KR101336609B1 - A Novel Microorganism(Bacillus vallismortis GG290) having Turfgrass-Diseases Antagonism, the Method of Cultivating the Novel microorganism, and a Microbial Composition comprising the same and a Method of Producing the Microbial Composition - Google Patents

Abstract

The present invention relates to Bacillus vallismortis , a novel strain having an antagonistic response to major diseases of grass, and more particularly, to the major diseases of grass. Bacillus , a strain that antagonizes against Rhizoctonia solani and Sclerotinia homoeocarpa It relates to vallismortis GG290 (Accession Number: KCCM11211P) and a culture method of the strain, and a microbial agent comprising the strain thereof as an active ingredient.
The novel strain of the present invention was isolated and cultured in the soil similar to the turf environment, it was confirmed that the excellent activity and sustained control effect against turf disease by improving the culture source such as carbon source, nitrogen source in the culture process of the strain. . In addition, the novel strains of the present invention are brown leaf blight ( Rhizoctonia solani ) and coin blight ( Sclerotinia ) most widely distributed among grass diseases. homoeocarpa ) and markedly antagonistic activity, in particular, the long-term control effect against the grass pathogens was confirmed to be long lasting, showing the possibility of a very economical and environmentally friendly microbial agent.

Description

Bacillus Balismortis ＧＧ290, a novel strain having an antagonistic reaction to the major diseases of turfgrass, cultivation method of the strain and microorganisms for control including the strain and preparation method thereof [A Novel Microorganism (Bacillus vallismortis GG290) having Turfgrass -Diseases Antagonism, the Method of Cultivating the Novel microorganism, and a Microbial Composition comprising the same and a Method of Producing the Microbial Composition}

The present invention relates to a new strain Bacillus vallismortis GG290 (Accession No .: KCCM11211P), which has an antagonistic response to the major diseases of grass, and more particularly, among the major diseases of grass, especially Rhizoctonia solani and coin blight. ( Sclerotinia homoeocarpa ) Bacillus vallismortis GG290 (Accession Number: KCCM11211P), a strain showing an antagonistic reaction, and a method for culturing the strain, and a microbial agent using the strain as an active ingredient.

The novel strain of the present invention was isolated and cultured in the soil similar to the turf environment, it was confirmed that the excellent activity and sustained control effect against turf disease by improving the culture source such as carbon source, nitrogen source in the culture process of the strain. . In addition, the novel strains of the present invention are brown leaf blight ( Rhizoctonia solani ) and coin blight ( Sclerotinia ) most widely distributed among grass diseases. homoeocarpa ), and showed a long-lasting control effect against the Rhizoctonia solani and Sclerotinia homoeocarpa , indicating the possibility of a very economical and environmentally friendly microbial agent. It was.

Increasing income levels of the Korean people and the spread of various leisure cultures have increased interest in golf and the number of golf course users is increasing every year. On the other hand, although golf is a sport that many people enjoy, there are still socially negative problems such as damaging the forests when constructing golf courses and excessively applying fertilizers or pesticides to manage the grass and adversely affect the environment. It can also occur.

In particular, the main diseases of the golf course grass in Korea, Brown Patch, Large Spot, Brown Patch, Brown Patch, Pythium, etc. For many years, the use of pesticides and fertilizers has not declined significantly to maintain their planting status. It is a method to control and manage major grass diseases by using antimicrobial microorganisms for the solution of drug resistant bacteria and environmentally friendly grass management for continuous use of pesticides.

Bacillus vallismortis GG290 a novel strain having antagonistic reaction to major diseases of grass: provide (Accession No. KCCM11211P), and in particular strains exhibit antagonistic reaction to a brown leaf blight (Rhizoctonia solani) and coin blight (Sclerotinia homoeocarpa) Bacillus By providing vallismortis GG290 (Accession No .: KCCM11211P) and the method of culturing the strains, and microorganisms using the strains as an active ingredient, antimicrobial microorganisms for the solution of drug-resistant bacteria for sustainable pesticide use and environmentally friendly lawn management To contribute to the control and management of major grass pests.

