KR20150057083A - Antimicrobial bacillus subtilis HC57 for cladobotryum mycophilum, and process for culturing thereof - Google Patents

Abstract

The present invention relates to Bacillus subtilis HC57, a microorganism, having antibacterial activity against Cladobotryum mycophilum. More specifically, the Bacillus subtilis HC57 has high compatibility with mushroom. Moreover, the optimum temperature for growth of the Bacillus subtilis HC57 is close to that of fungus. Therefore, when sprayed with fungus, the Bacillus subtilis HC57 can prevent growth of Cladobotryum mycophilum. Accordingly, the Bacillus subtilis HC57 is effective as an eco-friendly agent for pest control.

Description

Bacillus subtilis HC57 having antimicrobial activity against fluffy fungus, and a method for culturing the same,

The present invention relates to a Bacillus subtilis microorganism having an antibacterial activity against a fungus, and more particularly, to a microorganism having high affinity for fungi, The present invention relates to Bacillus subtilis HC57, which is effective as an environmentally friendly antiseptic to suppress the growth of fungal pathogens.

Mushroom cultivation, like the cultivation of other crops, causes not only a decrease in the production of mushroom but also a decrease in the quality of the mushroom due to various kinds of insects and germs. The damage caused by such insect pests and germs occurs during the growth, development, transportation and storage of mushrooms. In particular, the disease occurring during mushroom cultivation is largely divided into non-pathogenic disease and pathogenic disease depending on the cause of the disease . Non-pathogenic diseases are called abiotic diseases or non-infectious diseases and are mainly caused by inadequate environmental conditions of mushroom cultivation and poor cultivation facilities.

Pathogenic diseases are caused by the parasitism of harmful organisms, and they are usually infectious. Therefore, there are cases where the disease occurrence area and damage degree are markedly different. Such pathogenic diseases are called infectious diseases or biotic diseases. Pathogenic agents causing mushroom diseases are known as fungi, actinomycetes, bacteria, viruses and nematodes. Among these, mushroom diseases caused by fungi and bacteria include bacterial brown fungus, blue mold fungus, and fluffy fungus disease.

The bacterial brown foliage disease is a disease that causes quantitative and qualitative degradation of mushroom by changing the mushroom freshness to brown. It is caused by toxin called tolaasin which is mainly secreted by Pseudomonas tolaassi ≪ / RTI > Patent No. 10-0267752 discloses a technology for detecting and quantifying Pseudomonas tolashi bacteria causing mushroom bacterial galangal pathogens. However, studies on fluffy fungal diseases causing great damage to mushroom cultivated farms and their effective control There are few studies on biological control agents.

DISCLOSURE Technical Problem The present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide Bacillus subtilis HC7 which can be used as an environmentally friendly antiseptic agent because it can inhibit high affinity and growth of fungal pathogens against mushroom.

In addition, the present invention provides a microbial pesticide, a seed coating agent, a microbial pesticide for controlling a downy mildew fungus of a mushroom, and a non-sterilized microbicide for mushroom cultivation for the prevention of a fluffy fungus disease by spraying together with a mushroom fungus, Bacillus subtilis HC7, which can be added to additives, biofuels, microbiological nutrients, soil conditioners, composting homework, foliar spraying agents and traditional spraying agents.

The present invention relates to Bacillus altitudinis HC57, which comprises the nucleotide sequence of SEQ ID NO: 1 and which has the accession number KACC91891P.

According to a preferred embodiment of the present invention, the Bacillus subtilis HC57 may have an antimicrobial activity against a downy mildew disease caused by mushrooms.

According to another preferred embodiment of the present invention, the mushroom may be at least one mushroom of the group consisting of mushroom mushroom, oyster mushroom, and large mushroom mushroom.

