KR100489914B1 - Preparation method and antioomycete activity of a novel antibiotic produced by Lechevalieria aerocolonigenes strain VK-A9 against Phytophthora capsici - Google Patents

Abstract

The present invention relates to a novel Lechevalieria aerocolonygenes VK-A9 strain that produces and secretes thiobutasin, a new antibiotic having antibacterial activity against the pepper germ bacterium, which is a phytopathogenic bacterium, and a method for producing antibiotics produced from the strain. It is about.

Lechevalieria aerocolonygenes VK-A9 strains and antibiotics produced from these strains include Phytophthora capsici , Botrytis cinerea , Cladosporium cucumerinum It is effective in remarkably suppressing the growth of).

Description

Preparation method and antioomycete activity of a novel antibiotic produced by Lechevalieria aerocolonigenes strain VK-A9 against Phytophthora capsici}

The present invention relates to a method for producing Lechevalieria aerocolonigenes strain VK-A9 strain having antibacterial activity against phytopathogens and a novel antibiotic produced therefrom. A novel antimicrobial activity of Lechevalieria aerocolonygenes VK-A9 which produces and secretes thiobutasin, a new antibiotic having antimicrobial effect against phytopathogens such as cucumber gray fungus, and antibiotics produced from the strain It is about.

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

The red pepper disease occurs mainly in the roots and stems from the seedling stage to the entire growing season, but pathogens splash into the rainwater and also occur on the ground such as leaves, fruits, and branches. In the middle and later stages of growth, the diseased tree is wilted at first, then later turned yellow to dry.

Phytophthora capsici , a causative agent of red pepper disease, is a water-loving hemi-aquatic bacterium that easily forms a zygote sac in the water or on an incubator, where the spilled zygote swims through the water with two flagella. The causative agent of red pepper disease is the primary infectious agent after overwintering in the form of mycelium or follicle in the diseased plant tissues and becoming a primary infectious agent.

Red pepper plague occurs from seedlings to all growing seasons, and in plant cultivation throughout the year, and when the soil is excessively moist or poorly drained and flooded, the disease-producing environment is promoted. In particular, the pepper blight is also possible seed transmission, but most infectious agents are difficult to control because it is introduced from the soil.

Currently, cultivation and rotation of resistant varieties have been carried out for the control of red pepper disease, but until now, the most effective control method has been known to be chemical control using fungicides. The amount of fungicide used in pepper cultivation soil for the control of red pepper disease using chemical control is about 1,000 M / T in the packaging of 83,000 information, and the side effects on the environment and the killing by the organic synthetic pesticides used are severe. This is causing social problems.

From this point of view, it can be said that the development of a low toxicity and pollution-free fungicide that can be used for the control of pepper disease is urgently required.

Already, a large number of known antibiotics have been isolated from actinomycetes, particularly actinomycetes in Streptomyces , which include aminoglycosides, anthracyclines, glycopeptides and betalactams. It is well known to produce antibiotics with various chemical structures such as β-lactams, macrolides, nucleic acid sequences, peptides, and polyenes. Recently, however, a search for actinomycetes known as rare actinomycetes has been attempted as a way to search for new antimicrobial actives.

The taxonomic location of several species of Saccharothrix , one of the rare actinomycetes, has recently been renamed to Lechevalieria , and actinomycetes of the genus Lecevaleria and Saccharothrix Although it has recently been studied as a separate source, although there is a high possibility of producing various antimicrobial antibiotics, research on agricultural antibiotics has not been conducted so far. Actinomycetes in most Lecevalieria are known to be isolated mainly from general land soils with high organic content. To date, antibiotics found in Lecevariaria, including the genus Saccharotrix, include galacardin and saccharomicin, which have antibacterial activity, and karnamicin and antifungal activity. Formamicin, anticarcinogenic tetrazomine, and herbicidal phosphonothrixin are known. However, no new butanoic acid derivatives, such as thiobutacin, have been found in actinomycetes of the genus Lecevaria.

The present invention is to solve the problems and demands of the prior art as described above, the object is to provide a low-toxic and environmentally friendly new fungicide that can be used in the control of plant diseases, such as pepper blight, cucumber gray mold, etc. I would like to.

