KR101734159B1 - Antimicrobial Bacillus Methylotrophicus for Blue Mold, and Process for Culturing Thereof - Google Patents

Abstract

The present invention relates to the use of Bacillus methylotrophicus ( Bacillus sp. Methylotrophicus ), more specifically, it can effectively inhibit the growth of blue mold pathogens and has a high affinity for mushrooms, and the optimum temperature for growth is similar to that of mushroom bacteria, so that they can be sprayed together with mushroom bacteria , It is effective to provide an effective Bacillus methylotrophicus strain as an environment-friendly antiseptic agent.

Description

[0001] The present invention relates to a Bacillus methylotrophicus strain having antimicrobial activity against a blue fungal pathogen, and a method for culturing the Bacillus methylotrophicus for Blue Mold,

The present invention relates to a strain of Bacillus methylotrophicus having antimicrobial activity against a fungal pathogenic fungus, more particularly, to a strain having high affinity for fungi, The present invention relates to Bacillus methylotrophicus CH518, which is effective as an environmentally friendly antiseptic agent by spraying with bacteria to inhibit the growth of blue mold 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, transport and storage of mushroom. 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 have. Non-pathogenic diseases are said to be 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 blue fungus disease is a disease caused by artificial cultivation of all the mushrooms such as mushroom, oyster mushroom and grass mushroom which are artificial cultivated, and it is a collective term of a disease which has a white color or spore formed in the mycelial growth period, To kill mycelium or to secrete toxins to inhibit mycelial growth and mycelial growth.

The pathogenicity of blue fungus disease depends on the type of fungus, among which Trichoderma spp.) is the most frequently occurring, and the damage is most severe. This bottle is spontaneously spores on the mycelium. The blue color is incomplete generation. The complete generation belongs to the genus of Hypocrea. It is generally not formed well in the laboratory medium and various kinds (Mushroom cultivation technique; Park, Won-Mok, et al., Journal of the Korean Society of Plant Growth, v.31, no.2, 10 (2): 91, 1971).

Symptoms of blue fungus disease begin to occur in the pods and seedlings, and the hyphae of white color grow blue. The infected mushroom hyphae will die, brown spots will appear on the surface of fresh mushrooms, and brownish brown lesions will form on the surface of mushrooms. Symptoms of bacterial brown foliage disease are mushroom fruiting surface with brown spots and irregular large lesions of dark brown color, and if the cell destruction is severe, the part becomes dented. In the case of mushroom mushroom, blue mildew is not directly parasitized in a healthy mushroom, but when the mushroom absorbs the toxic metabolite of gliotoxin secreted by the bacterium, the mushroom is transformed from brown to brown, The entire mushroom will die.

Accordingly, the present inventors have made extensive efforts to select microorganisms having a high antimicrobial activity against pathogenic microorganisms causing mildew fungi. As a result, the inventors of the present invention selected Bacillus methylotrophicus strains having high antimicrobial activity, It has been confirmed that there is no precedent used for the strain of the genus Trichoderma, which is a causative organism of fungal diseases, and the present invention has been completed.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention provides a Bacillus methylotrophicus strain having antibacterial activity against a fungal pathogen.

A second object of the present invention is to provide a medium for growth of the Bacillus methylotrophicus strain.

A third object of the present invention is to provide a culture method for the growth of the Bacillus methylotrophicus strain.

A fourth object of the present invention is to provide a controlling agent for the control of blue fungus bacterium comprising the above-mentioned Bacillus methylotrophicus strain as an active ingredient.

In order to solve the first problem of the present invention described above, the present invention provides a method for producing a microorganism having Bacillus microtrophicus Methylotrophicus ).

According to a preferred embodiment of the present invention, the Bacillus methylotrophicus strain may be Bacillus methylotrophicus strain of Accession No. KACC91999P.