The present invention also relates to a pharmaceutical composition comprising a carbon source comprising at least one of the group consisting of Fructose, Soluble starch, Xylose and Dextrine; An organic nitrogen source comprising at least one of the group consisting of Soytone, Yeast extract and Malt extract; NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ and NaNO ₃ ; But it includes at least one of the group consisting of asparagine, methionine, lecine, glutamine, valine, arginine, histidine and threonine Amino acids; And at least one inorganic salt selected from the group consisting of KCl, BaCl ₂ , CaCl ₂ , MgSO ₄ , FeSO ₄ , Na ₂ MoO ₄ and KH ₂ PO ₄ ; Lt; RTI ID = 0.0 > HC57 < / RTI >

According to a preferred embodiment of the present invention, the carbon source includes at least one of the group consisting of Xylose and Dextrine, and the organic nitrogen source is selected from the group consisting of Soytone and yeast extract ), the inorganic, and include at least one of the group nitrogen source consisting of the NH ₄ H ₂ PO ₄ and (NH _{4) 2} include HPO least one of the group consisting of ₄ species, and the amino acid asparagine (asparagine), Mesa At least one member selected from the group consisting of methionine, arginine and histidine, and the inorganic salts include at least one member selected from the group consisting of CaCl ₂ , MgSO ₄ and KH ₂ PO ₄ .

According to another preferred embodiment of the present invention, the culture medium contains 100-200 parts by weight of an organic nitrogen source, 30-100 parts by weight of an inorganic nitrogen source, 10-80 parts by weight of amino acid and 0.1-1 parts by weight of inorganic salts . ≪ / RTI >

Further, the present invention relates to a Bacillus subtilis HC57 culture method comprising culturing the Bacillus subtilis HC57 containing the nucleotide sequence of SEQ ID NO: 1 and having the accession number KACC91891P at a culture temperature of 25 to 35 DEG C for 20 to 30 hours in the medium as described above .

According to a preferred embodiment of the present invention, the culture may be cultured at pH 5.0 to 7.0.

In addition, the present invention provides an antifungal agent for controlling a fungus including Bacillus subtilis HC57, which contains the nucleotide sequence of SEQ ID NO: 1 and has the accession number KACC91891P.

According to a preferred embodiment of the present invention, the controlling agent is selected from the group consisting of microbial pesticides, seed coating agents, microbial pesticides for controlling downy mildew of mushrooms, additives for preparing mushroom cultivation non-sterilized microbes, biosatto, microbial nutrients, soil conditioner, A homework agent, a foliar spray agent, or a root spray agent.

The present invention provides Bacillus subtilis HC57 having a high affinity for mushroom and capable of effectively inhibiting the growth of a fungal pathogen, and a method for culturing the same.

In addition, the present invention has an effect of providing Bacillus subtilis HC57 which is effective as an environment-friendly antiseptic agent because the temperature of growth is similar to that of mushroom bacteria and can be sprayed together with mushroom bacteria.

