrate that can be used by living things and provides a nitrogen source to help plant growth.

Plants require a variety of nutrients for growth and development, and obtain essential nutrients from soil, water, and air. Soil-derived macronutrients include N, P, K, S, Ca, and Mg, and micronutrients include B, Cl, Cu, Fe, Mn, Mo, Zn, and Ni. However, it is difficult to use because it exists in an insoluble form that is difficult for most plants to use.

Microorganisms are known to play an important role in solubilizing minerals such as phosphoric acid, silicon, and zinc, and as various insoluble minerals are solubilized by organic acids produced by microorganisms, availability to plants increases. let it

Therefore, strains having mineral solubilizing ability were used to screen strains that can directly or indirectly help promote plant growth in plant growth and development.

Phosphoric acid is a key nutrient for plants and is known to play an important role in all major metabolic processes such as photosynthesis, energy transmission, signal transmission, and biosynthesis of macromolecules. It occupies about 0.05% (w/w) in soil, but it is difficult to use because most of them exist in insoluble form. Phosphorus-solubilizing bacteria present in the rhizosphere solubilize and mineralize phosphorus present in the soil, effectively release phosphorus from organic and inorganic matter, and help plants use it mainly in the form of HPO ₄ ^2- and H ₂ PO ^4- .

The average calcium content of the earth's crust is about 3.6%, which is relatively more than other plant nutrients. Calcium (Ca) is mainly present in primary minerals such as plagioclase, plagioclase, amphibole, and oleophobite. As a result, there are many in the developed soil. Calcium is absorbed in the form of Ca ²⁺ and exists in the form of Ca ²⁺ in plant tissues. ^It is mainly present in cell walls in large quantities. It is also known to play this role.

In general, when calcium is deficient in crop plants, the permeability of the membrane is impaired, making it difficult to preserve the diffusible material in the cells, leaking from the membrane, and finally damaging the membrane structure.

Solubilization of calcite is achieved by various mechanisms such as acidification by the production of organic acids, inorganic acids, chelating substances, and extracellular polysaccharides produced by microorganisms. Among them, organic acid production is the most effective method for solubilizing calcite.

Silicon is the second most abundant element (27%) on Earth and is generally insoluble with aluminum (Al), magnesium (Mg), calcium (Ca), sodium (Na), potassium (K) and iron (Fe). It exists in the form of silicate (SiO ₃ ).

Although silicon is not yet recognized as an essential element for plant growth, its effects on plant growth, development, yield and disease resistance have been observed in a variety of plant species. In addition, it provides the potential to withstand biotic and abiotic stresses, and has the effect of improving resistance to disease from attack by insects and other pests. However, plants cannot use Si directly unless it is released by weathering or dissolved in soil water (which can be absorbed in the form of Si(OH) ₄ ).

Therefore, in order to increase the utilization rate of silicon, strains having silicon solubilizing ability are used to convert the silicon into a form usable by plants, thereby facilitating absorption. There are many microorganisms in the soil, but few silicon-solubilizing bacteria exist, and they can mainly decompose silicates such as Al ₂ SiO ₅ . In addition, silicon-solubilizing strains play an important role in dissolving insoluble forms of silicon as well as potassium and phosphate, which can increase soil fertility and improve harvest productivity.

On the other hand, siderophore is a substance that specifically binds to iron ion, which is an essential element for plant growth. It is a substance that directly helps plant growth and competitively inhibits plant pathogens from absorbing iron ions. A substance that can inhibit the growth of Therefore, the PGPR strain may help plant growth and exhibit a plant disease control effect by generating siderophores.

In addition, PGPR strains directly help plant growth by producing IAA (indole-3-acetic acid), a kind of plant hormone, and indirectly, plant growth through antibiotics, lytic enzymes, systemic induced tolerance, etc. It is known to help with

Ethylene, a plant hormone, has played an important role in agricultural quality control. During plant growth and development, the level of ethylene is as low as 0.05 μl/L, but during plant aging or fruit ripening period ethylene at a high concentration of 100 μl/L or more is synthesized.

Appropriate amounts of ethylene help germination and growth in the early stages of plant growth, but high concentrations of ethylene cause inhibition of root elongation. is increased, and aging and cell damage are accelerated.

ACC (1-aminocyclopropane-1-carboxylate) deaminase, produced by PGPR strains, decomposes ACC, a precursor of ethylene, into ammonia and α-ketobutyrate, thereby lowering the concentration of ethylene. It relieves stress and helps promote plant growth.

An exoenzyme or extracellular enzyme is an enzyme that is secreted by a cell and functions outside that cell. Hydrolytic enzymes such as amylase, protease, cellulase, nuclease, lipase, and phosphatase are included therein. Exoenzymes are produced by both prokaryotic and eukaryotic cells and have been shown to be important components in many biological processes. In most cases, these enzymes are involved in the breakdown of larger macromolecules. The breakdown of these large macromolecules is important for their constituents to cross the cell membrane and enter the cell. Small molecules produced by exoenzyme activity enter cells and are utilized for various cellular functions.

In addition, bacteria and fungi produce exoenzymes to digest nutrients in their environment, or perform degradative parasitism to degrade the cell walls of plant pathogenic fungi, so that strains have plant disease control activity. These organisms can be used to perform laboratory assays to confirm the presence and function of such exoenzymes.

Another important role served by microbial exoenzymes is in the natural ecology and bioremediation of terrestrial and marine environments.

Plant pathology is the scientific study of plant diseases caused by pathogens (infectious organisms) and environmental conditions (physiological factors). Organisms that cause infectious diseases include fungi, oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, protozoa, nematodes, and parasitic plants. Ectoparasites such as insects, mites, vertebrates or other pests that affect plant health by eating plant tissue are not included. In addition, plant pathology includes the study of pathogen identification, disease causes, disease cycles, economic impacts, plant disease epidemiology, plant disease resistance, plant disease effects on humans and animals, pathogenetics, and plant disease management.

Fungi, or fungi, play many roles in the Earth's ecosystem, interacting with various organisms. A representative example is the role of a decomposer, which decomposes organic matter to make inorganic matter. Certain fungi can cause disease by interacting with plants, and these are called phytopathogenic fungi.

Most plant pathogenic fungi belong to the ascomycetes and basidiomycetes. Fungi reproduce both sexually and asexually through the production of spores and other structures. Spores can be spread over long distances by air or water, or through the soil.

Many soil-dwelling fungi can live eutrophically, performing part of their life cycle in the soil. These are facultative sacrotrophs. Fungal diseases can be controlled through fungicides and other agricultural practices. However, new species of fungi that are resistant to various fungicides often evolve. Biotrophic fungal pathogens colonize living plant tissues and obtain nutrients from living host cells. Necrotrophic fungal pathogens infect and kill host tissue and extract nutrients from dead host cells.

