KR101346242B1 - Pseudomonas genus antibacterial microorganism HC5 of Pseudomonas tolaasii - Google Patents

Abstract

The present invention relates to a microorganism having an antimicrobial effect against mushroom bacterial antagonist disease, including the nucleotide sequence of SEQ ID NO: 1, and relates to a microorganism which is KACC91694P. Since the microorganism according to the present invention has an excellent antimicrobial effect against mushroom bacterial crab disease, when the microorganism according to the present invention is treated to mushrooms having mushroom bacterial crab disease, it is more environmentally friendly than the case of treating mushroom bacterial crab disease using chemical pesticides. It will produce mushrooms that are harmless to the human body.

Description

Pseudomonas genus antibacterial microorganism HC5 of Pseudomonas tolaasii} for Pseudomonas tolaci

The present invention relates to a microorganism having an antimicrobial activity against Pseudomonas tolaasii, which is a causative bacterium of bacterial crab disease occurring in mushrooms.

Mushroom Bacterial Calvary Disease refers to a disease in which the lampshade begins to turn brown first, and the growth of the mushroom stops, or the mushroom that turns brown after the lampshade turns brown and decays due to a heavy fishy.

Such bacterial bacterial crab disease may be caused by the growing environment, such as long periods of excessive humidification, poor circulation of air in the growing environment, humidification under low pressure, or repeated drying and humidification. . However, these bacterial bacterial gallbladders are usually caused by infection of a pathogen called Pseudomonas tolaasii under these circumstances.

Therefore, in order to prevent mushroom bacterial crab disease, it was common to suppress the activity of Pseudomonas tolaasii using chemical pesticides or to kill Pseudomonas tolaasii. However, when the chemical pesticide is used, pesticide residues harmful to the human body remain in the mushrooms, which is fatal to the human body, and there is a problem that it is not environmentally friendly. Thus, if microorganisms with antimicrobial activity against Pseudomonas tolaasii are found and used in the treatment of mushroom bacterial bran, it is expected that the problems caused by the chemical pesticides can be prevented. However, there is a lack of efforts to find microorganisms with antimicrobial activity against Pseudomonas tolaasii in order to prevent mushroom bacterial crab disease.

In order to solve the problems of the prior art as described above, an object of the present invention is to find a microorganism having excellent antimicrobial activity against Pseudomonas tolaasii bacteria causing mushroom bacterial crab disease, and to treat mushroom bacterial crab disease through these microorganisms. To provide technology. Therefore, to provide a technology for producing mushrooms more environmentally friendly and harmless to the human body when chemical pesticides are used through the treatment of the bacterial bacterial crab disease through the microorganism according to the present invention.

The antimicrobial microorganism for mushroom bacterial crab disease according to one feature of the present invention for solving the above problems is to include the nucleotide sequence of SEQ ID NO: 1 is KACC91694P. In addition, the microorganism is characterized in that the culture temperature is obtained by culturing at 10 ℃ ~ 20 ℃. In addition, the microorganism is characterized in that the pH is obtained by culturing at 5.7 to 11.0. In addition, the microorganism is one or more carbon sources selected from the group consisting of fructose, galactose, soluble stark, inositol, glycerol, xylose, dextrose, lactose, dextrin, adonitol, mannitol, mannose, maltose, raffinose and ethanol Characterized in that obtained by culturing. In addition, the microorganism is characterized in that obtained by culturing with one or more organic nitrogen source selected from the group consisting of peptone, soyton, casamino acid, tryptone, yeast extract and malt extract. In addition, the microorganism is one selected from the group consisting of NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , NH ₄ NO ₃ , NaNO ₃ and (NH ₄ ) ₂ SO ₄ . Or obtained by culturing with at least two inorganic nitrogen sources. In addition, the microorganism is characterized in that it is obtained by culturing with one or more amino acids selected from the group consisting of asparagine, mesionine, proline, leucine, glutamine, glutamic acid, histidine and threonine. In addition, the microorganism may be obtained by culturing one or two or more substances selected from the group consisting of KCl, BaCl₂, CaCl₂, Li ₂ SO₄, MnSO, MgSO₄, ZnSO₄, FeSO₄, Na ₂ MoO₄, KH ₂ PO₄ and FeCl₃. .

