KR100327510B1 - Antifungal activity and Preparation-method of antibiotics produced from Micromonospora coerulea Ao58 - Google Patents

Abstract

The invention pepper station pathogen, rice blast fungus, cucumber gray mold pathogen, such as an antibacterial effect against phytopathogenic fungi with antibiotics streptavidin tea midon (streptimidone) to generate micro-mono spokes of secreting new LA to Ko of Luria (Micromonospora coerulea ) Ao58 strain and a method for producing antibiotics produced from the strain.

The micromonospora coeruria Ao58 strain and the antibiotics produced from the strain include Phytophthora capsici and Rhizoctonia solani , Magnaporthe grisea , Capsicum anthrax It has the effect of significantly inhibiting the mycelial growth of bacteria such as Colletotrichum gloeosporioides , Botrytis cinerea , Cladosporium cucumerinum , and Didymella bryoniae .

Description

Micro monospora coerulio bio58 strain having antibacterial activity against phytopathogenic fungi and method for producing antibiotics from the strain {Antifungal activity and Preparation-method of antibiotics produced from Micromonospora coerulea Ao58}

The present invention relates to a micromonospora coerulea strain having a novel antifungal activity and a method for producing an antibiotic produced therefrom. Specifically, the present invention relates to pepper blight, cucumber gray fungus, and rice blast disease. Micromonospora coeruria Ao58 strain having novel antifungal activity by producing and secreting an antimicrobial substance, streptimidone, which has an antimicrobial effect against phytopathogenic fungi such as fungi and antibiotics produced from the strain It relates to a manufacturing method of.

In general, rice blast (Phytophthora blight) is an economically important plant disease, which will be described in detail as follows.

Rice febrile disease is the most infectious disease of rice cultivation, and occurs in the leaves, nodes, earwoods, ear branches and rice seed of rice. When infected with the leaves, there are grayish-white spots on the center and brown spots on the edges, and gray patterns appear on the nodes, and they break well. When infected by the trees, they turn white and become rice when they turn dark. . Therefore, it is impossible to expect a yield in the disease bottle infected with blast.

Magnaporthe grisea invades the epidermis in the form of conidia and overwinters in rice straw or rice seed in the form of mycelia or conidia. To control this disease, methods such as cultivating resistant varieties, refraining from excessive use of nitrogen fertilizers, removing diseased rice straw, using seed disinfectants such as homai and berates, and controlling organic synthetic pesticides are used. However, rice blast is a pathogen with a large genetic variation in which many races are known, and a number of strains resistant to fungicides have been reported, and the effectiveness of the control by the fungicides used in the past is gradually decreasing. For this reason, the development of a fungicide that is more effective in controlling rice blast, and environmentally friendly is urgently needed.

Cayenne pepper plague occurs from the seedling stage to the entire growing season, and mainly occurs in the roots and stems.However, pathogens spring up in the rainwater and occur on the ground, such as leaves, fruits, and branches. In the middle of the growing season and later stages of disease, the diseased tree is wilted at first and later turns reddish yellow. Phytophthora capsici , a causative agent of the causative pepper disease, is a water-loving hemi-aquatic bacterium that easily forms a zygote sac in water or on an incubator. . The pathogens of cayenne pepper disease are overwintered in the state of mycelia and follicles in diseased plant tissues and then germinated again to become the primary infectious agents. Red pepper plague occurs from seedlings to all growing seasons, and in plant cultivation year-round. In particular, pepper blight is also possible seed transmission, but since most of the infectious agents are introduced from the soil is difficult to control.

Therefore, cultivation, crop rotation, etc. of resistant varieties are currently performed for the control of red pepper disease, and the most effective control method so far is chemical control using a fungicide. The amount of fungicide used in pepper cultivation soil to control pepper pest disease using chemical control is about 1,000 M / T (1993) in the packaging of 83,000 information, and the adverse effects on the environment and the killing by the organic synthetic pesticides used are severe. It is a situation that is causing social problems at the level.

From this point of view, it can be said that there is an urgent need for the development of a low-toxic and pollution-free fungicide that can be used for controlling plant diseases such as pepper blight and rice fever.

