KR20220162899A - Microbacterium testaceum DDP398 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 microbacterium testaceum DDP398 strain having activities of promoting plant growth and controlling plant disease, and the use thereof. Specifically, the microbacterium testaceum DDP398 strain (KFCC 11880P) has phosphate solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixation ability, IAA production ability, ACC deaminase activity, siderophore production ability and extracellular enzyme activity, 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

Microbacterium testaceum DDP398 strain having plant growth promotion and plant disease control activity and its use {Microbacterium testaceum DDP398 having activities of promoting plant growth and controlling plant disease, and uses thereof}

It relates to a Microbacterium testaceum DDP398 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 to plants through nitrogen fixation, mineral fixation, and solubilization, or indole-3, a growth hormone. -Promotes plant growth by providing acetic acid (IAA) and siderophore generation, antibiotics, and extracellular enzymes, and helps plant growth by inhibiting 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 in plants. can

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, the availability of plants increases. .

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.

Zinc is an essential micronutrient for plant growth, and is involved in carbohydrate metabolism and Auxin metabolism in plants, and is known to have a significant antioxidant effect. Zinc deficiency in plants causes delayed growth, yellowing and reduced leaf size, and affects yield, pollen formation, root development, water intake and transport, as well as susceptibility to heat, light and fungal infection. Plants can absorb zinc in the form of divalent cations, but only a small amount is present in soil in water-soluble form, mostly in the form of insoluble complexes and minerals. Zinc-solubilizing bacteria are a potential alternative method of supplying zinc and can improve plant growth and development by converting insoluble zinc to a usable form.

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.

The present invention relates to a Microbacterium testaceum DDP398 strain having plant growth promotion and plant disease control activities, nitrogen fixation ability, IAA production ability, siderophore production ability, ACC desorption from the rhizosphere of plants. It has excellent amino enzyme activity and extracellular enzyme activity, shows antifungal effect against 5 kinds of plant pathogenic fungi, and also has various mineral solubilizing ability. The strain having the plant growth promoting and plant disease controlling activity was isolated and named as Microbacterium testaceum DDP398.

Microbacterium testaceum ( Microbacterium testaceum ) As a result of searching prior literature for plant growth promotion and plant disease control use of DDP398 strain with technical characteristics, the following contents of the same genus or species or the same use are disclosed.

First of all, the plant growth promotion (phosphate solubilization ability, siderophore production ability) antifungal activity and extracellular Plant growth promotion use and plant disease control use are disclosed through enzyme (cellulase, chitinase) activity, and secondly, Microbacterium testaceum belonging to the same species as the strain of the present invention ( Microbacterium testaceum ) strain (LAP2-26) It discloses the plant growth promotion (nitrogen fixation) use of, and thirdly, the antifungal activity of the Microbacterium aurum MA-8 strain belonging to the same genus as the strain of the present invention against Sclerotinia sclerotiorum It discloses the use of plant disease control through.

Although the above prior literature discloses the same use as the present invention, which is for plant growth promotion and plant disease control, it relates to the strain of the same or the same species as the strain of the present invention, and the Microbacterium testaceum DDP398 strain of the present invention There is no prior literature disclosing the use of the same strain for plant growth promotion and plant control activity.

Therefore, the present invention was completed by screening new strains for plant growth promotion and plant disease control of the novel Microbacterium testaceum DDP398 strain.

An object of the present invention is to provide a Microbacterium testaceum DDP398 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 Microbacterium testaceum DDP398 strain deposited under accession number KFCC 11880P.

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

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

The extract includes the strain and/or its culture.

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.

Microbacterium testaceum DDP398 strain of the present invention has phosphoric acid solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixation ability, IAA production ability, siderophore production ability, ACC deamination Since it has enzymatic activity and extracellular enzyme activity, a composition containing the strain, its culture or its extract as an active ingredient can be usefully used for plant growth promotion and plant disease control.