The culturing method of the microorganism including the nucleotide sequence of SEQ ID NO: 1 according to another feature of the present invention and KACC91694P is characterized in that the culture temperature is incubated at 10 ℃ ~ 20 ℃. In addition, the culture method of the microorganism is characterized in that the culture pH is cultured at 5.7 to 11.0. In addition, the method of culturing the microorganisms in the group consisting of fructose, galactose, soluble stark, inositol, glycerol, xylose, dextrose, lactose, dextrin, adonitol, mannitol, mannose, maltose, raffinose and ethanol during the culture It is characterized by culturing with one or more selected carbon sources. In addition, the method of culturing the microorganism is characterized in that the culture with one or two or more organic nitrogen source selected from the group consisting of peptone, soyton, casamino acid, tryptone, yeast extract and malt extract. In addition, the culture method of the microorganism is NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , NH ₄ NO ₃ , NaNO ₃ and (NH ₄ ) ₂ SO ₄ group. It is characterized by culturing with one or more inorganic nitrogen sources selected from.

In addition, the method for culturing the microorganism is characterized in that it is cultured with one or more amino acids selected from the group consisting of asparagine, mesionine, proline, leucine, glutamine, glutamic acid, histidine and threonine. In addition, the method of culturing the microorganism is characterized in that one or more materials selected from the group consisting of KCl, BaCl₂, CaCl₂, Li2SO2, MnSO, MgSOg, ZnSO₄, FeSO₄, Na ₂ MoO₄, KH ₂ PO₄ and FeCl₃.

Drug according to another aspect of the present invention comprises the microorganism comprising the nucleotide sequence of SEQ ID NO: 1 and KACC91694P.

In addition, the method for treating mushroom bacterial crab disease according to another feature of the present invention comprises a nucleotide sequence of SEQ ID NO: 1 and is made using a microorganism of KACC91694P.

Using the microorganism according to the present invention to cure mushroom bacterial crab disease is environmentally friendly compared to the case of using chemical pesticides, it is possible to produce mushrooms harmless to the human body. This contributes to an increase in the consumption of mushrooms by consumers, which in turn improves the income of mushroom farmers.

1 is a photograph showing the results obtained by replacing the culture with Pseudomonas tolasi according to the present invention.
Figure 2 is a graph showing the conditions of optimum temperature and optimum pH at the time of culturing microorganisms according to the present invention.
Figure 3 is a graph showing the conditions of the optimal carbon source when culturing the microorganism according to the present invention.
Figure 4 is a graph showing the conditions of the optimum organic nitrogen source when culturing the microorganism according to the present invention.
5 is a graph showing the conditions of the optimum inorganic nitrogen source in the culture of the microorganism according to the present invention.
Figure 6 is a graph showing the conditions of the optimum amino acid when culturing the microorganism according to the present invention.
Figure 7 is a graph showing the conditions of the optimal inorganic material in the cultivation of microorganisms according to the present invention.
Figure 8 is a photograph showing that the microorganism according to the present invention has an antimicrobial activity in oyster mushroom.
9 is a photograph showing that the microorganism according to the present invention has an antimicrobial activity in mushroom mushrooms.
10 is a photograph showing that the microorganism according to the present invention has an antimicrobial activity in the enoki mushroom.
11 is a genetic analysis of the microorganism according to the present invention.

Accordingly, the present inventors have made a thorough research for the identification and development of microorganisms for overcoming mushroom bacterial crab disease, which has been pointed out as a problem of the above-mentioned prior arts. The invention was completed.