To this end, the present invention provides a novel Lechevalieria aerocolonigenes strain VK-A9 strain isolated from soil and a method for producing a new antibiotic thioutacin isolated and extracted from the culture filtrate of the strain. It consists of

The present invention relates to a novel Lechevalieria aerocolonigenes VK-A9 strain (microbial accession number: KCCM 10461) having antibacterial activity. The present invention relates to a pollution-free pesticide containing a strain of Lechevalieria aerocolonigenes VK-A9 or a culture filtrate of the strain. The present invention is for the control of plant diseases caused by Phytophthora capsici or Botrytis cinerea containing Lechevalieria aerocolonigenes VK-A9 strain or its culture filtrate. It is about pollution-free pesticides.

The present invention relates to a novel antibiotic thiobutasine having the following structural formula produced from a novel Lechevalieria aerocolonigenes VK-A9 strain having antibacterial activity.

In the present invention, Lechevalieria aerocolonigenes VK-A9 strain is cultured in a liquid medium containing a carbon source and a nitrogen source, the filtrate is adsorbed to HP-20 resin and developed with methanol to extract with ethyl acetate After concentration, flash chromatography was performed on a column packed with C ₁₈ resin and silica gel, and the antimicrobial activity fraction was subjected to Sephadex LH-20 column chromatography. The eluate eluted with methanol was purified by HPLC for final antimicrobial activity. The present invention relates to a method for preparing thiobutacin, which prepares a novel antibiotic having the following structural formula from a Lechevalieria aerocolonigenes VK-A9 strain.

In the present invention, the strain is a method for producing thiobutacin which is cultured under aerobic conditions of 20 ° C to 40 ° C. In the present invention, methanol-water performing stepwise gradient elution in HP-20 column chromatography is a method for preparing thiobutacin which is 80:20 or 100: 0. In the present invention, methanol-water performing stepwise gradient elution in C ₁₈ reversed-phase flash column chromatography is a method for preparing thiobutacin which is 60:40 or 80:20. In the present invention, chloroform-methanol performing stepwise gradient elution in silica gel flash column chromatography is a method for producing thiobutacin which is 100: 0 or 90:10. In the present invention, the developing solvent in Sephadex LH-20 column chromatography is a method for preparing thiobutacin using methanol. In the present invention, the developing solvent in the HPLC method is a method for preparing thiobutacin, which is a mixed solution of acetonitrile-water to which trifluoroacetic acid is added. In the present invention, the liquid medium is a glycerol peptone broth.

The present inventors isolated about 1,500 actinomycetes from forest, lake, orchard and crop cultivation soils in Gyeonggi and Chungnam, Korea, and tested the potency of various plant pathogens on agar plate. Actinomycetes strain VK-A9, which produces a novel antimicrobial antibiotic thiobutacin with excellent antibacterial effect, was selected and identified.

Actinomycetes strain VK-A9 of the present invention was identical to Lechevalieria aerocolonigenes ( Lechevalieria aerocolonigenes ) physiological, biochemical properties and 16S rDNA base sequence was distinguished from the actinomycetes of other genera.

In other words, the present inventors have conducted research for the development of antifungal antibiotics from soil actinomycetes and bacteria for the last decade, and have shown that many antagonistic fungi that produce antifungal antibiotics exhibiting excellent antimicrobial activity against phytopathogenic fungi. After discovering, based on this, the present invention has been completed.

In other words, the present inventors isolated about 1,500 actinomycetes from forest, lake, orchard and crop cultivation soils in Gyeonggi and Chungnam, Korea, and tested the titer of various plant pathogens on agar plate. Actinomycetes strain VK-A9 with excellent antibacterial effect was found and deposited as an actinomycetes strain with antibacterial activity against phytopathogenic fungi (Microbial Accession No .: KCCM 10461).

Subsequently, through the continuous research, the actinomycetes strain VK-A9 was identified, and the chemical structure of the antibiotics with antimicrobial activity was purified from the culture filtrate, and the antimicrobial effect in vitro and greenhouses against various phytopathogenic fungi were identified. The control effect against pepper blight under conditions was bioassayed. As a result, it was the first time that the identified antibiotic, thiobutasin, was found as a natural substance. It was confirmed that it has antimicrobial activity.

Accordingly, the present invention is to provide a method for producing a new antibiotic thiobutacin produced and isolated from the culture filtrate of Lechevalieria aerocolonigenes strain VK-A9 strain isolated from the soil and the actinomycetes strains It is.