According to another preferred embodiment of the present invention, the blue fungus pathogen is Trichoderma harzianum , which can cause a blue fungus disease in at least one mushroom of the group consisting of mushroom mushroom, oyster mushroom and large mushroom .

In order to solve the second problem of the present invention, the culture medium for Bacillus methylotrophicus strain of the present invention is a culture medium containing Saccharose, Xylose, Mannitol, Cellobiose and Salicine A carbon source comprising at least one of the group consisting of; An organic nitrogen source comprising at least one of the group consisting of Soytone, Yeast extract and Malt extract; An inorganic nitrogen source comprising at least one member selected from the group consisting of ammonium monophosphate (NH ₄ H ₂ PO ₄ ), ammonium diphosphate ((NH ₄ ) ₂ HPO ₄ ) and sodium nitrate (NaNO ₃ ); An amino acid comprising at least one member selected from the group consisting of asparagine, valine, glutamic acid and histidine; And one of the group consisting of potassium chloride (KCl), barium chloride (BaCl ₂ ), calcium chloride (CaCl ₂ ), magnesium sulfate (MgSO ₄ ), sodium molybdate (Na ₂ MoO ₄ ) and potassium phosphate (KH ₂ PO ₄ ) And inorganic salts including the above.

According to a preferred embodiment of the present invention, the Bacillus methylotrophicus strain may be Bacillus methylotrophicus CH518 of Accession No. KACC91999P.

According to another preferred embodiment of the present invention, the carbon source comprises Saccharose, the organic nitrogen source comprises Soytone, and the inorganic nitrogen source is ammonium diphosphate ((NH ₄ ) ₂ HPO ₄ ), the amino acid includes glutamic acid, and the inorganic salt may include magnesium sulfate (MgSO ₄ ).

According to another preferred embodiment of the present invention, the 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 >

In order to solve the third problem of the present invention, the method for culturing a strain of Bacillus methylotrophicus according to the present invention comprises culturing in the medium for cultivation of Bacillus methylotrophicus CH518 at a culture temperature of 25 to 35 DEG C for 20 to 30 hours do.

According to a preferred embodiment of the present invention, the Bacillus methylotrophicus strain may be Bacillus methylotrophicus strain of Accession No. KACC91999P.

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

In order to solve the third problem of the present invention, the present invention includes a controlling agent for the control of blue fungus, comprising a Bacillus methylotrophicus strain having an antibacterial activity against a blue fungus hospital.

According to a preferred embodiment of the present invention, the Bacillus methylotrophicus strain may be Bacillus methylotrophicus strain of Accession No. KACC91999P.

According to another 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 mold of mushroom, additives for preparing mushroom cultivation non-sterilized microbes, biofabric, microbial nutrients, soil conditioner, A homework agent, a foliar spray agent, or a root spray agent.

The present invention provides a Bacillus methylotrophicus strain having a high affinity to mushroom and capable of effectively inhibiting the growth of a pathogenic fungus pathotype of mushroom mushroom and a method for culturing the same. The Bacillus methylotrophicus strain of the present invention has an excellent antimicrobial activity against blue fungus pathogens as compared to other Bacillus sp. Strains. The medium composition and culture method of the present invention are most effective for culturing Bacillus methylotrophicus have.