Specifically, the microorganism having the antimicrobial effect against the bacterial bacterial gallbladder according to the present invention is a microorganism having an accession number KACC91694P, and includes the nucleotide sequence of SEQ ID NO: 1. Therefore, the microorganism of Accession No. KACC91694P according to the present invention has an excellent antimicrobial effect against the bacterial bacterial crab disease.

In addition, as a culture method of the microorganism according to another feature of the present invention, the culture temperature is preferably incubated at 10 ℃ to 20 ℃. In addition, the pH of the culture is preferably cultured in the range of 5.7 to 11. The carbon source may be one or more selected from the group consisting of fructose, galactose, soluble stark, inositol, glycerol, xylose, dextrose, lactose, dextrin, adonitol, mannitol, mannose, maltose, raffinose and ethanol. Culture is preferred, and double adonitol is most preferred. In addition, the organic nitrogen source is preferably cultivated using one or two or more selected from the group consisting of peptone, soyton, casamino acid, tryptone, yeast extract and malt extract, and it is most preferable to use a double yeast extract. . The inorganic nitrogen source may be selected from the group consisting of NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , NH ₄ NO ₃ , NaNO _3, and (NH ₄ ) ₂ SO ₄ . Cultivation using one or more than two is preferred, of which NH ₄ H ₂ PO ₄ is most preferred. In addition, the amino acid is preferably incubated with one or two or more selected from the group consisting of asparagine, mesionine, proline, leucine, glutamine, glutamic acid, histidine and threonine, and most preferably double asparagine is used. . In addition, the inorganic material is preferably cultivated using one or more selected from the group consisting of KCl, BaCl₂, CaCl₂, Li2SO₄, MnSO, MgSO₄, ZnSO₄, FeSO₄, Na2MoO₄, KH ₂ PO₄ and FeCl₃, it is preferable to use MgSO₄ Most preferred.

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

< Example  1: Pseudomonas Tolasi ( Pseudomonas tolaasii Isolation and Identification of Bacteria>

Fruiting bodies showing the symptoms of bacterial bacterial crab disease were obtained in mushroom farms nationwide, and the diseased parts of these fruiting bodies were cut to 1 cm in size and crushed into 1 ml of EP tubes in sterile water. Subsequently, 100 μl of the supernatant was plated in nutrient agar through gradual dilution, and cultured in an incubator at 25 ° C., after which, a single colony was purified. The pure isolates were incubated with Pseudomonas reactanse in Pseudomonas agar F medium. Thus, white sedimentation line formation was confirmed, and 17 strains of Pseudomonas tolaasii were identified through genetic analysis and physiological and biochemical experiments. In order to investigate the pathogenicity of P. tolaasii bacteria isolated from mushrooms, pathogens were diluted in sterile water to 1.5 x 10 8 cfu / ml and used as inoculum. 50 μl of pathogens were inoculated on the surface of fresh mushrooms of Pleurotus eryngii and mushroom mushrooms, and placed in a thermostat at 25 ° C. and 95% to confirm pathogenicity. The pathogenicity was slightly different depending on the strains, but showed strong pathogenicity, which is typical of browning and tenderness in mushroom strains and fruiting bodies.

< Example  2: Selection of strains with antimicrobial activity and measurement of antimicrobial activity>

In order to isolate the microorganism with antimicrobial activity against bacterial crab disease, the waste medium and the mushroom mushroom compost of cultivated oyster mushroom were collected and used for each experiment. The microorganisms were separated and cultured step by step in R2A medium, and were separated independently from a plate forming 50 to 60 colonies. The pure microorganisms were incubated in R2A medium for 2 days, and the cells were collected and put in a 20% (v / v) glycerol solution and stored at? Antimicrobial activity was tested by purely isolated microorganisms using a paper disk method. In order to effectively assay a large number of isolated strains, one petri dish was inoculated at four intervals of the isolated bacterial strains. Pseudomonas tolaasii was incubated in R2A medium for 48 hours. Then, suspended in sterile water (5 × 106) and plated in petri-ish (petidish) and then inoculated 50 μl of the separated microorganisms on a paper disk (antimicrobial activity) for each strain depending on the degree of growth inhibition Evaluated.