Many known antibiotics have been isolated from actinomycetes. Among them, actinomycetes of Streptomyces genus are known to produce the most diverse antibiotics in terms of chemical structure and biological activity. Recently, however, as a way to search for new antimicrobial actives, the search for actinomycetes known as rare actinomycetes has been attempted. In particular, actinomycetes in the micromonospora spp . Among the rare actinomycetes, although there is a high possibility of producing various bioactive substances, a lot of studies on agricultural antibiotics have not been made yet.

Actinomycetes in the micromonospora are known to be distributed in soil samples of various environments and types. Until now, in the actinomycetes of the genus Micromonospora , the aminoglycoside antibacterial active substance gentamicin (gentamicin) is the micromonospora purpurea and micromonospora Echinospora ( M. ethinospora) Since its discovery in), various aminoglycoside-based antibacterial substances have been developed. Later, macrolide antibiotics megalomicin and rosaramycin juvenimicins were developed, iodinin, tetrenolin, and bottromycin. A variety of antibiotics have been found in several actinomycetes of the genus Micromonospora. However, no glutarimide antibiotics such as streptimidone have been found in actinomycetes in micromonospora. Most glutarimide antibiotics have high levels of antifungal activity. Reported.

It is an object of the present invention to provide a new strain having antifungal activity and antibiotics produced by the strain, which is used for the control of pepper blight, cucumber gray mold, rice blast, and the like, rice blast, pepper blight and To provide a biological, environmentally friendly medicine or control method for controlling cucumber gray mold.

To this end, the present invention consists of separating the bacteria from the soil, culturing the isolated bacteria on the medium, verifying the antimicrobial effect on the plant, and separating and identifying the antimicrobial active material.

1 is a scanning electron micrograph of the strain of micromonospora coeraria Ao58

Figure 2 is a graph showing the inhibitory effect on the pepper blight and rice blast fungi of the present invention according to the medium composition.

Figure 3 is a graph showing the inhibitory effect on the pepper blight and rice blast bacterium of the present invention according to the incubation time.

Figure 4 is a graph showing the mycelial growth inhibitory effect of red pepper blight and rice blast bacteria on agar medium according to the concentration of antibiotics passed through the XAD-2 chromatography column.

Figure 5 is a graph showing the effect of controlling pepper blight on pepper seedlings (greenhouse pot test) according to the concentration of antibiotics passed through the XAD-2 chromatography column.

Figure 6 is a block diagram showing the purification process of streptimidone in the culture filtrate of the present invention micromonospora coeruria Ao58.

The present inventors isolated about 1,000 species of actinomycetes from the tidal flat soil of the west coast of Korea, and tested the titer on agar plate and pepper plants under greenhouse conditions. Actinomycete strain Ao58 producing thymidone (streptimidone) was selected and identified.

Actinomycetes strain Ao58 of the present invention was distinguished from actinomycetes of different genera because of the same physiological and biochemical characteristics as Micromonospora coerulea .

The present inventors conducted research to develop antifungal antibiotics from soil microorganisms in Korea University Department of Agriculture and Biology since 1993, and found a number of antagonists that produce antifungal antibiotics showing excellent antimicrobial activity against phytopathogenic fungi. Based on this, the present invention was completed.

Specifically, the present inventors separated about 1,000 actinomycetes from soil samples of the tidal flat area of the west coast of Korea and tested the titer under agar medium and greenhouse conditions. It was found and deposited with excellent antimicrobial activity against hospital fungi. (Microbial Accession No .: KCTC 8982P)

In addition, as a result of pure separation of the material produced by the actinomycetes strain Ao58 showing high antifungal activity, it was confirmed that the glutarimide antibiotic streptimidone (streptimidone) is a glutarimide system in the micromonospora This is the first time that the antibiotic streptimidone has been found. The researchers found that the antibiotic had a significantly higher level of antimicrobial activity against plant pathogenic fungi, particularly the bacterium fungal bacterium, as well as cucumber gray fungus and rice blast fungi.

As described above, the present inventors have developed a novel micromonospora coerulea Ao58 strain that produces streptimidone having antimicrobial activity against phytopathogenic fungi, and thus, the present invention is isolated from soil. A novel Micromonospora coerulea Ao58 strain and a method for preparing streptimidone isolated and extracted from the culture medium of the novel strain can be provided.

