KR20200131785A - Novel Bacillus methylotrophicus DYGS-5 Strain and Use Thereof - Google Patents

Abstract

The present invention relates to a novel Bacillus methylotropiccus DYGS-5 strain and to a composition for controlling plant diseases comprising the same. More specifically, the novel Bacillus methylotrophicus DYGS-5 strain isolated from the soil has excellent antifungal activity against Fusarium oxysporum or Colletotrichum acutatum, as well as excellent cellulose decomposing ability, protein decomposing ability, and phosphate solubilizing ability, thereby being used as a microbial pesticide.

Description

Novel Bacillus methylotrophicus DYGS-5 Strain and Use Thereof}

The present invention relates to a novel Bacillus methylotrophicus DYGS-5 strain and its use, in particular to a plant disease control use.

The world's population increased rapidly after the industrial revolution in the mid-18th century, with the development of medicine and agriculture. According to the United Nations' World Population Prospects, the world population from 2 billion in 1927 has increased to about 7.6 billion as of 2017, and is predicted to reach 8.6 billion by 2030 (United Nations, World Population Prospects: The 2017 Revision). Modern agriculture has also made rapid progress in order to solve the problem of increasing the scale of food consumption due to the increase of the world population. Since the 1960s, mainly in the United States, research on crop variety improvement has been actively conducted, and agricultural production has increased dramatically (Green Revolution, Encyclopedia Britannica). It is true that the improved variety is highly productive, but as the dependence on chemical fertilizers of the multibiseong variety increased, it revealed a vulnerability to various pests, and the use of chemical pesticides naturally increased (Death GMO, Min Kyung-ryong, Kim Kook-il).

Chemical pesticides are effective quickly, are convenient to use, and have long-lasting medicinal effects to ensure crop yield and marketability, but they cause side effects such as accumulation of pesticide residues, the emergence of pesticide-resistant pathogens, and soil and water pollution. There is a growing social demand to limit its use. In addition, after the Rotterdam Convention (PIC) and the Stockholm Convention (POPs), international regulations on pesticides with high risk to humans and the environment have been strengthened. Accordingly, biopesticides using indigenous microorganisms existing in nature or using metabolites of microorganisms are attracting attention.

According to the Global Markets for Biopesticides (CHM029F) published by BCC Research, the global pesticide market is expected to grow by an annual average of 5.5% from USD 60.2 billion in 2016 to USD 77.8 billion in 2021, but about 6.7% of the global pesticide market in 2016. The value of the biopesticide market, which was only $4 billion, is expected to show an annual average growth rate (CAGR) of 14.1% to $7.7 billion by 2021 (Global Markets for Biopesticides, BCC Research, 2017). At this point, it is understood that the size of the biopesticide market is small compared to the chemical pesticide market, but the size of the biopesticide market is expected to reverse in the 2050s as preference for eco-friendly agricultural products increases and environmental regulations are strengthened year by year. Predicted (The Biopesticide Market, Lux Research, Sara Olson, 2015). The decrease in crop yield due to pests, increased resistance to pests against chemical pesticides, increased demand for eco-friendly agricultural products, and R&D costs, which are cheaper than chemical pesticides, are expected to be the driving force for the continued expansion of the market size of the biopesticide market.

The domestic eco-friendly agricultural material market (eco-friendly pesticides, eco-friendly fertilizers, eco-friendly animal feed additives) is expected to grow from 184 billion won in 2012 to 14.3% per year, growing to 315 billion won in 2016. Trend, Agriculture, Forestry and Fisheries Food Technology Planning and Evaluation Institute, 2011). In addition, as the new certification of low-pesticide agricultural products was discontinued from 2010 and completely abolished in 2015, interest in biological pesticides is expected to increase further (Act on Fostering Eco-Friendly Agriculture and Fisheries and Management and Support of Organic Food, etc.)