Fig. 1 shows the result of incubation of HC57 and downy fungi selected in Example 2 against each other.
FIG. 2 is a diagram showing the genetic relationship of HC57 analyzed in Experimental Example 1. FIG.
Fig. 3 shows the results of the control effect measured in Experimental Example 2. Fig.
FIG. 4 shows the result of measuring the growth activity according to the culture temperature change in Experimental Example 3. FIG.
FIG. 5 shows the result of measuring the growth activity according to the change of culture pH in Experimental Example 3. FIG.
6 shows the result of measuring the growth activity according to the type of carbon source in Experimental Example 3. FIG. 6: glycerol, 7: xylose, 8: dextrose, 9: lactose, 10: dextrin, 11: sodium starch glycolate, 2: galactose, 3: saccharose, 4: soluble starch, 14: mannose, 15: maltose, 16: raffinose, 17: cellobis, 18: ethanol, 19: solicin)
FIG. 7 shows the result of measuring the growth activity according to the concentration of xylose in Experimental Example 3. FIG.
FIG. 8 shows the result of measuring the growth activity according to the type of organic nitrogen source in Experimental Example 3. FIG. (1: peptone, 2: soitone, 3: urea, 4: casamino acid, 5: tryptone, 6: yeast extract,
FIG. 9 shows the result of measuring the growth activity according to the concentration of soytone in Experimental Example 3. FIG.
10 shows the result of measuring the growth activity according to the type of inorganic nitrogen source in Experimental Example 3. FIG. (NH ₄ Cl ₂ , (COONH ₄ ) ₂ , 3: NH ₄ H ₂ PO ₄ , 4: (NH ₄ ) ₂ HPO ₄ , 5: NH ₄ NO ₃ , 6: NaNO ₃ , ₄ ) ₂ SO ₄ )
11 shows the result of measuring the growth activity according to the concentration of NH ₄ H ₂ PO ₄ in Experimental Example 3. FIG.
FIG. 12 shows the result of measuring the growth activity according to the amino acid type in Experimental Example 3. FIG. 6: valine, 7: aspartic acid, 8: glutamic acid, 9: arginine, 10: histidine, 11: cysteine, 12: threonine)
FIG. 13 shows the result of measuring the growth activity according to the concentration of histidine in Experimental Example 3. FIG.
Fig. 14 shows the result of measurement of the growth activity according to the kinds of inorganic salts in Experimental Example 3. Fig. (1: KCl, 2: BaCl ₂ , 3: CaCl ₂ , 4: CoCl ₂ , 5: Li ₂ SO ₄ , 6: MnSO ₄ , 7: MgSO ₄ , 8: ZnSO ₄ , 9: FeSO ₄ , ₃ , 11: Na ₂ MoO ₄ , 12: KH ₂ PO ₄ , 13: FeCl ₃ )
FIG. 15 shows the result of measuring the growth activity according to the concentration of CaCl ₂ in Experimental Example 3. FIG.

Hereinafter, the present invention will be described in more detail.

As described above, studies on fungi and bacteria, especially bacterial brown foliage disease and blue fungi disease, are active in pathogen causing mushroom disease, but studies on fluffy fungal disease causing great damage to mushroom cultivation farms There is little research on biological control agents that can effectively control these diseases.

The present invention sought to solve the above-mentioned problem by providing Accession No. KACC91891P Bacillus subtilis HC57. Thus, it is possible to effectively inhibit the growth of downy fungus pathogens with high affinity to mushrooms and to provide effective bacillus subtilis HC7 as an environmentally friendly antiseptic agent because the temperature of growth is similar to that of mushroom bacteria and can be sprayed together with mushroom bacteria It is effective.

The present invention provides Bacillus subtilis HC57, which contains the nucleotide sequence of SEQ ID NO: 1 and has the accession number KACC91891P.

The Bacillus subtilis HC57 has antimicrobial activity against the downy mildew fungus caused by mushroom, thereby effectively suppressing the growth of the downy fungus pathogen.

The mushroom is not particularly limited as long as it is cultivated in a farmhouse. Preferably, the mushroom may be at least one mushroom of the group consisting of mushroom mushroom, oyster mushroom and large mushroom, more preferably mushroom mushroom.

The present invention also relates to a pharmaceutical composition comprising a carbon source comprising at least one of the group consisting of Fructose, Soluble starch, Xylose and Dextrine; An organic nitrogen source comprising at least one of the group consisting of Soytone, Yeast extract and Malt extract; NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ and NaNO ₃ ; But it includes at least one of the group consisting of asparagine, methionine, lecine, glutamine, valine, arginine, histidine and threonine Amino acids; And at least one inorganic salt selected from the group consisting of KCl, BaCl ₂ , CaCl ₂ , MgSO ₄ , FeSO ₄ , Na ₂ MoO ₄ and KH ₂ PO ₄ ; Lt; RTI ID = 0.0 > HC57 < / RTI > culture medium.

The culture medium for culturing Bacillus subtilis HC57 is not particularly limited as long as it is a carbon source, an organic nitrogen source, an inorganic nitrogen source, an amino acid and an inorganic salt mixture used in a conventional culture medium. Preferably, 100-200 parts by weight of organic nitrogen source , 30 to 100 parts by weight of an inorganic nitrogen source, 10 to 80 parts by weight of amino acid and 0.1 to 1 part by weight of inorganic salts.