An important fungal plant pathogen is Fusarium spp. (Fusarium wilt), Thielaviopsis spp. (Stem blight, black root rot, Tiellabiopsis root rot), Verticillium spp. (plant half-wilt), Magnaporthe grisea (rice blast), Sclerotinia sclerotiorum (down rot), etc., and Ustilago spp. (barley smut) belonging to the basidiomycetes, Rhizoctonia spp. (cutting off, root rot, eye blight, root rot), Phakospora pachyrhizi (soybean rust), Puccinia spp. (barley and wheat rust), Armillaria spp. (tree white rot root disease) and the like.

Regarding the risk of phytopathogenic fungi, when looking at serious cases of damage caused by plant diseases in the past, 17 out of 33 major diseases were caused by fungi. The detailed disease name is as follows:

Grain rust, grain smut, wheat and rye ergot, potato blight, rice seed blotch, corn seed blotch, grapevine powdery mildew, grapevine downy mildew, tobacco downy mildew, chestnut stem blight, elm wilt, pine stem rust, Mistletoe, coffee tree rust, banana tree spot, rubber tree leaf blight, and wheat red mold.

It is predicted that 9 out of 27 diseases with a high risk of outbreaks in the future will be caused by fungi. Candidates include potato and tomato blight, corn and sorghum downy mildew, wheat smut, soybean rust, cocoa pod rot, chrysanthemum white rust, sugar cane rust, tangerine black spot, and orange scab. They not only threaten human food, but also become a risk factor that can break the balance of the ecosystem.

The present invention relates to a novel Bacillus sp. strain having an antagonistic action against plant pathogenic fungi, and 10 plant pathogenic fungi ( Alternaria alternata, Botrytis cinerea, Colletotrichum acutatum, Corynespora cassiicola, Fusarium solani, Fusarium tricinctum) from soil , Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum ), and a new Bacillus sp. strain having antagonistic ability was isolated and named as Bacillus marisflavi SN56.

As a result of searching for prior literature on the use of Bacillus marisflavi SN56 for controlling plant diseases by technical characteristics, the following prior literatures of the same Bacillus genus or the same use were searched

Republic of Korea Patent No. 10-1936471 is a plant disease control through antibacterial activity against plant pathogenic microorganisms including Colletotrichum acutatum, Fusarium oxysporum, Sclerotium rolfsii, Rhizoctonia solani AG-2-2 and Rhizoctonia cerealis of Bacillus amyloliquefaciens strains Korean Registered Patent No. 10-1667541 discloses Cylindrocarpon destructans, Fusarium solani, Sclerotinia nivalis, Bipolaris oryzae, Rhizoctonia solani, Acidovorax avenae, Pyricularia grisea, Cercospora capcisi, Phytophthora capcisi, Sclerotinia sclerotiorum, Alternaria Plant disease control through antibacterial activity against plant pathogenic microorganisms including alternata, Botrytis cinerea, Streptomyces scabiei, Streptomyces acidiscabies and Clavibacter michiganensis , Republic of Korea Patent No. 10-2124060 discloses Tricothecieum roseum and Plant disease control through antibacterial activity against plant pathogenic microorganisms including Acremonium acutatum , Korean Registered Patent No. 10-1715749 includes Sclerotinia minor , Sclerotinia sclerotiorum , Scleromitrula shiraiana and Ciboria shiraiana of new Bacillus thuringiensis strains For the use of controlling plant diseases through antibacterial activity against plant pathogenic microorganisms, Korean Registered Patent No. 10-1845708 is a novel Bacillus bellegensis strain of Sercospora ( Cercospora sp.), Septoria ( S eptoria sp.), Colletotrichum sp., Sclerotinia sclerotiorum , Phoma sp., and Botrytis cinerea , including bacteria It discloses the use of plant disease control through antibacterial activity against plant pathogenic microorganisms.

Although the above prior literature discloses the same use as the present invention, which is the use of controlling plant diseases through antibacterial activity against phytopathogenic microorganisms, this relates to the same strain of the Bacillus genus, and plants of the same strain as the Bacillus marisflavi strain of the present invention There is no prior literature disclosing the use of growth promoting and plant control activities.

Therefore, the antifungal activity of the novel Bacillus marisflavi strain against phytopathogenic microorganisms and the resulting plant disease control use are technical features to solve the problems described above.

An object of the present invention is to provide a Bacillus marisflavi SN56 strain having plant growth promoting and plant disease control activities and uses thereof.

In order to achieve the above object, the present invention provides a Bacillus marisflavi SN56 strain deposited with accession number KFCC11878P.

In addition, the present invention provides a composition for promoting plant growth containing the strain, its culture, or a mixture thereof as an active ingredient.

In addition, the present invention provides a composition for controlling plant diseases containing the strain, its culture, or a mixture thereof as an active ingredient.

In addition, the present invention provides a plant growth promotion method comprising the step of treating a plant, its seed or its habitat with the composition for promoting plant growth.

In addition, the present invention provides a plant disease control method comprising the step of treating a plant, its seed or its habitat with the composition for controlling plant disease.

Bacillus marisflavi SN56 strain of the present invention has phosphate solubilizing ability, calcium carbonate solubilizing ability, magnesium silicate solubilizing ability, nitrogen fixing ability, IAA generating ability, ACC deaminase activity, siderophore generating ability, extracellular enzyme activity And since it has antifungal activity against various plant pathogenic fungi, a composition containing the strain, its culture, or a mixture thereof as an active ingredient can be usefully used for plant growth promotion and plant disease control.

1a shows SN56, DDP343 and ANG42 This is a graph of the result of measuring the ability of the strain to produce indole-3-acetic acid (IAA).
Figure 1b shows SN56, DDP343 and ANG42 This is a picture evaluating the siderophore production ability of the strain.
2a shows SN56, DDP343 and ANG42 This is a photograph evaluating the phosphoric acid solubilization ability of the strain.
2b shows SN56, DDP343 and ANG42 This is a photograph evaluating the ZnO, Zn ₃ (PO ₄ ) ₂ ·4H ₂ O _{, and} ZnCO ₃ solubilizing ability of the strain.
2c shows SN56, DDP343 and ANG42 This is a photograph evaluating the calcium carbonate solubilization ability of the strain.
2d shows SN56, DDP343 and ANG42 It is a photograph evaluating the silicon solubilizing ability of the strain.
Figure 3 is a phylogenetic diagram created through 16s rRNA sequencing analysis of Bacillus marisflavi SN56 strain.
Figure 4 is a scanning electron microscope (Scanning electron microscope, SEM) picture of Bacillus marisflavi SN56 strain.