Thus, among the isolated strains, primary antimicrobial active bacteria were selected by a paper disk method. In order to effectively test the isolated strains selected in this way, four Petri strains were inoculated at equal intervals in one petri dish. Pseudomonas tolaasii, a pathogen obtained according to Example 1, was incubated in R2A medium for 48 hours, suspended in sterile water (5 × 10 6), plated in petrishes, and then isolated. 50 μl each was inoculated on a paper disk. Thus, the antimicrobial activity of each strain was evaluated according to the degree of growth hypolipidemia.

Among the microorganisms showing the strongest antimicrobial activity among them, a total of three microorganisms named HC5, HC6, HC7 were selected. Figure 1 is a photograph showing the results obtained by replacing the three microorganisms with Pseudomonas tolaasii (Pseudomonas tolaasii). According to FIG. 1, it was confirmed that the reaction of HC5 occurred over the widest surface area, showing the highest antibacterial activity among the three strains.

As described above, the microorganism HC5 according to the present invention was confirmed to have an antimicrobial effect against bacterial bacterial cranial disease. Thus, the microorganism HC5 according to the present invention was deposited in the National Agricultural Genetic Resource Center, and the accession number is a microorganism having a nucleotide sequence of SEQ ID NO: 1 as a microorganism having a KACC91694P.

< Example  3: HC5 Optimal Culture Conditions>

As a result of examining the optimum conditions for cultivation through the temperature, pH, carbon source, organic nitrogen source, inorganic nitrogen source, amino acid, and minerals of selected microorganism HC5, the temperature started to increase rapidly from 5 ℃ to 10 ~ 20 ℃. This was maintained. It was also confirmed that the optimum antimicrobial activity at 15 ℃. However, after 20 ℃, the antibacterial activity began to drop rapidly again, and it was confirmed that the antimicrobial activity was almost absent at 25 ℃. In addition, pH started to increase rapidly from 5 to 11, the activity was maintained until 11, the growth was the best from 6 to 9. Thus, it showed strong inhibitory activity against the pathogen Pseudomonas tolasi in the temperature and pH range. Types of carbon sources are fructose, galactose, saccharose, soluble stark, inositol, glycerol, xylose, dextrose, lactose, dextrin, Na-CMC, adonitol, mannitol, mannose, maltose, raffinose, cellobis, ethanol , Solysine, double actives from protose, galactose, soluble stark, inositol, glycerol, xylose, dextrose, lactose, dextrin, adonitol, mannitol, mannose, maltose, raffinose and ethanol Among them, the optimal carbon source was adonitol, which showed the highest activity in culturing HC5 at a concentration of 0.6%. The types of organic nitrogen sources were peptone, soyton, urea, casamino acid, tryptone, yeast extract, and malt extract. Among them, double peptone, soyton, casamino acid, tryptone, yeast extract, and malt extract were used. The activity was confirmed, and the optimum source of organic nitrogen was yeast extract (Yeast extract), the highest activity in the culture of HC5 at 1.5% concentration. The inorganic nitrogen sources were NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , (NH 4) 2 HPO 4, NH ₄ NO ₃ , NaNO ₃ , and (NH ₄ ) ₂ SO ₄ . The activity was confirmed in NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , NH ₄ NO ₃ , NaNO ₃ and (NH ₄ ) ₂ SO ₄ , of which the optimal inorganic Nitrogen source was NH ₄ H ₂ PO _{4 It} was the highest activity in the culture of HC5 at 1.0% concentration. The amino acids were asparagine, mesionine, proline, leucine, glutamine, valine, aspartic acid, glutamic acid, arginine, histidine, cysteine, and threonine, among which asparagine, mesionine, proline, Activity was confirmed in leucine, glutamine, glutamic acid, histidine and threonine, and the optimal amino acid was asparagine, which showed the highest activity in the culture of HC5 at a concentration of 0.2%. In addition, minerals include KCl, BaCl ₂ , CaCl ₂ , CoCl ₂ , Li ₂ SO ₄ , MnSO, MnSO ₄ , ZnSO ₄ , FeSO ₄ , AgNO ₄ , Na ₂ MoO ₄ , KH ₂ PO ₄ , FeCl ₃ Among them, KCl, BaCl₂, CaCl₂, Li2SO₄, MnSO, MgSO₄, ZnSO₄, FeSO₄, Na ₂ MoO₄, KH ₂ PO₄ and FeCl₃ were found to be active. Among them, the optimal inorganic material was 5nM MgSO₄, when incubating HC5 at 5nM. The activity was the best. Table 1 below shows the optimum culture results.