1a is a graph showing the results of measuring indole-3-acetic acid (IAA) producing ability of DDP77 and DDP398 strains.
Figure 1b is a photograph evaluating the siderophore production ability of DDP77 and DDP398 strains.
Figure 2a is a photograph evaluating the phosphoric acid solubilization ability of DDP77 and DDP398 strains.
Figure 2b is a photograph of the evaluation of ZnO, Zn ₃ (PO ₄ ) ₂ 4H ₂ O _, ZnCO ₃ solubilizing ability of DDP77 and DDP398 strains.
Figure 2c is a photograph evaluating the calcium carbonate solubilization ability of DDP77 and DDP398 strains.
Figure 2d is a photograph evaluating the silicon solubilization ability of DDP77 and DDP398 strains.
3 is a phylogenetic diagram created through 16s rRNA sequencing analysis of Microbacterium testaceum DDP398 strain.
4 is a scanning electron microscope (SEM) picture of Microbacterium testaceum DDP398 strain.

Hereinafter, the present invention will be described in detail.

The classification of Microbacterium testaceum is as follows:

Inverse: Bacteria,

Phylum: Actinobacteria,

Class: Actinomycetia,

Order: Micrococcales,

Family: Microbacteriaceae

Genus: Microbacterium,

Species: Testaceum

The genus Microbacterium belongs to the class Actinobacteria with high GC content. Members of the genus Microbacterium can be isolated from a wide range of environments, including soil, insects, human clinical specimens, marine environments, plants, dairy products, and the like.

Microbacterium testaceum is an endogenous bacterium that resides within plant hosts without causing disease symptoms.

The present invention provides a Microbacterium testaceum DDP398 strain deposited under accession number KFCC 11880P.

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 phosphate solubilization ability, zinc phosphate 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 selected from the group consisting of

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.

The strain can grow at 10 to 60 ° C, preferably 10 to 50 ° C, more preferably 10 to 35 ° C, grow at pH 5 to 11, and have a salt (NaCl) concentration of 0 to 10%. Can grow on phosphorus medium.

In addition, the present invention provides a composition for promoting plant growth containing, as an active ingredient, Microbacterium testaceum DDP398, a culture thereof, or an extract thereof deposited under accession number KFCC 11880P.

The Microbacterium testaceum ( Microbacterium testaceum ) DDP398 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 a plant disease control activity.

The culture is a culture medium in which the Microbacterium testaceum DDP398 strain is cultured or filtered by a physical method.

The composition is a mixture by mixing an inert carrier with the strain, its culture or extract thereof, and formulating the mixture into an emulsion, emulsion, glidant, wettable powder, granulated wettable powder, powder, granule, etc. It is prepared by adding a surfactant and other necessary auxiliary agents to.

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 (butylhydroxytoluene).

In addition, the present invention provides a composition for controlling plant diseases containing, as an active ingredient, the Microbacterium testaceum DDP398 strain deposited under accession number KFCC 11880P, a culture thereof, or an extract thereof.

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.

As an example, the composition is a composition containing a Microbacterium testaceum DDP398 strain, a culture thereof, or an extract thereof deposited under accession number KFCC 11880P 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, liquid treatment agent dripping), examples of places to be treated include planting holes, furrows, around planting holes, around planting furrows, the entire surface of growing areas, areas between soil and plants, areas between roots, It includes the lower part of the stem of the plant, the main furrow, the growing soil, the seed bed, the seedling box, the seedling tray, 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.

In a specific embodiment of the present invention, the present inventors isolated strains from soil and plant roots of 10 points in Korea, and tested phosphoric acid solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability, and magnesium silicate solubilization ability for the isolated strains. , Nitrogen fixation ability, IAA production ability, siderophore production ability, ACC deaminase activity and extracellular enzyme activity were evaluated to select two strains.

Microbacterium testaceum ( Microbacterium testaceum ) Both DDP77 and DDP398 strains showed moderate nitrogen fixation ability, IAA production ability, siderophore production ability and ACC deaminase activity (Table 2).

In addition, in terms of mineral solubilization ability, zinc phosphate, calcium carbonate, and magnesium silicate solubilization ability were moderate, and in particular, strain DDP77 showed moderate phosphate solubilization ability, whereas strain DDP398 showed strong phosphate solubilization ability (Table 3).

In addition, the three types of extracellular enzyme activity showed similar levels, and the protease showed a concentration of 0.85 mg / ml for DDP398, which was 68% higher than that of 0.58 mg / ml for DDP77 (Table 4).