The present invention for achieving the technical problem as described above provides a Bacillus vallismortis GG290 (Accession Number: KCCM11211P), which is a grass disease antagonistic strain isolated from the soil, culture method and microbial agent that can maximize the control effect of the strain By providing a, it is intended to contribute to the control of grass diseases using microbial agents without using pesticides.

Hereinafter, the configuration of the present invention will be described in detail.

First, they are isolated and cultivated in the soil, and have an antagonistic response to turf diseases, and in particular, antagonistic responses to turf diseases of Rhizoctonia solani and Sclerotinia homoeocarpa . Grass disease antagonist strain Bacillus vallismortis GG290 (Accession Number: KCCM11211P) is provided.

Second, Bacillus In the method for culturing the strain of vallismortis GG290 (Accession No .: KCCM11211P), it provides a culture method characterized in that the antimicrobial activity of the antagonistic response to the disease of grass cultivation is excellent.

The culture medium which makes the antimicrobial activity of the antagonistic reaction to the disease of the grass excellent,

The carbon source is sorbitol, the nitrogen source is yeast and bactotryptone, and the inorganic salts have excellent antimicrobial activity against antagonism of grass by using a culture source consisting of MgSO ₄ 7H ₂ O, K ₂ HPO ₄ , KH ₂ PO ₄ . It provides a culture method characterized in that the culture.

Third, Bacillus antagonistic strain Microorganisms comprising vallismortis GG290 (Accession No .: KCCM11211P) as an active ingredient, and having a treatment and prevention effect of turfgrass diseases caused by Rhizoctonia solani and Sclerotinia homoeocarpa To provide.

As described above, the present invention provides Bacillus vallismortis GG290 (Accession No .: KCCM11211P), a strain having an antagonistic action against grass diseases, in particular brown leaf blight and coin blight, and provides a method of culturing the bacteria and a high control effect. By providing a culture condition that can be represented, and by providing a microbial agent of the strain and a method for producing the same can greatly contribute to the effective control of grass pests without pesticides.

1 is Bacillus Picture of vallismortis GG290.
2 is Bacillus A graph showing the optimal culture temperature of vallismortis GG290, where the x-axis represents the temperature (° C).
3 is Bacillus It is a graph showing pH (pH) suitable for the culture of vallismortis GG290.
4 is Bacillus This photo shows the cellulase degradation effect of vallismortis GG290.
5 is Bacillus This is a picture confirming the control effect of vallismortis GG290 Dallas Spot pathogen.
6 is Bacillus Photograph showing the control effect of grass brown leaf blight of vallismortis GG290.
7 is Bacillus This picture shows the control effect of coin blight of vallismortis GG290.
8 is Bacillus The antimicrobial effect of vallismortis GG290 brown leaf blight, anticoin blight and spring blight.
9 is Bacillus 16s rRNA sequence of vallismortis GG290.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The implementation of the present invention is described in detail by dividing into Examples 1 to 3 as follows.

First, Example 1, Bacillus vallismortis GG290 strain temperature and acidity suitable for isolation, identification and culture.

Example 1

(1) Isolation of Strains

In January 2008, the paddy soil of Ibbuk-dong, Gwon-gu, Suwon-si, Gyeonggi-do, Korea was collected, weighed 1 g of the collected soil and suspended in 10 ml of sterile water, and then 1 ml of suspension was diluted 10-fold in a test tube containing 9 ml of sterile water. It was. 200 μl of sterile water diluted in the suspension was taken, placed in the center of a NA (Nutrient Agar) solid plate medium, and evenly pushed with a sterile spread and incubated in a 25 ° C. thermostat for 3 days. The colonies formed in each shape were taken, labeled with strain numbers, and selected for effective strains using the antifungal reaction.

(2) Identification of Strains

Brown leaf blight ( Rhizoctonia solani ) and grass blight ( Sclerotinia) In order to identify strains showing antagonistic response to homoeocarpa ), morphological characteristics were observed with a scanning electron microscope (HITACHI S-3000) (FIG. 1), and the BCH card of the bacteriostat VITEK compact II was used. The biochemical reaction assay was confirmed and the types of bacteria were identified (Table 1), and the sequencing of the 16s rRNA was analyzed (FIG. 9). Then, NCBI blast was searched for strain identification. As a result, the strain was identified as Bacillus vallismortis as a result of the identification of VITEK compact BCH card and the sequencing analysis. The isolated bacterium was named Bacillus vallismortis GG290. The bacterium was deposited with the Korea Microorganism Conservation Center under accession number KCCM11211P, as indicated in the attached microbial deposit at the time of filing.