According to the U.S. Environmental Protection Agency, Biological Pest Control Agents (PCA) are largely classified into Biorational Pesticides (biological pesticides) and Other living PCA (natural enemies, nematodes, parasites). Biological pesticides include microbial pesticides (Microbial PCA) using bacteria, fungi, viruses, and protozoa, and biochemical pesticides (Biochemical PCA) using proteins, antibiotics, pheromones, etc. , EPA Code of Federal Regulations 40, USA).

The effect of biological pesticides is largely due to the biological control effect by the antibiotics produced by antagonists and the mechanism of action of decomposing ability through the decomposition of organic matter in the soil by various decomposing enzymes. Antibiotics include antibiotics, which are secondary metabolites, and bacteriocin, which is a protein biosynthesis, and have direct antifungal effects against plant pathogens. Some microorganisms used as biological pesticides are in symbiosis or heterotroph with plants in the Rhizosphere of plants, and these microbial groups are called Plant Growth Promoting Rhizobacteria (PGPR) (Kloepper). et al., Nature 284:885-886, 1980, Lugtenberg and Kamilova, Annu. Rev. Microbiol. 63:541-556, 2009, Vacheron et al., Front. Plant Sci. 4:356, 2013). PGPR is known to inhibit the growth of plant pathogens while parasitic on the surface or inside of plant roots, as well as protect plants from the invasion of infectious diseases in the soil, and produce antibiotics, induced systemic resistance (ISR), It has useful functions in promoting plant growth and controlling plant diseases, such as the ability to generate organic matter degrading enzymes, nitrogen fixing ability, and phosphoric acid solubilization ability (Park et al., Microbiology 29:48-53, 2001, Ramamoorty et al., Crop Protection 20:1). -11, 2001, Van Loon LC, Eur. J. Plant Pathol. 119:243-254, 2007, Spaepen et al., FEMS Microbiol. Rev. 31:425-448, 2007, Kumar et al., Microbiol. Res. 167:493-499, 2012, Kavamura et al., Microbiol. Res. 168:183-191, 2013).

Representative microorganisms currently registered for use as microbial agents include Bacillus sp., Candida sp., Lactobacillus sp., Lactococcus sp., and Rhodobacter genus ( Rhodobacter sp.), Pseudomonas sp., and the like. Currently, as research on useful microorganisms continues, more and more microorganisms are registered as microorganisms that can be used as microbial pesticides every year. Most of the strains that can be developed as products are caused by soil transmission, such as Fusarium solani ( Mozalok disease, root rot), Botrytis cinerea (Gray fungal disease), Rhizoctonia solani ( Smallloc disease ), Stemphylium herbarum (rot disease, leaf blight), Diaporthe ambigua (stem Blight), Colletotrichum acutatum (Anthrax), Phytophthora cactorum (Plague), Phytophthora capsici (Plague), Didymella bryoniae (Vine Blight), etc.It was developed and marketed as a product for controlling plant diseases, but it has not yet occupied a large market. to be.

In the present invention, a new strain of Bacillus methylotrophicus was isolated and identified, and the antifungal activity of this strain against plant pathogenic bacteria was confirmed to investigate the possibility of development as a microbial pesticide for controlling plant diseases.

It is an object of the present invention to provide a new strain of Bacillus methylotropicus.

Another object of the present invention is to provide a composition for controlling plant diseases using the new strain.

Another object or specific object of the present invention will be presented below.

In the present invention, as confirmed in the Examples and Experimental Examples below, a new strain was isolated from a soil sample in Damyang-gun using a TSA (Tryptic soy agar) medium containing cyclohexamide, and the 16s rRNA gene sequence was blastn and As a result of analysis using cluster omega, it was identified as DYGS-5, a new strain of Bacillus methylotropicus species, and the new strain has excellent antifungal activity against Fusarium oxysporum or Colletotrichum acutatum , and cellulose It was completed by confirming that it has resolution, protein resolution and phosphoric acid solubilization ability.

Considering the above, the present invention can be understood as a novel Bacillus methylotrophicus DYGS-5 strain.

The novel Bacillus methylotrophicus DYGS-5 strain of the present invention specifically comprises the sequence of the 16S rRNA gene sequence disclosed in SEQ ID NO: 1, and its antifungal activity, enzyme activity and phosphoric acid solubilization ability It can be understood as a strain having the characteristics identified in the experimental examples below.