In addition, Figure 2 is a graph showing the results of the experiment on the temperature and pH for the optimal culture of HC5. In addition, Figure 3 is a graph showing the optimum culture conditions of the carbon source, Figure 4 is a graph showing the optimum culture conditions of the organic nitrogen source, Figure 5 is a graph showing the optimum culture conditions of the inorganic nitrogen source, Figure 6 is the optimum culture of amino acids It is a graph showing the conditions, Figure 7 is a graph showing the optimum culture conditions of the mineral.

Experimental Example

< Experimental Example  One: HC5  Mushroom Bacteria of Isolated Strains Branch disease  Control Activity Measurement>

Experiments to investigate the control activity of HC5 according to the embodiment for bacterial antagonism were carried out separately. After inoculation of Pseudomonas tolaasii bacteria obtained from Example 1 to Pleurotus eryngii, HC5 treatment resulted in 93.8% morbidity in untreated but 27.8% morbidity in HC5 treatment. 71%. The mushrooms showed 56.3% morbidity in the non-treated mushrooms but 15.3% morbidity in HC5 treatment, and the control effect was 73%. The top mushroom showed 89.8% morbidity in the untreated group, but the HC5 treatment showed 19.5% morbidity, and the control effect was 78%. The above results suggest that HC5 strain has a high control effect on almost all mushrooms causing bacterial crab disease. Thus, HC5 was confirmed to show a high control activity against mushroom bacterial crab disease. The results are shown in Table 2 below. In addition, Figure 8 is a photograph showing the results of the experiment on the antimicrobial activity in oyster mushroom, Figure 9 is a photograph showing the results of the experiment on the antimicrobial activity in mushroom mushrooms, Figure 10 is the antimicrobial activity in the mushroom Photo shows the experimental results.

< Experimental Example  2: HC5 Experiment to Determine Physiological Characteristics of

An experiment was conducted to determine the physiological characteristics of HC5, and the results as shown in Table 3 below were obtained.

< Experimental Example  3: HC5 Experiment to find genetic relationship of

Experiments were conducted to find out which genus and species belong to which HC5. Thus, the same result as in FIG. 11 was obtained. That is, although the HC5 according to the present invention is a microorganism belonging to the genus Pseudomonas, it was confirmed that the microorganism belongs to a new species different from the existing microorganisms among the microorganisms belonging to the Pseudomonas genus.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. It is natural.

Agricultural Biotechnology Research Institute KACC91694P 20111219

Attach an electronic file to a sequence list

Claims (17)