Hereinafter, the present invention will be described in detail.

The classification of Bacillus marisflavi is as follows:

Inverse: Bacteria,

System: Eubacteria,

Phylum: Firmicutes,

River: Bacilli,

Order: Bacillales,

Genus: Bacillus,

Species: Marisflavi

Bacillus maris flavi is a bacterium isolated from tidal flats in the Yellow Sea of Korea. Found primarily in soil, the organism can live in extreme environments such as high pH, high temperature and high salinity. The organisms are grown on Nutrient Agar (NA) at 30-35°C.

Gram-positive Bacillus marisflavi is rod-shaped, about 1.5-3.5 micrometers, positive for endospore formation. Aerobic colonies are round, slightly irregular, shiny yellow, slightly elevated. Also, Bacillus marisflavi is a motile organism.

The organism has a 16S rRNA sequence.

The cell wall of the organism is composed of teichoic acid.

The biochemical properties of Bacillus marisflavi are as follows:

Positive results for gelatin hydrolysis, DNA hydrolysis, trisaccharide agar medium, eosin methylene agar medium, MacConkey medium, decarboxylation, hydrogen peroxide lyase, mannitol saline medium, antibacterial sensitivity and disinfection sensitivity.

Phenol Red Broth, Starch Hydrolysis, Casein Hydrolysis, Lipid Hydrolysis, Methyl Red, Foggies Proscauer, Citrate Test, SIM Test, Nitrate Reduction Test, Urea Hydrolysis, Oxidase, Hektoen Enteric ( enteric) agar, phenylalanine deaminase, blood agar and phenylethyl alcohol agar.

Bacillus marisflavi has been found to cause Bacillus bacteremia, which can rarely cause local infections such as pneumonia, intestinal abscesses, and tissue necrosis. Antibiotics such as clindamycin and vancomycin are useful in treating these infections.

The present invention provides a Bacillus marisflavi SN56 strain deposited with accession number KFCC11878P.

The strain has the 16S rRNA sequence of SEQ ID NO: 1.

The strain is isolated from soil or plant roots.

The strain has plant growth promoting activity and has plant disease control activity.

The plant growth promoting activity of the strain is selected from the group consisting of phosphoric acid solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixation ability, IAA production ability, siderophore production ability, ACC deaminase activity and extracellular enzyme activity It is due to any one or more activities that become.

The plant disease control activity of the strain is due to the ability to produce siderophores.

Plant disease control activity of the strain is due to extracellular enzyme activity.

Plant disease control activity of the strain is Alternaria alternata KACC 40019 causing black blotch, Botrytis cinerea KACC 40573 causing gray mold, Colletotrichum acutatum KACC 40042 causing anthrax, Corynespora cassiicola KACC 44719 causing leaf blight, wilt disease Fusarium solani KACC 41092 causing wilt, Fusarium tricinctum KACC 42097 causing wilt, Fusarium oxysporum f.sp. lactucae KACC 42795, Phytophthora capsici KACC 40180 causing pepper blight, Rhizoctonia solani KACC 40124 causing root rot, and Sclerotinia sclerotiorum KACC 41065 causing sclerotinosis. It is an inhibitory activity.

The strain can grow at 10 to 60 ° C, preferably 10 to 50 ° C, more preferably 15 to 40 ° C, grow at pH 6 to 11, and have a salt (NaCl) concentration of 0 to 15%. Can grow on phosphorus medium.

In addition, the present invention provides a composition for promoting plant growth containing Bacillus marisflavi SN56 deposited under accession number KFCC11878P, a culture thereof, or a mixture thereof as an active ingredient.

The Bacillus marisflavi ( Bacillus marisflavi ) SN56 strain may have the characteristics as described above. As an example, the strain has a 16S rRNA sequence of SEQ ID NO: 1.

In addition, the strain may be isolated from soil or plant roots.

In addition, the strain may have a plant growth promoting activity, and has a plant disease control activity.

The culture is a culture medium in which the Bacillus marisflavi SN56 strain is cultured or filtered by a physical method.

The composition is prepared by mixing an inert carrier with the strain, its culture, or a mixture thereof, and the mixture can be formulated into an emulsion, emulsion, glidant, wettable powder, granulated wettable powder, powder, granule, etc. It is prepared by adding a surfactant and other adjuvants as required to the mixture.

Examples of liquid carriers that may be used in the formulation include water; alcohols such as methanol and ethanol; ketones such as acetone and methyl ethyl ketone; aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene and methylnaphthalene; aliphatic hydrocarbons such as hexane, cyclohexane, kerosene and light oil; esters such as ethyl acetate and butyl acetate; nitriles such as acetonitrile and isobutyrnitrile; ethers such as diisopropylether and dioxane; acid amides such as N,N-dimethyl formamide and N,N-dimethylacetamide; halogenated hydrocarbons such as dichloromethane, trichloroethane and carbon tetrachloride; dimethyl sulfoxide; and vegetable oils such as soybean oil and cottonseed oil.

Examples of solid carriers that can be used in the formulation include fine powders or granules such as minerals such as kaolin clay, attapulgite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and talcite; natural organic substances such as corn stalk meal and walnut husk meal; synthetic organic substances such as urea; salts such as calcium carbonate and ammonium sulfate; synthetic inorganic materials such as synthetic hydrated silicon oxide; As liquid carriers, aromatic hydrocarbons such as xylenes, alkylbenzenes and methylnaphthalenes; alcohols such as 2-propanol, ethylene glycol, propylene glycol and ethylene glycol monoethyl ether; ketones such as acetone, cyclohexanone and isophorone; vegetable oils such as soybean oil and cottonseed oil; petroleum aliphatic hydrocarbons, esters, dimethylsulfoxide, acetonitrile and water.

Examples of surfactants include anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalenesulfonate form aldehyde polycondensates; and nonionic surfactants such as polyoxyethylene alkyl aryl ethers, polyoxyethylene alkylpolyoxypropylene block copolymers and sorbitan fatty acid esters, and cationic surfactants such as alkyltrimethylammonium salts.

Examples of other formulation auxiliaries are water-soluble polymers such as polyvinyl alcohol and polyvinylpyrrolidone, polysaccharides such as gum arabic, alginic acid and its salts, CMC (carboxymethyl-cellulose), xanthan gum, inorganic materials such as aluminum magnesium silicate and alumina sol ( alumina sol), preservatives, colorants and stabilizers such as PAP (acid phosphate isopropyl) and BHT (butylhydroxyltoluene).

In addition, the present invention provides a composition for controlling plant diseases containing the Bacillus marisflavi SN56 strain, a culture thereof, or a mixture thereof deposited under accession number KFCC11878P as an active ingredient.

