KR102106802B1 - Beijerinckia fluminensis ANG34 having activities of promoting plant growth and controlling plant disease, and uses thereof - Google Patents

Abstract

The present invention relates to Baijerinckia fluminensis ANG34 strains having activities of promoting plant growth and controlling plant diseases, and uses thereof. Specifically, the Baijerinckia fluminensis ANG34 strains (KFCC11823P) of the present invention have phosphate solubility, nitrogen immobilization capacity, IAA production capacity, siderophore production capacity, ACC deaminase activity, extracellular enzyme activity, and antifungal activity against various plant pathogenic fungi. Accordingly, a composition containing the strain, a culture thereof, or a mixture thereof as an active ingredient can be useful for promoting plant growth and controlling plant diseases.

Description

Beijerinckia fluminensis ANG34 having activities of promoting plant growth and controlling plant disease, and uses thereof}

The present invention relates to a Beijerinckia fluminensis ANG34 strain having a plant growth promotion and plant disease control activity and uses thereof.

Plant growth promoting rhizobacteria (PGPR) is a soil microorganism that promotes growth in plants while being present in the root zone of the plant, which directly or indirectly helps the growth and development of plants (Glick BR et al. J. .Theor. Biol. 190 (1): 63-68, 1998). Phosphoric acid is an essential nutrient that plays an important role in all major metabolic processes such as photosynthesis of plants, energy transfer, signal transduction, and biosynthesis of polymer materials, and the PGPR strain can directly aid in plant growth by solubilizing phosphoric acid. In addition, nitrogen in the atmosphere exists in a form that is not available to living organisms, but the PGPR strain assists in plant growth by providing a nitrogen source by changing it to a form of nitrate available to living organisms.

On the other hand, siderophore (siderophore) is a substance that specifically binds to iron ions, which is an essential element for plant growth, and is a material that directly helps plant growth, but also competitively inhibits plant pathogens from absorbing iron ions. It is a substance that can inhibit the growth of. Therefore, the PGPR strain may help to grow plants by generating sidelopores, and at the same time may exhibit plant disease control effects. In addition, the PGPR strain directly supports plant growth by generating IAA (indole-3-acetic acid), a type of plant hormone, and indirectly, plants grow through antibiotics, lytic enzymes, and systemic induction resistance. It is known to help.

Phytohormone, ethylene, has played an important role in agricultural quality control, and while the plant is growing and developing, the level of ethylene is low at 0.05 μl / L, but the process of aging the plant or the period during which the fruit ripens. In it, high concentration ethylene of 100 µl / L or more is synthesized. An adequate amount of ethylene helps germination and growth in the early stages of plant growth, but high concentrations of ethylene cause inhibition of root elongation, which is the concentration of ethylene when frequently exposed to environmental stresses such as extreme temperature changes, moisture, UV light, and disease. Increases, and aging and cell damage are accelerated. The ACC (1-aminocyclopropane-1-carboxylate) deaminoase produced by the PGPR strain degrades the ethylene precursor ACC into ammonia and alpha-ketobutyrate, thereby lowering the concentration of ethylene. Relieve stress and promote plant growth.

An object of the present invention is to provide a bay jerin Escherichia flu minen sheath (Beijerinckia fluminensis) ANG34 strain and its use having plant growth promotion and plant disease control activity.

In order to achieve the above object, the present invention Provided is a Beijerinckia fluminensis ANG34 strain deposited with accession number KFCC11823P.

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

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

In addition, the present invention provides a plant growth promoting method comprising the step of treating the plant growth promoting composition to a plant, its seeds or its habitat.

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

Beijerinckia fluminensis ANG34 strain (KFCC11823P) of the present invention is a phosphate solubilizing ability, nitrogen immobilizing ability, IAA generating ability, siderophore generating ability, ACC deaminase activity, extracellular enzyme activity and various plant pathogenic fungi Since it has an antifungal activity against, the composition containing the strain, a culture thereof or a mixture thereof as an active ingredient can be usefully used for promoting plant growth and controlling plant diseases.

Figure 1 is a graph showing the results of measuring the ability to generate IAA (indole-3-acetic acid) of six strains (ANG4, ANG14, ANG16, ANG25, ANG28, and Baizerinkia fluminensis ANG34).
Figure 2 is an ANG4 (A), ANG14 (B), ANG16 (C), ANG25 (D), ANG28 (E) and Bayeserinkia fluminensis ANG34 (F) strain sideropore (siderophore) production ability of the strain was evaluated It is a picture.
3 is a genealogy diagram prepared through 16s rRNA sequencing of Bayeserincia fluminensis ANG34 strain.
FIG. 4 is a scanning electron microscope (SEM) photograph of the Baizerinkia fluminensis ANG34 strain.