TABLE 1 Biochemical Properties

(3) Proper culture temperature

In order to confirm the proper culture temperature of the isolated strain, 2 ml of preculture was inoculated into 200 ml of TSB (Triptic Soy Broth) medium and incubated at 180 rpm for 24 hours at 15, 20, 25, 30, 35, and 40 ° C for each temperature. Afterwards, the culture medium was irradiated with UV spectrophotometer (BECKMAM DU730) at a wavelength of 600 nm to check the growth of bacteria. As a result, as shown in Figure 2 was the best growth when incubated at 30 ℃ for 24 hours.

(4) proper pH

In order to confirm the acidity suitable for the growth of this strain, 200 ml of TSB medium was adjusted to 3, 4, 5, 6, 7, 8 with NaOH and HCl, and shaken at 180 rpm for 24 hours and 48 hours. The absorbance at UV spectrophotometer 600nm wavelength was investigated to compare the growth of bacteria. Results As shown in FIG. 3, the growth of the strain was generally good at pH7.

Next, Example 2, Bacillus The vallismortis GG290 strain cultivation method characterized in that the antimicrobial activity of the antagonistic response to turfgrass disease and the control effect against turfgrass disease.

Example 2

(1) Optimal Medium Nutrient and Antimicrobial Activity Assay

Minimum medium (K ₂ HPO ₄ 0.7%, KH ₂ PO ₄ 0.2%, Glucose 0.5%, (NH ₄ ) ₂ SO ₄ 0.1%, MgSO ₄ 7H ₂ O 0.01%) After the preparation, the Glucose was excluded from the carbon source, and 1% of each carbon source was added thereto, followed by incubation at 180 rpm at 25 ° C. for 24 hours. In order to determine the nitrogen source, 0.5% of each nitrogen source was added instead of 0.1% of (NH ₄ ) ₂ SO ₄ in the minimum medium, and K ₂ HPO ₄ 0.7%, KH ₂ , the inorganic salt in the minimum medium, to confirm the proper inorganic salt. Instead of 0.2% of PO _{4 and} 0.01% of MgSO ₄ 7H ₂ O, 5 mM of each inorganic salt was added thereto, followed by incubation at 180 rpm for 24 hours at 25 ° C. Antimicrobial activity was measured by inoculating 100 ㎕ of the culture medium incubated with each carbon source, nitrogen source, and inorganic salts in a PDA medium on paper discs, and substituting the main turfogenic pathogens for 25 days and 7 days. As a result, as shown in Table 2, when sorbitol was used as a carbon source, the culture was well, and the antibacterial activity against the main grass pathogens was the best. Nitrogen source was well cultured in yeast extract as shown in Table 3, the best antibacterial activity when using yeast and bactotryptone. Inorganic salts were good antimicrobial activity of the strain when MgSO ₄ 7H ₂ O, K ₂ HPO ₄ , KH ₂ PO ₄ was used as a medium source (Table 4).

[Table 2] Optimum Carbon Source and Antimicrobial Activity Assay

[Table 3] Optimum Nitrogen Source and Antimicrobial Activity Assay

[Table 4] Optimum inorganic salt source and antibacterial activity assay

(2) Antifungal assay for phytopathogenic bacteria of antagonists

Sclerotinia to determine the antimicrobial activity against major phytopathogens as well as turf fungi of isolated antagonistic microorganisms sclerotiorum , Botrytis cinerea , Fusarium oxysporum , Alternaria panax , Colletotrichum sp. Rhizoctonia sp., Phytophthora capsici It was replaced by paper disc method. Brown leaf blight, Rhizoctonia , a grass pathogen solani AG2-2, R. solani AG1, coin blight Sclerotinia In addition to homoeocarpa , it showed high antimicrobial activity against major diseases such as gray mold, wilted disease and anthrax (Table 5).

