KR101476265B1 - Alcaligenes HC12 having anti-bacterial activity - Google Patents

Abstract

The present invention relates to a novel strain of the genus Alcaligenes , and more particularly to a novel strain of the genus Alcaligenes HC12 which has excellent antimicrobial activity isolated from the mussel medium.

Description

Alcaligenes HC12 having anti-bacterial activity < RTI ID = 0.0 >

The present invention relates to a novel strain of the genus Alcaligenes , and more particularly to a novel strain of the genus Alcaligenes HC12 which has excellent antimicrobial activity isolated from the mussel medium.

Mushroom mushroom ( Agaricus bisporus ) is an edible mushroom belonging to the neck of the wrinkled mushroom, and is excellent in taste and fragrance and is widely consumed worldwide. Mushroom mushroom is a mushroom widely grown and used in many countries of the world because it has higher protein content and superior meat quality than the commonly known oyster mushroom, shiitake mushroom, and top mushroom. Mushroom mushroom has a similar amount of protein to milk, so it is very useful for cancer patients who have high protein consumption in the body. It is known to be good for hypertension and anemia because it contains vitamin D, vitamin B2 and folic acid. In 1955, mushroom mushroom was first tested and cultivated in Korea in 1955. Mushroom mushroom was introduced from USA and Japan in 1961. Since then, as the cultivation area of mushrooms has been increased, the disease damage has been increased along with a large amount of production.

Pseudomonas agarici is known to be the causative organism of the disease, which is a bacterial gray pattern disease which causes much damage when growing mushrooms.

The present invention provides a novel strain of the genus Alcaligenes having antibacterial activity, particularly a novel strain having antimicrobial activity against bacterial gray fleck disease, an antimicrobial agent containing the antimicrobial agent, an antifungal agent for environmentally friendly mushroom, and a biological control method . This enables the environmentally friendly cultivation of mushrooms.

The present inventors have conducted extensive research to develop an effective biological control agent against bacterial gray mold disease. As a result, the present inventors isolated a pathogenic Pseudomonas agarici from the fruiting body of mushroom mushroom and found HC12 Microorganisms were selected. This strain has a high affinity for mushroom, and the optimum temperature for growth is similar to that of mushroom, so that the microorganism is sprayed to inhibit the growth of the disease and is effective for the environmentally friendly control of mushroom.

Thus, the present invention provides an Alcaligenes sp. Strain HC12 (accession number: KACC91774P).

The strain HC12 of the present invention exhibits a very excellent antibacterial activity and exhibits a strong antibacterial activity against Pseudomonas agarici which is a causative agent of bacterial gray mold disease of mushroom mushroom.

The present invention also provides an antibacterial agent comprising an Alcaligenes sp. Strain HC12.

The present invention also provides a biological control agent for bacterial gray mold disease comprising an alkaline genus strain HC12. The bacterial gray pattern bottle may be a bacterial gray pattern bottle for mushroom.

The present invention also provides a method of controlling a bacterial gray mold disease using an Alkali genus strain HC12.

The present invention can provide a novel strain of Alcaligenes , an antimicrobial agent containing the same, an antifungal agent for environmentally friendly mushroom, and a method for controlling harmful bacteria against mushroom. Accordingly, the present invention provides a biological control method for a bacterial gray foliar disease, which is a problem in mushrooms, thereby enabling environmentally friendly cultivation of mushrooms. In addition, it can contribute to increase consumer confidence and consumption through production of environmentally friendly mushrooms.

Fig. 1 is a view showing the results of confluent cultivation of the strain of the present invention and P. agarici .
2 is a view showing the antimicrobial effect of the strain HC12 of the present invention against mushroom mushroom.
Fig. 3 is a diagram showing a genetically flexible relationship by 16S rDNA analysis of the strain HC12 of the present invention. Fig.
4 is a graph showing the influence of temperature and pH of the strain HC12 of the present invention.
5 is a graph showing the effect of the carbon source of the strain HC12 of the present invention.
Fig. 6 is a graph showing the effect of the organic matter of the strain HC12 of the present invention. Fig.
Fig. 7 is a graph showing the effect of the mineral source of the strain HC12 of the present invention. Fig.
8 is a graph showing the effect of the amino acid of the strain HC12 of the present invention.
9 is a graph showing the effect of the strain HC12 of the present invention on inorganic salts.

According to one aspect of the present invention, the present invention provides a novel strain of Alcaligenes sp. HC12. This strain was deposited on December 17, 2012 at the Korean Microorganism Conservation Center (Accession No .: KACC91774P).