Hereinafter, the present invention will be described in detail.

The present invention provides a Baizerinkia fluminensis ANG34 strain deposited with accession number KFCC11823P.

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

The strain may be isolated from soil or plant roots.

The strain may have a plant growth promoting activity, and may have a plant disease control activity.

The plant growth promoting activity of the strain may be due to any one or more activities selected from the group consisting of phosphate solubilizing ability, nitrogen immobilizing ability, IAA generating ability, siderophore generating ability, extracellular enzyme activity and ACC deaminase activity. .

The plant disease control activity of the strain may be due to sidelophore generating ability.

The plant disease control activity of the strain may be due to the ability to generate extracellular enzymes.

The plant disease control activity of the strain is a control against plant diseases derived from any one or more bacteria selected from the group consisting of Fusarium genus causing wilting disease, Colletotrichum acutatum causing anthrax , and Corynespora cassiicola causing leaf blight Can be active.

The strain can grow at 10 to 60 ° C, preferably 10 to 50 ° C, more preferably 10 to 40 ° C, and can grow at pH 5 to 9, and the salt (NaCl) concentration is 0 to 4% It can be grown in phosphorus medium.

In addition, the present invention provides a composition for promoting plant growth containing Beijerinckia fluminensis ANG34 deposited with accession number KFCC11823P, a culture thereof, or a mixture thereof as an active ingredient.

The Baizerinkia fluminensis ANG34 strain may have the characteristics as described above. In one example, the strain may have the 16S rRNA sequence of SEQ ID NO: 1.

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

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

The culture may be a culture medium in which the Baizerinkia fluminensis ANG34 strain is cultured or filtered by a physical method.

The composition is mixed with an inert carrier in the strain, its culture, or a mixture thereof, so that the mixture can be formulated as an emulsion, emulsion, fluidizing agent, wettable powder, granulated wettable powder, powder, granule, etc. It can be prepared by adding surfactants and other necessary adjuvants to the mixture.

Examples of liquid carriers that can 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 oils; Esters, such as ethyl acetate and butyl acetate; Nitriles such as acetonitrile and isobutyronitrile; Ethers, such as diisopropyl ether 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, aterpulgite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and talcite; Natural organic materials such as corn leaf powder and walnut shell powder; Synthetic organic materials 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 xylene, alkylbenzene and methylnaphthalene; 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 are anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalene sulfonate form Aldehyde polycondensates; And nonionic surfactants such as polyoxyethylene alkyl aryl ether, polyoxyethylene alkylpolyoxypropylene block copolymers and sorbitan fatty acid esters and cationic surfactants such as alkyltrimethylammonium salts.

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

In addition, the present invention provides a composition for controlling plant diseases containing Bayeserinkia fluminensis ANG34 strain deposited with accession number KFCC11823P, a culture thereof, or a mixture thereof as an active ingredient.

The Baizerinkia fluminensis ANG34 strain may have the characteristics as described above. In one example, the strain may have the 16S rRNA sequence of SEQ ID NO: 1.

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

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

The plant disease control activity of the strain is a control against plant diseases derived from any one or more bacteria selected from the group consisting of Fusarium genus causing wilting disease, Colletotrichum acutatum causing anthrax, and Corynespora cassiicola causing leaf blight Can be active.

The culture may be a culture medium in which the Baizerinkia fluminensis ANG34 strain is cultured or filtered by a physical method.

The composition is mixed with an inert carrier in the strain, its culture, or a mixture thereof, so that the mixture can be formulated as an emulsion, emulsion, fluidizing agent, wettable powder, granulated wettable powder, powder, granule, etc. It can be prepared by adding surfactants and other necessary adjuvants to the mixture.

Examples of liquid carriers that can 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 oils; Esters, such as ethyl acetate and butyl acetate; Nitriles such as acetonitrile and isobutyronitrile; Ethers, such as diisopropyl ether 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, aterpulgite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and talcite; Natural organic materials such as corn leaf powder and walnut shell powder; Synthetic organic materials 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 xylene, alkylbenzene and methylnaphthalene; 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 are anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalene sulfonate form Aldehyde polycondensates; And nonionic surfactants such as polyoxyethylene alkyl aryl ether, polyoxyethylene alkylpolyoxypropylene block copolymers and sorbitan fatty acid esters and cationic surfactants such as alkyltrimethylammonium salts.