The Bacillus marisflavi ( Bacillus marisflavi ) SN56 strain has the characteristics as described above.

As an example, the strain has a 16S rRNA sequence of SEQ ID NO: 1.

In addition, the strain is isolated from soil or plant roots.

In addition, the strain has a plant growth promoting activity, and has a plant disease control activity.

Plant disease control activity of the strain is Alternaria alternata KACC 40019 causing black blotch, Botrytis cinerea KACC 40573 causing gray mold, Colletotrichum acutatum KACC 40042 causing anthrax, Corynespora cassiicola KACC 44719 causing leaf blight, wilt disease Fusarium solani KACC 41092 causing wilt, Fusarium tricinctum KACC 42097 causing wilt, Fusarium oxysporum f.sp. lactucae KACC 42795, Phytophthora capsici KACC 40180 causing pepper blight, Rhizoctonia solani KACC 40124 causing root rot, and Sclerotinia sclerotiorum KACC 41065 causing sclerotinosis. It is an inhibitory activity.

The culture is a culture medium in which the Bacillus marisflavi SN56 strain is cultured or filtered by a physical method.

The composition is prepared by mixing an inert carrier with the strain, its culture, or a mixture thereof, and the mixture can be formulated into an emulsion, emulsion, glidant, wettable powder, granulated wettable powder, powder, granule, etc. It is prepared by adding a surfactant and other adjuvants as required to the mixture.

In addition, the present invention provides a plant growth promotion method comprising the step of treating a plant, its seed or its habitat with the composition for promoting plant growth.

The composition for promoting plant growth has the same characteristics as described above.

In one example, the composition is a composition containing a Bacillus marisflavi SN56 strain, a culture thereof, or a mixture thereof as an active ingredient deposited under accession number KFCC11878P.

The treatment is to spray the composition directly on the plant, spray on the soil in which the plant is growing, or spray on the medium for culturing the plant.

Methods of spraying the composition directly on plants include application on plant surfaces, such as spraying stems and leaves, for example.

A method of spraying the composition to the soil in which plants are growing, for example, spraying on soil, mixing with soil, spraying a liquid treatment agent into the soil (irrigation of a liquid treatment agent, injection into the soil, dropping of a liquid treatment agent) ) may be included, examples of places to be treated include planting holes, furrows, around planting holes, around planting furrows, the entire surface of the growing area, the part between the soil and the plant, the part between the roots, and the plant body It includes the lower part of the stem, the main furrow, the growing soil, the seed bed, the box for growing seedlings, the tray for growing seedlings, and the seedling bed.

In addition, the present invention provides a plant disease control method comprising the step of treating a plant, its seed or its habitat with the composition for controlling plant disease.

The composition for controlling plant diseases has the same characteristics as described above.

In one example, the composition is a composition containing a Bacillus marisflavi SN56 strain deposited under accession number KFCC11878P, a culture thereof, or a mixture thereof as an active ingredient.

The treatment is to spray the composition directly on the plant, spray on the soil in which the plant is growing, or spray on the medium for culturing the plant.

Methods of spraying the composition directly on plants include application on plant surfaces, such as spraying stems and leaves, for example.

A method of spraying the composition to the soil in which plants are growing, for example, spraying on soil, mixing with soil, spraying a liquid treatment agent into the soil (irrigation of a liquid treatment agent, injection into the soil, dropping of a liquid treatment agent) ) may be included, examples of places to be treated include planting holes, furrows, around planting holes, around planting furrows, the entire surface of the growing area, the part between the soil and the plant, the part between the roots, and the plant body It includes the lower part of the stem, the main furrow, the growing soil, the seed bed, the box for growing seedlings, the tray for growing seedlings, and the seedling bed.

In a specific embodiment of the present invention, the present inventors subculture colonies of different shapes and colors from soil and plant roots of 10 points in Korea to isolate 510 strains, and phosphoric acid solubilization for the isolated strains , calcium carbonate solubilizing ability, magnesium silicate solubilizing ability, nitrogen fixing ability, IAA generating ability, sideropore generating ability, ACC deaminase activity and extracellular enzyme activity were evaluated to select three strains, and to treat various plant pathogenic fungi. SN56 strains having antifungal activity against were finally selected (Tables 1 to 4).

In addition, the present inventors identified the selected SN56 strain and named it Bacillus marisflavi SN56 strain (FIG. 3) and deposited it with the Korea Microorganism Conservation Center under the accession number KFCC11878P, and the morphological and physiological characteristics of the strain was confirmed (FIG. 4 and Table 8).

Therefore, Bacillus marisflavi SN56 strain (KFCC11878P) of the present invention has phosphoric acid solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixation ability, IAA production ability, sideropore production ability, ACC deaminase activity , It has extracellular enzyme activity and antifungal activity against various plant pathogenic fungi, so it can be usefully used for plant growth promotion and plant disease control.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.

Isolation of novel strains from soil

In order to isolate new strains from rhizosphere soil, reed fields in Samnak Park (Busan Metropolitan City, South Korea), Dadaepo Beach reed fields and beaches (Busan Metropolitan City, South Korea), hills near Silla University (Busan Metropolitan City, South Korea), Daejeo Tomato Soil and plant roots from 10 points: a field (Busan Metropolitan City, South Korea), a pear field in Gijang-gun (Busan Metropolitan City, South Korea), a hill and vegetable garden near Changwon-si (Gyeongsangnam-do, South Korea), and Norimae Park and nearby beaches in Jeju-si (Jeju Island, South Korea) were each collected.

The collected soil and roots were air-dried in the shade for 2 days, and each 1 g soil sample containing plant roots was suspended in 9 mL of sterilized physiological saline. After serially diluting each suspension, 5 plate media, BA (Bennet agar, 1% glucose, 0.2% peptone, 0.1% beef extract, 0.1% yeast extract, 1.5% agar), GMSA (glucose minimal salts agar, 0.2 % glucose, 1% yeast extract, 0.2% KH ₂ PO ₄ , 0.3% K ₂ HPO ₄ , 0.01% MgSO ₄ 7H ₂ O, 0.0002% MnSO ₄ , 1.5% agar), PDA (potato dextrose agar, Difco ^TM , 0.4% potato starch, 2% dextrose, 1.5% agar), TSA (tryptic soy agar, Difco ^TM , 1.7% pancreatic digest of casein, 0.3% papaic digest of soybean, 0.25% dextrose, 0.5% sodium chloride, 0.25% dipotassium phosphate , 1.5% agar) and HV (humic-vitamin agar, 0.1% humic acid, 0.05% KCl, 0.05% Na ₂ HPO ₄ , 0.05% MgSO ₄ , 0.002% CaCO ₃ , 0.002% FeSO ₄ , 0.1% VB stock solution, 1.5% agar) was smeared by 0.1 mL each and incubated at 30 ° C for 48 hours.