According to an embodiment of the present invention, the carbon source includes at least one kind selected from the group consisting of Xylose and Dextrine, and the organic nitrogen source is selected from the group consisting of Soytone and Yeast extract, Wherein the inorganic nitrogen source comprises at least one member selected from the group consisting of NH ₄ H ₂ PO ₄ and (NH ₄ ) ₂ HPO ₄ , wherein the amino acid is selected from the group consisting of asparagine, Methionine, arginine and histidine. The inorganic salts may include at least one of the group consisting of CaCl ₂ , MgSO ₄ and KH ₂ PO ₄ . have.

If xylose and / or methyl lin are used as a carbon source, high growth can be confirmed as shown in Fig.

If soitone and / or yeast extract were used as an organic nitrogen source, high growth was observed as seen in FIG.

If NH ₄ H ₂ PO ₄ and / or (NH ₄ ) ₂ HPO ₄ were used as an inorganic nitrogen source, high growth was confirmed as shown in FIG. 10.

When cultured using asparagine, mesaionine, arginine and / or histidine as an inorganic nitrogen source, high growth was confirmed as shown in FIG. 12.

When CaCl ₂ , MgSO ₄ and / or KH ₂ PO ₄ were used as inorganic salts, high growth was observed as shown in FIG. 14.

Further, the present invention provides a method for culturing Bacillus subtilis HC57, which comprises the nucleotide sequence of SEQ ID NO: 1 and has the accession number KACC91891P.

The culturing conditions include culturing at a culture temperature of 22 to 35 ° C for 20 to 30 hours, more preferably at 25 to 31 ° C for 22 to 27 hours (see FIG. 4).

If the culture is performed at a temperature lower than 22 캜, mycelial growth may be very slow and long-term cultivation may occur. When the culture is performed at a temperature exceeding 35 캜, rapid growth of mycelial growth and death May occur.

According to an embodiment of the present invention, the culture can be performed at pH 5.0 to 7.0, more preferably at pH 5.7 to 6.8 (see FIG. 5).

In addition, when cultured at a pH lower than pH 5.0, there is a problem that the growth is slow and the cultivation time becomes long, and when cultured at a pH exceeding 7.0, the growth is drastically slowed, Lt; / RTI >

In addition, the present invention provides an antifungal agent for controlling a fungus including Bacillus subtilis HC57, which contains the nucleotide sequence of SEQ ID NO: 1 and has the accession number KACC91891P.

The antiseptic agent containing Bacillus subtilis HC57 is not particularly limited as long as it is a conventional anticancer agent used for the prevention and treatment of fluffy fungi. Preferably, the anticancer agent, seed coating agent, microbial pesticide for controlling mildew fungus disease, Microbial nutrients, soil remediation agents, composting homework agents, foliar spraying agents or route spraying agents, for the production of a mushroom-free sterilization medium.

In particular, it has an excellent effect in inhibiting the growth of Cladobotryum mycophilum, which is a problem in cultivation of mushroom mushroom, and is useful as a microbial pesticide for controlling fungal diseases of mushroom, an additive for producing mushroom cultivation non-sterile medium, Can be utilized.

The microbial pesticide is not particularly limited as long as it is produced by a conventional method including Bacillus subtilis HC57.

For example, it may include Bacillus Subtilis HC57 and a biomatrix as a microorganism carrier, and the biomatrix may include a plant resource polymer material, a microbial biopolymer material, a nutrient, an auxiliary material, and an additive.

In addition, the plant resource polymer material may include potato powder, soy flour, starch powder and the like. The microbial biopolymer may include mucopolysaccharide, poly-beta hydroxy-alkanoate, and coagulant. Further, the nutrient may include glucose, peptone, and inorganic salts, and the auxiliary material may include vermiculite, rice hull powder, and the like, and the additive may include an extender, a UV blocker, and a surfactant.