In another aspect, the present invention relates to a method of culturing a novel Bacillus methylotrophicus DYGS-5 strain.

The method of the present invention includes (a) preparing a medium containing a carbon source, a nitrogen source, and a trace element, and (b) inoculating the medium with a novel Bacillus methylotrophicus DYGS-5 strain, and It comprises a step of culturing.

The carbon source is preferably a sugar such as a monosaccharide, disaccharide or polysaccharide. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, starch hydrolyzate, and the like can be used. Complex compounds such as molasses or by-products of the sugar refining process may also be used. In some cases, oils such as soybean oil and sunflower oil, organic acids such as acetic acid, and glycerol may be preferable as the carbon source. These carbon sources may be included in the medium alone or in a mixture of two or more, and whether included in the medium alone or as a mixture of two or more, 2% (w/w) to 20% (w/w) of The range can be included in the medium. When natural or processed natural products such as starch or starch hydrolyzate are used as carbon sources, these carbon sources can be sterilized to prevent unwanted fermentation by other microorganisms. Sterilization may be performed using a method known in the art such as high temperature and high pressure sterilization method, ultraviolet irradiation.

As the nitrogen source, it may be usually an inorganic nitrogen compound, an organic nitrogen compound, or a composite compound containing these compounds. Examples of inorganic nitrogen compounds include ammonia, ammonium salts (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.), nitrates, and the like, and organic nitrogen compounds include urea and amino acids. As a complex compound, yeast extract, soytone, peptone, tryptone, malt extract, meat juice, soybean powder, soybean bean curd powder, cheonggukjang powder, and soybean paste And natural nitrogen sources such as powder. One or more of these nitrogen sources may be mixed and used, and may be included in the medium in the range of 0.1% (w/w) to 8.0% (w/w). When using a natural nitrogen source, it is preferable to use it after sterilization for the same reason as described in relation to the case of using a natural carbon source.

Trace elements include calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, iron, phosphorus, sulfur, etc. These trace elements can be added to the medium in the form of salt compounds, and the medium of these trace elements In order to increase the solubility and maintain a solution state, a chelating agent such as catechol, citric acid, etc. may be added together. Examples of the salt compound form include sodium hydrogen phosphate, magnesium sulfate, iron chloride, calcium salt, manganese salt, cobalt salt, molybthenate salt, and chelate metal salt. These trace elements may be used by mixing one or more, and may be included in the medium in the range of 0.001% (w/w) to 3.0% (w/w).

In addition, the medium may optionally be prepared by including a growth promoter such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate, and pyridoxine.

The medium is preferably an ordinary medium (nutrient broth), as used in the experimental examples below, and contains an extract containing carbon sources, nitrogen sources, and trace elements such as beef extract and yeast extract. Can be used.

In another aspect, the present invention relates to a composition for controlling plant diseases comprising the novel Bacillus methylotrophicus DYGS-5 ( Bacillus methylotrophicus DYGS-5) strain or a culture thereof as an active ingredient.

In the present specification, the term "active ingredient" refers to an ingredient capable of exhibiting a desired activity alone, or capable of exhibiting activity together with a carrier that is not itself active.

In addition, in the present specification, the "strain" of the present invention may be used as a probiotic, and preferably, may be used in the form of a liquid probiotic or a probiotic powder.

On the other hand, Bacillus methyl trophy coarse DYGS-5 (Bacillus methylotrophicus DYGS- 5) ' culture "of the strain Bacillus methyl trophy for containing a carbon source, a nitrogen source, trace elements in the foregoing medium-coarse DYGS 5 (Bacillus methylotrophicus DYGS- 5) It refers to the product obtained after inoculation and culture of the strain, and includes a cultured strain or a form in which the cultured strain has been removed.