In addition, the present invention has an effect of providing an effective Bacillus methylotrophicus strain 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 is a schematic diagram of Trichoderma harzianum) trophy kusu (Bacillus strain as Bacillus and methyl Methylotrophicus ) CH518.
Fig. 2 is data showing genetic relationship (A) and nucleotide sequence information (B) of 16S rRNA by 16S rRNA analysis of the selected microorganism CH518.
Fig. 3 shows the antimicrobial effect of CH518 against mushroom mushroom.
FIG. 4 shows the results of measuring the growth activity of CH518 according to the culture temperature (A) and the culture pH (B).
5 shows the results of measuring the growth activity (A) of CH518 and the growth activity (B) of CH518 according to the concentration of saccharose according to the type of carbon source (1; fructose, 2: galactose, 3: saccharose, 4: Starch 5: inositol 6: glycerol 7: xylose 8: dextrose 9: lactose 10: dextrin 11: Na-CMC 12: adonitol 13: mannitol 14: mannose 15: Maltose, 16: raffinose, 17: cellobiose, 18: ethanol, 19: salicin).
FIG. 6 shows the results of measurement of the growth activity (B) of CH518 according to the organic matter (A) and the concentration of soytone (1: peptone, 2: soitone, 3: urea, Minothio, 5: tryptone, 6: yeast extract, 7: malt extract).
7 is a result of measuring the growth activity (B) of the CH518 according to the concentration of the growth activity (A) and NH ₄ H ₂ PO ₄ in CH518 according to the kind of the inorganic nitrogen source is _{(1: 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 , 7: (NH ₄ ) ₂ SO ₄ ).
FIG. 8 shows the results of measuring the growth activity (B) of CH518 according to the growth activity (A) and glutamic acid concentration of CH518 according to the kinds of amino acids (1: asparagine, 2: methionine, 3: proline, 5: glutamine, 6: valine, 7: aspartic acid, 8: glutamic acid, 9: arginine, 10: histidine, 11: cysteine, 15: threonine).
9 shows the results of measurement of the growth activity (B) of CH518 according to the growth activity (A) and MgSO ₄ concentration of CH518 according to the inorganic salts (1: KCL, 2: BaCl ₂ , 3: CaCl ₂ , 10: AgNO ₂ , 11: Na ₂ MoO ₄ , 12: KH ₂ PO ₄ , 13: CoCl ₂ , 5: Li ₂ SO ₄ , 6: MnSO ₄ , 7: MgSO ₄ , 8: ZnSO ₄ , 9: FeSO ₄ , FeCl _2).

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

As described above, studies on a strain having a resistance to mushroom disease caused by a strain of Trichoderma spp. Have been carried out, but the mushroom blue mold disease causing a great damage to the mushroom cultivation farm There is little research on biological control agents that can be effectively controlled.

The present invention relates to the use of Bacillus methylotrophicus Methylotrophicus ) to solve the above-mentioned problems. It has high affinity to mushroom and can effectively inhibit the growth of blue mushroom pathogenic bacterium. It can spray with mushroom bacteria because the temperature of growth is similar to that of mushroom bacteria. Therefore, it is effective as an environmentally friendly anti-bacterial agent. Bacillus methylotrophicus Thereby providing a strain.

The present invention relates to the use of Bacillus methylotrophicus ( Bacillus sp. Methylotrophicus ).

FIG. 1 shows that the strain selected in the present invention has a high antimicrobial activity against a blue fungal pathogen, as a result of confluence of Trichoderma harzianum , one of the fungal pathogens of the blue mold, and the strain selected in the present invention. Respectively. Also, FIG. 2A shows that the strain of the present invention corresponds to Bacillus methylotrophicus species by genetic relationship between 16S rRNA analysis of strains selected in the present invention, and the strain was transformed with Bacillus methylotrophicus CH518 Respectively.

Thus, the strain is a Bacillus methylotrophicus strain containing the nucleotide sequence of SEQ ID NO: 1 (FIG. 2B) and having the accession number KACC91999P.

The above-mentioned Bacillus methylotrophicus CH518 has an antimicrobial activity against the blue fungus disease caused by mushroom, and it is confirmed that the growth of blue fungus pathogens can be effectively inhibited. Therefore, the Bacillus methylotrophicus CH518 of the present invention is superior to the other Bacillus methylotrophicus strains belonging to the same species and has an antimicrobial activity against blue fungus pathogens, as well as Bacillus subtilis ( Bacillus subtilis ) ( Bacillus spp.) Or Streptomyces spp., Which contains the antimicrobial activity of the strain Bacillus spp.