Hereinafter, with reference to the accompanying drawings the technical idea of a micromonospora coerulea Ao58 strain having an antifungal activity of the present invention configured as described above and an antibiotic production method produced from the strain The explanation is as follows.

In order to select antagonists, soil 10 cm deep from topsoil was taken from the tidal flats of Yeongjong Island, and 5 g of sample soil was mixed with 50 ml of sterile water and shaken with a rotary shaker (150 rpm) for 30 minutes. Prepared. The suspension was filtered through Whatman 1 filter paper and diluted with sterile water in a 1: 100 (v: v) ratio. Humic acid vitamin agar: 1.0 g humic acid, 0.5 g Na ₂ HPO ₄ , 1.71 g KCl, 0.05 g MgSO ₄ · 7H ₂ O, 0.01 g FeSO ₄ 7H ₂ O, 0.02 g CaCO ₃ , 0.5 mg thiamine-HCl, 0.5 mg riboflavin, 0.5 mg niacin, 0.5 mg pyridoxin-HCl, 0.5 mg inositol, 0.5 mg Ca-pantothenate, 0.5 mg p-aminobenzoic acid, 0.25 mg biotin , 50 mg cycloheximide, 18 g agar, 1 liter H ₂ O, adjusted to pH 7.2; vitamins and cycloheximide were filter-sterilized and malt extract agar: 3 g malt extract, 0.5 g peptone, 0.5 g glucose, 0.85% NaCl, 18 g agar, 1 liter H ₂ O), and the inoculated agar plates were incubated at 28 ° C. for 14 days until colonies were generated.

As described above, the bacterial flora, which appeared to be actinomycetes, was selected among the resulting floras and tested for antimicrobial activity against red pepper bacterium, apple anthrax, rice leaf blight and rice blast bacteria. The selected actinomycetes were streaked 30 mm away from the plate of V8 juice agar (V8A) and incubated for 36 hours at 27 ° C. Mycelial disks (mycelial disk, 7 mm in diameter) were removed from the cultures of phytopathogenic fungi such as fungi and red pepper bacterium.

On the other hand, the storage method of the selected antagonists are as follows. For daily use, inoculated with 0.3% TSA medium in a glass test tube with a lid (10 × 28 ㎜). For long-term storage, pure cultures were grown in 0.3% TSB medium and mixed with 15% glycerol. And stored at -70 ° C.

Antibiotic crude extracts were extracted and prepared in the following manner to examine the antimicrobial activity of the antibiotics produced by the selected antagonists. The crude extract is used to test whether the material produced by this strain is actually effective in plant control in order to save time and cost for the complete purification of antibiotics produced by microorganisms. In addition, if the culture filtrate of the microorganism is used as it is, the medium component added for the culture of the microorganism may be left, which may cause a disease promoting effect. Organic solvent extraction is used.

Starch medium to each of the antagonistic actinomycetes in shake flask culture (SSB: 10 g of soluble starch , 10 g of glycerol, 3.0 g of (NH 4) 2 SO 4, 2.0 g of yeast extract, 1 g of MgSO 4 · 7H 2 O, 1 L of distilled water, pH 7.0-7.2 before autoclaving, 3% (w / v) Amberlite XAD-16 hydrophobic aromatic resin (Sigma), fermented for 14 days at 28 ° C, and then cultured 7000 g The culture filtrate recovered by centrifuge was extracted three times with n-butanol. The separated organic solvent layer was concentrated in a vacuum distillation vessel at 40 ° C., and the remaining residue was dissolved in a minimum amount of dichloromethane and stored at −20 ° C. The bioassay was performed in the following manner with the crude extract of each of the antagonistic fungi prepared in this way.

Biological assays of antibiotic crude extracts were performed using the paper disk method on agar plates inoculated with P. capsici and M. grisea . The explanation is as follows.

First, in case of red pepper disease, 10 ml of V8A medium was mixed with 10 ml of red pepper bacteriospoon suspension, and in case of rice blast, 10 ml of suspension of spores and segmented mycelium was added to liquid potato deck. A black plate was prepared by mixing with 10 ml of stratos agar medium (potato dextrose agar) (PDA, potato dextrose 20g, agar 18g, H ₂ O 1L). Then, the sterilized filter paper strips (7 mm) moistened with the crude crude extract were dried at room temperature for 2 hours, placed on a seeded medium inoculated with the target pathogen, and the plate was plated at 28 ° C for 42 hours. The size of the low support was evaluated by the diameter of the low support produced after incubation for 72 hours.