In the present specification, "control" of the plant disease means the prevention, treatment, or alleviation of symptoms of plant disease. Preferably, it is meant to include control of plant diseases caused by infection with Fusarium oxysporum or Colletotrichum acutatum as confirmed in the examples below, and the plant disease is Fusarium Oxysporum ( Fusarium oxysporum ) or Colletotrichum acutatum ( Colletotrichum acutatum ) is a wilt disease or red pepper anthrax caused by infection.

Melon vine disease (Fusarium wilt) is caused by Fusarium oxysporum f. sp. melonis (FOM) and is one of the diseases that cause enormous damage to melon cultivation worldwide (Beckman CH, APS Press, St. Paul, MN, 1987, Katan et al., Phytopathology 84:153-157, 1994). In vine-splitting disease, the hyphae of F. oxysporum invades through the epidermis, cortex, and endodermis tissues of the roots of the plant and reaches the xylem, and then a large amount of conidiospore is produced. It proliferates and causes wilt symptoms, resulting in plant death (Bishop and Cooper, Physiol. Plant Pathol. 23:323-343, 1983, Beckman and Roberts, Adv. Bot. Res. 21:35-77, 1995, Di Pietro et al. ., Mol. Plant Pathol. 4:315-325, 2003, Michielse and Rep, Mol. Plant Pathol. 10:311-324, 2009). When infected with vine pecking disease, pathogens invade the water pipe of the stem through the roots, so the whole vegetation gradually withers and turns yellow and dies. Lee Byung-joo shows the typical symptoms of vine prickly disease, where the soil surface cracks and a brown essence appears, and if it is humid, it can be observed that pink mold is dense on the diseased area.

Anthracnose is a plant disease caused by Filamentous fungus of the genus Colletotrichum sp. It occurs on the leaves, stems, and fruits of useful plants such as cereals, vegetables, and fruit trees, and makes brown lesions or leaf necrosis. When the lesion is old, pinkish viscous matter appears in the darkened area, which are conidiophores and scatter in the air to become a secondary source of transmission. Anthrax is particularly damaging to gourds and crops such as peppers, watermelons, and melons. In Korea, it is estimated that the annual loss due to capsicum anthrax ( colletotrichum acutatum ) is estimated to be more than 100 million dollars, which is equivalent to 10% of total annual production, causing serious damage to pepper farmers (Kim et al., Plant Pathol). J. 24(1): 17-23, 2008).

The control composition of the present invention may contain the active ingredient in an arbitrary amount within the range of 0.001 to 99.999% by weight based on the total weight of the control composition of the present invention, its formulation, the object to be applied, and the site. Will be included in the range of 0.01 to 30% by weight, in the liquid formulation is usually 0.1 to 20% by weight, in the powder is usually included in the range of 1 to 40% by weight.

The composition for control of the present invention is prepared by diluting or powdering the active ingredient as it is, or mixed with an agrochemically acceptable carrier and formulated into emulsions, solutions, suspensions, wettables, flow agents, powders, granules, dispersants, etc. Can be.

In the above, "agrochemically acceptable carrier" is blended with the active ingredient of the control agent composition of the present invention to promote spraying, storage and/or transport of the active ingredient without inhibiting the activity of the active ingredient and acceptable to plants. It refers to any substance that does not exhibit the above toxicity.

Carriers that can be used include soil natural minerals (e.g. kaolin, clay, talc, chalk), soil synthetic minerals (e.g. highly disperse silica, silicates), carbon-based materials (charcoal, bitumen, etc.), sulfur, natural or synthetic Resins, fertilizers, cellulose based materials (sawdust and corncob), water, aromatic solvents (e.g. Solveso Products, xylene), paraffins (e.g. mineral oil fraction), alcohols (e.g. methanol, butanol, pentanol, Benzyl alcohol), ketones (e.g. cyclohexanone, methyl hydroxybutyl ketone, diacetone alcohol, mesityl oxide, isophorone), lactones (e.g. gamma-butyrolactone), pyrrolidone (pyrrolidone , N-methylpyrrolidone, N-ethylpyrrolidone, n-octylpyrrolidone), acetate (glycol diacetate), glycol, dimethyl fatty acid amide, fatty acid, fatty acid ester, and mixtures thereof.