In order to separate endophytic fungi from the collected plant roots, each plant root sample was sterilized with a surfactant (Tween 80) and bleach (perchloric acid 1.0%), washed with sterile water, and then subjected to sonication. The sonicated root samples were placed on each of the five plate media and cultured at 30° C. for 48 hours. 510 strains were isolated by subculturing colonies of different shapes and colors on a culture medium in which microorganisms were grown.

Experimental Example 1. Antifungal activity of the isolated strain against plant pathogenic fungi

In order to evaluate the plant disease control activity of the isolated strain, the antifungal activity against 10 plant pathogenic fungi was measured by a well diffusion method using a PDA.

Alternaria alternata KACC 40019 causing black blotch, Botrytis cinerea KACC 40573 causing gray mold, Colletotrichum acutatum KACC 40042 causing anthracnose, Corynespora cassiicola KACC 44719 causing leaf blight, Fusarium solani KACC 41092 causing wilt, wilt disease and Fusarium oxysporum f.sp. lactucae KACC 42795, Phytophthora capsici KACC 40180 causing root rot, Rhizoctonia solani AG-2-1 KACC 40124 causing root rot, and Sclerotinia sclerotiorum KACC 41065 causing scleroderma. 100 μl of the supernatant of the isolated strain was dispensed in PDA medium and subcultured at 25 ° C. with the test strain. After culturing, the distance between plant pathogenic fungi and useful microorganisms was measured and converted into an inhibition rate (%) according to the following formula.

[formula]

Inhibition rate (%) = (R - r) / R × 100

(R: maximum radius between colonies of plant pathogenic fungi and colonies of useful microorganisms; and

r: Radius between plant pathogenic fungal colonies and beneficial microbial colonies on the opposite side)

As a result, the SN56 strain showed antifungal activity against 10 species, 51.5% against Alternaria alternata and 51.5% against Botrytis cinerea . 41.5% , against Colletotrichum acutatum 65.1% , against Corynespora cassiicola 65.0% , against Fusarium solani 42.1% , against Fusarium tricinctum 49.1% against Fusarium oxysporum 42.8% against Phytophthora capsici 45.9%, 47.7% against Rhizoconia solani , and 29.4% against Sclerotinia sclerotiorum . DDP343 strain against Colletotrichum acutatum It showed 33.7% inhibitory activity, and in the case of ANG42, 35.0% against Alternaria alternata and against Botrytis cinerea 16.1% , against Colletotrichum acutatum 44.3% , against Corynespora cassiicola It was confirmed that it had an inhibitory activity of 40.4% and 26.7% against Fusarium oxysporum . This suggests that strain SN56, despite being the same Bacillus marisflavi, has excellent antifungal activity against 10 plant pathogenic fungi (Table 1).

division SN56 DDP343 ANG42 Alternaria alternata 51.5% - 35.0% Botrytis cinerea 41.5% - 16.1% Colletotrichum acutatum 65.1% 33.7% 44.3% Corynespora cassiicola 65.0% - 40.4% Fusarium solani 42.1% - - Fusarium tricinctum 49.1% - - Fusarium oxysporum 42.8% - 26.7% Phytophthora capsica 45.9% - - Rhizoctonia solani 47.7% - - Sclerotinia sclerotiorum 29.4% - -

Experimental Example 2. Plant growth promoting activity of the isolated strain

2-1. Confirmation of nitrogen fixation ability of the isolated strain

Fixing nitrogen in the atmosphere and providing a nitrogen source in the form of nitrate that can be used by organisms can help plant growth, so separation The nitrogen fixation capacity of the strains was measured.

Specifically, NFB (Nitrogen Free Bromothymol blue) for isolation of nitrogen-fixing bacteria [(HO ₂ CCH ₂ CH(OH)CO ₂ H, 0.05% K ₂ HPO ₄ , 0.001% MgSO ₄ 7H ₂ O, 0.002% NaCl, 0.005 % FeSO ₄ 7H ₂ O, 0.0002% Na ₂ MoO ₄ , 0.001% MnSO ₄ 7H ₂ O, 0.001% CaCl ₂ , 0.4% KOH, 0.2% bromothymol blue (in 0.5% alcohol), 0.175% agar, pH 6.8 ] medium was used.

The medium contains only inorganic salts and a carbon source and lacks a nitrogen source, so that only strains capable of fixing nitrogen in the atmosphere can grow.

Put 5 mL of NFB medium in a test tube, inoculate the precultured strain isolated in Example 1 with a loop, and incubate at 30 ° C for 2 days, when the color of the medium turns blue, the strain can grow by fixing nitrogen in the atmosphere. It was determined that it was a possible strain.

As a result, SN56, DDP343 and ANG42 It was confirmed that the strain had nitrogen fixation ability (Table 2).

division SN56 DDP343 ANG42 Nitrogen fixation ability + + + Ability to produce IAA + + ++ Ability to generate sideropores + + ++ ACC deaminase activity + + +

++: strong plant growth promoting activity; +: Moderate plant growth promoting activity

2-2. Confirmation of IAA-producing ability of the isolated strain

Since indole-3-acetic acid (IAA) is a plant growth hormone that promotes plant elongation and promotes root elongation and fruit formation, the ability of the isolated strains to produce IAA was measured.

Specifically, the precultured strain isolated in Example 1 was inoculated into KB (King's B) medium supplemented with 0.1% tryptophan, and cultured at 30° C. at 130 rpm for 2 days. After separating the supernatant by centrifuging the culture medium (10,000 rpm, 15 minutes), Salkowski's reagent (35% HClO ₄ 50 mL, 0.5 M FeCl ₃ 1 mL) was added 1:2 (v/ v), and allowed to stand at room temperature for 30 minutes while pink color was developed. Then, the absorbance of each mixed solution was measured at 530 nm using an absorbance photometer. After performing the experiment in the same manner as above using IAA as a standard material, a calibration curve was prepared to convert the concentration of IAA in each reaction solution.

As a result, SN56, DDP343 and ANG42 It was confirmed that the strain showed the ability to produce IAA (Table 2, Fig. 1a).

2-3. Confirmation of the siderophore production ability of the isolated strain

Siderophores directly help plant growth by specifically binding to iron ions, and inhibit growth by interfering with the absorption of iron ions by plant pathogens. separated by The siderophore-producing ability of the strains was measured.