The blue fungus pathogen is preferably Trichoderma harzianum , but the present invention is not limited thereto. The Bacillus methylotrophicus strain of the present invention may be a fungus causing fungal diseases other than tricodelma arsenium Lt; / RTI >

The blue mold pathogen is a pathogenic bacterium that induces a blue fungus disease in mushroom. The mushroom is not particularly limited as long as it is cultivated in a farmhouse. Preferably, the mushroom is one or more of mushroom, oyster mushroom and big mushroom Mushroom, and 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 Saccharose, Xylose, Mannitol, Cellobiose and Salicine; An organic nitrogen source comprising at least one of the group consisting of Soytone, Yeast extract and Malt extract; An inorganic nitrogen source comprising at least one member selected from the group consisting of ammonium monophosphate (NH ₄ H ₂ PO ₄ ), ammonium diphosphate ((NH ₄ ) ₂ HPO ₄ ) and sodium nitrate (NaNO ₃ ); An amino acid comprising at least one member selected from the group consisting of asparagine, valine, glutamic acid and histidine; And one of the group consisting of potassium chloride (KCl), barium chloride (BaCl ₂ ), calcium chloride (CaCl ₂ ), magnesium sulfate (MgSO ₄ ), sodium molybdate (Na ₂ MoO ₄ ) and potassium phosphate (KH ₂ PO ₄ ) Inorganic salts including the above; And a culture medium for culturing Bacillus methylotrophicus strain.

The culture medium for culturing Bacillus methylotrophicus strain 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, but is preferably 100-200 parts by weight 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.

5 to 9 show the degree of growth of Bacillus methylotrophicus according to the type of carbon source, organic nitrogen source, inorganic nitrogen source, amino acid, and inorganic salt. According to one embodiment of the present invention, wherein the organic nitrogen source comprises Soytone and the inorganic nitrogen source comprises ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ), wherein the amino acid comprises glutamic acid , and the inorganic salts may include magnesium sulfate (MgSO _4).

((NH ₄ ) ₂ HPO ₄ ) was added as an inorganic mineral substance when cultured in the presence of saccharose as a carbon source (FIG. 5) In the case of culturing (FIG. 7), when glutamic acid is added as an amino acid and cultured (FIG. 8), magnesium sulfate (MgSO ₄ ) is added as inorganic salts and cultured. As shown in the respective figures, It was possible to confirm the high growth of the strain.

Further, the present invention provides a method for culturing a Bacillus methylotrophicus strain having an antimicrobial activity against a fungal pathogenic fungus, wherein the Bacillus methylotrophicus strain comprises a nucleotide sequence of SEQ ID NO: 1 and is selected from the group consisting of Bacillus subtilis strain KACC91999P Methyltropicus strain.

FIG. 4 shows the results of measurement of the growth activity of Bacillus methylotrophicus strain according to the culture temperature and culture pH. The culture conditions are as follows: culture at a temperature of 22 to 35 ° C for 20 to 30 hours, And culturing at 25 to 31 DEG C for 22 to 27 hours. Also, according to an embodiment of the present invention, the culture may be cultured at a pH of 5.0 to 7.0, more preferably at a pH of 5.7 to 6.8.

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.

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 a controlling agent for the control of blue fungus including a Bacillus methylotrophicus strain having an antimicrobial activity against a blue fungal pathogen, wherein the Bacillus methylotrophicus strain comprises the nucleotide sequence of SEQ ID NO: 1 And Bacillus methylotrophicus strain, accession number KACC91999P.

FIG. 3 shows the antibacterial effect of the Bacillus methylotrophicus strain of the present invention on the mushroom of the present invention. According to one embodiment of the present invention, a pathogen spore suspension is sprayed on the mushroom fruiting body and then the bacillus methyltropicus strain of the present invention As a result of the spraying of the suspension of kus, the mating percentage was 87.6% in the untreated group, but 19.5% in the group treated with the antibacterial microorganism CH518 showed a high control effect of 77.7%.