Hereinafter, Lechevalieria aerocolonigenes strain VK-A9 strain having the antimicrobial activity of the present invention configured as described above ( Lechevalieria aerocolonigenes strain VK-A9) and attached to the technical idea of a novel antibiotic production method produced from the strain It will be described in detail with reference to.

In order to select antagonists, 10-20 cm of soil was collected from the surface layer and air-dried at room temperature for one week. 1 g of the dried soil sample was mixed with 10 ml of sterile water and shaken for 1 hour in a rotary shaker (170 rpm) to prepare a soil suspension. The suspension was left at room temperature for 1 hour and the supernatant was taken and diluted with sterile water in a ratio of 1: 1000 (v: v). 1 ml of each dilution was added to 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 for the actinomycetes selection medium). 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 , 0.25mg biotin, 50mg cycloheximide, 18g agar, 1 liter H ₂ O, adjusted to pH 7.2; vitamins and cycloheximide were filter-sterilized) and spread the agar plate medium at 30 ° C for 4 weeks Incubate until development occurs.

Among the germs generated by culturing as described above, the germs that appear to be actinomycetes, that is, unlike general bacteria, form mycelia (substrate mycelium) and aerial mycelium (aerial mycelium) to form a mycelium on the agar medium to select Antimicrobial activity against red pepper bacterium, cucumber anthrax bacterium, rice blast bacterium, tomato wilted bacterium, rice leaf blight blight, and apple polyposis was tested.

To this end, selected actinomycetes strains were single-streaking in the middle of a plate of V8-juice agar (20% V8-juice, 18 g agar, 1 L distilled water, pH 6.4) and 4 at 28 ° C. After incubation for 10 days, the mycelial disk (5 mm in diameter) removed from the culture of phytopathogenic fungi, such as red pepper bacillus and cucumber anthrax, was placed 30 mm away from the center of the medium. The jersey length was then measured.

On the other hand, the storage method of the selected antagonistic strains is as follows. For daily use, it was inoculated with yeast-malt extract agar agar medium (10g malt extract, 4g yeast extract, 4g glucose, 18g agar) contained in a glass test tube (10 × 28 mm), and yeast-malt extract for long-term storage. After propagation in broth, pure cultures were mixed with 15% glycerol and stored in a cryogenic cooler at -70 ° C.

In order to test the antimicrobial activity of the antibiotics produced by the selected antagonistic strains in advance, crude extracts of the antibiotics were prepared by the following method.

The reason for extracting crude extracts is that it takes time and money to fully purify the antibiotics produced by microorganisms. Therefore, it is necessary to test whether the antibiotics produced by the test strain at the initial stage of the assay actually have a control effect in plants. It is for. At this time, if the culture filtrate of the microorganism is used as it is, the medium component added for the culture of the microorganism may remain, and thus may have a disease promoting effect. Thus, the ethyl acetate may be removed to remove the medium component and recover the pure secondary metabolite produced by the microorganism. Organic solvent extraction method is used.

The antagonistic bacteria were fermented in glycerol-peptone medium (GPB: 20 g glycerol, 10 g polypeptone, 5 g meat extract, 1 L distilled water, pH 7.0 before autoclaving) by shaking flask culture, and incubated at 28 ° C. for 10 days. The culture solution was centrifuged at 8000 g or filtered through a filter-paper to remove the cells from the culture filtrate three times with the same volume of ethyl acetate. In addition, the separated ethyl acetate layer was concentrated in a vacuum distillation at 35-40 ° C., and the remaining residue was dissolved in a minimum amount of methanol 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 the crude crude extracts were performed using the paper disk-agar diffusion method on agar plates inoculated with red pepper bacillus ( Phytophthora capsici ) and cucumber anthrax ( colletotrichum orbiculare ). Explained as follows.

In the case of red pepper bacterium, a bioassay plate medium was prepared by mixing a red pepper juice agar medium with a liquid vitreous juice agar medium (10 ⁶ shakers / ml), and in the case of cucumber anthrax, a conidia spore suspension (10 ⁶ spores). / Ml) was mixed with liquid potato dextrose agar (potato dextrose agar; 20 g potato dextrose broth, 10 g agar, H ₂ O 1 L) to make a black plate. Then, the sterilized filter paper (paper disk, 7 mm) moistened with the crude crude extract was dried at room temperature for 2 hours and placed on a seeded agar media inoculated with the target pathogen. The degree of inhibition was evaluated by the diameter of the low support produced after 72 hours of incubation.