In the present invention, "strain HC12" should be understood to include said deposit microorganism and its variants. Such mutants include mutant strains or recombinant strains, which can be produced by known processes. Conventional processes include irradiation with radiation, treatment at a high temperature, and treatment with a mutagenic source. Variant strains may be other naturally occurring isolates and / or sub-isolates of the genus described herein. Recombinant strains can be produced by any method known in molecular biology for the expression of an exogenous gene or an alternative to the function or expression of an exogenous gene.

Table 1 below shows general characteristics of the selective microorganism HC12 of the present invention.

Characteristic HC12 Characteristic HC12 Grow at 42 ℃ + L-serine + Nitrite reduction - Malonate + Gelatin liquefaction - Valerate + Urease - Propionate + L-histidine + Proline - L-tryptophan + N-acetylglucosamine + L-ornithine + Potassium gluconate -

The Alcaligenes sp. Strain HC12 of the present invention exhibits an excellent antibacterial activity and has an excellent antimicrobial activity against Pseudomonas agarici which is a causative agent of bacterial gray fleck disease of mushroom mushroom.

In the present invention, " antimicrobial activity " means that the plant has a controlling effect against harmful microorganisms, and it can exhibit especially a controlling effect against the harmful bacteria of mushroom.

The present invention also provides a biological control agent for bacterial gray mold disease comprising an alkaline genus strain HC12.

The above-mentioned alkali genus strain HC12 used in the biological control agent of the present invention can be used as it is without adding any other components. If necessary, it may be prepared by adding a composition prepared by combining with various carriers such as a solid carrier or a liquid carrier or a preparation auxiliary agent such as an additive to prepare a microorganism or a microorganism in the form of a wettable powder, a solution, a suspension, a granule, The formulations obtained by formulating them into formulations can be used.

These preparations usually contain about 0.1 to 99% by weight of the alkali genus strain HC12 of the present invention (the bacterial weight is the wetting amount). It is preferred that the preparation contains the inventive alkaline genus strain HC12 of about 10 ³ to about 10 ¹⁰ colony forming units (hereinafter referred to as CFU) per gram of the preparation.

Examples of the solid carrier used in the formulation include mineral powders such as kaolin clay, bentonite, diatomaceous earth, synthetic silicon oxide, talc, quartz, vermiculite and pearlite; inorganic salts such as ammonium sulfate, ammonium phosphate, (Wheat flour, soybean powder, wheat bran, chitin, rice bran, skim milk powder and whole milk powder), activated carbon and calcium carbonate. Examples of the liquid carrier include water, glycerol, vegetable oils such as soybean oil and rapeseed oil, liquid animal oils such as fish oil, ethylene glycol, poly (ethylene glycol), propylene glycol and poly (propylene glycol) .

Examples of the adjuvant for the preparation include additives such as casein, gelatin, polysaccharides such as starch powder, gum arabic, cellulose derivatives and alginic acid, lignin derivatives, bentonites, sugars, vegetable oils, synthetic oils, synthetic water- ), Poly (acrylic acid), etc.), propylene glycol, ethylene glycol and the like; Defoaming agents such as silicone compounds; Thickeners such as natural polysaccharides (e.g., xanthan gum), inorganic substances (e.g., aluminum and bentonite), and synthetic water-soluble polymers (e.g., poly (acrylic acid) and the like).

The controlling agent of the present invention may be used in combination with or without mixing with an insecticide, a nematicide, a fungicide, a fungicide, a bactericide, a herbicide, a plant growth regulator, an electrodeposit agent, a fertilizer, a microbial material, .

In the plant disease control according to the present invention, the application amount of the alkali genus strain HC12 used is usually about 0.1 g to about 10,000 g, preferably about 10 g to about 1000 g, per 10 arcs.

The biological control agent of the present invention has a very good control agent and can be used by spraying it when cultivating mushroom. The application amount and the application concentration at the time of spraying treatment may be increased or decreased depending on the kind of the preparation, application time, application place, application method, kind of cultivation method, degree of damage, have.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Isolation and Pathogenicity of Pathogens

In the mushroom cultivation area of the mushroom mushroom, the affected part of the fruiting body showing gray patterned disease was cut into 1 cm size, put in an EP tube of sterilized water 1 ml, and then subjected to stepwise dilution, and 100 μl of the supernatant was applied to a nutrient agar, And cultured in an incubator to isolate single colonies. And when a pure isolated strains Pseudomonas Agaricus through genetic analysis, physiological and biochemical tests (Pseudomonas agarici ) were identified and used for the experiment.

In order to investigate the pathogenicity of P. agarici isolated from the mussel medium, the pathogen was diluted to 5 × 10 ⁷ / ml in sterilized water and used as an inoculation source. 50 μl of the pathogen was inoculated on the surface of fresh mushroom mushroom, and the mushroom was placed in a constant temperature incubator at 25, 95% for 2 days to confirm the presence of pathogen.