Pseudomonas azotoformans HC5 (Pseudomonas azotoformans HC5), which includes the nucleotide sequence of SEQ ID NO: 1 and has accession number KACC91694P, and which has antimicrobial activity against mushroom bacterial plaque, which occurs in one or more mushrooms in the group consisting of a mushroom and an enoki mushroom. The Pseudomonas azotoperformance HC5 according to claim 1, wherein the Pseudomonas azotoforms HC5 is obtained by culturing at a culture temperature of 10 ° C to 20 ° C. The method of claim 1, wherein the Pseudomonas azotoforms HC5 Pseudomonas azotoforms HC5, characterized in that obtained by culturing at a pH of 5.7 to 11.0. The method according to claim 1, wherein the Pseudomonas azotoperformance HC5 is made of fructose, galactose, soluble stark, inositol, glycerol, xylose, dextrose, lactose, dextrin, adonitol, mannitol, mannose, maltose, raffinose and ethanol. Pseudomonas azotoperformance HC5, characterized in that obtained by culturing with one or more carbon sources selected from the group consisting of. The method of claim 1, wherein the Pseudomonas azotopics HC5 is obtained by culturing with one or more organic nitrogen sources selected from the group consisting of peptone, soyton, casamino acid, tryptone, yeast extract and malt extract Pseudomonas Azoto Performance HC5. The method according to claim 1, wherein the Pseudomonas azotopics HC5 is NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , NH ₄ NO ₃ , NaNO ₃ and (NH ₄ ) Pseudomonas azotoform HC5, which is obtained by culturing with one or two or more inorganic nitrogen sources selected from the group consisting of ₂ SO ₄ . The method of claim 1, wherein the Pseudomonas azotopics HC5 is obtained by culturing with one or two or more amino acids selected from the group consisting of asparagine, mesionine, proline, leucine, glutamine, glutamic acid, histidine and threonine Pseudomonas azotoperformance HC5 to make. The method of claim 1, wherein the Pseudomonas azotopics HC5 is obtained by culturing one or more materials selected from the group consisting of KCl, BaCl2, CaCl2, Li2SO₄, MnSO, MgSO₄, ZnSO₄, FeSO₄, Na2MoO₄, KH ₂ PO₄ and FeCl₃. Pseudomonas azototoxic HC5 characterized by the above-mentioned. delete The method for culturing microorganisms according to claim 1, wherein the Pseudomonas azototoxic HC5 is pH 5.7 to 11.0 and is cultured at 10 ° C to 20 ° C. Pseudomonas azotoperformance HC5 according to claim 1, wherein the culture of fructose, galactose, soluble stark, inositol, glycerol, xylose, dextrose, lactose, dextrin, adonitol, mannitol, mannose, maltose, raffinose and ethanol Microbial culture method, characterized in that cultured with one or two or more carbon sources selected from the group consisting of. A method for culturing microorganisms according to claim 1, wherein the Pseudomonas azotoperformance HC5 is cultivated with one or two or more organic nitrogen sources selected from the group consisting of peptone, soyton, casamino acid, tryptone, yeast extract and malt extract. Pseudomonas azototoxic HC5 according to claim 1 is NH ₄ Cl, (COONH ₄ ) ₂ , NH ₄ H ₂ PO ₄ , (NH ₄ ) ₂ HPO ₄ , NH ₄ NO ₃ , NaNO ₃ and (NH ₄ ) ₂ SO Microorganism culture method characterized in that the culture with one or two or more inorganic nitrogen source selected from the group consisting of _four . Microorganism culture, characterized in that the Pseudomonas azototoxic HC5 according to claim 1 is incubated with one or more amino acids selected from the group consisting of asparagine, mesionine, proline, leucine, glutamine, glutamic acid, histidine and threonine. Way. Claim the culturing of one or more than one material selected from the group consisting of Pseudomonas azo Topo Month HC5 according to claim 1 as KCl, BaCl₂, CaCl₂, Li2SO₄, MnSO, MgSO₄, ZnSO₄, FeSO₄, Na 2 MoO₄, KH 2 PO₄ and FeCl₃ Microbial culture method characterized in that. A drug for treating bacterial bacterial bran disease, which occurs in at least one mushroom in the group consisting of Prunus ulcer and Enoki mushroom, comprising Pseudomonas azotoperformance HC5 according to any one of claims 1 to 8. A method for treating a bacterial bacterial gallbladder disease which occurs in at least one mushroom in the group consisting of a mushroom and an enoki mushroom using the Pseudomonas azotoperformance HC5 according to any one of claims 1 to 8.

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