The composition for controlling of the present invention may also contain one or more adjuvants such as a wetting agent, a dispersing agent, an antifreeze agent, a thickener, a preservative, and an electrodeposition agent.

These adjuvants may be used by selecting appropriate ones from those known in the art.For example, alkylnaphthalene sulfonate (especially diisopropylnaphthalenesulfonate or diisobutylnaphthalenesulfonate) may be used as a wetting agent, and nonionic Dispersants (ethylene oxide/propylene oxide block polymer, alkylphenol polyglycol ether, tristyrylphenol polyglycol, etc.), anionic dispersants (ligninsulfonic acid, naphthalenesulfonic acid, phenolsulfonic acid, dibutylnaphthalenesulfonic acid, alkylarylsulfonate, alkyl alcohol) Pate, alkylsulfonate, etc.) or a mixture thereof, and as an antifreezing agent, methanol, ethanol, isopropanol, butanol, glycol, glycerin, diethylene glycol, etc. may be used, and as a thickener, a cellulose derivative, a polyacrylic acid derivative, and xanthan , Modified clay, etc. can be used, dichlorophene, isothiazolene, benzyl alcohol, etc. can be used as preservatives, and the electrodeposition agent is polyvinyl alcohol, polyvinylpyrrolidone, polyacrylate, polymethacrylate, polybutene , Polyisobutylene, polystyrene, polyethyleneamine, polyethyleneamide, polyethyleneimine, polyether, and the like can be used.

The composition for control of the present invention may further contain other known components, such as a herbicidal component, an insecticidal component, and a bactericidal component, depending on the purpose of use and the environment of the cultivated land.

In another aspect, the present invention relates to a method for controlling wilt disease or red pepper anthrax using the control composition of the present invention as described above.

The control method of the present invention comprises the step of contacting the control composition of the present invention with the causative agent of wilt disease or red pepper anthrax.

The contacting step is usually performed by spraying on plants with wither disease or pepper anthrax disease, but is performed by spraying or mixing in the soil, which is the habitat environment of the plant, or spraying on their seeds or seedlings, or the growth environment of the plant. It can also be done by spraying or mixing on the medium or soil.

Alternatively, the contacting may be performed by mixing other fungicides, pesticides, herbicides, fertilizers, and the like.

The amount of the active ingredient of the composition of the present invention to be used in plants with wilt disease or pepper anthrax disease is determined by comprehensively considering the growth degree of the plant, the degree of disease, soil conditions, rainfall, and the type of active ingredient to be used. Will be decided. Usually, it is used in an amount of 10 to 500 g, preferably 30 to 250 g per hectare, and in the case of spraying and mixing in soil, it is used in an amount of 10 to 1000 g, preferably 100 to 500 g.

As described above, according to the present invention, a novel Bacillus methylotropicus DYGS-5 strain can be provided.

The strain of the present invention can be usefully used as a composition for controlling pepper anthrax or wilt disease.

1 is a phylogenetic tree of the Bacillus methylotropicus DYGS-5 strain of the present invention.
FIG. 2 is a result of confirming the antifungal activity of the isolated strains of Damyang-gun soil samples, including the Bacillus methylotropicus DYGS-5 (Gasan-ri 5) strain of the present invention.
3 is a result of confirming the cellulose resolution, protein resolution, and phosphoric acid solubilization ability of the Damyang-gun soil sample isolate strain including the Bacillus methylotropicus DYGS-5 (Gasanri 5) strain of the present invention.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example> Isolation and identification of a new strain DYGS-5

<Example 1> Isolation of new strains

In order to select microorganisms having antifungal activity against fungi that cause plant diseases, soil samples were collected in the whole area of Damyang-gun. Samples were collected at a depth of about 15 centimeters in the topsoil layer, and foreign substances such as weed roots and fallen leaves were removed, and then placed in a zipper bag and stored in a low-temperature storage (4℃).