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

In addition, the present invention provides a plant growth promoting method comprising the step of treating the plant growth promoting composition to a plant, its seeds or its habitat.

The composition for promoting plant growth may have the characteristics as described above. In one example, the composition may be a composition containing as an active ingredient Bayeserinkia fluminensis ANG34 strain deposited with accession number KFCC11823P, a culture thereof, or a mixture thereof.

The treatment may be to spray the composition directly to the plant, to the soil where the plant is growing, or to a medium for cultivation of the plant.

Application of the composition directly to the plant may include application on the plant surface, for example spraying on stems and leaves.

As a method of spraying the composition on the soil in which the plant is growing, for example, spraying on the soil, mixing with the soil, spraying of the liquid treatment agent into the soil (irrigation of the liquid treatment agent, injection into the soil, dropping of the liquid treatment agent) ) May be included, and examples of places to be treated include replanting holes, furrows, replanting areas, surrounding planting furrows, the entire surface of the growth site, parts between soil and plants, areas between roots, plants Includes the base of the stem, the main furrow, growing soil, nails, a box for seedlings, a tray for seedlings, and a bed.

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

The composition for controlling plant diseases may have the characteristics as described above. In one example, the composition may be a composition containing as an active ingredient Bayeserinkia fluminensis ANG34 strain deposited with accession number KFCC11823P, a culture thereof, or a mixture thereof.

The treatment may be to spray the composition directly to the plant, to the soil where the plant is growing, or to a medium for cultivation of the plant.

Application of the composition directly to the plant may include application on the plant surface, for example spraying on stems and leaves.

As a method of spraying the composition on the soil in which the plant is growing, for example, spraying on the soil, mixing with the soil, spraying of the liquid treatment agent into the soil (irrigation of the liquid treatment agent, injection into the soil, dropping of the liquid treatment agent) ) May be included, and examples of places to be treated include replanting holes, furrows, replanting areas, surrounding planting furrows, the entire surface of the growth site, parts between soil and plants, areas between roots, plants Includes the base of the stem, the main furrow, growing soil, nails, the box for seedling, the tray for seedling, and the bed.

In a specific embodiment of the present invention, the present inventors separate 66 strains from soil and plant roots in various parts of the Republic of Korea, and phosphate solubilization capacity, nitrogen immobilization capacity, IAA generation capacity, sidelopore generation capacity, and cells for the separated strains 6 strains are selected by evaluating other enzyme activity and ACC deaminase activity, and their growth conditions can be checked to grow under a wide range of temperature, pH and salt concentration conditions, and antifungal activity against various plant pathogenic fungi ANG34 strain was finally selected (see Tables 1 to 6, and FIGS. 1 to 2).

In addition, the present inventors identified the selected ANG34 strain and named it Baizerinkia fluminensis ANG34 strain and deposited it with accession number KFCC11823P at the Korea Microbial Conservation Center, and confirmed the morphological and physiological characteristics of the strain (Table 7 And Figure 4).

Therefore, the Bayerelin Kia fluminensis ANG34 strain (KFCC11823P) of the present invention has phosphate solubility, nitrogen immobilization ability, IAA production ability, siderophore production ability, extracellular enzyme activity, ACC deaminase activity and various plant pathogenic fungi. Since it has antifungal activity, it can be usefully used for promoting plant growth and controlling plant diseases.

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

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Separation of new strains from soil

To separate new strains from the rhizosphere soil, Yasan near Silla University (Busan Metropolitan City, Korea), Daejeo Tomato Field (Busan Metropolitan City), Gijang-gun Pear Field (Busan Metropolitan City), Yasan and Garden near Changwon City (Korea), Jeju City Soil and plant roots were collected from Norimae Park and the nearby beaches (South Korea) at 7 points. The collected soil and roots were air dried for 2 days in the shade, and 1 g of each soil sample containing plant roots was suspended in 9 mL of sterile saline. Each suspension was serially diluted to give each of 25 concentrations, followed by 6 plate media (bennet agar (BA), glucose minimal salts agar (GMSA), potato dextrose agar (PDA), tryptic soy agar (TSA), AIA ( Actinomycetes isolation agar) and HV (humic-vitamin agar) were plated in 0.1 mL increments and incubated at 30 ° C for 24 hours. To separate endogenous 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 sonicated. The sonicated root samples were placed on each of the six plate media and incubated at 30 ° C for 24 hours. Colonies of different shapes or colors were subcultured on a medium grown with microorganisms to isolate 66 strains.