[Table 5] Antimicrobial Activity of Major Grass Diseases of GG290

(3) Cellulase Degradation Effects of Antagonists

In addition to the antimicrobial effect of the isolated antagonist microorganisms, it was confirmed that the cellulase decomposing effect that can decompose the moor layer formed by the mowed grass of golf courses. In order to confirm the cellulolytic function, which is the main component of the plant cell wall, microorganisms prepared by separating DCAB medium were incubated at 30 ° C. for 3 days, treated with 0.1% congo red for 30 minutes, and treated with NaCl 1 M for 15 minutes to confirm that cellulose was degraded. (FIG. 4). Once a golf course is established, its planting style is maintained for many years, and the disease is encouraged by wounds caused by frequent mowing, stress caused by pressure, and excessive formation of excess layers, causing more disease than other plants and increasing pesticide use due to difficulty in controlling. easy to do. For this reason, microorganisms with antimicrobial function and cellulose decomposition function, which is the main ingredient of grass, are treated on the golf course to reduce excessive generation of excess grass piled with mowed grass, and also to eliminate the habitat of pathogens, thereby increasing the antimicrobial function of microorganisms. .

(4) Antibacterial potency test of antagonist microorganism

GG290 microorganisms were cultivated in a plastic box and cultured at 24 ° C. for 24 hours at 30 ° C. and 180 rpm, and then cultured at 1 × 10 ⁷ cfu / ml at a concentration of 1 L / m ² three times a week. Three days after the first treatment, 1 g of Dallas Spot pathogen incubated in oatmeal sand medium was inoculated in four places in the box for 20 days to confirm the onset of disease. There was a prophylactic effect (Table 6, Figure 5).

[Table 6] Effect of GG290 on Dallas Spot Pathogen Control

(5) packaging assay of antagonists

In order to confirm the antimicrobial effect of GG290 against large patch disease, 1 × 10 ⁷ cfu / ml of GG290 microorganism cultured at 180 rpm in TSB medium for 24 hours at the spot where large patch disease occurs in Korean grass in Vivaldi Golf Course, Hongcheon, Korea every year. After adjusting to the concentration, 1 L / m ² was sprayed 5 times a week from the end of August, and the progress of large patch lesions was investigated. The antibacterial effect of GG290 against the coin blight was the creeping bent glass packaging in the Sky 72 golf course. In the same method as in the above cultured and sprayed to investigate the lesions generated in the package. Results Large patch bottles had 82% control effect compared to no treatment when GG290 was treated, and coin blight bottle had 73% control effect when GG290 was treated (Table 7, Table 8).

Bacillus as a result of the above The vallismortis GG290 microorganism can be used as an excellent microorganism that can prevent major diseases occurring in other crops, including brown leaf blight and coin blight, which is a problem in grass (FIG. 6, FIG. 7 and FIG. 8).

[Table 7] Effects of GG290 on Grass Brown Leaf Blight

[Table 8] Effect of GG290 Coin Blight Control

Next, Example 3, Bacillus The present invention relates to a method for preparing a microbial agent for control, including the vallismortis GG290 strain.

Example 3

Preparations using microorganisms must be well established in the antagonism of pathogens and roots of the host, to compete with other microorganisms and to adapt to a variety of environmental conditions. To use microorganisms in a stabilized state, 5% vegetable oil ethylene oxide (1%) and 1% citric acid were added to GG290 microorganism culture cultured at 180 rpm in TSB medium for 24 hours, and then diluted 100-fold and creased once every five times. When treated with graphajang, the occurrence of coin blight showed about 78% control effect compared to no treatment (Table 9, Figure 7).

[Table 9] Effect of GG290 Coin Drying Bottle Control

Korea Microorganism Conservation Center KCCM11211P 20111010

Claims (6)

delete A new turbid antagonist strain Bacillus vallismortis GG290, which is isolated from soil and cultured in a medium, and has an antagonistic response to the grass diseases of Rhizoctonia solani and Sclerotinia homoeocarpa . : KCCM11211P). delete delete delete Grass disease antagonist strain Bacillus Microorganisms comprising vallismortis GG290 (Accession Number: KCCM11211P) as an active ingredient, and having a treatment and prevention effect of turf disease caused by Rhizoctonia solani and Sclerotinia homoeocarpa .

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