10 g of a soil sample was suspended in 100 ml of sterile water and shaken at 28° C. and 160 rpm for 30 minutes to prepare a soil suspension. After diluting this suspension to 10 to ⁵ times, 40ul of the diluted solution was spread on TSA (Tryptic soy agar, MBcell, Korea) medium in which cyclohexamide (Cyclohexamide, Sigma, SL, USA) is mixed at a concentration of 50 mg/L. Incubated for 2 days at 30 ℃. The resulting colonies were separated by subculture in LB (Luria-Bertani, Difco, MD, USA) solid medium, respectively, and the pure isolated strain was cultured in NB (Nutrient broth, MBcell, Korea) medium mixed with 40% glycerol. , Stored at -80°C. The isolated strains were all 10 species: Goseo 1, Goseo 3, Saminsan 1, Gasanri 1, Gasanri 5, Gasanri 7, Ssangtaeri 2, Ssangtaeri 8, Ssangtaeri 10, and Ssangtaeri 12.

<Example 2> Identification of new strains

In order to identify the isolated strain, 16s rRNA sequencing was requested to Solgent (Solgent Co., Ltd, Korea).

The obtained nucleotide sequence was analyzed using the blastn program of NCBI (https://www.ncbi.nlm.nih.gov/) and Clustal omega (http://www.clustal.org/omega/) software. Sequence homology was compared. In addition, a phylogenic tree was created using a neighbor-joining (NJ) algorithm.

As a result of 16s rRNA sequencing, Gasanri 5 (SEQ ID NO: 1 was identified as Bacillus methlytrophicus ) (99% sequence homology with similar strains in the tree), and the remaining 9 species were also identified as Bacillus genus. 10 species Among the strains of, Gasanri 5, which has excellent antifungal activity, was named as Bacillus methlytrophicus strain DYGS-5 and deposited with the Microbial Bank of the National Academy of Agricultural Sciences, and was given the accession number KACC 92217P. Bacillus methyl The results of the phylogenetic diagram of the Rotropicus DYGS-5 strain are shown in FIG. 1.

<Experimental Example> Characterization of the new strain DYGS-5

Bacillus methylotropicus DYGS-5 and 9 other species stored at -80°C were plated on NA (Nutrient agar, Difco, MD, USA) medium and incubated at 30°C for 24 hours to reactivate. A single colony of the reactivated strain was inoculated in NB (Nutrient broth) medium and incubated at 30° C. and 130 rpm for 24 hours. The total number of bacteria of the cultured isolate was counted using a flow cytometer (BD AccuriTMC6Plus, BD, CA, USA), and a cell suspension of 1 X 10 ⁶ cfu/ml was prepared.

<Experimental Example 1> Confirmation of antifungal activity

Subjects to investigate the antifungal activity of 9 species including Bacillus methylotropicus DYGS-5 and strains of plant pathogens such as Capsicum bacterium ( Coletotrichum acutatum ) and wilting bacteria ( Fusarium oxysporum ) strains It was used as a strain. The pathogen hyphae (1 X 1 cm ² ) was placed in the center of the PDA (Difco, MD, USA) medium, and 30 ul (1 X 10 ⁶ cfu) of the culture suspension of the isolated strain was inoculated at a point 30 mm away from the pathogen. Incubated for 5 to 6 days at ℃. The antifungal activity of the isolated strain was investigated by measuring the growth inhibition distance of the plant pathogen, and the results are shown in [Fig. 2] and [Table 1].

Plant pathogens C. acutatum (capsicum anthracnose) and F. oxysporum (withered disease) were incubated with the isolated strain for 6 days to investigate the growth inhibition distance. Among the isolated strains, DYGS-5 (Gasan-ri 5) strains were C. acutatum and F. For oxysporum , the average growth inhibition distance was 7.38 mm and 4.63 mm, respectively, which was confirmed to have high growth inhibition activity. On the other hand, it was investigated that the strains of Gasanri 7 and Ssangtaeri had no antifungal activity against two types of plant pathogens.