The microbial pesticide may be used as an antifungal microbial pesticide, a seed coating agent, a microbial nutrient, a soil improving agent, a compostant homework, a leaf surface spraying agent, or a root spraying agent.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[ Example ]

Preparation Example . Fluffy fungus  Isolation and identification

In the mushroom cultivation area of mushroom mushroom, the affected part of the fruiting body showing the symptoms of downy fungal disease was surface sterilized and then cut into 1 cm size and impregnated with a potato dextrose agar (PDA) medium and cultured in a thermostat at 25 ° C for 3 to 4 days The hyphae of the hyphae were cut into 5 mm size using a flame sterilized knife, and cultured in PDA to isolate the pathogens. The genetic analysis of the internally transcribed spacer (ITS) region of the rDNA amplified for general purpose primer ITS1 (5'-TCCGTAGGTGAACCTGCG-3 ') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3' Clomobacterium mycophilum ( C. mycophilum ) was identified by mycological characteristics such as physiology, biochemistry and morphology, and pathogenicity test through bioassay .

In order to investigate the pathogenicity of C. mycophilum , the spore density of the pathogen was diluted to 5 × 10 ⁵ / ㎖ in sterilized water and sprayed with 25% ℃, 95% thermostat. After 2 days, pathogenicity was confirmed.

As a result, there was a slight difference according to the strains, but the pathogenicity of the typical fluffy fungus disease symptoms in the mushroom mushroom strain and fruiting body was confirmed, and it was confirmed that it is a pathogenic C. mycophilum.

Example  1. Isolation of useful microorganisms

In order to isolate useful microorganisms, three mushroom cultivars of mushroom mushroom cultivated from mushroom cultivated mushroom were collected and used for the experiment. The medium was diluted stepwise with serial dilution using sterilized water, streaked on R2A solid medium, and cultured in a 28 ° C incubator for 24 hours to isolate single colonies from 50-60 colony-forming plates And used as a microorganism.

The microorganisms were 1,500 strains. The strains were cultured in R2A medium for 2 days, and the cells were collected and stored in a 20% (v / v) glycerol solution at -70 ° C.

Example  2. Selection of antimicrobial microorganisms

Antibacterial microorganisms among the strains isolated in Example 1 were tested in a confluent culture method.

Specifically, in order to effectively test a large number of isolated strains, four strains of the strains isolated in Example 1 were inoculated at the same intervals in one petri dish. The flora of the fungus which can be used for confluent culture was confirmed in the preparation example. The fungus was cultivated in the PDA medium for 5 days, and then the edge was collected using a cork borer. The fungus was placed in the center of the PDA medium for assay, Were evaluated for antimicrobial activity against each strain.

As a result of the evaluation of the antibacterial ability, 3 kinds of antibacterial microorganisms HC57 and HC94 showing the strongest antimicrobial activity among 1,500 strains isolated in Example 1 were selected. Among them, HC57 showed the highest antimicrobial activity and HC57 was selected as an antibacterial microorganism. The results of the confluent culture of HC57 are shown in Fig.

Experimental Example  1. Identification of Selected Antimicrobial Microorganisms

For the identification of the antimicrobial microorganism HC57 selected in Example 2, genetic flexibility was analyzed.

DNA was isolated using Quiagen Genomic DNA Isolation Kit (Quiagen, USA) and PCR amplification was performed with Techne thermocycler (Techne LTD, Duxford, Cambridge, UK). The PCR mixture was diluted with 1 x buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl, 2.5 mM MgCl ₂ , 0.01% gelatin and 0.1% Triton X-100), deoxyribonucleotide triphosphates (dATP, dCTP, dTTP ), 0.6 U Taq DNA polymerase (Molecular Biochemicals, Mt Wellington, Auckland, New Zealand), a forward primer fD1 (5'-AGAGTTTGATCCTGGCTCAG-3 ') and a reverse primer rP2 5'-ACGGCTACCTTGTT ACGACTT-3 ')] and 10 ng template DNA. PCR was performed at 94 ° C for 1 minute, at 56 ° C for 1 minute, and at 72 ° C for 30 minutes. After the reaction, primers and dNTPs were amplified using High Pure PCR Product Purification Kit (Bioneer Co., Chungbuk, Korea) PCR product.