Specifically, 60.5 mg of CAS was dissolved in 50 mL of distilled water, 10 mL of Fe(III) solution (FeCl ₃ .6H ₂ O 27 mg, HCl 83.3 μl/100 mL distilled water) was added, and 72.9 mg of HDTMA ( hexa decyltrimethyl ammonium bromide) was dissolved in 40 mL of distilled water and then added to prepare a CAS indicator solution. A medium was prepared by adding 10 g of trytone, 5 g of yeast extract, 5 g of NaCl, and 15 g of agar to 900 mL of distilled water, mixed with the CAS indicator solution, adjusted to pH 6.8, and sterilized. After cutting the center of the sterilized CAS blue agar plate using a cork borer, inoculating the supernatant of the isolated strain and incubating it at 30 ° C for 4 days, observing the formation of an orange halo in the blue medium, the sideropore The production ability was measured.

As a result, SN56, DDP343 and ANG42 It was confirmed that an orange circle (Orange halo) appeared in the strain and had the ability to generate sideropores (Table 2, Fig. 1b) .

2-4. Confirmation of ACC deaminase production ability of the isolated strain

ACC deaminase can help plant growth by decomposing ACC, a precursor of ethylene that inhibits plant growth, into ammonia and α-ketobutyrate. The ACC deaminase activity of the strain was measured.

Specifically, 5 mL of TSB (tryptic soy broth) medium was placed in a test tube, and each of the precultured strains isolated in Example 1 was inoculated, cultured at 30 ° C. at 130 rpm for 48 hours, and then centrifuged to recover the cells. . The recovered cells were DF (Dworkin-Foster) salt medium (KH ₂ PO ₄ 0.4%, Na ₂ HPO ₄ 0.6%, MgSO ₄ 7H ₂ O 0.02%, FeSO ₄ 0.0001%, H ₃ BO ₃ 0.000001% , MnSO ₄ H ₂ O 0.00001%, ZnSO ₄ 0.000007%, CuSO ₄ 0.000005%, MoO ₃ 0.000001%, glucose 0.2%, C ₆ H ₁₂ O ₇ 0.2%, C ₆ H ₈ O ₇ 0.2%, pH 7.3), washed twice, and inoculated into DF salt medium supplemented with 3 mM ACC as a nitrogen source, followed by incubation at 30 ° C and 130 rpm for 5 days. As a control, a DF salt medium to which nothing was added was used as a nitrogen source, and the ACC deaminase activity was measured by measuring the absorbance at 600 nm to confirm the degree of increase in the cells grown in the medium to which ACC was added.

As a result, SN56, DDP343 and ANG42 It was confirmed that the strain could grow in DF salt medium to which only ACC was added as a nitrogen source, and it was confirmed that there was an ACC deaminase activity (Table 2).

Experimental Example 3. Mineral solubilization ability of the isolated strain

Since mineral solubilization in soil can promote plant growth, in order to evaluate the mineral solubilization ability of the isolates, the zinc solubilization ability, calcium carbonate solubilization ability, and magnesium silicate solubilization ability were measured for each culture time.

Specifically, the phosphoric acid solubilization ability was measured in a solid PVK (pikopvskaya) agar medium (0.05% yeast extract, 1% glucose, 0.5% calcium phosphate, 0.05% ammonium sulphate, 0.02% calcium phosphate, 0.02% calcium phosphate). potassium chloride, 0.01% magnesium sulphate, 0.00001% manganese sulphate, 0.00001% ferrous sulphate 1.5% agar) After cutting the center with a cork borer, 10 μl of the supernatant of the pure separated strain was inoculated. After inoculation, the solubilization ability of sparingly soluble phosphoric acid was determined by examining the formation of a clear zone in the medium while culturing at 30° C. for 4 days.

Zinc solubilization was measured with Tris-minimal salt (1% D-glucose, 0.606% Tris-HCl, 0.468% NaCl, 0.149% KCl, 0.107% NH ₄ Cl, 0.043% Na ₂ SO ₄ , 0.02% MgCl ₂ 2H ₂ O , 0.003% CaCl ₂ 2H ₂ O, 1.5% agar, pH 7.0) by adding 0.1244% ZnO, 0.1998% Zn ₃ (PO ₄ ) ₂ 4H ₂ O, 0.1728% ZnCO ₃ to each zinc oxide (ZnO ), zinc phosphate (Zn ₃ (PO ₄ ) ₂ 4H ₂ O), zinc carbonate (ZnCO ₃ ) solubilization ability wanted to check. After cutting with a cork borer in the center of the solid medium, inoculate the pure separated pre-cultured isolate, and then incubate at 30 ° C for 4 days, examining the formation of a clear zone around the cells to obtain ZnO, Zn ₃ (PO ₄ ) ₂ ·4H ₂ O _, ZnCO ₃ Solubilizing ability was measured.

에서 4일간 배양하면서 균체주위의 clear zone 형성 유무를 조사하여 CaCO₃ 가용화능을 측정하였다.Calcium carbonate solubilization ability was measured in Deveze-Bruni agar (0.5% D-glucose, 0.1% yeast extract, 0.1% peptone, 0.04% K ₂ HPO ₄ , 0.001% MgSO ₄ , 0.5% NaCl, 0.005% (NH ₄ ) ₂ SO ₄ , 0.5% CaCO ₃ , 1.5% agar, pH 6.8) medium was used. After cutting the center of the solid medium using a cork borer, inoculate the pure separated pre-cultured isolate, and then 30

CaCO ₃ solubilizing ability was measured by examining the formation of a clear zone around the cells while culturing for 4 days.

To investigate the silicon solubilizing ability, a silicate agar (1% D-glucose, 0.25% magnesium trisilicate, 1.5% agar, pH 6.8) medium was used. The silicon solubilizing ability was measured by examining the formation of a clear zone around the cells while culturing at 30 ℃ for 4 days after inoculating the pure separated pre-cultured isolate after cutting it out using a cork borer in the center of the solid medium.

As a result, it was confirmed that SN56, DDP343 and ANG42 had phosphoric acid solubilizing ability, calcium carbonate solubilizing ability and magnesium silicate solubilizing activity, and in the case of DDP343 and ANG42, it was confirmed that they also had zinc phosphate solubilizing ability (FIGS. 2a to 2d and Table 3).

division SN56 DDP343 ANG42 Phosphoric acid solubilization ability + + ++ Zinc oxide (ZnO) solubilization ability - - - Zinc phosphate (Zn ₃ (PO ₄ ) ₂ 4H ₂ O) solubilization ability - + + Zinc carbonate (ZnCO ₃ ) solubilization ability - - - Calcium carbonate (CaCO ₃ ) solubilization ability + + + Magnesium silicate (Mg ₂ Si ₃ O ₈ ) solubilizing ability + + +

++: strong mineral solubilization activity; +: moderate mineral solubilizing activity; -: no activity

Experimental Example 4. Confirmation of extracellular enzyme activity of the isolated strain

Enzymes such as amylase, cellulase, protease and xylanase perform degradative parasitism to degrade the cell wall of plant pathogenic fungi so that the strain has plant disease control activity. The extracellular enzyme activity of the isolated strains was measured.