In addition, the inhibitory effect of the antibiotic extract of antagonist Ao58 strain against the infection of pepper blight in pepper plants was evaluated as follows. First, red pepper seeds (type: Hanbyeol), which is a disease of red pepper disease, were sown in plastic pots containing steam sterilized sand and loam soil (1: 1, v: v). Six seedlings grown up to the fourth leaf were implanted into each plastic pot containing steam sterilized loam, sand and peat (1: 1: 1, v: v: v). Thereafter, the crude extract was mixed with 30 ml of the red pepper yeast strain (10 ⁶ zoospores / ml) and placed in the soil of the pot containing the red pepper plant of the first branch. The disease severity was 0 when no symptom of diseased pepper was completely diseased and 0 when no symptom of disease was completely died.

The present invention configured as described above will be described in more detail with reference to the following examples. These examples are intended to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Physiological and Biochemical Properties of Antagonist Ao58

Actinomycetes strain Ao58 of the present invention selected as described above has the characteristics of actinomycetes that produce substrate mycelia on a medium, and shows Gram positive results as a result of Gram staining. Physiological, biochemical and morphological tests for identification of the actinomycetes strain Ao58 were all performed by the method described in the Burgy's manual of systematic bacteriology.

As shown in Table 1, the actinomycetes strain Ao58 forms a substrate mycelia on the medium, but the aerial mycelium is not formed, which is distinguished from the actinomycetes in the Streptomyces genus. In addition, the mycelium deposited green pigments, but there were no pigments spreading out on the medium, and electron microscopic observation showed typical characteristics of the micromonaspora actinomycetes that produced only one spore in one sporophore ( 1). As a result of testing the chemical properties of the genus of the strain, the chemical composition of the cell wall contained the diamino pimelic acid of meso form, and the gyros in the total cell hydrolysis solution. (xylose) and arabinose were identified. The main menaquinone components were found to be MK-10 (H4) and MK-10 (H6).

These features distinguish the genus of Actinomycetes from the genus Micromonospora spp. , Indicating that Actinomycetes strain Ao58 belongs to the genus Micromonospora .

We recorded the results of physiological, biochemical characteristics and carbon source utilization tests, as shown in Table 2, for species classification. As described above, the results of physiological and biochemical analysis of actinomycetes strain Ao58 were compared with those described in the Burgess manual cystematic bacterium, and the actinomycetes strain Ao58 was compared with the micromonospora coerulea. ) Ao58.

Physiological Characteristics of Antagonistic Strain Ao58 Test Items Result (+ = positive,-= negative) Gram dyeing + Aerial hyphae formation - Mycelium Pigment green Pigment Formation on Medium - Spore Count Single spore Shape of spores Spherical or egg-shaped Spore motility - Cell wall chemotype Type II Sugar Components in Cell Hydrolyzate Gyros and Arabinos Master Menaquinone Form MK-10 (H4) and MK-10 (H6)

Biochemical Characteristics and Carbon Source Availability of Ao58 Test Items Result (+ = positive,-= negative) Growth in Czapek-sucrose - Growth in Potato Slices + Carbon source availability L-arabinose - D-arabinose - D-fructose + D-galactose + Glycerol + Inositol - β-lactose + D-mannitol + α-melibioose + Raffinose + L-Ramnose - D-Ribose - Glycosidase activity α-galactosides + α-mannosides + β-gyrosides - Nitrate Reduction - Resolution Cellulose + gelatin + Starch + Flameproof (%, W / W) 3% Intracellular 3-hydroxydiaminopimelate -

Example 2 Micromonospora coerulea

Usefulness of Ao58

1) Antibacterial effect against various phytopathogenic fungi and bacteria

As shown in Table 3, the antimicrobial activity of the micromonospora coerria Ao58 strain on the V8A medium was shown in Table 3, including M. grisea and P. capsici . Botrytis cinerea , Fujium oxysporum f.sp. Fusarium oxysporum f.sp.cucumerinum , Sclerotinia sclerotiorum , Rhizoctonia solani , and choletotricum grooseporis , pathogens that cause apple anthrax The mycelial growth of Oedes ( Colletotrichum gloeosporioides ) was significantly inhibited.