Experimental Example 1. Phosphoric acid solubility of separated strains confirmed

Solubilization of the phosphoric acid in the soil can promote plant growth, so the poorly soluble phosphoric acid solubilizing ability of the strains isolated in Example 1 was measured.

Specifically, after cutting the center of a solid PVK (pikopvskaya) agar medium to which 0.5% calcium phosphate was added with cork borer, 20 µl of the purely isolated preculture strain in Example 1, respectively Thick inoculation. After inoculation, while culturing at 30 ° C for 4 days, the presence or absence of a clear zone in the medium was investigated to determine the solubility of poorly soluble phosphoric acid.

Strain name Phosphoric acid soluble capacity ANG4 + ANG14 + ANG16 ++ ANG25 ++ ANG28 + ANG34 ++

+: Diameter of the clear zone is 1.0 cm or more; And ++: the diameter of the clear zone is 3.0 cm or more.

As a result, strains having a clear zone diameter of 1.0 cm or more were identified as six strains of ANG4, ANG14, ANG16, ANG25, ANG28 and ANG34 (Table 1).

Experimental Example 2. Confirmation of nitrogen immobilization capacity of isolated strains

Fixing nitrogen in the atmosphere to provide a nitrogen source in the form of nitrate that can be used by organisms can help plant growth, and thus the nitrogen immobilization capacity of the strains isolated in Example 1 was measured.

Specifically, nitrogen free bacteria separation NFB (Nitrogen Free Bromothymol blue) [(HO ₂ CCH ₂ CH (OH) CO ₂ H 5.0 g, K ₂ HPO ₄ 0.5 g, MgSO ₄ · 7H ₂ O 0.01 g, NaCl 0.02 g _{, FeSO 4 · 7H 2 O 0.05} g, Na 2 MoO 4 0.002 g, MnSO 4 · 7H 2 O 0.01 g, CaCl 2 0.01 g, KOH 4.0 g, 2.0 mL Bromothymol Blue / 1L 0.5% alcohol, agar 1.75 g, pH 6.8] A medium was used, which is a selective medium that can grow only strains capable of immobilizing nitrogen in the atmosphere because it contains only inorganic salts and carbon sources and lacks nitrogen sources. When the culture medium color changed to blue while inoculating the pre-culture strain isolated in Example 1 with a loop and incubating at 30 ° C for 2 days, it was determined that the strain was a strain capable of growing by fixing nitrogen in the atmosphere.

Strain name Nitrogen immobilization capacity ANG4 ++ ANG14 ++ ANG16 ++ ANG25 + ANG28 + ANG34 +

+: Medium nitrogen immobilization capacity; And ++: strong nitrogen immobilization capacity.

As a result, it was confirmed that six strains of ANG4, ANG14, ANG16, ANG25, ANG28 and ANG34 have nitrogen immobilization capacity (Table 2).

Experimental Example 3. IAA (indole-3-acetic acid) generation ability of the isolated strains confirmed

IAA is a plant growth hormone that promotes the growth of plants and promotes root elongation and fruit formation, and thus, the ability to generate IAA of the strains isolated in Example 1 was measured.

Specifically, the pre-culture strain isolated in Example 1 was inoculated into KB (King's B) medium to which 0.1% tryptophan was added, and cultured for 2 days at 30 ° C and 130 rpm. After centrifuging the cultured medium (10,000 rpm, 15 minutes) to separate the supernatant, Salkowski's reagent (35% HClO ₄ 50 mL, 0.5 M FeCl ₃ 1 mL) 1: 2 (v / v), and stood at room temperature for 30 minutes while pink was developed. Thereafter, the absorbance of each mixture was measured at 530 nm using an absorbance photometer. After experimenting in the same way as above using IAA as a standard, a calibration curve was prepared to convert the concentration of IAA in each reaction solution.

As a result, the ANG14 strain produced 96.76 μg / mL of IAA, and the ANG16 and ANG28 strains generated 40.84 μg / mL and 46.99 μg / mL of IAA, respectively, and the ANG4, ANG25, and ANG34 strains 20.02 μg / mL, respectively. 20.02 μg / mL and 8.53 μg / mL of IAA were produced to show excellent IAA production capacity (FIG. 1).

Experimental Example 4. Confirmation of the ability to generate siderophores of isolated strains

Sidelophore binds specifically to iron ions and directly helps plant growth, and inhibits growth by inhibiting the absorption of iron ions by plant pathogens, so it uses the CAS agar method (chrome azurol S blue agar plate assay). By measuring the sidelopore production ability of the strains isolated in Example 1 above.