Specifically, in the case of amylase activity, the strain was inoculated into a nutrient medium containing starch, incubated at 30 ° C. for 2 days, and then centrifuged (3000 rpm, 10 minutes). The culture supernatant and 1% starch solution (50 mM sodium phosphate buffer) were mixed 1:1 and reacted at 20 ° C for 3 minutes, and then the color reagent solution was mixed and heat-treated at 100 ° C for 15 minutes did After cooling with ice, the absorbance was measured at 540 nm using a spectrophotometer (Multiskan GO, ThermoScientific, Vantaa, Finland).

The enzyme activity was expressed in terms of the amount of enzyme that produced reducing sugar corresponding to 1 mg of D-maltose from starch under the above test conditions.

In the case of cellulase activity, the strain was inoculated into a nutrient medium containing carboxymethyl cellulose (CMC), incubated at 30 ° C for 2 days, and then centrifuged (3000 rpm, 10 minutes). The centrifuged supernatant was mixed with 1% CMC (50 mM sodium phosphate buffer) and reacted at 50 ° C for 10 minutes, followed by 3,5-dinitrosalicylic acid (DNS) reagent. It was added and cooled after heat treatment at 100 ° C. for 10 minutes. Absorbance was measured at 540 nm using a spectrophotometer (Multiskan GO, Thermo Scientific, Vantaa, Finland). The enzyme activity was expressed in terms of the amount of enzyme that produced reducing sugar corresponding to 1 mg of glucose from CMC under the above test conditions.

In the case of protease activity, the strain was inoculated into a nutrient medium containing skim milk, incubated at 30 ° C. for 2 days, and then centrifuged (3000 rpm, 10 minutes). The centrifuged supernatant was mixed with 0.65% casein (50 mM sodium phosphate buffer) and reacted at 37° C. for 10 minutes. After that, 0.44M of trichloroacetic acid was added and the protein was coagulated at 37°C for 30 minutes. After filtering the coagulated protein using a 0.45 μm filter, the filtrate and 3-fold diluted Folin-ciocalteu's phenol reagent were mixed and reacted at 37° C. for 30 minutes. Absorbance was measured at 660 nm using a spectrophotometer (Multiskan GO, Thermo Scientific, Vantaa, Finland).

The enzyme activity was expressed in terms of the amount of enzyme that liberated 1 mg of tyrosine from casein under the above test conditions.

In the case of xylanase activity, the strain was inoculated into a nutrient medium containing beechwood xylan, incubated at 30 ° C. for 2 days, and then centrifuged (3000 rpm, 10 minutes). The centrifuged supernatant was mixed with 1% xylan (50 mM acetic acid buffer) and reacted at 50° C. for 10 minutes. After the reaction, 3,5-dinitrosalicylic acid (DNS) reagent was added, heat-treated at 100° C. for 10 minutes, and then cooled.

Absorbance was measured at 540 nm using a spectrophotometer (Multiskan GO, Thermo Scientific, Vantaa, Finland).

The enzyme activity was expressed in terms of the amount of enzyme that produced reducing sugar corresponding to 1 mg of D-xylose from xylan under the above test conditions.

As a result, strain SN56 showed activities of 0.23 mg/mL, 0.16 mg/mL, 0.57 mg/mL, and 0.15 mg/mL of amylase, cellulase, protease, and xylenase, respectively, and strain DDP343 showed activities of 0.09 mg/mL, respectively. , 0.58 mg/mL and 0.12 mg/mL of cellulase, protease, and xylenase activities were shown. In addition, strain ANG42 exhibited cellulase, protease, and xylenase activities of 0.13 mg/mL, 0.48 mg/mL, and 0.13 mg/mL (Table 4).

According to the above results, a wide range of The SN56 strain was finally selected in consideration of plant disease control activity, plant growth promotion, mineral solubilization ability, and extracellular enzyme activity.

division SN56 DDP343 ANG42 amylase 0.23 mg/mL - - cellulase 0.16 mg/mL 0.09 mg/mL 0.13 mg/mL protease 0.57 mg/mL 0.58 mg/mL 0.48 mg/mL xylanase 0.15 mg/mL 0.12 mg/mL 0.13 mg/mL

Example 5. Confirmation of growth conditions of strain SN56

Growth at various temperatures, pH and salt concentrations by selecting SN56 strains having antifungal activity, nitrogen fixation ability, IAA generating ability, sideropore generating ability, ACC deaminase activity and extracellular enzyme activity through Experimental Examples 1 to 4 The ability was confirmed (Table 5).

division SN56 Temperature range (℃) 15-40 pH range 6-11 Salt concentration range (NaCl %) 0-15

As a result, it was confirmed that SN56 grew at a temperature of 15-40 ° C., and it was confirmed that the strain could grow in the pH range of 6-11. In addition, it was confirmed that the strain could grow in the range of 0-15% in the salt concentration range (NaCl %) (Table 5).

Experimental Example 6. Bacillus marisflavi Identification of strain SN56

Of the SN56 strain finally selected in Experimental Example 4 For taxonomic identification, 16S rRNA sequencing was performed.

6-1. Isolation and purification of the 16S rRNA gene segment of strain SN56

Cells of the separated strain were inoculated into TSB medium, cultured at 30° C. for 48 hours, and centrifuged to recover cells. Genomic DNA was extracted from the cells recovered using a DNA extraction kit, and a polymerase chain reaction (PCR) was performed using the extracted DNA as a template. 2.5 units of DNA polymerase (i-StarTaqTM DNA polymerase, iNtRON Biotechnology, Korea), 5 μl of DNA sample, 4.0 μl each of 2.5 mM dNTPs, 10 pmol of primer, 5.0 μl of buffer , and sterile distilled water to a total volume of 50 μl. As primers, the universal primers in Table 6 were used, and PCR was performed under the reaction conditions in Table 7.

Primer name Sequence (5' → 3') 27F AGA GTT TGA TCM TGG CTC AG 1492R GGT ATC CTT GTT ACG ACT T

division Temperature (℃) hour number of cycles pre-denaturation 94 5 minutes One denaturation 94 1 minute 30 annealing 55 1 minute 30 extension 72 1 minute 30 final height 72 10 minutes One

The amplified PCR product was stained with EtBr (ethidium bromide) for 15 minutes, checked on a 1.0% agarose gel, and purified using a PCR purification kit (Millipore PCR cleanup kit, Millipore, USA).