Antimicrobial Effect of Micromonospora coerulea Ao58 against Various Phytopathogenic Fungi Phytopathogenic fungi Low support length (mm) Colletotricum Geroosporioides 17.5 Magnaporte Gracia 26 Phytopsora Capshisai 24 Raiatonia Solani 27 Botrytis Cinema 25 Fujium oxysporum f.sp. Cucumerum 15.5 Screlotini Screlotrium 9.5

The micromonospora coerria Ao58 is Bacillus subtilis and E. coli. E. coli and Pseudomonas solanacearum have no antimicrobial activity.

2) Inhibitory Effect of Crude Extracts Produced by Micromonospora Coeraria Ao58

The filtrate of antagonist Ao58 cultured in 2% TSB for 5 days was extracted with butanol to prepare an antibiotic crude extract, and the antimicrobial activity was compared using a paper disk method. The results are shown in Table 4. The assay plate used was prepared by mixing the yeast suspension of red pepper bacterium and the mycelium suspension of rice blast bacteria with V8A medium.

Inhibitory Effect of Crude Extracts Produced by Micro Monospora Coerulia Ao58 Strains on Mycelial Growth ^* division Chilli Plague Rice Bacteria Diameter of low support 42.0 ± 1.2 29.5 ± 1.2

* Paper slices (7 mm) moistened with 5 ml of antibiotic extract based on the culture filtrate.

Diameter) was placed on the assay medium.

3) Effect of Crude Extracts Produced by Micro Monospora Coeraria Ao58 on the Control of Pepper Disease

We investigated the effects of pest control by soil irrigation of the antibiotic extract of antagonist Ao58 along with the fermenter suspension of pepper blight bacteria in pepper greenhouse. After treatment with the antibiotic extract of the antagonist Ao58, six replicates of pepper seedlings compared with the untreated pepper severity showed excellent inhibitory effect of pepper blight. At this time, the degree of disease was evaluated in 0 to 5 units as shown below when the untreated red pepper seedlings were completely diseased and died. It is 0 when it is not visible at all, while 5 when pepper seedlings are ill and die.

0. No Onset

1.Slightly wilted and necrotic lesions begin to appear

2. About 30-50% of whole plants

3. About 50-80% of all plants

4. About 80-90% of all plants

5. Death of the plant

In addition, the results of the greenhouse test (greenhouse pot test) of the late blight control effect by the crude antibiotic extract is shown in Table 5.

Inhibitory Effects of Micromonospora Coeraria on Pepper Diseases by Crude Extracts division Throughput of Antibiotic Crude Extract (ml) ^* 0 10 50 100 Disease damage 4.9 ± 1.2 2.5 ± 0.8 0.0 ± 0.0 0.0 ± 0.0

^* Amount of culture filtrate

^** Each value is (mean ± standard deviation) for 6 replicates.

Example 3 Culture Conditions

Micromonospora coerulea Ao58 strains are cultured under aerobic conditions in a water-soluble nutrient medium containing an assimilable carbon source, nitrogen source, and inorganic salt to produce various antibiotics such as streptimidone. Since the production amount of these antibiotics varies depending on the culture conditions such as the type of incubator, the incubation temperature and the oxygen supply rate, in order to find out the type and culture time of the antagonistic Ao58 strain to optimally produce the antibiotic, Four mediums were selected for the production of materials and easy to feed. After 21 days of incubation, the culture filtrate separated from each medium was extracted with butanol and the antimicrobial activity was compared by the paper disk method. As a result, the inhibitory effect on the antagonistic actinomycetes Ao58 strain culture filtrate according to the composition of the medium against red pepper bacterium and rice blast bacterium, as shown in Figure 2, was relatively high in the starch liquid medium (SSB).

In addition, the inhibitory effect of the antagonistic actinomycete strain Ao58 culture filtrate with the culture time against the pepper blight and rice blast bacterium was high in both pepper blight and rice blast bacteria during 21 days of culture. At this time, the vertical line on the drawing represents the standard deviation.

Specific composition of the medium used for the antibacterial test of the antagonistic Ao58 strain according to the medium composition is as follows.