Next will be described in more detail with reference to the embodiment of the present invention configured as described above.

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 Identification of Antagonist VK-A9 by Physiological, Biochemical Characteristics and 16S rDNA Sequencing

The selected antagonistic actinomycetes strain VK-A9 has the characteristics of actinomycetes that generate aerial mycelia and matrix mycelia on the medium, and showed Gram positive results as a result of Gram staining test. Morphological, cytochemical, physiological and biochemical tests for identification of the actinomycetes strain VK-A9 are all carried out in Burgy's manual of systematic bacteriology and Burgess manual of determinant. It was performed according to the method described in the Bergey's manual of determinative bacteriology.

In order to identify 16S rDNA sequencing of the actinomycetes strain VK-A9, 16S rDNA was purely separated from chromosomal DNA, and nucleotide sequence was analyzed. The nucleotide sequence of the analyzed 16S rDNA was identified in the actinomycetes strain VK-A9 compared to the NCBI GenBank database, the base sequence of 16S rDNA of the actinomycetes strain VK-A9 was EMBL / Genebank Deposited in (EMBL / GenBank) database (EMBL / GenBank Accession number: AY196702).

The culture characteristics of the antagonist VK-A9 strain were evaluated using six ISP (International Streptomyces Project) media and nutrient agar and Bennett's agar media.

Antagonist VK-A9 strains showed good growth in all media except peptone-yeast extract iron agar (ISP 6 medium). White or light yellow aerial mycelium was formed in all the medium, and the color of the substrate mycelia formed was light yellow or light brown. Tyrosine agar (ISP 7 medium) produced light brown water-soluble pigments, but the formation of water-soluble pigments was not observed in the other medium.

Spore chain shape of the VK-A9 strain was confirmed by scanning electron microscopy (see Figure 1A). VK-A9 strain forms long cylindrical spores of 0.3-0.6 × 0.8-1.6 μm on aerial hyphae, and spore chains contain 8-14 spores in a straight or lightly flexuous type. Was observed. In addition, unusual structures such as pseudosporangium were also observed (see FIG. 1B).

In order to identify the diaminopimelic acid (DAP) isomer, one of the cell wall components of actinomycetes, the cell wall components of the VK-A9 strain were analyzed.The only amino acid of peptidoglycan was meso- It was confirmed that it contained DAP. Sugars such as galactose, rhamnose, and mannose were detected in the mycelium extract of the VK-A9 strain, but arabinose, madurose, xylose, and xylose were detected. And the like were not detected. These results show that the VK-A9 strain corresponds to cell wall type III and whole cell sugar pattern type E in cell components.

As a result of analyzing menaquinone, tetrahydrogenated menaquinone [MK-9 (H ₄ )] having nine isoprene units was detected as the major menaquinone of the VK-A9 strain. Mycolic acid was not detected. This fact suggests that the VK-A9 strain belongs to the genus Lechevalieria , a genus of rare actinomycetes that is distinct from the well-known actinomycetes Streptomyces .

VK-A9 strains were degradable against casein, esculin, gelatin, hypoxanthine, starch, tyrosine, and urea, but not hydrolyzed against adenine, hippurate, and xanthine. Nitrate reduction was observed in KNO ₃ -added medium, and phosphatase activity was detected. The VK-A9 strain did not grow at 45 ° C, was resistant to 4% NaCl, and had the ability to use citrate, but the ability to use oxalate and DL-tartrate was not observed. Acid production from L-arabinose, cellobiose, dextrin, glucose, glycerol, inositol, lactose, melibiose, L-rhamnose, salicin, sucrose, and D-xylose were observed.

Based on the above morphological and culture, cytochemical, physiological and biochemical properties, the antagonistic VK-A9 strain was the best match with Lechevalieria aerocolonigenes . These results were again confirmed through 16S rDNA sequencing of the VK-A9 strain.

Phylogenetic tree determined by the cluster method shows that the VK-A9 strain is the best match with Lechevalieria aerocolonigenes (FIG. 2). Of the 1476 16S rDNA sequences of the VK-A9 strain, 1474 corresponded to Lechevalieria aerocolonigenes ISP 5034 (EMBL / GenBank Accession number: AB020030), with 99.9% base sequence similarity of the two strains. It was confirmed that.

Based on the above morphological, physiological and biochemical characteristics and the results of 16S rDNA sequencing, the antagonistic strain VK-A9 was identified as Lechevalieria aerocolonigenes strain VK-A9. .