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 added to prepare a CAS indicator solution. Make King's B medium by adding 20.0 g of proteose peptone, 1.5 g of K ₂ HPO _4, 1.5 g of MgSO ₄ · 7H ₂ O, 10.0 mL of glycerol, and 15 g agar in 900 mL of distilled water. The indicator solution was mixed to adjust the pH to 6.8 and sterilized. After the center of the sterilized CAS blue agar plate was cut using a cork borer, the pre-culture strain isolated in Example 1 was inoculated and incubated at 30 ° C for 4 days, and the presence or absence of orange circles (orange halo) was observed in the blue medium. The sidelopore generation ability was measured.

As a result, an orange circle with a diameter of 1 cm or more appeared in each medium inoculated with 6 strains of ANG4, ANG14, ANG16, ANG25, ANG28, and ANG34, and it was confirmed that there was a sidelopore generation ability (FIG. 2).

Experimental Example 5. ACC (1-aminocyclopropane-1-carboxylate) deaminase activity of the isolated strains Confirm

ACC deaminase can help plant growth by decomposing ACC, a precursor of ethylene that inhibits plant growth, into ammonia and alpha-ketobutyrate, so the strain isolated in Example 1 Their ACC deaminase activity was measured.

Specifically, 5 mL of TSB (tryptic soy broth) medium was added to a test tube, and the pre-culture strains isolated in Example 1 were inoculated, followed by incubation at 30 ° C at 130 rpm for 48 hours, followed by centrifugation to recover the cells. . The recovered cells were DF (Dworkin-Foster) salt (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 _{(glucose) 0.2%, C 6} H 12 O 7 0.2%, C 6 H 8 O 7 0.2%, pH 7.3) twice and inoculated in a DF salt medium to which 3 mM ACC was added as a nitrogen source, and incubated at 30 ° C for 130 hours at 130 rpm. As a control, a DF salt medium with nothing added as a nitrogen source was used, and by measuring absorbance at 600 nm, the increase in the number of cells grown in the medium with ACC was measured to measure ACC deaminase activity.

Strain name ACC deamination enzyme activity ANG4 + ANG14 + ANG16 + ANG25 + ANG28 + ANG34 +

+: Moderate ACC deaminase activity.

As a result, it was confirmed that the six strains of ANG4, ANG14, ANG16, ANG25, ANG28, and ANG34 were able to grow in DF salt medium in which only ACC was added as a nitrogen source, indicating that it had ACC deaminase activity (Table) 3).

Experimental Example 6. Confirmation of extracellular enzyme activity of Bayerincia pluminensis ANG34 strain

The enzymes of amylase, cellulase, protease and xylanase perform degradative parasitism that breaks down the cell wall of plant pathogenic fungi, so that the strain has plant disease control activity, The extracellular enzyme activity of the ANG34 strain was measured.

Specifically, in the case of amylase activity, the strain was inoculated into a nutrient medium containing starch and cultured at 30 ° C for 2 days, followed by centrifugation (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 mixed with a color reagent solution to heat treatment at 100 ° C for 15 minutes. Did. After cooling to room temperature, absorbance was measured at 540 nm using a spectrophotometer (spectrophotometer, Multiskan GO, Thermo Scientific, Vantaa, Finland). One unit of enzyme activity was expressed in terms of the amount of enzyme that produces a reducing sugar corresponding to 1 μg of di-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 heat treated at 100 ° C for 10 minutes and then cooled. Absorbance was measured at 540 nm using a spectrophotometer (spectrophotometer, Multiskan GO, Thermo Scientific, Vantaa, Finland). One unit of enzyme activity was expressed in terms of the amount of enzyme that produces a reducing sugar corresponding to 1 μg 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 and cultured at 30 ° C for 2 days, followed by centrifugation (3000 rpm, 10 minutes). The supernatant centrifuged was mixed with 0.65% casein (50 mM sodium phosphate buffer) and reacted at 37 ° C for 10 minutes. Thereafter, 0.44M of trichloroacetic acid was added to coagulate the protein at 37 ° C for 30 minutes. After the coagulated protein was filtered 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 (spectrophotometer, Multiskan GO, Thermo Scientific, Vantaa, Finland). One unit of enzyme activity was expressed in terms of the amount of enzyme that liberates 1 μg of tyrosine from casein under the above test conditions.