The purified PCR products were cloned into a pT7 blue vector (Novagen Co., Madison, WI, USA) and sequenced using Big Dye Terminator Kit and ABI Prism 310 Genetic Analyzer (Perkin Elmer, New Jersey, USA). The ClustralW analysis program was used to determine species similarity with other nucleotide sequences in GenBank. We used the Neighbor-joining method of MEGA 4 to construct the tree structure. The stability of the tree was investigated by bootstrap analysis of 1000 iterations.

After 16S rDNA analysis, the results were analyzed by various biochemical tests and microbiological classification (Bergey's manual of systematic bacteriology index). The result of the identification is shown in FIG. 2, and the 16S rDNA nucleotide sequence of the HC57 strain of the present invention is shown in SEQ ID NO: 1.

As can be seen in FIG. 2, HC57 was found to be a species of Bacillus altitudinis.

In order to understand the biological characteristics of HC57, growth was observed under various conditions described in Table 1 below. The growth results are shown in Table 1 below.

Experimental Example  2. Control effect of antimicrobial microorganisms

In order to test the control effect of HC57 on the fungus, mushroom cultivated in a box (30 cm x 20 cm) was used in the experiment. In a suspension prepared by spraying a pathogenic spore suspension with a pathogenicity confirmed in the preparation example in a mushroom of mushroom mushroom and stored at -70 ° C in a 20% (v / v) glycerol solution prepared in Example 1 30 minutes later, An HC57 suspension was sprayed and cultivated at a temperature of 17 ° C and a humidity of 87% in a growth chamber. The results of the control effect are shown in Table 2 and FIG.

As can be seen in Table 2 and FIG. 3, the control group showed 88.7% morbidity rate, but in the group treated with the antimicrobial microorganism HC57, the morbidity rate was 25.5%, showing a high control effect of 71.2%.

Experimental Example  3. Culture condition investigation

In order to investigate the optimal culture condition of HC57, growth and antibacterial activities were investigated using M9 Minimal medium (MBcell) at pH 3 ~ 10, culture temperature 10 ~ 45 ℃, carbon source, nitrogen source, Respectively.

Specifically, the growth was inoculated into R2A liquid medium at a concentration of 1 × 10 ⁷ cfu / ml and incubated at 30 ° C. for 24 hours at 180 rpm. The absorbance was measured with a spectrophotometer (US / Spectramax Plus 38, USA) The antimicrobial activity was measured by inoculating the PDA medium with the fungal pathogens and inoculating the antimicrobial microorganisms and incubating for 3 to 5 days. The results of growth and antibacterial activity according to the culture conditions are shown in Figs. 4 to 15.

As can be seen in FIG. 4, the growth temperature was rapidly increased from 20 ° C to 40 ° C, and the growth was abruptly lowered. It was also confirmed that the optimum growth was observed at 25 ° C even within this temperature range.

In addition, as shown in FIG. 5, the activity increased sharply from 5.0 at the culture pH to 7.0, and the highest growth was observed at pH 6.0.

Therefore, it showed a strong inhibitory effect against the pathogenic fungus at the culture temperature of 22 ~ 35 ℃ and pH 5.0 ~ 7.0.

Further, as shown in FIG. 6, the kind of the carbon source is selected from the group consisting of proctose, galactose, saccharose, soluble starch, inositol, glycerol, xylose, dextrose, lactose, dextrin, Na-CMC, adonitol, mannitol, mannose , Maltose, raffinose, cellobis, ethanol, and solicin. As a result, it was confirmed that fructose, soluble starch, Xylose and Dextrine were active. there was. In addition, the optimum carbon source was xylose, and as shown in FIG. 7, it was confirmed that the optimum concentration was 1.5% xylose, and the activity was most excellent when HC57 was cultured.