Table 1 Morphological and cytochemical characteristics of antagonistic VK-A9 strains

[Table 2] Physiological and biochemical characteristics of antagonist VK-A9 strain

Example 2 Antibacterial Effect Test

    1) Antibacterial effect against various phytopathogenic fungi and oomycete

The antimicrobial activity of Lechevalieria aerocolonigenes strain VK-A9 on V8-juice agar medium, as shown in Table 3, was investigated in apples, including the red pepper blight Phytophthora capsici . Alternaria mali , a double fungus, Colletotrichum orbiculare , which causes cucumber anthrax, Colletotrichum gloeosporioides , which causes apple anthrax, and rice crust Magnaporthe grisea , and Rhizoctonia solani , which causes rice leaf blight, significantly inhibited mycelial growth.

[Table 3] Antimicrobial Effects of Lechevalieria Aerocolonygenes Strain VK-A9 against Various Plant Pathogenic Fungi and Fungi

The Lechevalieria aerocolonigenes strain VK-A9 is Bacillus subtilis , E. coli. Bacteria, such as E. coli and Ralstonia solanacearum , have no antimicrobial activity.

2) Antimicrobial Effect of Antibiotic Crude Extract Produced by Lechevalieria aerocolonigenes Strain VK-A9

A crude extract of the antibiotics produced by extracting the culture filtrate of Lechevalieria aerocolonygenes strain VK-A9 incubated in glycerol-peptone medium for 10 days with ethyl acetate. Bioassay was compared by the paper disk method to compare the antimicrobial activity, and the results are shown in Table 4 below. Antibiotic crude extract produced by Rechevaleria aerocolonigenes strain VK-A9 showed distinct antimicrobial activity against pepper germ and cucumber anthrax in vitro .

[Table 4] Inhibitory Effect of Crude Extracts of Antibiotics Produced by Lechevalieria aerocolonigenes Strain VK-A9

* A paper slice (7 mm in diameter) moistened with 5 ml of antibiotic crude extract was placed on the assay medium.

Example 3 Culture Conditions of Lechevalieria Aerocolonygenes Strain VK-A9 for Antibiotic Production

In order to determine the optimal production conditions of the antimicrobial material produced by the antagonistic strain VK-A9, the production changes of the antibiotics were investigated for each culture time using four different media. The medium used here is starch-casein broth (SCB), starch glucose broth (SGB), glycerol dextrin broth (GDB) and glycerol peptone broth (GDB). , GPB).

After inoculation, the culture filtrate was recovered from each medium at 2 days intervals from 4 days to 14 days, and then adsorbed onto Diaion HP-20 resin and extracted with methanol to prevent the antimicrobial activity against cucumber anthrax, red pepper bacterium, and lyxonia solani. Assay.

As a result, the production of antibiotics increased as the incubation time in each medium, especially in the glycerol-peptone medium showed the highest antimicrobial activity when cultured for 8 days, the highest amount of antimicrobial material is produced It was found (see Figure 3).

On glycerol-peptone medium, the pH was increased and decreased repeatedly during the incubation period, and there seems to be some correlation between the secretion of the antimicrobial substance and the change of pH. Cell mass was observed to increase continuously until 14 days of culture (see FIG. 4).

Through the above results, it was determined that the culture conditions for the production of antibiotics from Lechevalieria aerocolonygenes strain VK-A9 were cultured in glycerol-peptone medium for 8 days, and a total of 78 liters were cultured.

The specific composition of the medium used for the production of antibiotics of Lechevalieria aerocolonigenes strain VK-A9 according to the medium composition is as follows.

Example 4 Isolation and Purification of the Antibiotic Thiobutasin

Lechevalieria aerocolonigenes strain VK-A9 of the present invention was incubated in a glycerol peptone liquid medium for a total of 78 liters at 28 ° C for 8 days, and the culture filtrate was removed 6L of Diaion HP-20 resin Was adsorbed on to develop an HP-20 column with 12 liters each from 0% to 100% methanol. Thereafter, the purification of antibiotics was performed through the process shown in FIG. 6.