The controlling agent comprising the Bacillus methylotrophicus strain of the present invention is not particularly limited as long as it is a conventional controlling agent used for the prevention and treatment of blue fungus disease. Preferably, the controlling agent is a microbial pesticide, a seed coating agent, Microbial nutrients, soil remediation agents, composting homework agents, foliar spraying agents, or cross-linking spraying agents, for example, for controlling microbial pesticides for control, microbial pesticides for controlling microbes,

Particularly, it has been reported that blue mold fungus ( Trichoderma harzianum ), and thus can be used for the production of microbial pesticides for controlling mildew fungus, additives for the production of mushroom-free microbicides, and bio-soil.

The microbial pesticide is not particularly limited as long as it is produced by a conventional method including the Bacillus methylotrophicus strain of the present invention.

For example, the biomatrix may include a Bacillus methylotrophicus strain 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 . Blue fungus  Isolation and identification

In the mushroom cultivation area of the mushroom cultivar, the affected part of the fruiting body showing the symptoms of blue fungus was surface-sterilized and then cut into 1 cm size and placed in a potato dextrose agar (PDA) medium, cultured in a thermostat at 25 ° C for 3 to 4 days The hyphae were cut into 5 mm size using a flame-sterilized knife and re-cultured on PDA to obtain seven The pathogens were separated pure. Genes of purely isolated pathogenic bacterium were extracted and analyzed for genetic analysis, physiology, biochemistry and morphology of ITS (internal transcribed spacers) region of rDNA amplified by the universal primer sequence (Genocell, Korea) ( Trichoderma harzianum ) was identified through pathogenicity test through the biochemical test.

Primer sequences for gene amplification of pathogens Sequence information SEQ ID NO: forward ITS1 5 '- TCC GTA GGT GAA CCT GCG G-3' SEQ ID NO: 2 reverse ITS4 5 '- TCC TCC GCT TAT TGA TAT GC-3' SEQ ID NO: 3

In order to investigate the virulence of P. tumefaciens isolates, the microorganisms were diluted to a concentration of 5 × 10 ⁵ / ml of pathogenic microorganisms in sterilized water and sprayed with microbial spores diluted on the surface of fresh mushroom After incubation at 25 ° C and 95% in a thermostat, 2 days later, pathogenicity was confirmed.

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

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 blue fungus used in the incubation culture was confirmed to have pathogenicity in the preparation example. After culturing for 5 days in the PDA medium, the edge was collected using a cork borer, and it was placed in the center of the PDA medium for assay, Were evaluated for antimicrobial activity against each strain.

As a result of the above antibacterial ability evaluation, three kinds of antibacterial microorganisms CH12, CH20 and CH518 showing the strongest antimicrobial activity among the 1,500 strains isolated in Example 1 were selected. Among them, CH518 showed the highest antimicrobial activity and CH518 was selected as an antibacterial microorganism Respectively. The results of the confluent culture of CH518 are shown in Fig.

Experimental Example  1. Identification of Selected Antimicrobial Microorganisms

In order to isolate the antibacterial microorganism CH518 selected in Example 2, the 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 and reverse primer (primer of Table 2; . 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.

Primer sequences for gene amplification of antimicrobial microorganisms Sequence information SEQ ID NO: forward (fD1) 5'-AGAGTTTGATCCTGGCTCAG-3 ' SEQ ID NO: 4 reverse (rP2) 5'-ACGGCTACCTTGTT ACGACTT-3 ' SEQ ID NO: 5

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. 2A, and the nucleotide sequence of the 16S rDNA of the CH518 strain of the present invention is shown in SEQ ID NO: 1 (FIG. 2B).

As will be confirmed from FIG. 2, CH518 is was confirmed that species trophy kusu (Bacillus Methylotrophicus) of the Bacillus methyl, was named the strain as Bacillus trophy kusu CH518 as methyl.