6-2. Identification of strain SN95 through 16S rRNA sequencing

The similarity between the nucleotide sequence of the purified gene and the existing bacteria was analyzed using NCBI's BLAST, and a phylogeny was created using the MEGA 7.0 program's NJ (Neighbor-joining) algorithm, followed by phylogenetic analysis At this time, bootstrapping was repeated 2,000 times.

As a result, the SN56 strain was found to have 99% homology with the Bacillus marisflavi strain. In response, SN56 The strain was named Bacillus marisflavi SN56, and on February 4, 2021, it was deposited with the Korea Microorganism Conservation Center under accession number KFCC11878P.

6-3 Bacillus marisflavi Confirmation of morphological characteristics of strain SN56

Bacillus marisflavi strain SN56 was photographed with a scanning electron microscope (SEM) to confirm morphological characteristics.

As a result, it was confirmed that the Bacillus marisflavi SN56 strain had a bacillus form (FIG. 4).

Experimental Example 7. Bacillus marisflavi SN56 Identification of physiological characteristics of the strain

In order to confirm the physiological characteristics of the Bacillus marisflavi strain SN56, 19 enzyme activities of the strain were measured using a kit (API ZYM kit, bioMιrieux, France) according to the manufacturer's instructions.

As a result, it was confirmed that Esterase (C8) and Naphtol-AS-BI-phosphohydrolase enzyme activities were shown in Bacillus marisflavi strain SN56 (Table 8).

enzyme name temperament SN56 One Control - 2 Alkaline phosphatase 2-naphthyl phosphate - 3 Esterase (C4) 2-naphthyl butyrate - 4 Esterase (C8) 2-naphthyl caprylate + 5 Lipase (C14) 2-naphthyl myristate - 6 Leucine arylamidase L-leucyl-2-naphthylamide - 7 Valine arylamidase L-valyl-2-naphthylamide - 8 Crystine arylamidase L-cystyl-2-naphthylamide - 9 Trypsin N-benzoyl-DL-arginine-2-naphthylamide - 10 α-chymotrypsin N-glutaryl-phenylanine-2-naphthylamide - 11 Acid phosphatase 2-naphthyl phosphate - 12 Naphtol-AS-BI-phosphohydrolase Naphthol-AS-BI-phosphate + 13 α-galactosidase 6-Br-2-naphthyl-αD-galactopyranoside - 14 β-glucuronidase 2-naphthyl-βD-galactopyranoside - 15 β-glucosidase Naphthol_AS-BI-βD-glucuronide - 16 α-glucosidase 2-naphthyl-αD-glucopyranoside - 17 β-glucosidase 6-Br-2-naphthyl-βD-glucopyranoside - 18 N-acetyl-β-glucosaminidase 1-naphthyl-N-acetyl-βD-glucosaminide - 19 α-mannosidase 6-Br-2-naphthyl-αD-mannopyranoside - 20 α-fucosidase 2-naphthyl-αL-fucopyranoside -

+: enzyme activity; -: no enzyme activity

Korea Microbial Conservation Center (Korea) KFCC11878P 20210204

<110> ANGEL, CO., LTD. <120> Bacillus marisflavi SN56 having activities of promoting plant growth and controlling plant disease, and uses its <130> 2021P-02-016 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1422 <212> DNA <213> artificial sequence <220> <223> 16s rRNA of Bacillus marisflavi SN56 <400> 1 gcagtcgagc ggatcgatgg gagcttgctc cctgagatca gcggcggacg ggtgagtaac 60 acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct aataccggat 120 aacacctacc cccgcatggg ggaaggttga aaggtggctt cggctatcac ttacagatgg 180 acccgcggcg cattagctag ttggtgaggt aatggctcac caaggcgacg atgcgtagcc 240 gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300 cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360 agaaggtttt cggatcgtaa aactctgttg ttagggaaga acaagtgccg ttcgaatagg 420 gcggcgcctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480 taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagcgcgc gcaggtggtt 540 tcttaagtct gatgtgaaag cccacggctc aaccgtggag ggtcattgga aactggggaa 600 cttgagtgca gaagaggaaa gtggaattcc aagtgtagcg gtgaaatgcg tagatatttg 660 gaggaacacc agtggcgaag gcgactttct ggtctgtaac tgacactgag gcgcgaaagc 720 gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780 tgttagaggg tttccgccct ttagtgctgc agctaacgca ttaagcactc cgcctgggga 840 gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 900 tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaacc 960 ctagagatag ggctttcccc ttcgggggac agagtgacag gtggtgcatg gttgtcgtca 1020 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080 ccagcattca gttgggcact ctaagatgac tgccggtgac aaaccggagg aaggtgggga 1140 tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacggta 1200 caaagggctg caagaccgcg aggtttagcc aatcccataa aaccgttctc agttcggatt 1260 gtaggctgca actcgcctac atgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380 cccgaagtcg gtgaggtaac cttttggagc cagccgccta ag 1422

Claims (13)

Bacillus marisflavi deposited with accession number KFCC11878P ( Bacillus marisflavi ) SN56 strain. According to claim 1, wherein the strain Bacillus Marisflavi characterized in that it can grow at 15 to 40 ℃ ( Bacillus marisflavi ) SN56 strain. According to claim 1, wherein the strain is Bacillus marisflavi characterized in that it can grow at pH 6 to 11 ( Bacillus marisflavi ) SN56 strain. The Bacillus marisflavi SN56 strain according to claim 1, wherein the strain can grow in a medium having a salt (NaCl) concentration of 0 to 15%. The method of claim 1, wherein the strain is Bacillus marisplata, characterized in that derived from soil and plant roots collected from one or more points selected from the group consisting of reed fields, beaches, fields, tomato fields, pear fields, vegetable gardens and parks Rain ( Bacillus marisflavi ) SN56 strain. According to claim 1, wherein the strain is Bacillus marisflavi having a 16s rRNA sequence of SEQ ID NO: 1 ( Bacillus marisflavi ) SN56 strain. According to claim 1, wherein the strain is Bacillus marisflavi having a plant growth promoting activity ( Bacillus marisflavi ) SN56 strain. According to claim 1, wherein the strain is Bacillus marisflavi having a plant disease control activity ( Bacillus marisflavi ) SN56 strain. A composition for promoting plant growth containing the strain of claim 1, its culture, or a mixture thereof as an active ingredient. A composition for controlling plant diseases containing the strain of claim 1, its culture, or a mixture thereof as an active ingredient. A method for promoting plant growth comprising the step of treating a plant, its seed or its habitat with the composition of claim 9. A plant disease control method comprising the step of treating a plant, its seed or its habitat with the composition of claim 10. Contaminated soil recovery method comprising the step of treating the composition of claim 1 to soil contaminated with metal salts or inorganic salts.

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