Starch liquid medium (SSB, Soluble starch broth)

10 g of soluble starch

10 g of glycerol

(NH ₄ ) ₂ SO ₄ , 3.0 g

2 g of yeast extract

MgSO ₄ 7H ₂ O 1 g

1,000ml of distilled water

Tryptic soy broth

20 g of tryptic soy

1,000 ml of distilled water

Soybean glucose starch broth

25 g soy flour

20 g of glucose

10 g of starch

1 g of meat extract

4 g of dry yeast

NaCl 2 g

K ₂ HPO ₄ .3H ₂ O 0.05 g

1,000 ml of distilled water

Potato dexrose broth

Potato-dextrose 20 g

1,000 ml of distilled water

Example 4 Isolation and Purification of Antibiotic Streptimidone

Mycelia and XAD-16 resin were isolated from the culture of antagonist Ao58, and each was extracted with a mixed solvent of methanol and acetone (50:50), and filtered with Whatman No. 2 filter paper. The extract was concentrated to a minimum amount (about 3 L) under reduced pressure and extracted with 5 L of n-butanol. The extracted organic solvent layer was concentrated again under reduced pressure and dried. The dried samples were dissolved in methanol and tested for antimicrobial activity by paper-disk diffusion analysis. The antibiotic powder showed high levels of antimicrobial activity against red pepper germ and rice blast. As shown in Figure 4 and Figure 5, agar medium and greenhouse pot test showed a significant inhibitory effect against red pepper blight bacteria.

As a next purification step of the sample, the crude antibiotic extract was subjected to flash column chromatography using C18 and inorganic silica gel. Crude extracts of antibiotics were placed in a C18 resin filled in a glass column of 150x200 mm and water and methanol (v: v, 100: 0, 80:20, 60:40, 40:60, 20:80, 0: 100) The mixed solvent was eluted with a concentration gradient. The antimicrobial activity was identified by the paper disk method, concentrated under reduced pressure and replaced with a mixture of chloroform and methanol and passed through a silica gel resin filled in a glass column. Eluted with a mixed solvent of chloroform and methanol, the active fraction was passed through a Sephadex LH-20 column for separation according to molecular weight. The active fraction of the same molecular weight band was eluted with methanol by scraping off a single active band using a silica gel preparative TLC method with a developing solvent of chloroform-methanol (9: 1). It was concentrated and completely purified by preparative high pressure liquid phase analyzer (HPLC), which was named antibiotic Ao58A.

Example 5 Structural Determination of Antibiotic Streptimidone

Purely isolated antibiotic Ao58A showed an Rf value between 0.58-0.63 when developed with chloroform-methanol (9: 1) using TLC. Under the HPLC conditions below, antibiotic Ao58A exhibits a retention time of 35.4 minutes.

Column: SymmetryPrep-C18 (7.8 × 300 mm)

Solvent: Elution with a linear gradient of 50% from 30% methanol to 70% methanol

Detection wavelength: 232 nm

Flow rate: 2 ml / min

The ultraviolet absorption spectrum of the antibiotic Ao58A dissolved in methanol shows the maximum absorption at 232 nm, and the spectral data of ¹ H NMR (CD ₃ OD) and ¹³ C NMR (CD ₃ OD) of the antibiotic Ao58A are shown below. . The structural analysis test (1H, 13C, COSY, HMQC, HMBC) using a nuclear magnetic resonance (NMR) results are shown in Table 6 below.

Analysis based on the data shows that 16 carbons (C) and 23 hydrogen (H) are used, and that the antibiotics were analyzed by a negative FAB mass spectrum. 294 Da, and the molecular formula is C ₁₆ H ₂₃ NO ₄ .

Ultraviolet spectrum and mass spectrometry of antibiotic Ao58A and DEPT, HH COSY, HMQC, HMBC, NOESY experimental results show that the antibiotic Ao58A is 4- (2-hydroxy-5,7-dimethyl-4oxo-6. , 8-non-adedenyl) -2,6-piperidinedione structure was identified as glutarimide antibiotic streptimidone.

Example 6 Antifungal Activity of Antibiotic Strepthymidone

Mycelial growth inhibitory effect on phytopathogenic fungi was compared in V8A medium supplemented with a series of concentrations of purely isolated antibiotic streptimidone. Based on the mycelial growth in the V8A medium without the addition of drugs, the concentration of the drug that started to suppress mycelial growth in the antibiotic-treated medium was evaluated as the minimum inhibitory concentration (MIC), and the results are shown in Table 7 below.