80% and 100% methanol fractions showing antimicrobial activity among the fractions in which HP-20 column was developed from 0% to 100% methanol were collected, concentrated under reduced pressure, and extracted with the same volume of ethyl acetate to collect only the organic solvent layer. This was again concentrated in small amounts and subjected to reverse phase flash chromatography with C ₁₈ resin. Among the fractions developed from 0% to 100% methanol, 60% and 80% methanol fractions showed antimicrobial activity. As a result of collecting silica gel flash chromatography, the antimicrobial activity was observed in 100% and 90% chloroform fractions. This active fraction was partially purified by running with 100% methanol using a Sephadex LH-20 column. Sephadex LH-20 column chromatography was performed again from the 31 to 52 fractions showing antimicrobial activity among the 300 fractions.

Partially purified antimicrobial material (Section 57-81, Figure 5) was purified through Sephadex LH-20 column chromatography by preparative HPLC using a C ₁₈ reversed phase column at UV 227 nm. Among the peaks fractionated in a linear gradient solvent development system from 10% acetonitrile to 50% acetonitrile with 0.1% trifluoroacetic acid (TFA) added, the substance detected at the peak of 24.03 minutes retention time was identified as Phytophthora capsici. Mycelial growth inhibitory effect on). This antimicrobial was developed with several other organic solvents on analytical HPLC and showed a single peak.

All of the isolated antimicrobial activity peaks were collected and concentrated using a preparative HPLC system, and finally 9.8 mg of a brown viscous antimicrobial active material was obtained. Pure anti-microbial active material was dissolved in methanol and the UV spectral pattern was found to show maximum absorbance at wavelengths of 227nm, 260nm, and 360nm.

Example 5 Determination of Chemical Structure of Thiobutasin

NMR spectra were investigated using a Bruker AMX 500 NMR spectrometer for chemical structure analysis of purely isolated antimicrobial actives.

The ¹ H NMR and ¹³ C NMR spectra were dissolved in CD ₃ OD, and the DEPT spectroscopy experiments were conducted to determine the binding state of carbon and the correlation between hydrogen and carbon atoms. Two-dimensional NMR spectroscopy was performed.

Two-dimensional NMR spectroscopy was performed with ¹ H- ¹ H and ¹ H- ¹³ C COZY spectroscopy, HMBC, HMQC, HSQC, ROESY and NOESY spectroscopy by dissolving the antimicrobial in CD ₃ OD. High resolution mass spectrometry was performed to determine the composition ratios of nitrogen, oxygen and oxygen atoms. Mass spectra were identified by EI method using J EOL HX 100A-HX100A Tandem mass spectrometer. The chemical structure of the antimicrobial active material was identified by analyzing the spectra obtained using the above organic spectroscopic method.

As a result of analyzing the antimicrobial substance by high resolution EI mass spectrometry, the molecular formula was estimated to be C ₁₁ H ₁₃ NO ₃ S and the molecular weight was 239.2951. In addition, ¹ H, ¹³ C NMR and DEPT spectra were analyzed to confirm that 13 hydrogen and 11 carbon is present. Table 5 shows the results of NMR analysis of the antibiotic thiobutasin.

[Table 5] NMR analysis results of the antibiotic thiobutasin produced by Lechevalieria aerocolonygenes strain VK-A9

^a chemical shift in 125 MHz, ppm

^b Chemical shift in 500 MHz, ppm

^c Abbreviation for Signal multiplicity, s: singlet, dd: doublet of doublets, ddd: doublet of doublet of doublets

Hydrogen NMR spectra confirmed the presence of methine groups at the peaks located at 6.60, 6.73, 7.23, and 7.78 ppm. The carbon NMR spectra confirmed the presence of aromatic carbons at the peaks located at 115.0, 117.0, 130.8, and 134.3 ppm and the presence of amine groups at the peak located at 151.4 ppm. Carbonyl groups were also detected at the peak of the carbon NMR spectrum located at 199.2 ppm. These results suggest that the chemical structure of the antimicrobial material is that the amine group and the carbonyl group are bonded to the aromatic group. Hydrogen NMR and carbon NMR spectral analysis confirmed the presence of methylene groups at the peaks at 3.27 and 3.66 ppm and 41.0 ppm, respectively. The presence of methyl groups bound to sulfur ions was confirmed by hydrogen NMR peaks at 2.23 ppm and carbon NMR peaks at 13.2 ppm.