<Experimental Example 2> Confirmation of other properties

In order to check the cellulose resolution, protein resolution, and phosphoric acid solubilization ability of the isolated strain, cultivate in a solid medium containing carboxymethyl cellulose (CMC), skim milk, and insoluble phosphoric acid (Ca ₃ (PO ₄ ) ₂ ) and surrounding The size of the ring formed in was measured and analyzed. Analysis targets include DYGS-5 (Gasan-ri 5), a new strain of the present invention, and 10 strains isolated from Damyang-gun soil (Goseo 1, Goseo 3, Saminsan 1, Gasan-ri 1, Gasan-ri 5, Gasan-ri 7, Ssang-ta-ri 2, Ssang-ta-ri 8 , 10 twins, 12 twins.

<Experimental Example 2-1> Cellulose resolution

Cellulose degradation is related to the ability of the cell wall to break down the cell wall of phytopathogenic bacteria composed of cellulose. Accordingly, 10 strains isolated from Damyang-gun soil, including DYGS-5 (Gasan-ri 5) (Goseo 1, Goseo 3, Saminsan 1, Gasan-ri 1, Gasan-ri 5, Gasan-ri 7, Ssang-ri 2, Ssang-ta-ri 8, Ssang-ta-ri 10, Ssang-ta-ri 12 ) Of the cellulase (Cellulase) was confirmed.

CYEA (Casein 5g/L, Yeast extract 2.5g/L, Glucose 1g/L, Agar 18g/L) and 1% CMC (Carboxymethyl cellulose sodium salt 10g/L) sterilized in a medium mixed with paper disc Was raised, 30ul (1 X 10 ⁶ cfu) of the culture suspension of the isolate was inoculated, and then incubated at 30° C. for 40 hours. The solid medium was dyed with 0.1% Congo-red for 20 minutes at room temperature, followed by bleaching with 1M NaCl, and measuring the size of the yellow halo formed around the inoculum to measure the cellulose resolution. (Teather and Wood, 1982). The results of confirming the cellulose decomposition activity of Bacillus methylotropiccus DYGS-5 and 9 types are shown in [Fig. 3], and the size of the ring through the measurement of two repeated experiments is shown in [Table 1].

As can be seen in [Fig. 3] and [Table 1], the ring size of DYGS-5 (Gasanri 5) was found to be the largest with an average of 26.625 mm, which can be understood to be the best in cellulose resolution.

<Experimental Example 2-2> Protein resolution

An experiment to confirm the proteolytic activity of the isolated strain was performed as follows.

Put a sterilized paper disc on a medium mixed with 7% skim milk (70g/L) in CYEA (Casein 5g/L, Yeast extract 2.5g/L, Glucose 1g/L, Agar 18g/L) , 30ul (1 X 10 ⁶ cfu) of the culture suspension of the isolate was inoculated, and then incubated at 30° C. for 40 hours. After cultivation, protease activity can be confirmed by measuring the size of a clear zone formed around the inoculum (Kumar et al. 2005). The results of confirming the activities of 9 types other than Bacillus methylotropiccus DYGS-5 are shown in [Fig. 3] and [Table 1].

As a result of the above experiment, it was confirmed that the size of the transparent ring formed around DYGS-5 (Gasan-ri 5) was 25.5 mm, which is larger than the size of other strains. Accordingly, it can be seen that the protein resolution of DYGS-5 is excellent.

<Experimental Example 2-3> Phosphoric acid solubilization ability

Checking whether various phosphates accumulated in a large amount in an insoluble form, which are difficult for plants to absorb in the soil, are solubilized can be helpful in the development of multifunctional microbial preparations that function as biopesticides and biofertilizers. Accordingly, an experiment to confirm the phosphate solubilization of the isolated strain was performed as follows.