For the activity of xylanase, the strain was inoculated into a nutrient medium containing beechwood xylan, incubated at 30 ° C for 2 days, and centrifuged (3000 rpm, 10 minutes). The supernatant centrifuged was mixed with 1% xylan (50 mM acetic acid buffer) and reacted at 50 ° C for 10 minutes. After the reaction, a 3,5-dinitrosalicylic acid (DNS) reagent was added, followed by heat treatment at 100 ° C for 10 minutes and cooling. Absorbance was measured at 540 nm using a spectrophotometer (spectrophotometer, Multiskan GO, Thermo Scientific, Vantaa, Finland). One unit of enzyme activity was expressed in terms of the amount of enzyme that produces a reducing sugar corresponding to 1 μg of di-xylose from xylan under the above test conditions.

As a result, the ANG34 strain of the present invention showed the activity of amylase, protease, and xylenase at 49.33 U / mL, 7.26 U / mL, and 72.06 U / mL, respectively. (Table 4)

 Strain  ANG34 Amylase 49.33 U / mL Cellulase - Protease 7.26 U / mL Xylenase 72.06 U / mL

Experimental Example 7. Antifungal activity of isolated strains against plant pathogenic fungi Confirm

In order to evaluate the plant disease control activity of the strains isolated in Example 1, antifungal activity against seven plant pathogenic fungi was measured by a well diffusion method using PDA.

Specifically, as plant pathogenic fungi, Rhizoctoni solani AG-2-1 KACC 40124 causing root rot, Sclerotinia sclerotiorum KACC 40457 causing mycosis, KACC 40241 from Fusarium causing wilting disease, Botrytis cinerea KACC causing gray mold 40574, Colletotrichum acutatum KACC40801 causing anthrax, Corynespora cassiicola KACC 44719 causing leaf blight and Phytophthora capsici KACC 40180 causing pepper blight. The pre-culture strains isolated in Example 1 were dispensed into PDA medium at 20 µl and replaced with assay strains at 25 ° C. After cultivation, the distance between the plant pathogenic fungi and useful microorganisms was measured and converted into an inhibition rate (%) according to the following calculation formula.

[formula]

Inhibition rate (%) = (R-r) / R × 100

(R: maximum radius between plant pathogenic fungal colonies and useful microbial colonies; and

r: radius between plant pathogenic fungal colonies and useful microbial colonies on the other side)

As a result, the ANG4 and ANG14 strains showed antifungal activity against the genus Fusarium , the ANG28 strain inhibited 14.3% against Rhizoctoni solani , 16.7% against Botrytis cinerea , 19.0% against Colletotrichum acutatum , and 23.1% against Corynespora cassiicola . The ANG34 strain showed 23.7% against Fusarium genus, 33.0% against Colletotrichum acutatum , and 34.8% against Corynespora cassiicola . This suggests that the ANG34 strain has antifungal activity against various plant pathogenic fungi.

Experimental Example 8. Confirmation of the growth conditions of the selected 6 strains

6 strains (ANG4, ANG14, ANG16, ANG25, ANG28 and ANG34) having nitrogen immobilization ability, IAA production ability, siderophore production ability, and ACC deaminase activity were screened through Experimental Examples 1 to 4 above, at various temperatures and pHs And viability at salt concentration.

Specifically, 5 mL TSB medium was added to the test tube as a pre-culture medium, and 20 μl of the pre-culture solution of the selected 6 strains was inoculated and used as an inoculum of the present culture. After incubation for 2 days at a temperature of 10 to 50 ° C, a pH of 4 to 12, and a concentration of 0 to 10% salt (NaCl), the absorbance was measured at 600 nm, and the absorbance of TSB medium without inoculation of the strain was controlled. It was used as, and the inoculation amount of the strain was the same under all experimental conditions.

Strain name Temperature range (℃) pH range Salt concentration range (%) ANG4 20 ~ 40 4 ~ 10 0 ~ 10 ANG14 20 ~ 50 5 ~ 10 0 ~ 10 ANG16 20 ~ 35 5 ~ 10 0 ~ 6 ANG25 20 ~ 35 5 ~ 9 0 ~ 3 ANG28 20 ~ 40 5 ~ 9 0 ~ 6 ANG34 20 ~ 40 5 ~ 9 0-4

As a result, it was confirmed that 4 strains of ANG4, ANG14, ANG28, and ANG34 were grown at 40 ° C or higher, and that 4 strains of ANG4, ANG14, ANG16, and ANG28 could be grown in a medium containing 6% or more salt (( Table 5). ANG34 strains capable of growing under a wide range of temperature, pH and salt concentration conditions were finally selected in consideration of utilization and plant disease control activity according to various environmental stress factors.

