KR100769365B1 - 131 ~ 133 Burkholderia gladioli NIAB 131 ~ 133 strains having antibiotic activation and the inhibiting method of Colletotrichum gloeosporioides by using the culture solution thereof - Google Patents

Abstract

Burkholderia gladioli NIAB 131-133 strains and culture solutions thereof are provided to show antibacterial activity specifically on Collectotrichum gloeosporioides and very excellent antibacterial vitality, maintain antagonism without changing of the antibacterial activity after heat-treatment and be used as it is without concentration due to very strong antibacterial vitality, thereby being industrially very useful as a controlling agent of the Collectotrichum gloeosporioides. A Burkholderia gladioli NIAB 131 strain having antibiotic activity is deposited as a deposition no. KACC 91226. A Burkholderia gladioli NIAB 132 strain having antibiotic activity is deposited as a deposition no. KACC 91227. A Burkholderia gladioli NIAB 133 strain having antibiotic activity is deposited as a deposition no. KACC 91228. An agent for controlling Collectotrichum gloeosporioides comprises a culture solution of one of the Burkholderia gladioli NIAB 131-133 strains.

Description

(Burkholderia gladioli NIAB 131 ~ 133 strains having antibiotic activation and the inhibiting method of Colletotrichum gloeosporioides by using the culture solution }

1 is a photograph showing the antagonistic activity against pepper anthrax of the strains of Burcolderia gladioli NIAB 131 ~ 133 provided according to the present invention.

Figure 2 shows the results of assaying the antibiotic resistance of strains of Burcolderia gladiolus NIAB 131 ~ 133.

Figure 3 is a result of examining the biochemical properties of strains of Burcolderia gladiolus NIAB 131 ~ 133 provided according to the present invention.

Figure 4 shows the results of assaying the antimicrobial activity after the heat treatment for strains of Burcolderia gladiolus NIAB 131 ~ 133 provided according to the present invention.

The present invention relates to a strain of antagonistic bacteria Burcolderia Gladioli NIAB 131 ~ 133 ( Burkholderia gladioli NIAB 131 ~ 133) isolated from the crop root zone soil, more specifically, the anthrax bacterium Collectoriumium Gloosporioi death member kolde Ria geulradi up NIAB 131 ~ 133 strain and the control of Kiwoom glow Eos Poly cucumber des (Collectotrichum gloeosporioides) call rekto tree as a sterile culture medium in which the strain is produced having a specific with antagonistic ability for (Collectotrichum gloeosporioides) It is about how to. The strain specifically exhibits antagonistic activity only to the pepper anthrax bacterium Collectotrichum gloeosporioides , and its antimicrobial activity is very good, and the culture medium of the strain is changed even after sterilization. Not only maintain strong antagonism, but also very strong antibacterial activity can be used as it is without concentrating the culture. Therefore, it is very useful in terms of its effectiveness and economical efficiency as a control agent for pepper anthrax.

With the recent expansion of agricultural imports, increasing the competitiveness of agriculture is becoming more important than ever. The continuous increase in the use of chemical fertilizers is a real problem that is not only a problem in Korea. The use of chemical fertilizers not only results in extreme environmental pollution, but according to the basic ecosystem principles of the food chain, its evil function has a serious impact on the health of human consumers. In addition, conventional chemical pesticides are being used more and more each year due to the increased resistance of microorganisms, causing health problems for consumers as well as farmers. In addition, since the development of new pesticides takes a lot of time and research personnel, and causes enormous environmental pollution, it is urgently required to develop pollution-free biological pesticides that can overcome this problem.

The international community's efforts to avoid the overuse of chemical fertilizers and the resulting calamities are also intensifying. The United Nations Environment Development Council (UNCED) encourages countries to reduce their use of chemical pesticides by 25 percent, and Korea has announced that by 2010, it will reduce its consumption by 40 percent for chemical fertilizers and 50 percent for synthetic pesticides. Since it is inevitable to develop alternative pesticides to significantly reduce the amount of chemical and synthetic pesticides while maintaining agricultural productivity, pollution-free biological pesticides are the only alternative to chemical fertilizers and synthetic pesticides. Thus, since the 1980s, some of the world's leading companies have invested heavily in the development of guaranteed microbial pesticides. In Korea, biopesticide sales account for only 0.16% of the total pesticide market, but the share of biopesticides is expected to increase rapidly.

In particular, as the Green Round Convention on Environmental Protection and Ecosystem Conservation becomes the subject of international concern, the abuse of excessive pesticides is a serious problem and the development of biocide for plant control that replaces it is becoming international. As a result, competition is emerging as a key field. In order to overcome the environmental pollution problem caused by conventional pesticides, it is very urgent to select antagonistic microorganisms that are easy to produce, store and apply and effectively inhibit pathogens and to separate biologically active substances. In particular, antifungal actives are less toxic to mammals than conventional pesticides, no harm to plants, excellent in activity and less residual, and do not affect rotation, and should not contaminate groundwater, soil and river water.