After confirming their growth conditions, DDP398 strains, which can grow in a wide range of temperature from 10 to 35 ° C, pH from 5 to 11 and salt concentration from 0 to 10%, and have various plant growth promoting activities, were finally selected. .

In addition, the present inventors identified the selected DDP398 strain, named it Microbacterium testaceum DDP398 strain, and deposited it with the Korea Microorganism Conservation Center under the accession number KFCC 11880P, and the morphological and physiological characteristics of the strain confirmed

It was confirmed that the DDP398 strain had the form of rod-shaped bacillus (FIG. 4).

In addition, alkaline phosphatase, esterase (C4), esterase (C8), leucine arylamidase, valine arylamidase, crystaline arylamidase, α-chymotrypsin, acid phosphatase, naphtol-AS-BI-phosphohydrolase, α-glucosidase, β-glucuronidase, α-glucosidase and β-glucosidase enzyme activities, among which strong enzyme activities were shown in leucine arylamidase and α-glucosidase (Table 7).

Therefore, the Microbacterium testaceum DDP398 strain (KFCC 11880P) of the present invention has phosphoric acid solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability, magnesium silicate solubilization ability, nitrogen fixing ability, IAA production ability, sideways Since it has pore-forming ability, ACC deaminase activity and extracellular enzyme activity, 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 Microbacterium testaceum DDP77 strain of the present invention was mainly isolated from the reed field soil of Dadaepo Beach, Busan, and the Microbacterium testaceum DDP398 strain was isolated from the ultrasonically treated Dadaepo Beach reed field root sample.

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 to make 25 concentrations, 5 types of plate medium 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% NaCl , 0.25% K ₂ HPO ₄ , 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) were smeared in 0.1 mL each and incubated at 30 ° C for 24 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 24 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. Plant growth promoting activity of the isolated strain

1-1. 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, it was confirmed that the DDP77 and DDP398 strains had nitrogen fixation ability (Table 1).

Microbacterium testaceum ( Microbacterium testaceum ) Plant growth promoting activity of DDP77 and DDP398 division DDP77 DDP398 Nitrogen fixation ability + + Ability to produce IAA + ++ Ability to generate sideropores + + ACC deaminase activity + +

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

1-2. 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 the experiment was conducted 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, it was confirmed that the DDP77 and DDP398 strains showed IAA producing ability (Table 1 and FIG. 1a).

1-3. 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, an orange halo appeared in the DDP77 and DDP398 strains, confirming that they had the ability to generate sideropores (Table 1 and FIG. 1b).

1-4. 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, it was confirmed that the DDP77 and DDP398 strains could grow in DF salt medium supplemented with only ACC as a nitrogen source, confirming that they had ACC deaminase activity (Table 1).

Experimental Example 2. 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 poorly 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.

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 with a cork borer in the center of the solid medium, the pure separated pre-cultured isolate was inoculated and incubated at 30 ° C for 4 days, and the presence or absence of a clear zone around the cells was examined to measure the CaCO ₃ solubilization ability.

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 strains DDP77 and DDP398 had phosphate solubilization ability, zinc phosphate solubilization ability, calcium carbonate solubilization ability and magnesium silicate solubilization activity in common, and in the case of strain DDP398, phosphate solubilization ability showed higher activity (Fig. 2a to 2d and Table 2).

Microbacterium testaceum ( Microbacterium testaceum ) Mineral solubilizing activity of DDP77 and DDP398 division DDP77 DDP398 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 3. 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, 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. 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.

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

As a result, the DDP77 strain showed activities of 0.18 mg/mL, 0.08 mg/mL, 0.58 mg/mL, and 0.13 mg/mL of amylase, cellulase, protease, and xylenase, respectively, and strain DDP398 showed activities of 0.18 mg/mL, respectively. , 0.09 mg/mL, 0.85 mg/mL, and 0.16 mg/mL of amylase, cellulase, protease, and xylenase activities were shown (Table 3).

According to the above results, the DDP398 strain was finally selected in consideration of plant growth promotion, mineral solubilization ability, and extracellular enzyme activity.