Pikovaskaya's medium (PVK; Glucose 10g/L, (NH ₄ ) ₂ SO ₄ 0.5g/L, KCl 0.2g/L, MgSO ₄ 0.1g/L) containing insoluble phosphoric acid (Ca ₃ (PO ₄ ) ₂ 5g/L) L, MnSO ₄ 0.002g/L, FeSO ₄ 0.002g/L, Yeast extract 0.5g/L, Agar 18g/L) on a sterilized paper disc, 30ul of the culture suspension of the isolate (1 X 10 ⁶ cfu) was inoculated and then incubated at 30° C. for 7 days. Then, the ability of phosphoric acid solubilization was determined according to the formation of a clear zone around the inoculum (Kumar et al. 2005). The results of confirming the phosphoric acid solubilization activity of Bacillus methylotropiccus DYGS-5 (Gasanri 5) and 9 types are shown in [Fig. 3] and [Table 1].

As can be seen in [Fig. 3] and [Table 1], in the antifungal activity results of Experimental Example 1, DYGS-5 (Gasanri 5) exhibited excellent phosphoric acid resolution with an average of 15.375 mm.

As can be seen from the above results, the new strain DYGS-5 of Bacillus methylotropicus of the present invention has excellent cellulose resolution, protein resolution, and phosphoric acid solubilization ability in addition to antifungal activity.

<110> Damyang-gun <120> Novel Bacillus methylotrophicus DYGS-5 Strain and Use Thereof <130> PP18-000-Damyang-Novel Bacillus <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1403 <212> DNA <213> Unknown <220> <223> Bacillus methylotrophicus <400> 1 agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac 60 gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg 120 ttgtttgaac cgcatggttc agacataaaa ggtggcttcg gctaccactt acagatggac 180 ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga 240 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300 gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360 aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt caaatagggc 420 ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480 atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 540 ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact 600 tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga 660 ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt 720 ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780 ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt 840 acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900 tggtttaatt cgaagcaacg cgaagaaccc ttaccaggtc ttgacatcct ctgacaatcc 960 tagtgatagg acgtcccctt ctgggggcag agtgacaggt ggtgcatggt tgtcgtcagc 1020 tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccaagat cttagttgcc 1080 agcattcagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1140 acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca 1200 aagggcagcg aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc 1260 agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg 1320 gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1380 cgaagtcggt gaggtaacct tta 1403

Claims (9)

A novel Bacillus methylotrophicus DYGS-5 strain (accession number KACC 92217P).
The method of claim 1,
The strain is a novel Bacillus methylotrophicus DYGS-5 ( Bacillus methylotrophicus DYGS-5) strain, characterized in that the 16S rRNA gene sequence comprises the sequence disclosed in SEQ ID NO: 1.
The method of claim 1,
The strain has antifungal activity, cellulose resolution, protein resolution or phosphoric acid solubilization ability,
The antifungal activity is a novel Bacillus methylotrophicus DYGS-5 ( Bacillus methylotrophicus DYGS-5) strain, characterized in that it is antifungal activity against wilt disease bacteria ( Fusarium oxysporum ) or capsicum anthrax ( Coletotrichum acutatum ).
(a) preparing a medium containing a carbon source, a nitrogen source, and a trace element, and (b) inoculating and culturing the Bacillus methylotropicus DYGS-5 strain in the medium.
Bacillus methylotrophicus DYGS-5 strain ( Bacillus methylotrophicus DYGS-5) culture method.
Bacillus methylotrophicus DYGS-5 strain ( Bacillus methylotrophicus DYGS-5) culture according to the method of claim 4.
The novel Bacillus methylotrophicus DYGS-5 ( Bacillus methylotrophicus DYGS-5) strain (accession number KACC 92217P) or its culture containing as an active ingredient, Fusarium oxysporum ( Fusarium oxysporum ) or Coletotricum acu Tartum ( Colletotrichum acutatum ) a composition for controlling plant diseases.
The method of claim 6,
The plant disease is a plant disease control composition, characterized in that the wither disease or red pepper anthrax.
The method of claim 6,
The composition further comprises an agrochemically acceptable carrier and at least one adjuvant selected from the group consisting of a wetting agent, a dispersant, an antifreeze agent, a thickener, a preservative and an electrodeposition agent.
A method for controlling wilt disease or red pepper anthrax, comprising the step of contacting a plant with the composition according to any one of claims 6 to 8.

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