As shown in FIG. 8, peptone, soytone, urea, casamino acid, tryptone, yeast extract, and malt extract were used as organic nitrogen sources. Soiton, yeast extract and malt extract High activity was confirmed. Further, as shown in FIG. 9, the optimum concentration of organic nitrogen source was found to be the best when the HC57 was cultured at an optimum concentration of 2% soytone.

In addition, as shown in FIG. 10, the types of inorganic nitrogen sources include NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , NH ₄ NO ₃ , NaNO ₃ , ₄ ) ₂ SO ₄ , and NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ and NaNO ₃ activity were confirmed. Among them, NH ₄ H ₂ PO ₄ was the optimal inorganic nitrogen source , the optimum concentration is the culture when the activity of HC57 was the most excellent in 1% NH ₄ H ₂ PO ₄ as identified in Figure 11.

Furthermore, as shown in Fig. 12, the types of amino acids were asparagine, mesaionine, proline, leucine, glutamine, valine, aspartic acid, glutamic acid, arginine, histidine, cysteine and threonine The activity was confirmed in asparagine, methionine, leucine, glutamine, valine, arginine, histidine and threonine. In addition, as shown in FIG. 13, the optimum amino acid was the most active at the concentration of 0.5% as histidine in the culture of HC57.

Further, as shown in FIG. 14, the kind of the inorganic substance is selected from the group consisting of KCl, BaCl ₂ , CaCl ₂ , MgSO ₄ , ZnSO ₄ , FeSO ₄ , AgNO ₃ , Na ₂ MoO ₄ , KH ₂ PO ₄ , FeCl ₃ , CoCl ₂ , Li ₂ SO ₄ and MnSO ₄ were investigated. Among them, activity was confirmed in KCl, BaCl ₂ , CaCl ₂ , MgSO ₄ , FeSO ₄ , Na ₂ MoO ₄ and KH ₂ PO ₄ . In addition, the optimum mineral was CaCl ₂ , and as shown in FIG. 15, the activity of HC57 was the highest at 7 mM CaCl ₂ .

Table 3 below shows the optimal culture results.

As can be seen from the above Examples and Experimental Examples, the antimicrobial microorganism HC57 of the present invention has high affinity to mushroom and the temperature of growth is similar to that of mushroom, so that it is sprayed together with mushroom bacteria to inhibit the growth of downy fungus pathogens And it was confirmed that it is suitable as an environmentally friendly antiseptic.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Antimicrobial bacillus subtilis HC57 for cladobotryum mycophilum,          and process for culturing thereof <130> 1040145 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1366 <212> DNA <213> Agaricus bisporus <400> 1 ccgacttcgg gtgttacaaa ctctcgtggt gtgacgggcg gtgtgtacaa ggcccgggaa 60 cgtattcacc gcggcatgct gatccgcgat tactagcgat tccagcttca cgcagtcgag 120 ttgcagactg cgatccgaac tgagaacaga tttgtgggat tggcttaacc tcgcggtttc 180 gctgcccttt gttctgtcca ttgtagcacg tgtgtagccc aggtcataag gggcatgatg 240 atttgacgtc atccccacct tcctccggtt tgtcaccggc agtcacctta gagtgcccaa 300 ctgaatgctg gcaactaaga tcaagggttg cgctcgttgc gggacttaac ccaacatctc 360 acgacacgag ctgacgacaa ccatgcacca cctgtcactc tgcccccgaa ggggacgtcc 420 tatctctagg attgtcagag gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat 480 taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat tcctttgagt ttcagtcttg 540 cgaccgtact ccccaggcgg agtgcttaat gcgttagctg cagcactaag gggcggaaac 600 cccctaacac ttagcactca tcgtttacgg cgtggactac cagggtatct aatcctgttc 660 gctccccacg ctttcgctcc tcagcgtcag ttacagacca gagagtcgcc ttcgccactg 720 gtgttcctcc acatctctac gcatttcacc gctacacgtg gaattccact ctcctcttct 780 gcactcaagt tccccagttt ccaatgaccc tccccggttg agccgggggc tttcacatca 840 gacttaagaa accgcctgcg agccctttac gcccaataat tccggacaac gcttgccacc 900 tacgtattac cgcggctgct ggcacgtagt tagccgtggc tttctggtta ggtaccgtca 960 aggtaccgcc ctattcgaac ggtacttgtt cttccctaac aacagagctt tacgatccga 1020 aaaccttcat cactcacgcg gcgttgctcc gtcagacttt cgtccattgc ggaagattcc 1080 ctactgctgc ctcccgtagg agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc 1140 tctcaggtcg gctacgcatc gttgccttgg tgagccgtta cctcaccaac tagctaatgc 1200 gccgcgggtc catctgtaag tggtagccga agccaccttt tatgtttgaa ccatgcggtt 1260 caaacaacca tccggtatta gccccggttt cccggagtta tcccagtctt acaggcaggt 1320 tacccacgtg ttactcaccc gtccgccgct aacatcaggg agcaag 1366