Minimum Inhibitory Concentration of Phytopathogenic Fungi of Streptimidone Purely Isolated from Micromonospora Coerria Ao58 Strains Phytopathogenic fungi Minimum inhibitory concentration (㎍ / mL) Alternaria Brassica 50 Alternaria Solani 50 Botryosparia Tochidia 25 Botrytis Cinema 10 Cylindro carpon destructurals 25 Mycosperella Melonis 5 Magnaporte Gracia 5 Phytopsora Capshisai 5 Raiatonia Solani 50 Screlotini Screlotrium 50

Streptimidone is 5 to about P. capsici , Magnaporte grisea , B. cinerea , and M. melonis . 10㎍ / ㎖ showed the mycelial growth inhibitory effect of high levels in the drug concentration in the culture medium, Alterna Ria solani (A. solani), Alterna Ria Brassica (Alternaria brassica), boat Rios L Ria lightness Dia (B. dothidea ) and cylindrocarpon destructans ( C. destructans ) showed antimicrobial activity in 25-50 μg / ml of drug medium. However, R. solani and Sclerotinia sclerotiorm did not show mycelial growth inhibition.

Example 7 Preparation of Antibiotic Streptimidone

First, inoculate the inoculum of the micromonospora coeruria Ao58 strain preincubated at 28 ° C. for 6 days into 500 mL starch medium (SSB) in a 1 L flask, and then mix at 150 rpm for 21 days at 28 ° C. Fermented while giving. The culture medium (90 L) thus cultured was centrifuged at 7,000 g for 15 minutes to separate the filtrate, the XAD-16 resin and the mycelium, and then each was extracted with a mixed solvent of methanol and acetone (50:50), and then Whatman. Filtered with No.2 filter paper. The extract was concentrated to a minimum amount (about 3 liters) under reduced pressure and extracted with 5 L of n-butanol. The extracted organic solvent layer was concentrated again under reduced pressure and dried. After dissolving the dried sample in methanol and testing the antimicrobial activity through paper-disk diffusion analysis, it showed high level of antimicrobial activity against red pepper bacillus and rice blast bacterium. It showed a significant inhibitory effect on.

For bioassay of antifungal activity, lyophilized antibiotic crude extract was dissolved in a small amount of methanol and added to 40 ° C V-pal juice agar (V8A) medium at concentrations of 0, 1, 10, 100, 1000 μg / ml. . Each 5 ml of the drug medium was poured into a Petri dish having a diameter of 60 mm, the mycelial disks of red pepper germ and rice blast bacteria were removed and placed in the center of the flat medium to which the crude extract was added. The growth inhibition rate of the red pepper bacterium and rice blast bacterium by the crude crude extract was evaluated by comparison with the control medium without the drug.

To evaluate the inhibitory effect of late blight on red pepper seedlings, the freeze-dried XAD-2 eluate dissolved in methanol was 50, 500, 1000 ㎍ / ml, and the suspension of the red pepper blight (10 ⁶ yugi / ml). Together with the soil of pepper seedlings. At this time, after inoculation of the pepper blight, disease damage was evaluated based on the same criteria.