On the other hand, certain microorganisms are antagonistic against phytopathogenic fungi, and various metabolites and cell wall degrading enzymes they produce are known to have antagonism against pathogens.

Microbial metabolites involved in disease inhibition through biological control methods known to date include antifungal agents, including siderophores, NH3 enzymes, hydrogen cyanide, and the like. Antagonistic activity. Microbial fungicides used for biological control of fungi reported so far are derived from bacteria such as Pseudomonas, Bacillus, Streptomyces, Agrobacterium and Bercholeria, and fungi such as Trichoderma, Zirocladium, and Fusarium Etc. are known.

However, products currently commercially available for controlling phytopathogenic fungi still have insufficient market share. In addition, most of the pathogen microbial control agents attempted up to now have been used directly after using the isolated functional strain or after separating and purifying the product of the strain, and then artificially synthesized by analyzing the structure.

The present inventors have found that specific only to raise the antagonistic bacteria member kolde Ria geulradi separated from the crop rhizosphere NIAB 131 ~ 133 (Burkholderia gladioli NIAB 131 ~ 133) strain is red pepper anthracnose fungus of call rekto tree Kiwoom glow Eos Poly cucumber des (Collectotrichum gloeosporioides) Antagonistic activity, the antimicrobial activity is also very good, and the culture medium of the strain is not only maintain a strong antagonism without change in antimicrobial activity even after sterilization, and the antimicrobial activity is very strong so that the culture medium can be used as it is without concentration. It has been found that the present invention can be completed.

Therefore, an object of the present invention is an antagonistic bacterium gladioluria NIAB 131 ~ 133 which exhibits a particularly strong antimicrobial activity only to Collectotrichum gloeosporioides , the pepper anthrax bacterium isolated from the crop root zone soil. ( Burkholderia gladioli NIAB 131 ~ 133) It is to provide a method for controlling pepper anthrax by sterilizing the strain and the culture medium produced by the strain as it is.

In order to achieve the above object, in the present invention, antagonistic bacterium Burcholderia gladioli NIAB 131 ~ 133 ( Burkholderia gladioli NIAB 131 ~) having antibacterial activity against the pepper anthrax bacterium Collectorrichum gloeosporioides 133) strains are provided.

The present invention also provides a control agent for Collectorrichum gloeosporioides , which is a pepper anthrax bacterium, characterized by containing a culture solution of the strain.

In addition, the present invention provides a method for controlling Collectorrichum gloeosporioides , which is a pepper anthrax bacterium used by sterilizing the culture medium produced by the strain as it is.

Hereinafter, the present invention will be described in detail.

The present invention has an inhibitory effect on the strain Burkholderia gladioli NIAB 131 ~ 133 ( Burkholderia gladioli NIAB 131 ~ 133) and the pepper anthrax produced by the strain Contains antimicrobials. Isolation and propagation of this strain is possible by conventional methods and the culture can be sterilized or boiled.

The present invention is a TSB liquid medium containing 1% tryptone, 0.5% yeast extract, and 1% NaCl, various antagonists isolated from the crop root zone of the agricultural technology center package of the Rural Development Administration 1-4 days at 28 ℃ After shaking to culture, each culture filtrate was analyzed for Colletotrichum gloeosporioides Antimicrobial activity was measured. Among them, three strains of NIAB 131-133 which strongly inhibit the growth of pepper anthrax were selected.

The present inventors have found that the microbial strain of the present invention is Burkholderia gladioli After confirming that the strain belongs to, Burkholderia gladioli NIAB 131 ~ 133 ( Burkholderia gladioli NIAB 131 ~ 133) and named the strain on April 3, 2006, deposited in the Korea Agricultural Microbiological Resources Center The numbers KACC 91226, KACC 91227 and KACC 91228 have been assigned.

The present inventors have indicated six fungi causing serious disease in plants in order to check the antimicrobial spectrum of the antimicrobial substances produced by the strain Burkholderia gladioli NIAB 131-133. In vitro antimicrobial assays were performed.

In addition, the antibacterial activity produced by the strain Burkholderia gladioli NIAB 131 ~ 133 of the present invention was tested for specific antimicrobial activity against pepper anthrax, and the change of antimicrobial activity was observed after heat treatment. It was.

Hereinafter, the structure of the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Isolation and Identification of Burkholderia gladioli NIAB 131-133 Strains

A variety of antagonists isolated from crop root zone soils of agricultural technology center under the Rural Development Administration were cultured with TSB liquid medium containing 1% tryptone, 0.5% yeast extract and 1% NaCl for 1-4 days at 28 ℃. Each culture filtrate was then analyzed for Colletotrichum gloeosporioides, a pepper anthrax bacterium. Antimicrobial activity was measured.

As shown in FIG. 1, the antibacterial activity assay was performed on pepper anthrax bacteria using a culture medium of 11 strains selected first. Among them, three strains (131 to 133 strains) having potent activity that inhibit the growth of the plant pathogenic fungi were selected.