In order to understand the biological characteristics of Bacillus methylotrophicus CH518 (hereinafter referred to as " CH518 "), growth was observed under various conditions described in Table 3 below.

Characteristics of Bacillus methylotrophicus CH518 Characters CH518 Characters CH518 Anaerobic growth -  alpha -galactosidase - Oxidase +  β-galactosidase - Growth in 10% NaCl +  β-glucuronidase - Maximum growth temp (10) -  α-glucosidase - Minimum growth temp (50) +  β-glucosidase - Utilization of  N-acetyl-b-glucosaminidase -   Ethanol +  a-mannosidase -   Methanol +  a-fucosidase - Nitrate reduction to nitrite +  Trisodium citrate + Alkaline phosphatase +  Capric acid - Esterase (C4) +  Phenylacetic acid - Arginine dihydrolase -  Tryptophane - b-Galactosidase -  Gelatine + Esterase lipase (C8) +  D-glucose + Naphthol-AS-BI-phosphohydrolase +  L-arabinose + Valine arylamidase +  D-mannose + Lipase (C14) -  D-mannitol + Leucine arylamidase -  N-acetyl-glucosamine + Trypsin -  Maltose + Acid phosphatase +  Potassium gluconate + Cystine arylamidase -  Adipic acid - alpha-chymotrypsin -  Malic acid +

The above strain was deposited at the National Institute of Agricultural Science and Technology on October 23, 2014 (Accession No. KACC91999P).

Experimental Example  2. Control effect of antimicrobial microorganisms

In order to test the control effect of CH518 on blue mold, mushroom 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, A CH518 suspension was sprayed and cultivated in a growth chamber at a temperature of 17 ° C and a humidity of 87%. The results of the control effect are shown in Table 4 and FIG.

Item name Processing name Disease rate (%) Control (%) Mushroom mushroom No treatment 87.6 Antagonistic CH518 19.5 77.7

As shown in Table 4 and FIG. 3, the control group showed 87.6% morbidity rate in the control group but 19.5% in the group treated with the antibacterial microorganism CH518, showing a high control effect of 77.7%.

Experimental Example  3. Culture condition investigation

In order to investigate optimal culture conditions of CH518, growth and antimicrobial activities were investigated using M9 Minimal medium (MBcell) at pH 3 ~ 10, culture temperature 10 ~ 45 ℃, carbon source, nitrogen source, amino acid and inorganic salts Activity.

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 by a spectrophotometer (US / Spectramax Plus 38, USA).

3-1: Culture temperature and pH  According to the change CH518 Degree of growth  Confirm

CH518 was cultured by the culture method of Experimental Example 3 at a culture temperature of 10 to 45 ° C and a pH of 3 to 10 to measure the absorbance change by strain growth.

As can be seen in FIG. 4A, the growth of CH518 began to increase from 15 ° C after the incubation temperature and remained at 45 ° C. However, it was confirmed that the growth of CH518 gradually decreased after 25 ° C. That is, it was confirmed that the optimum growth was observed at 25 ° C even within this temperature range.

In addition, as shown in FIG. 4B, the growth rapidly increased from pH 4.0 after 4.0 to the growth of 7.0, and the highest growth was observed at pH 6.0.

As a result, it showed a strong inhibitory effect against the pathogenic bacteria, blue mold, at a temperature of 22 to 35 ° C and a pH of 5.0 to 7.0.

3-2: On carbon  Following CH518 Degree of growth  Confirm

Examples of the carbon source include fructose, galactose, saccharose, soluble starch, inositol, glycerol, xylose, dextrose, Lactose, Dextrine, Na-CMC, Adonitol, Mannitol, Mannose, Maltose, Raffinose, Cellobiose, Ethanol ) And Salicine, respectively, and CH518 was cultured by the culture method of Experimental Example 3 to measure the absorbance change according to the strain growth.