As a next purification step of the sample, the crude antibiotic extract was subjected to flash column chromatography using C18 and inorganic silica gel. Crude extracts of antibiotics were placed in a C18 resin filled in a glass column of 150x200 mm and water and methanol (v: v, 100: 0, 80:20, 60:40, 40:60, 20: 80,0: 100) The mixed solvent was eluted with a concentration gradient. As a result of confirming the antimicrobial activity by the paper disk method, the antimicrobial activity was confirmed in the fraction eluted with 20% and 40% methanol. The fractions were concentrated under reduced pressure, replaced with a mixture of chloroform and methanol, and passed through a silica gel resin filled in a glass column. As a developing solvent, a mixed solvent of chloroform and methanol was used. The active fraction (chloroform: methanol, 9: 1) was concentrated and passed through a Sephadex LH-20 (Sephadex LH-20, C26 / 100) column for molecular weight separation. Using methanol as the developing solvent and developing at a flow rate of 0.15 mL per minute, 3 mL of the eluate was collected, and the antimicrobial activity was confirmed in the fraction of the same molecular weight (81-101 fractions). All of these active fractions were combined and purified by TLC method for silica gel preparative. After developing with a developing solvent of chloroform-methanol (9: 1), a single active band having an Rf value of 0.58-0.63 was scraped and eluted with methanol. It was concentrated and completely purified by preparative high pressure liquid phase analyzer (HPLC). The sample-injected column (SymmetryPrep-C18, 7.8 × 300 mm) was detected at a wavelength of 232 nm while eluting in a straight concentration gradient at a flow rate of 2 mL per minute from 30% methanol to 70% methanol for 50 minutes. At the peak of antimicrobial activity was identified. The aliquots were collected and concentrated in a vacuum distillation unit to obtain 402.7 mg of antibiotic Ao58A, and it was shown above that the structure of streptimidone was obtained through organic spectrochemical analysis for structure determination. In addition, the antifungal activity of streptimidone against various plant phytopathogenic fungi, including red pepper bacillus, was determined through the minimum inhibitory concentration (MIC). As shown in Table 7, the antifungal effect was excellent. there was.

As described above, by providing a micro-monospora coeruria Ao58 strain having an antifungal activity of the present invention and a method for producing antibiotics from the strain, rice fever bacillus, red pepper blight and cucumber ash fungus It can be used to control the back.

In addition, the present invention provides a method for producing an antibiotic, streptimidone, produced from the strain together with a strain having antifungal activity, thereby enabling biological, environmentally friendly drugs or control methods to control rice blast, pepper blight and cucumber gray mold. This could be provided.

Claims (10)

Novel Micromonospora coerulea Ao58 strain with antifungal activity. (Microorganism Accession No .: KCTC 8982P) A pollution-free pesticide characterized by containing the micromonospora coerulea Ao58 strain according to claim 1 or a culture filtrate of the strain. Phytophthora capsici , Magnaporthe grisea , Botryry, characterized in that the micromonospora coerulea Ao58 strain according to claim 1 or a culture filtrate thereof is used. Botrytis cinerea , Didymella bryoniae , Alternaria solani , Alternaria brassica , Botryosphaeria dothidea , Cylindro A method for controlling plant fungal diseases by Carlindrocarpon destructans . The micromonospora coerulea Ao58 strain according to claim 1 was cultured in a liquid medium containing a carbon source, a nitrogen source, and an inorganic salt, in which XAD-resin was added, and the mycelium and XAD-resin were recovered from the culture medium. After separation, the mixture was extracted with methanol and acetone mixture (v: v, 50:50), concentrated, flash chromatography was performed on a column filled with C18 resin and silica gel, and the active fraction was Sephadex LH-20 column chromatography. Purified using TLC method, eluted with methanol, and then eluted with HPLC, characterized in that it is produced from Micromonospora coerulea Ao58 strain having novel antifungal activity. Method for producing antibiotics. The method of claim 4, wherein The strain is characterized in that the culture under aerobic conditions of 20 ℃ to 40 ℃, characterized in that the production of antibiotics produced from Micromonospora coerulea Ao58 strain having a novel antifungal activity. The method of claim 4, wherein Novel antifungal activity Micromonospora coerulea , characterized in that the ratio of the methanol-water mixture performing the step gradient elution in C18 flash column chromatography is 20:80 or 40:60. Method for producing antibiotics produced from Ao58 strain. The method of claim 4, wherein The developing solvent in the TLC method is a mixed solution of chloroform-methanol, characterized in that the antibiotic production produced from Micromonospora coerulea Ao58 strain having a novel antifungal activity. The method of claim 4, wherein Process for producing antibiotics produced from the micromonospora coerulea Ao58 strain having a novel antifungal activity, characterized in that the developing solvent in the HPLC method is a methanol-water mixed solution. The method of claim 4, wherein The liquid medium is a starch broth (soluble starch broth), characterized in that the production of antibiotics produced from the micromonospora coerulea Ao58 strain having a novel antifungal activity. The method according to claim 4, wherein The antibiotic is a antibiotic produced from a micromonospora coerulea Ao58 strain having novel antifungal activity, characterized in that it is a streptimidone, a glutarimide antibiotic. Manufacturing method.