KR100737735B1 - Novel Bacillus sp. WRD-2 isolated from soil and antifungal agent composition thereof - Google Patents

Abstract

The present invention relates to a novel Bacillus sp. WRD-2 ( Bacillus sp. WRD-2, Accession No. KCTC 0869BP) isolated from soil with antifungal activity and an antifungal composition using the same, wherein the soil 10 cm deep from the soil surface is used as a sample. Three strains were collected and screened first, followed by cultivation in an optimal medium to measure antifungal activity against Fusarium oxysporum , a soil infectious pathogen that causes many obstacles to plant growth. The highest activity strain was isolated from the genus Bacillus WRD-2, the strain of the present invention is the most excellent growth and antifungal activity of the cells against Fusarium oxysporum at pH 6.0-8.0, 12 to 15 by incubation time Best among time, growth by carbon source is best with galactose addition and antifungal activity is not affected by addition of carbon source , The other growth in the nitrogen source is the best in the yeast extract addition and the antifungal activity is the best in the addition of malt extract. The WRD-2 and the antifungal composition using the same are excellent antifungal activity against soil infectious microorganisms or phytopathogens, especially Fusarium oxysporum It works.

Bacillus genus WRD-2, Fusarium oxysporum, antifungal composition, antifungal activity,

Description

Novel Bacillus sp. Novel Bacillus sp. WRD-2 isolated from soil and antifungal agent composition             

1 is a photograph showing the antifungal activity of the genus Bacillus WRD-2 against Fusarium oxysporum KCTC 16332,

2 is a graph showing the growth and antifungal activity of Bacillus sp. WRD-2 against Fusarium oxysporum KCTC 16332 according to culture time;

3 is a graph showing the growth and antifungal activity of WRD-2 of the genus Bacillus according to pH for Fusarium oxysporum KCTC 16332,

4 is a graph showing the growth and antifungal activity of the genus Bacillus WRD-2 according to the carbon source for Fusarium oxysporum KCTC 16332,

Figure 5 is a graph showing the growth and antifungal activity of the genus WRD-2 according to the nitrogen source for Fusarium oxysporum KCTC 16332.

The present invention relates to a novel Bacillus genus WRD-2 isolated from the soil and an antifungal composition using the same, and more specifically, three kinds of strains are primarily selected by collecting soil as a sample, which causes many obstacles to plant growth. The present invention relates to a bacterium WRD-2 isolated from cultivation in an optimal medium for measuring antifungal activity against the soil infectious fungus Fusarium oxysporum, and then examined by antifungal activity and an antifungal composition using the same.

In recent years, as international agreements on environmental protection and ecosystem conservation have become the subject of international concern, the use of excessive synthetic pesticides to control pests of crops has emerged as a serious problem internationally. The development of environmentally friendly control techniques has been proposed as a solution to these problems (Lewis, W. J. et al., 1997, A total system approach to sustainable pest management. Proc. Nat. Acad. Sci. USA 94: 12243-122481). Development using microbial secondary metabolites or antagonists as pesticides that are readily degraded in nature and selective for the pathogen of interest is one such alternative.

Biological control using antagonistic microorganisms is a method of artificially controlling interactions such as competition, parasitics, predation or antibiotic activity that can occur between two different species in a natural ecosystem. Since the biological control method has been reported since the interaction of microorganisms in the soil since the 1970s, it has been actively performed as part of a comprehensive control method to isolate antagonists belonging to many kinds of actinomycetes, bacteria and fungi. (Cook, RJ, 1990, Twenty-five years of progress towards biological control of soil-borne plant pathogens, CAB International, Wallingford, UK .; Handelsman J., Stabb, EV, 1996, Biocontrol of soilborne plant pathogens, Plant Cell , 8: 1855-1969). Soil infectious diseases are very difficult to control even with pesticides, and since they are almost impossible to treat, they are actively researching biological control against various soil infectious pathogens (Cook, RJ, 1990, Twenty-five years of progress). towards biological control of soil-borne plant pathogens, CAB International, Wallingford, UK.). In particular, it is most effective to isolate and control specific antagonistic microorganisms with selective antifungal activity against phytopathogens (Kyu-Young Kim, Sang-Dal Kim, 1997, Pyricularia oryzae of the metabolite of Bacillus sp. Biological Control, Kor.J. Appl.Microbiol.Biotechnol.25 (4): 396-402; Kim Sam-sun et al., 1997, Antifungal Activity and Biological Control of Bacillus sp. Microbiol.Biotechnol. 25 (5): 527-536; Lee Yong-se et al., 1999, Isolation of antagonists for the biological control of pepper blight, Kor. J. Life Science, 9 (1): 1-7).