Claims (10)

Bacillus altitudinis HC57, which contains the nucleotide sequence of SEQ ID NO: 1 and which has the accession number KACC91891P. The bacillus subtilis HC57 according to claim 1, wherein the Bacillus subtilis HC57 has an antibacterial activity against a downy mildew fungus caused by mushroom. The bacillus subtilis HC57 according to claim 2, wherein the mushroom is at least one mushroom of the group consisting of mushroom mushroom, oyster mushroom and large mushroom. A carbon source comprising at least one of the group consisting of Fructose, Soluble starch, Xylose and Dextrine;
An organic nitrogen source comprising at least one of the group consisting of Soytone, Yeast extract and Malt extract;
NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ and NaNO ₃ ;
But it includes at least one of the group consisting of asparagine, methionine, lecine, glutamine, valine, arginine, histidine and threonine Amino acids; And
Inorganic salts including at least one member selected from the group consisting of KCl, BaCl ₂ , CaCl ₂ , MgSO ₄ , FeSO ₄ , Na ₂ MoO ₄ and KH ₂ PO ₄ ;
Lt; RTI ID = 0.0 &gt; HC57 &lt; / RTI &gt; culture medium. 5. The method of claim 4, wherein the carbon source comprises at least one of the group consisting of Xylose and Dextrine, wherein the organic nitrogen source is selected from the group consisting of Soytone and Yeast extract the inorganic nitrogen source, and including at least one of the NH ₄ H ₂ PO ₄ and (NH _{4) 2} include one or more of the group consisting of HPO _4, wherein the amino acid is asparagine (asparagine), mesa EO Nin (Methionine ), arginine (arginine), and histidine (including at least one of the group consisting of histidine), and the inorganic salts Bacillus, including at least one of the group consisting of CaCl _2, MgSO _4, and KH ₂ PO ₄ sub Tirith HC57 Culture medium. The culture medium according to claim 4, wherein the culture medium comprises 100 to 200 parts by weight of an organic nitrogen source, 30 to 100 parts by weight of an inorganic nitrogen source, 10 to 80 parts by weight of amino acid and 0.1 to 1 part by weight of inorganic salts based on 100 parts by weight of carbon source As a medium for culture of Bacillus subtilis HC57. Bacillus subtilis HC57 containing the nucleotide sequence of SEQ ID NO: 1 and having the accession number KACC91891P is cultivated at a culture temperature of 25 to 35 DEG C for 20 to 30 hours in a medium for culturing Bacillus subtilis HC57 of claim 5 or 6, Tirith HC57 culture method. 8. The method according to claim 7, wherein the culture is performed at a pH of 5.0 to 7.0. A control agent for controlling a fungus comprising Bacillus subtilis HC57, which contains the nucleotide sequence of SEQ ID NO: 1 and has the accession number KACC91891P. The antifungal agent according to claim 9, wherein the controlling agent is at least one member selected from the group consisting of a microbial pesticide, a seed coating agent, an additive for producing a mushroom-free non-sterilizing medium, a leaf side spraying agent, and a root spraying agent.

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