Microbacterium testaceum ( Microbacterium testaceum ) Extracellular enzyme activity of DDP77 and DDP398 division DDP77 DDP398 amylase 0.18 mg/mL 0.18 mg/mL cellulase 0.08 mg/mL 0.09 mg/mL protease 0.58 mg/mL 0.85 mg/mL xylanase 0.13 mg/mL 0.16 mg/mL

Experimental Example 4. Confirmation of growth conditions of DDP398 strain

Through Experimental Examples 1 to 4, DDP398 strains having antifungal activity, nitrogen fixation ability, IAA generating ability, siderophore generating ability, ACC deaminase activity and extracellular enzyme activity were selected for growth at various temperatures, pH, and salt concentrations. ability was confirmed.

Growth conditions of strain DDP398 division DDP398 Temperature range (℃) 10-35 pH range 5-11 Salt concentration range (NaCl %) 0-10

As a result, it was confirmed that DDP398 grew at a temperature of 10-35 ° C., and it was confirmed that the strain could grow in the pH range of 5-11. In addition, it was confirmed that the strain could grow in the range of 0-10% in the salt concentration range (NaCl %) (Table 4).

Experimental Example 5. Microbacterium testaceum Identification of strain DDP398

The DDP398 strain selected in Experimental Example 1 For taxonomic identification, 16S rRNA sequencing was performed.

5-1. Isolation and purification of the 16S rRNA gene segment of strain DDP398

Cells of the separated strain were inoculated into TSB medium, cultured at 30° C. for 24 hours, and centrifuged to recover cells. Genomic DNA was extracted from the recovered cells 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, universal primers in Table 5 were used, and PCR was performed under the reaction conditions in Table 6.

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

PCR reaction conditions 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).

5-2. Identification of DDP398 strain 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 DDP398 strain was found to have 99% homology with the Microbacterium testaceum strain. Accordingly, the DDP398 The strain was named Microbacterium testaceum DDP398, and on February 23, 2021, it was deposited with the Korea Microorganism Conservation Center under accession number KFCC 11880P.

5-3 Microbacterium testaceum ( Microbacterium testaceum ) Confirmation of morphological characteristics of strain DDP398

Microbacterium testaceum ( Microbacterium testaceum ) DDP398 strain was photographed with a scanning electron microscope (Scanning electron microscope, SEM) to confirm morphological characteristics.

As a result, it was confirmed that the Microbacterium testaceum DDP398 strain had a rod-shaped bacillus form (FIG. 4).

Experimental Example 6. Microbacterium testaceum ( Microbacterium testaceum ) Confirmation of physiological characteristics of DDP398 strain

In order to confirm the physiological characteristics of the Microbacterium testaceum DDP398 strain, 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, Microbacterium testaceum ( Microbacterium testaceum ) DDP398 Alkaline phosphatase, Esterase (C4), Esterase (C8), Leucine arylamidase, Valine arylamidase, Crystine arylamidase, α-chymotrypsin, Acid phosphatase, Naphtol-AS-BI-phosphohydrolase, α-glucosidase, β-glucuronidase, α-glucosidase in strains and β-glucosidase enzyme activities, among which strong enzyme activities were shown in leucine arylamidase and α-glucosidase (Table 7).

Microbacterium testaceum ( Microbacterium testaceum ) Physiological characteristics of strain DDP398 enzyme name temperament DDP398 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 1-naphthyl-N-acetyl-βD-glucosaminide + 19 α-mannosidase 6-Br-2-naphthyl-αD-mannopyranoside - 20 α-fucosidase 2-naphthyl-αL-fucopyranoside -

+++: strong enzymatic activity, ++: moderate enzymatic activity, +: enzymatic activity; -: no enzyme activity