Experimental Example 9. Identification of ANG34 strain

For the taxonomic identification of the ANG34 strain selected in Experimental Example 8, 16S rRNA sequencing was performed.

9-1. Isolation and purification of 16S rRNA gene segment of ANG34 strain

The cells of the isolated ANG34 strain were inoculated in TSB medium, incubated at 30 ° C for 24 hours, and then centrifuged to collect the cells. Genomic DNA was extracted from the recovered cells using a DNA extraction kit, and polymerase chain reaction (PCR) was performed using the extracted DNA as a template. 2.5 units of DNA polymerase (i-StarTaq ^TM DNA polymerase, iNtRON Biotechnology, Korea), 5 μl of DNA sample, 4.0 μl of 2.5 mM dNTPs each, 10 pmol of primer, pyrobest buffer 5.0 The µl and sterile distilled water were mixed to make the total volume 50 µl. As a primer, a universal primer of Table 6 was used, and PCR was performed under the reaction conditions of Table 7.

Primer name Sequence (5 '→ 3') Sequence number 27F AGA GTT TGA TCM TGG CTC AG 2 1492R GGT ATC CTT GTT ACG ACT T 3

division Temperature (℃) time Cycle number 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, and then confirmed on a 1.0% agarose gel, and purified using a PCR purification kit (Millipore PCR cleanup kit, Millipore, USA).

9-2. Identification of ANG34 strain through 16S rRNA sequencing

The similarity between the base sequence of the purified gene and the existing bacteria was analyzed using NCLAST's BLAST, and a systematic diagram was prepared after performing a systematic analysis using the NJ (Neighbor-joining) algorithm of the MEGA 7.0 program. Bootstrapping was repeated 2,000 times (FIG. 3).

As a result, the ANG34 strain was found to have 99% homology with the Beijerinckia fluminensis strain. Thus, the ANG34 strain was named Baizerinkia fluminensis ANG34 strain and deposited with the Korea Microbial Conservation Center with accession number KFCC11823P as of February 27, 2019.

Experimental Example 10. Confirmation of the morphological characteristics of the Baererinkia pluminensis ANG34 strain

The morphological properties were confirmed by photographing the Baizerinkia fluminensis ANG34 strain with a scanning electron microscope (SEM).

As a result, it was confirmed that the Baererinchia fluminensis ANG34 strain has a form of bacillus (FIG. 4).

Experimental Example 11. Confirmation of physiological properties of Baizerinkia fluminensis ANG34 strain

In order to confirm the physiological properties of the Baizerinkia fluminensis ANG34 strain, 19 enzyme activities of the strain were measured according to the manufacturer's instructions using a kit (API ZYM kit, Biomerieux, France).

number Enzyme name activation One Alkaline phosphatase - 2 Esterase (C4) ++ 3 Esterase lipase (C8) + 4 Lipase (C14) - 5 Leucine arylamidase +++ 6 Valine arylamidase - 7 Cystine arylamidase - 8 Trypsin + 9 α-chymotrypsin - 10 Acid phosphatase ++ 11 Naphtol-AS-BI-phosphohydrolase + 12 α-galactosidase - 13 β-galactosidase - 14 β-glucuronidase - 15 α-glucosidase +++ 16 β-glucosidase - 17 N-acetyl-β-glucosaminidase + 18 α-mannosidase - 19 α-fucosidase -

+++: Very strong enzyme activity; ++: Strong enzyme activity;

+: Moderate enzyme activity; And

-: No enzyme activity.

As a result, the Baizerinkia fluminensis ANG34 strain showed a very strong activity of two enzymes (Leucine arylamidase and α-glucosidase), a strong activity of two enzymes (esterase, Acid phosphatase), and four enzymes ( Esterase lipase, Trypsin, naphtol-AS-BI-phosphohydrolase, N-acetyl-β-glucosaminidase) activity was moderate (Table 8).