In addition, Fusarium solani , a plant pathogen, Fusarium solani (Kim, Seong-il, Lee Min-woong, 1994, Root-microbe and Soil Disease Control-Effects of Ginseng Root Pathogens on the Ginseng Root Hospital). Korean Journal of Mycology 22 (1): 50-61) Phytophthora capsici (Kim Chang-jin, et al ., 1993, Anticancer Disease Activity of Concanamycin B Produced by Streptomyces neyagawaensis 38D10. Journal of the Industrial Microbial Society 21 (4): 322-328), Pyricularia oryzae (Kim, BS, Hwang, BK, 1993, Production, purification and antifungal activity of antibiotic substances produced by Pseudomonas aeruginosa strain B5.J. Microbiol.Biotechnol. 3 (1): 12-18), Cucumber ash fungus Botrytis cinerea (Yunbongsik et al., 1996, Bacillus subtilis Production of Iturin Antifungal Lipopeptide from Korean Journal of Biotechnology 9 (2): 224-229), Fusarium oxysporum (Kim, YS, Kim, SD, 1994, Antifungal mechanism and properties of antibiotic substances produ ced by Bacillus subtilis YB-70 as biological control agent.J. Microbiol.Biothechnol. 4 (4): 296-304; So, IY, Kim, HM, 1980, On the occurrence and control of the rhizome rot of the common ginger carsed by Fusarium oxysporum f. zingiberi.Kor.J. Microbiol. 18 (4): 172-179).

In particular, Fusarium oxysporum is a soil infectious pathogen that causes a lot of obstacles to various plant growth and damages to farms, and there is a lack of control techniques for this, and thus there is no need for prevention or treatment of the onset of disease. , RJ, 1990, Twenty-five years of progress towards biological control of soil-borne plant pathogens, CAB International, Wallingford, UK.)

The inventors of the present invention have been made in view of the above points for the purpose of effective control of Fusarium oxysporum screening strains having excellent antifungal activity against Fusarium oxysporum from the soil, and for the Fusarium oxysporum The present invention has been completed by determining culture conditions that can further increase antifungal activity.                         

Accordingly, it is an object of the present invention to provide a novel microorganism isolated from soil having antifungal activity against Fusarium oxysporum and an antifungal composition using the same.

The object of the present invention is to inoculate the YMA medium with the Fusarium oxysporum, which is a soil infectious pathogen that causes a lot of obstacles to plant growth by taking 10 cm deep soil as a sample from the soil surface and inoculated in the incubator at 30 ° C. After 4 days of cultivation, three large species of large growth zone (clear zone or halo) were first selected and purely cultured, and the antifungal activity and growth of cells were selected from the selected strains using the paper disk method. This best strain was achieved by final selection and microbiological identification.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Isolation and Identification of Strains Used

In order to select strains with antifungal activity from soil, samples were collected from paddy and field soils in Gyeongnam area. At this time, 10cm deep soil was collected from the soil surface, and the isolates were immediately taken. Fusium oxysporum KCTC 16332, widely known as a phytopathogen, was used as a test bacterium to confirm antifungal activity. The test bacterium Fusarium oxysporum KCTC 16332 was incubated at 10 ⁴ to 10 ⁵ cfu / ml, and then YMA medium (bacto yeast extract 3g / l, bacto malt extract 3g / l, bacto peptone 5g / l, bacto dextrose 10g / 1 liter, bacto agar 20 g / l). After the sample was properly diluted with sterile water, the test bacteria were sequentially spread on YMA medium plated with the test bacteria and incubated for 3 to 4 days in an incubator at 30 ° C. Subsequently, three large-growth growth-lowering rings formed by having antifungal activity against Fusarium oxysporum KCTC 16332 were first selected and purely cultured, and the antifungal activity was selected among the strains selected above using the disk filter method. The best strains were grown and named as WRD-2. 1 is a photograph showing the antifungal activity of WRD-2 against Fusarium oxysporum KCTC 16332. Strains were lyophilized using sterile glycerol mother liquor (1.5% glycerol) and stored in ampule form. In order to maintain the antifungal activity of the strain used in the experiment was used while subcultured in LB medium (tryptophan 10g / L, yeast extract 1.5g / L, NaCl 1.5g / L, agar 15g / L).

WRD-2 was positive in Gram Staining, spores were present in Spore Staining and had a circular colony.

For identification of WRD-2, it was identified as API 50CHB kit by the Biotechnology Research Center Gene Center and identified as Bacillus. Bacillus sp. It was named WRD-2 and deposited with the institution on accession number KCTC 0869BP on 2 October 2000. The physiological characteristics of the microbial strains of the present invention are shown in Table 1.