HMBC spectral analysis confirmed the correlation between carbonyl carbon (199.2 ppm, C-7) and aromatic hydrogen (7.78 ppm, H-6). The correlation between the carbonyl carbon (199.2 ppm, C-7) and methylence hydrogen (3.27, 3.66 ppm, H-8) was also confirmed. Correlation between carboxylic acid carbon (174.9 ppm) and methylence hydrogen (3.27, 3.66 ppm, H-8) and methyl carbon containing elemental sulfur (13.2 ppm) with hydrogen (H-9) at 3.71 ppm position It was also confirmed through HMBC spectral analysis.

Based on the analysis and synthesis of all the above spectral results, the purely isolated antimicrobial agent is 4- (2-aminophenyl) -4-oxo-2-methylthio, a novel butanoic acid derivative that has not been reported as a natural or synthetic substance. It was found to be -butanoic acid, and we named this novel antibiotic thiobutacin.

[Chemical structure of thiobutacin]

Several butanoic acid derivatives with bioactive activity have been reported to be isolated from natural substances or organically synthesized. 3- (3-indolyl) -butanoic acid has been reported to have antibacterial effect against Ralstonia solanacearum , which causes bacterial wilt disease, and 2-amino-2-methyl-butanoic acid is known to have anticancer effect. However, the antifungal and anti-oomycete activity of butanoic acid derivatives has not been reported so far, and the new antibiotic butoic acid thiobutacin has been isolated from actinomycetes of Lechevalieria .

Example 6 Antimicrobial Activity of the Novel Antibiotic Thiobutasin

Growth inhibitory effects on phytopathogenic fungi, crude fungi and yeasts, and bacteria were compared in potato dextrose medium or nutrient medium to which freshly isolated new antibiotic thiobutasin was added in a series of concentrations. Based on the microbial growth in the medium without antibiotics, the concentration at which growth inhibition began to occur in the antibiotic-treated medium was evaluated as the minimum inhibitory concentration (MIC), and the results are shown in Table 6 below.

TABLE 6 Minimum Inhibitory Concentrations for Various Microbial Growth of Novel Antibiotic Thiobutasin Isolated from Lechevalieria Aerocolonygenes Strain VK-A9

Between the novel antibiotics thio butanone worn pie Saratov seashell kaepsi (Phytophthora capsici), beam-trimethyl teeth on cine Liao (Botrytis cinerea), Cloud FIG sports Solarium Cuckoo Mary num (Cladosporium cucumerinum), (Saccharomyces serevisiae ) in my process serenity busy in Saccharomyces High growth inhibition effect.

Example 7 Preparation of New Antibiotic Thiobutasin

After inoculating 5 ml of the inoculum of Lechevalieria aerocolonygenes strain VK-A9 pre-cultured in yeast-malt extraction medium (100 ml) at 28 ° C. for 2 days in 500 ml glycerol peptone medium in a 1 L flask And fermentation in a shaker incubator at 28 ℃ for 10 days at a rotational speed of 150 rpm. The culture solution (78 L) was centrifuged at 30,000 g for 10 minutes, the culture filtrate and mycelium were separated, and then adsorbed onto 6 liter Diaion HP-20 resin, which was then HP with 12 liters of 0% to 100% methanol. -20 columns were developed. Each developed fraction was concentrated again to dryness under reduced pressure. The dried samples were dissolved in a small amount of methanol and tested for antimicrobial activity by the paper disk-agar diffusion method, and showed high levels of antimicrobial activity against pepper germ and cucumber anthrax.

Subsequently, 80% and 100% methanol fractions showing antimicrobial activity were collected and concentrated under reduced pressure, and the organic solvent layer was extracted with the same volume of ethyl acetate. Was collected. This was again concentrated in small amounts and subjected to reverse phase flash chromatography with C ₁₈ resin. Among the fractions developed from 0% to 100% methanol, 60% and 80% methanol fractions showed antimicrobial activity. As a result of collecting silica gel flash chromatography, the antimicrobial activity was observed in 100% and 90% chloroform fractions. This active fraction was partially purified by running with 100% methanol using a Sephadex LH-20 column. Sephadex LH-20 column chromatography was performed again from the 31 to 52 fractions showing antimicrobial activity among the 300 fractions.