Further, as shown in FIG. 5A, it was confirmed that the growth of CH518 was high in saccharose, xylose, mannitol, cellobiose, and salicin. In addition, the optimum carbon source was saccharose, and as shown in Fig. 5B, it was confirmed that the growth of CH518 was the most excellent in 3.0% saccharose.

3-3: Depending on organic matter CH518 Degree of growth  Confirm

A culture medium containing peptone, Soytone, Urea, Casamino acid, Tryptone, yeast extract and Malt extract as an organic substance, CH518 was cultured in accordance with the culture method of Experimental Example 3, and the absorbance change according to the strain growth was measured.

As can be seen in FIG. 6A, high strain growth was observed in soytone, yeast extract and malt extract, and it was confirmed that the best strain growth was obtained when soiton was added as an organic nitrogen source. As shown in FIG. 6B, it was confirmed that the growth of CH518 was the most excellent in 1.5% soytone.

3-4: According to mineral demand CH518 Degree of growth  Confirm

Ammonium chloride as inorganic nitrogen source _{(NH 4 Cl), (COONH} 4) 2, first ammonium phosphate _{(NH 4 H 2 PO 4)} , the second ammonium phosphate _{((NH 4) 2 HPO 4} ), ammonium nitrate (NH ₄ NO ₃ ), sodium nitrate (NaNO ₃ ) and ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) were cultured in the same manner as in Example 3, and the absorbance of the strain was measured.

As shown in FIG. 7A, high strain growth was confirmed in NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ and NaNO ₃ , and when NH ₄ H ₂ PO ₄ was added as an inorganic nitrogen source, And the growth rate was observed. The growth of CH518 in 1% NH ₄ H ₂ PO ₄ as identified in Figure 7B was confirmed that the best.

3-5: Depending on the amino acid CH518 Degree of growth  Confirm

Amino acids include asparagine, methionine, proline, leucine, glutamine, valine, aspartic acid, glutamic acid, arginine, histidine, CH518 was cultured in the culture medium containing Histidine, Cysteine, and Threonine, respectively, by the culture method of Experimental Example 3, and the absorbance changes according to the strain growth were measured.

As can be seen from FIG. 8A, high strain growth was observed in asparagine, valine, glutamic acid, and histidine, and it was confirmed that the best strain growth was observed when glutamic acid was added as an amino acid. As shown in Fig. 8B, it was confirmed that the growth of CH518 was the most excellent in glutamic acid at a concentration of 2.0%.

3-6: According to inorganic salts CH518 Degree of growth  Confirm

Potassium chloride as an inorganic salt (KCl), barium chloride (BaCl _2), acid calcium (CaCl _2), cobalt chloride (CoCl _2), lithium sulfate (Li ₂ SO _4), manganese sulfate (MnSO _4), magnesium sulfate (MgSO ₄ ), zinc sulfate (ZnSO _4), ferrous sulfate (FeSO _4), silver nitrate (AgNO _3), sodium molybdate (Na ₂ MoO _4), potassium phosphate (KH ₂ PO _4), including ferric chloride (FeCl _3), respectively CH518 was cultured in the culture medium of Experimental Example 3 to measure the absorbance change according to the strain growth.

As can be seen in FIG. 9A, high strain growth was confirmed in KCl, BaCl ₂ , CaCl ₂ , MgSO ₄ , Na ₂ MoO ₄ and KH ₂ PO _{4. When} MgSO ₄ was added as inorganic salts, Respectively. In 10mM MgSO ₄ in the growth of CH518 was confirmed that the most excellent, as identified in FIG. 9B.

Table 5 below shows the optimal culture results.

Incubation temperature   25 ℃ pH    6.0 Incubation time    24 hours     Nutrient source    Carbon source    3.0% Saccharose    Organic matter    1.5% Soytone    Inorganic matter 1% NH ₄ H ₂ PO ₄    amino acid    2.0% Glutamic acid    Inorganic salts 10 mmol < _{RTI ID =}

As can be seen from the above Examples and Experimental Examples, the antimicrobial microorganism CH518 of the present invention has a 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 blue mold pathogens And it was confirmed that it is suitable as an environmentally friendly antiseptic.