Physiological Characteristics of WRD-2 of the Genus Bacillus Glycerol + Salicine + Erythritol - Cellobiose + D-Arabinose - Maltose + L-Arabinose + Lactose + Ribose + Melibiose + D-Xylose + Saccharose + L-Xylose - Trehalose - Adonitol - Inuline - β-Methyl-xyloside - Melezitose - Galactose - D-Raffinose + D-Glucose + Amidon + D-Fructose + Glycogen + D-Mannose + Xylitol - L-Sorbose - β-Gentibiose + Rhamnose - D-Turanose - Dulcitol - D-Lyxose - Inositol + D-Tagatose - Manitol + D-Fucose - Sorbitol + L-Fucose - α-Methyl-D-mannoside - D-Arabitol - α-Methyl-D-glucoside + L-Arabitol - N-Acetyl glucosamine - Gluconate - Amygdaline + 2-Keto gluconate - Arbutine + 5-Keto gluconate - Esculine +

Example 2: antifungal activity according to growth time

As a pre-culture of WRD-2 to investigate antifungal activity according to growth time, one cell was added to 50 ml of LB (Luria-Bertani) medium (tryptophan 10g / L, yeast extract 1.5g / L, NaCl 1.5g / L). After inoculation, shaking culture was carried out at 30 ° C. for 24 hours at 200 rpm. The growth curve was measured at 660 nm with a spectrophotometer (HP 8452A) for 24 hours at intervals of 3 hours after inoculating 1% pre-culture with 100 ml of LB medium. In order to measure the antifungal activity of the WRD-2 strain, the test bacteria were prepared with 10 ⁴ to 10 ⁵ spores / ml of spores using 10 ml of 0.85% NaCl in precultured Fusarium oxysporum medium. 100 μl of the prepared spore solution was smeared on PDA (potato infusion 200 g / l, dextrose 20 g / l, agar 15g / l) medium, and 50 μl of supernatant supernatant was centrifuged at 12,000 rpm for 15 minutes. Antifungal activity was determined by subtracting the size of the disk filter using the disk filter method (Lancini, G. et al., 1995, Antibiotics A Multidisciplinary Approach 2nd ed., Plenum Press, NY). The results are shown in FIG. The growth of WRD-2 in the genus Bacillus rapidly increased after 6 hours, showing the highest growth at 15 hours, and then decreasing again. Antifungal activity was highest at 12 hours. Zuber et al. Reported that many antimicrobial substances were produced when the nutrients in the culture were depleted and accumulate at stationary stages. antibiotics.In " Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology and molecular genetics" Abraham, L. Sonenshein, JA, Hoch, RL (eds.), American Society for Microbiology, pp. 897-916.).

Example 3: Comparison of Antifungal Activity According to pH

In order to measure the cell growth and antifungal activity according to the pH of WRD-2, 50 ml of LB medium were adjusted to pH 4.0 to 10.0, respectively, and incubated for 24 hours at 30 ° C. at 200 rpm after inoculating 1% of the preculture. The results are shown in FIG.

Growth and antifungal activity of WRD-2 was not observed at initial pH 4.0 and pH 10.0, was good at pH 6.0-8.0, and maximum at pH 7.0.

Example 4 Comparison of Antifungal Activity According to Carbon Sources

In order to investigate the cell growth and antifungal activity according to the carbon source of WRD-2, 1% of glucose, galactose, maltose and sucrose were added to 50 ml of LB medium as a carbon source, and 1% of the preculture was inoculated at 30 ° C. Incubated at 200 rpm for 24 hours. The results are shown in FIG.

Growth of the cells was good for all cultures containing carbon source, especially when galactose was used. The growth of the hypolipidemic ring was 1.56∼1.84cm, and there was no significant difference, so the carbon source did not affect the antifungal activity.

Example 5 Comparison of Antifungal Activity According to Nitrogen Sources

In order to investigate the cell growth and antifungal activity according to the nitrogen source of WRD-2, peptone, yeast extract, malt extract, (NH ₄ ) ₂ SO ₄ and NH ₄ Cl were replaced with the same concentrations instead of tryptophan, a nitrogen source in LB medium. . pH was adjusted to 7.00, the initial pH of LB medium. After 1% inoculation of the preculture was incubated for 24 hours at 30 ℃ 200 rpm. The results are shown in FIG.

The growth of cells was the best in yeast extract, but the antifungal activity was the highest at 1.85cm in malt extract with the lowest growth of organic nitrogen. In addition, the antifungal activity was generally better when using an organic nitrogen source than when using an inorganic nitrogen source in the form of ammonia.

As described above, the Bacillus genus WRD-2 of the present invention has excellent antifungal activity against Fusarium oxysporum, a soil infectious pathogen causing many obstacles to plant growth. The antifungal composition used is a very useful invention in the biopesticide industry.

Claims (2)

delete Bacillus sp. ( Bacillus sp.) Cultured under shaking at 30 ° C., 6-15 hours, and 200 rpm in an LB medium of pH 6.0-8.0 consisting of malt extract 10g / l, yeast extract 1.5g / l and NaCl 1.5g / l. ) Treat the WRD-2 KCTC 0869BP strain culture solution for Fusarium oxy sports rooms (Fusarium oxysporum), how to control the Fusarium oxy sports rooms (Fusarium oxysporum).

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