Partially purified antimicrobial material (fractions 57-81) via Sephadex LH-20 column chromatography was completely purified by preparative HPLC using a C ₁₈ reversed phase column at UV 227 nm. Among the peaks fractionated in a linear gradient solvent development system from 10% acetonitrile to 50% acetonitrile with 0.1% trifluoroacetic acid (TFA) added, the substance detected at the peak of 24.03 minutes retention time was identified as Phytophthora capsici. Mycelial growth inhibitory effect on). This antimicrobial was developed with several other organic solvents on analytical HPLC and showed a single peak. All of the antimicrobial activity peaks separated using a preparative HPLC system were collected and concentrated, and finally, 9.8 mg of the brown viscous antibiotic thiobutasin was obtained.

Through the organic spectrochemical analysis for the determination of chemical structure, it was suggested above that the thiobutasin is an antibiotic of a new structure that has not been reported so far. In addition, the antimicrobial activity of thiobutacin against various kinds of microorganisms, including pepper blight, was determined through the minimum inhibitory concentration (MIC). As shown in Table 6, anti-oomycete activity was increased. It was found to be excellent.

As described above, the present invention Lechevaleriaria aero colony genes VK-A9 strain and a method for producing a new antibiotic material produced from this strain is to produce and secrete a new antibiotic thiobutacin having an antimicrobial effect against pepper blight bacteria By providing a novel Lechevalieria aerocolonygenes VK-A9 strain, it is possible to use the antibiotic produced from the Lechevalieria aerocolonygenes VK-A9 strain for the control of pepper blight. Thus, by completing the present invention, it is possible to provide a biological, environmentally friendly drugs and control methods that can be used for pepper blight.

1 is a scanning electron micrograph of the spore chain and pseudosporangia of the Lechevalieria aerocolonygenes VK-A9 strain.

Figure 2 is a schematic diagram showing the taxonomic location through 16S rDNA sequencing of the Lechevalieria aerocolonygenes VK-A9 strain.

Figure 3 is a graph showing the inhibitory effect on the phytopathogens of the present invention according to the medium composition and incubation time.

Figure 4 is a graph showing the inhibitory effect on antibiotic production and phytopathogens with the incubation time in the antibiotic production medium of glycerol-peptone medium of the present invention.

FIG. 5 is a graph showing the antimicrobial effect against red pepper bacterium according to the fraction of antibiotics passed through the Sephadex LH-20 chromatography column.

Figure 6 is a block diagram showing the process of purifying thiobutacin from the culture filtrate of the Lechevalieria aerocolonygenes VK-A9 strain of the present invention.

Claims (12)

Novel Lechevalieria aerocolonigenes VK-A9 strain with antimicrobial activity (Microbial Accession No .: KCCM 10461). A plant disease control agent comprising the Lechevalieria aerocolonigenes VK-A9 strain according to claim 1 or a culture filtrate of the strain. Phytophthora capsici or Botrytis cinerea , characterized by containing the Lechevalieria aerocolonigenes VK-A9 strain or the culture filtrate thereof according to claim 1 Control of plant diseases caused by. Novel antibiotic thiobutasine having the following structural formula produced from a novel Lechevalieria aerocolonigenes VK-A9 strain having antibacterial activity. The Lechevalieria aerocolonigenes VK-A9 strain according to claim 1 was incubated in a liquid medium containing a carbon source and a nitrogen source, and the culture filtrate was adsorbed onto HP-20 resin and developed with methanol to obtain ethyl acetate. After extraction and concentration, flash chromatography was performed on a column packed with C ₁₈ resin and silica gel, and the antimicrobial active fraction was subjected to Sephadex LH-20 column chromatography. The eluate eluted with methanol was purified by HPLC for final antibacterial activity. A method for producing thiobutacin , characterized by preparing a novel antibiotic of the following structural formula from a novel Lechevalieria aerocolonigenes VK-A9 strain having activity. The method of claim 5, wherein the strain is cultured under aerobic conditions of 20 ℃ to 40 ℃. The method of claim 5, wherein the methanol-water performing stepwise gradient elution in HP-20 column chromatography is 80:20 or 100: 0. 6. The process of claim 5, wherein the methanol-water undergoing stepwise gradient elution in C ₁₈ reverse phase flash column chromatography is 60:40 or 80:20. The method of claim 5, wherein the chloroform-methanol which is subjected to step gradient gradient in silica gel flash column chromatography is 100: 0 or 90:10. The method of claim 5, wherein the developing solvent in Sephadex LH-20 column chromatography is methanol. The method of claim 5, wherein the developing solvent in the HPLC method is a mixed solution of acetonitrile-water to which trifluoroacetic acid is added. The method of claim 5, wherein the liquid medium is glycerol peptone broth.