National Institute of Agricultural Science KACC91999P 20141023

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Antimicrobial Bacillus Methylotrophicus CH518 for Blue Mold, and          Process for Culturing Thereof <130> 1040903 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 1366 <212> DNA <213> Bacillus Methylotrophicus CH518 <400> 1 tagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta 60 agactgggat aactccggga aaccggggct aataccggat ggttgtttga accgcatggt 120 tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag 180 ttggtgaggt aacggctcac caaggcgacg atgcgtagcc gacctgagag ggtgatcggc 240 cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg 300 caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa 360 agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt gacggtacct 420 aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg 480 ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct gatgtgaaag 540 cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga 600 gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag 660 gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta 720 gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc 780 ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa 840 ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa 900 cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct 960 tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg 1020 ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct 1080 aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc 1140 ttatgacctg ggctacacac gtgctacaat ggacagaaca aagggcagcg aaaccgcgag 1200 gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt 1260 gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct 1320 tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagt 1366 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS1 forward primer <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS4 reverse primer <400> 3 tcctccgctt attgatatgc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward (fD1) primer <400> 4 agagtttgat cctggctcag 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse (rP2) primer <400> 5 acggctacct tgttacgact t 21

Claims (11)

Bacillus methylotrophicus CH518 strain having antibacterial activity against a blue mold pathogen (Accession No .: KACC91999P National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology).
delete [Claim 2] The method according to claim 1, wherein the blue fungus pathogen is Trichoderma harzianum , characterized by inducing a blue fungal disease in a mushroom comprising at least one member selected from the group consisting of mushroom mushroom, oyster mushroom and large mushroom Of Bacillus methylotrophicus strain CH518.
A carbon source comprising at least one of the group consisting of Saccharose, Xylose, Cellobiose and Salicine;
An organic matter which is a Soytone;
(NH ₄ H ₂ PO ₄ ) and ammonium diphosphate ((NH ₄ ) ₂ HPO ₄ );
An amino acid comprising at least one member selected from the group consisting of asparagine, valine, glutamic acid and histidine; And
At least one of the group consisting of potassium chloride (KCl), barium chloride (BaCl ₂ ), calcium chloride (CaCl ₂ ), magnesium sulfate (MgSO ₄ ), sodium molybdate (Na ₂ MoO ₄ ) and potassium phosphate (KH ₂ PO ₄ ) Inorganic salts including;
(Accession No .: KACC91999P, National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology) Culture medium for cultivation of Bacillus methylotrophicus CH518.
delete The method of claim 4, wherein the carbon source comprises Saccharose, the organic source comprises Soytone, and the inorganic source comprises a second ammonium phosphate ((NH ₄ ) ₂ HPO ₄ ). , The amino acid includes glutamic acid, and the inorganic salts include Bacillus methylotrophicus CH518 strain (accession number: KACC91999P National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology) culture medium containing magnesium sulfate (MgSO ₄ ) .
The culture medium according to claim 4, wherein the culture medium contains 100 to 200 parts by weight of an organic substance, 30 to 100 parts by weight of an inorganic filler, 10 to 80 parts by weight of an amino acid and 0.1 to 1 part by weight of an inorganic salt based on 100 parts by weight of carbon source (Accession No .: KACC91999P, National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology) Culture medium for culture.
delete delete Bacillus methylotrophicus strain CH518 (accession number: KACC91999P National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology) having antimicrobial activity against blue fungus pathogens.
The antifungal agent according to claim 10, wherein the controlling agent is at least one member selected from the group consisting of microbial pesticides, seed coating agents, additives for the production of a mushroom-free non-sterilizing medium, a leaf side spraying agent and a root spraying agent.

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