Korea Microbial Conservation Center (Korea) KFCC11880P 20210223

<110> ANGEL, CO., LTD. <120> Microbacterium testaceum DDP398 having activities of promoting plant growth and controlling plant disease, and uses its <130> 2021P-02-029 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1391 <212> DNA <213> artificial sequence <220> <223> 16s rRNA of Microbacterium testaceum DDP398 <400> 1 gcagtcgaac ggtgaagcca agcttgcttg gtggatcagt ggcgaacggg tgagtaacac 60 gtgagcaacc tgccctggac tctgggataa gcgctggaaa cggcgtctaa tactggatat 120 gagcccctat cgcatggtgg gggttggaaa gattttttgg tctgggatgg gctcgcggcc 180 tatcagcttg ttggtgaggt aatggctcac caaggcgtcg acgggtagcc ggcctgagag 240 ggtgaccggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaatattgca caatgggcga aagcctgatg cagcaacgcc gcgtgaggga tgacggcctt 360 cgggttgtaa acctctttta gcagggaaga agcgaaagtg acggtacctg cagaaaaagc 420 gccggctaac tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tatccggaat 480 tattgggcgt aaagagctcg taggcggttt gtcgcgtctg ctgtgaaatc ccgaggctca 540 acctcgggcc tgcagtgggt acgggcagac tagagtgcgg taggggagat tggaattcct 600 ggtgtagcgg tggaatgcgc agatatcagg aggaacaccg atggcgaagg cagatctctg 660 ggccgtaact gacgctgagg agcgaaaggg tggggagcaa acaggcttag ataccctggt 720 agtccacccc gtaaacgttg ggaactagtt gtggggacca ttccacggtt tccgtgacgc 780 agctaacgca ttaagttccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa 840 ttgacgggga cccgcacaag cggcggagca tgcggattaa ttcgatgcaa cgcgaagaac 900 cttaccaagg cttgacatat acgagaacgg gccagaaatg gtcaactctt tggacactcg 960 taaacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1020 aacgagcgca accctcgttc tatgttgcca gcacgtaatg gtgggaactc atgggatact 1080 gccggggtca actcggagga aggtggggat gacgtcaaat catcatgccc cttatgtctt 1140 gggcttcacg catgctacaa tggccggtac aaagggctgc aataccgtga ggtggagcga 1200 atcccaaaaa gccggtccca gttcggattg aggtctgcaa ctcgacctca tgaagtcgga 1260 gtcgctagta atcgcagatc agcaacgctg cggtgaatac gttcccgggt cttgtacaca 1320 ccgcccgtca agtcatgaaa gtcggtaaca cctgaagccg gtggcccaac ccttgtggag 1380 gagccgtcga a 1391

Claims (14)

Microbacterium testaceum deposited with accession number KFCC 11880P ( Microbacterium testaceum ) DDP398 strain. According to claim 1, wherein the strain is characterized in that it can grow at 10 to 35 ℃ Microbacterium testaceum ( Microbacterium testaceum ) DDP398 strain. According to claim 1, Microbacterium testaceum characterized in that the strain can grow at pH 5 to 11 ( Microbacterium testaceum ) DDP398 strain. According to claim 1, Microbacterium testaceum characterized in that the strain can grow in a medium having a salt (NaCl) concentration of 0 to 10% ( Microbacterium testaceum ) DDP398 strain. According to claim 1, wherein the strain has a 16s rRNA sequence of SEQ ID NO: 1 Microbacterium testaceum ( Microbacterium testaceum ) DDP398 strain. According to claim 1, wherein the strain is Microbacterium testaceum having a plant growth promoting activity ( Microbacterium testaceum ) DDP398 strain. According to claim 1, wherein the strain has a plant disease control activity Microbacterium testaceum ( Microbacterium testaceum ) DDP398 strain. The method of claim 6, wherein the plant growth promoting activity is phosphate solubilizing ability, zinc phosphate solubilizing ability, calcium carbonate solubilizing ability, magnesium silicate solubilizing ability, nitrogen fixing ability, IAA generating ability, siderophore generating ability, ACC deaminase activity and cell Microbacterium testaceum characterized in that by any one or more activities selected from the group consisting of other enzyme activities ( Microbacterium testaceum ) DDP398 strain. The method of claim 7, wherein the plant disease control activity is characterized in that by any one or more activities selected from the group consisting of sideropore producing ability and extracellular enzyme activity Microbacterium testaceum ( Microbacterium testaceum ) DDP398 strain . A composition for promoting plant growth containing the strain of claim 1, a culture thereof, or an extract thereof as an active ingredient. A composition for controlling plant diseases containing the strain of claim 1, a culture thereof, or an extract 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 10. A plant disease control method comprising the step of treating a plant, its seed or its habitat with the composition of claim 11. 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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