A total of 196 strains were isolated from fruiting bodies collected from mushroom cultivated farmers and identified 8 strains of P. agarici by gene analysis and physiological and biochemical characteristics. Although pathogenicity was slightly different depending on the strains, mushroom strain and fruiting body showed typical gray pattern symptom pathogenicity.

Selection of antagonistic bacteria and measurement of antimicrobial activity

In order to isolate the microorganisms useful in the present invention, three mushroom cultivated mushroom cultivars were collected from each farmhouse and used in the experiment. Separation of the microorganisms was carried out by stepwise dilution in R2A medium to isolate from 50 to 60 colony-forming plates independently. The pure microorganisms were cultured in R2A medium for 2 days, and the cells were collected and stored in a 20% (v / v) glycerol solution and stored at -70, and used as a test sample. The antimicrobial activity was examined by using paper disk method. In order to effectively test a large number of isolated strains, four strains of isolate bacteria were inoculated at the same intervals in one petridish. Bacterial pathogens were cultured in R2A medium for 48 hours, suspended in sterile water (5 × 10 ⁶ ), plated on petri dishes, and 50 μl of the isolating microorganisms were inoculated on a paper disk to determine the antagonistic effect of each strain Ability was evaluated.

1,500 strains were isolated from the mushroom mushroom culture medium and selected by the paper disk method. The antagonistic microorganism HC12, which has the strongest antimicrobial activity, was selected. Antagonistic microorganism HC12 showed the highest antimicrobial activity against P. agarici , a bacterial gray-colored pathogen (see top of FIG. 1).

In a bacterial gray pattern bottle  Bio-assay for

Mushroom mushroom cultivated in a box (30 × 20 cm) was used for the experiment to check the control effect of bacterial gray pattern disease. The growth rate of the disease was investigated by spraying the suspension of the pathogenic bacteria in the fruiting body of mushroom mushroom and spraying the suspension of the antagonistic microorganism after 30 minutes and cultivating in a growth chamber having a temperature of 16 and a humidity of 95%. The results are shown in Table 2 and FIG.

As can be seen from Table 2 below, HC12 treatment after the inoculation of the bacterial gray-colored pathogens in the mushroom showed 68.5% morbidity in the control group but 25.3% in the HC12 treatment, showing 63% control effect there was.

Item name Processing name Disease rate (%) Control (%) Mushroom mushroom No treatment 68.5 Antagonistic HC12 25.3 63

Identification of antagonistic bacteria Cultivated  Investigate characteristics

16S rDNA was analyzed after PCR amplification using general purpose primer for identification of selected antagonistic bacteria and final identification was made using various biochemical markers and 'Bergey's manual of systematic bacteriology' index. Growth and antimicrobial activities of the antagonistic antagonists were investigated by using the basal medium according to pH (3 ~ 10), temperature (10 ~ 45), carbon source, nitrogen source and proper amount of inorganic salts.

As a result of the 16S rDNA analysis of the strain HC12 of the present invention, it was found that it was an Alcaligenes genus strain (see FIG. 3).

The optimal culture conditions of the strain HC12 of the present invention are shown in Table 3 below.

Incubation temperature
   30 ℃     pH    9.0 Incubation time
24 hours
Nutrient source




Carbon source
   0.5% dextrin Organic matter
   1.5% yeast extract    Inorganic matter 1% NaNO ₃ amino acid
   1.5% L-asparagine    Inorganic salts 0.5% KH ₂ PO ₄

The effect of temperature and pH of the strain HC12 of the present invention, the effect of carbon source, the effect of organic matter, the effect of inorganic matter, the effect of amino acid and the effect of inorganic salt are shown in FIGS.

The growth temperature of the microorganism HC12 of the present invention was good at 20 to 40 DEG C and was able to grow at a wide range of pH (6 to 11). The optimum conditions for growth of the strain HC12 of the present invention were as follows: temperature 20 ° C, pH 9, dextrin 0.5% in carbon source, 1.5% yeast extract in organic matter, 1% NaNO _{3 in} mineral wax _, 1.5% L-asparagine in mineral wool, 0.5% KH ₂ PO ₄ showed the best growth and antagonism.

Claims (7)

Alcaligenes HC12, which has an antimicrobial activity against Pseudomonas agarici , KACC91774P. delete An antimicrobial agent having antimicrobial activity against Pseudomonas agarici including Alcaligenes HC12 of claim 1. delete A biological control agent for bacterial gray pattern disease comprising the alkaline genus HC12 of claim 1. 6. The biological control agent according to claim 5, wherein the biological control agent is a bacterial gray mold disease mushroom. A method of controlling a bacterial gray pattern disease using the alkali genus HC12 of claim 1.

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