To identify the strain, the 16S rDNA region was denatured at 94 ° C for 5 minutes using fD1 (5'-AGAGTTTGATCCTGGCTCAG-3 ') of SEQ ID NO: 4 and rP2 (5'-ACGGCTACCTTGTTACGACTT-3') primer of SEQ ID NO: 5 Then, PCR was performed in 34 cycles of 1 minute at 94 ° C, 1 minute 30 seconds at 30 ° C, and 2 minutes at 72 ° C. The base sequence was determined by cloning the PCR 16S rDNA fragment. As a result, the microbial strain of the present invention showed a homology of 98% to 99% with Burkholderia gladioli .

This strain is a representative Gram-positive bacillus that secretes yellow pigments at the beginning of growth on TSB solid medium and forms viscous colonies. 28 ° C. is also the optimal growth temperature and is sensitive to most antibiotics other than empicillin (FIG. 2).

In order to investigate the biochemical characteristics of the strain NIAB 131 ~ 133 of the present invention, the strain was incubated for 24 hours at 28 ° C of growth temperature, and then a single clone was collected and suspended in 0.85% NaCl buffer, based on standards buffer. After adjusting the OD values, the biochemical characteristics of the test strains using the API20 NE Kit (bioMerieux Inc.) showed that 131 and 132 had the same characteristics, and 133 strains were almost identical in terms of sugar availability. (FIG. 3)

According to the above results, the present inventors have found that the microbial strain of the present invention is Burkholderia gladioli . After confirming that the strain belongs to, the strain was named Burkholderia gladioli NIAB 131 ~ 133 (burkholderia gladioli NIAB 131 ~ 133), and the strain was deposited on April 3, 2006 to the Korea Agricultural Microbiological Resources Center The numbers KACC 91226, KACC 91227 and KACC 91228 have been assigned.

[Example 2] Antimicrobial activity spectrum of the strains of Burcolderia gladiole NIAB 131 ~ 133

In order to confirm the antimicrobial spectrum of the antimicrobial substances produced by the strains of Burcolderia gladioli NIAB 131 ~ 133 of the present invention, in vitro antimicrobial assays were carried out with indicators of six fungi causing serious diseases in plants. Was performed. As shown in Table 1, the culture solution of the present invention showed a particularly strong antifungal activity only against the pepper anthrax bacterium, Colletotrichum gloeosporioides .

            Disclosure Strain Pathogens 32 41 61 131 132 133  Collectoriumium Gloeosporioides ++ + + +++ +++ +++  Trichoderma sp. ++ + + - - -  Fusarium ozysporum + + + - - -  Rhizoctonia solani ++ + + - - -  Phytophthora sp. ++ + + - - -  Penicillium sp. ++ ++ ++ - - -

 * +++: 10 ~ 15 mm, ++: 5 ~ 10 mm, +: below 5 mm

[Example 3] Antibacterial activity assay against pepper anthrax after heat treatment of strains of Burcolderia gladiolis NIAB 131 ~ 133

The strains of Burcolderia gladioli NIAB 131-133 of the present invention were incubated in TS Broth containing 1% tryptone, 0.5% yeast extract, and 1% NaCl for 4 days, and then centrifuged to obtain only the supernatant. The supernatant was heat-treated in boiling water at 100 ° C. for 30 minutes and then tested for antimicrobial activity against red pepper anthrax. As a result, cultures of 7 strains including strain 31 lost antimicrobial activity and had poor heat resistance. Had weak antimicrobial activity. However, the excellent activity of 131 ~ 133 strain did not change at all even after the heat treatment (Fig. 4).

As described above, the culture medium of the strains of Burcolderia gladioli NIAB 131 ~ 133 of the present invention has a specific antimicrobial activity only to the pepper anthrax bacterium Collectorrichum gloeosporioides , the antibacterial Vitality is also very good, the culture medium of the strain not only maintains a strong antagonism without change in the antimicrobial activity even after heat treatment, the antimicrobial activity is very strong and can be used as it is without concentration, industrially as a pepper anthrax control agent Very useful.

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Claims (5)

Antagonistic bacterium Burkholderia gladioli NIAB 131 strain (Accession No. KACC 91226). Antagonistic bacterium Burkholderia gladioli NIAB 132 strain (Accession No. KACC 91227). Antagonistic bacterium Burkholderia gladioli NIAB 133 strain (Accession No. KACC 91228). 4. The caustic anthrax bacterium Colletotrium gloesporioi comprising the culture medium of Burkholderia gladioli NIAB 131 to 133 of any one of claims 1 to 3. Death ( Collectotrichum gloeosporioides ) control agent. Burkholderia gladioli of any one of claims 1 to 3 NIAB 131 ~ 133 NIAB 131 ~ 133) A method for controlling the collectotrichum gloeosporioides , a pepper anthrax bacterium, using a sterile culture medium of the strain.

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