Korea Microbial Conservation Center KFCC11823P 20190227

<110> LEE, MUN HYUN          LEE, Kwang Hui          ANGEL, CO., LTD. <120> Beijerinckia fluminensis ANG34 having activities of promoting          plant growth and controlling plant disease, and uses thereof <130> 2019p-02-021 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> Beijerinckia fluminensis <400> 1 tgcagtcgaa cgccccgcaa ggggagtggc agacgggtga gtaacgcgtg ggaacatacc 60 ctttcctgcg gaatagctcc gggaaactgg aattaatacc gcatacgccc tacgggggaa 120 agatttatcg gggaaggatt ggcccgcgtt ggattagcta gttggtgggg taaaggccta 180 ccaaggcgac gatccatagc tggtctgaga ggatgatcag ccacattggg actgagacac 240 ggcccaaact cctacgggag gcagcagtgg ggaatattgg acaatgggcg caagcctgat 300 ccagccatgc cgcgtgagtg atgaaggcct tagggttgta aagctctttc accggagaag 360 ataatgacgg tatccggaga agaagccccg gctaacttcg tgccagcagc cgcggtaata 420 cgaagggggc tagcgttgtt cggaattact gggcgtaaag cgcacgtagg cggatattta 480 agtcaggggt gaaatcccag agctcaactc tggaactgcc tttgatactg ggtatcttga 540 gtatggaaga ggtaagtgga attccgagtg tagaggtgaa attcgtagat attcggagga 600 acaccagtgg cgaaggcggc ttactggtcc attactgacg ctgaggtgcg aaagcgtggg 660 gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgaatg ttagccgtcg 720 ggcagtatac tgttcggtgg cgcagctaac gcattaaaca ttccgcctgg ggagtacggt 780 cgcaagatta aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 840 taattcgaag caacgcgcag aaccttacca gctcttgaca ttcggggtat gggcattgga 900 gacgatgtcc ttcagttagg ctggccccag aacaggtgct gcatggctgt cgtcagctcg 960 tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac cctcgccctt agttgccagc 1020 atttagttgg gcactctaag gggactgccg gtgataagcc gagaggaagg tggggatgac 1080 gtcaagtcct catggccctt acgggctggg ctacacacgt gctacaatgg tggtgacagt 1140 gggcagcgag acagcgatgt cgagctaatc tccaaaagcc atctcagttc ggattgcact 1200 ctgcaactcg agtgcatgaa gttggaatcg ctagtaatcg cagatcagca tgctgcggtg 1260 aatacgttcc cgggccttgt acacaccgcc cgtcacagcc atgggagttg gttttacccg 1320 aaggtagtgc gctaaccgca aggaggcagc taa 1353 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beijerinckia fluminensis <400> 2 agagtttgat cmtggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Beijerinckia fluminensis <400> 3 ggtatccttg ttacgactt 19

Claims (8)

Phosphoric acid solubilizing ability, nitrogen immobilizing ability, IAA ((indole-3-acetic acid) generating ability, siderophore generating ability, amylase, protease and xylanase (xylanase) It has extracellular enzyme activity and ACC (1-aminocyclopropane-1-carboxylate) deaminoase activity,
It has a control activity against plant diseases derived from any one or more bacteria selected from the group consisting of Colletotrichum acutatum causing anthrax and Corynespora cassiicola causing leaf blight,
Beijerinckia fluminensis ANG34 strain deposited with accession number KFCC11823P, having the 16S rRNA sequence of SEQ ID NO: 1.
delete The strain of claim 1, wherein the strain has a plant growth promoting activity, Beijerinckia fluminensis ANG34 strain.
The strain of claim 1, wherein the strain has a plant disease control activity, Baijerinckia fluminensis ANG34 strain.
Claim 1 selected from the group consisting of phosphate solubility, nitrogen immobilization capacity, IAA generation capacity, sidelophore generation capacity, amylase, protease and xylanase, containing the strain of claim 1, a culture thereof, or a mixture thereof as an active ingredient 1 A composition for promoting plant growth by any one or more activities selected from the group consisting of at least one extracellular enzyme activity and ACC deaminase activity.
Plant disease derived from any one or more bacteria selected from the group consisting of Colletotrichum acutatum causing anthrax and Corynespora cassiicola causing anthrax, comprising the strain of claim 1, a culture thereof, or a mixture thereof as an active ingredient Control composition.
A composition selected from the group consisting of phosphate solubilizing ability, nitrogen immobilizing ability, IAA generating ability, siderophore generating ability, amylase, protease and xylanase, comprising the step of treating the composition of claim 5 in a plant, its seed or its habitat. Method for promoting plant growth by any one or more activities selected from the group consisting of the above-mentioned extracellular enzyme activity and ACC deaminoase activity.
A plant disease derived from any one or more fungi selected from the group consisting of Colletotrichum acutatum causing anthrax and Corynespora cassiicola causing anthrax, comprising treating the composition of claim 6 on a plant, its seed or its habitat. Control method.

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