KR100333039B1 - Novel antagonistic strain Promicromonospora sp. KH-28 and proess for production of antifungal antibiotic therefrom - Google Patents

Abstract

The present invention relates to a novel antagonistic actinomycete strain Promicromonospora sp.KH-28 (Accession No. KCTC8946P) and to a method for producing antifungal antibiotics using the same, which comprises chitinase and antibiotics. Simultaneous production of phytopathogenic fungi Fusarium solani , Fusarium oxysporum , Alternaria mali , Phytophthora capsici , and Altera Kiki Alternaria kiki ) strongly inhibits the KH-28 from Promicromonospora genus, which is strongly inhibited, and is isolated from cultivated soils. The strain has a molecular weight of 503 daltons when incubated at pH 7.0 at 30 ° C for 5 days. It has an excellent effect of producing a large amount of antifungal antibiotics composed of alicyclic derivatives, and is particularly effective in controlling pepper blight and pepper seedling disease. And it is.

Description

Novel antagonistic strain Promicromonosporus ＫH-28 and production method of antifungal antibiotics using the same KH-28 and proess for production of antifungal antibiotic therefrom}

The present invention relates to a novel antagonistic actinomycete strain Promicromonospora sp. KH-28 (Accession No. KCTC8949P) and a method for producing antifungal antibiotics using the same. More specifically, the present invention relates to a novel antagonistic actinomycetes promicromonosporus KH-28 which simultaneously produces chitinase and antifungal antibiotics and a method for producing antifungal antibiotics produced by the strain. .

Today, as the development of human life is enhanced by industrial development, the problem of environmental pollution is getting serious. In particular, the development of agriculture increases the use of chemical fertilizers and chemical pesticides, causing more serious environmental problems. Due to the overuse of chemical pesticides, the damage of phytopathogens against chemical pesticides, residual toxicity, acidification of soil, poisoning of livestock, impact on natural enemies, pollution of drinking water sources and destruction of ecosystems are becoming more serious. Therefore, as one of the alternative methods to escape from the severe side effects caused by such chemical pesticides, a biocontrol method, which is a method of inhibiting phytopathogens by antagonistic microorganisms in soil, has been studied (Siegal. M. and Sisler, H.D. 1977.).

Biocontrol methods using microorganisms can decompose substances such as chitin, β-1,3-glucan, and mannan, which are the cell wall components of fungal phytopathogens. Method of using hydrolase such as chitinase, glucanase, mannase, etc. (Julieta U. Correa, Elango, N., Polacheck, L., and Cabib, E. 1982. , Sivan, A. and I. Chet. 1989., Skujus, JJ, HJ Potgieter and M. Alexander. 1965.) and direct inhibition of the growth of plant pathogens by secreting antibiotics, specific secondary metabolites of antagonists. There is an antibiotic mechanism (Brown, JG and AM Boyle. 1944.). Most of these antibiotics are streptomyces (Streptomyces) It is produced by the genus and is widely used for the development of medical antibiotics, but industrialization as agricultural microbial pesticides has many possibilities. In addition, metal salts necessary for the growth of phytopathogens in soil environment (Fe²⁺We are also studying biocontrol using a mechanism that inhibits the growth of pathogenic fungi by producing a material called siderophore that competitively chelates (Neilands, JB 1984., Teintze, M., MB). Hossain, CL Barnes, J. Leong, and DV Helm. 1981.). However, until now, it is true that antagonistic strains producing single membrane hydrolase or antibiotic as a biological control method using microorganisms have not had a great effect. Selecting a multifunctional antagonist that produces both chitinase and antifungal antibiotics at the same time would be a very effective biocontrol method.

The present inventors have isolated antagonistic microorganisms that specifically inhibit pathogenic fungi of soil, from low diseased arable soil to control soil infectious plant pathogens by Fusarium oxysporum , etc. By simultaneously producing chitinase and antifungal antibiotics, KH-28 ( Promicromonospora sp. KH-28), a multifunctional soil antagonist that has two antagonists, was identified and selected by antagonistic strains. The present invention was completed by investigating the optimum conditions for producing the substances, and after estimating the structure of the antibiotics by separating and purifying the produced antibiotics, and confirming the inhibitory effect in real soil by in vivo experiments.

An object of the present invention is to provide a novel antagonistic actinomycetes Promicromonospora genus KH-28 ( Promicromonospora sp. KH-28) which simultaneously produces chitinase and antifungal antibiotics. Another object of the present invention to provide an antibiotic production method from the culture medium of the novel antagonistic actinomycetes.

The object of the present invention is to select antagonistic actinomycetes that simultaneously produce chitinase and antifungal antibiotics that have strong growth inhibitory activity against phytopathogenic fungi Fusarium oxysporum from soils of low disease farmland nationwide. Taxonomically identified, cultured and physiologically analyzed to identify a novel antagonistic actinomycetes and named it as KH-28 (accession number KCTC 8946P) of the genus Promicromonospora, and then to 12 strains of phytopathogens of the strain. The antimicrobial activity of KH-28 in the antagonistic actinomycetes promicromonospora of the present invention was investigated. Then, in fact, the plant's biological control ability of the novel antagonistic actinomycetes promicromonospora genus KH-28 It was achieved by OK.

Hereinafter, the configuration and operation of the present invention.

1 is a graph showing chitinase productivity of several strains to select antifungal antagonist strains that produce chitinase from root zones in low diseased farmland soil.

Figure 2 is an electron micrograph showing the spores of the genus antagonistic actinomycetes promicromonospora KH-28 incubated on ISP4 medium for 14 days.

Figure 3 shows the results of cellulose thin-film chromatography to investigate the amino acid in the cell wall and diaminopimelic acid (DAP) isomer constituting the cell wall of the present invention antagonistic actinomycetes promicromonospora genus KH-28 .

Figure 4 shows the results of the cellulose thin membrane chromatography of the sugars for the whole cell extract of the genus antagonistic actinomycetes promicromonospora genus KH-28.

5 is a graph showing the effect of pH in the production of antibiotics of the genus KH-28 antagonistic actinomycetes promicromonospora.

Figure 6 is a graph showing the effect of temperature on the production of antibiotics of the genus antagonistic actinomycetes promicromonospora KH-28.

Figure 7 is a graph showing the effect of incubation time in the production of antibiotics KH-28 genus antagonistic actinomycetes micromonospora of the present invention.

Figure 8 shows the ¹ H-NMR spectrum of the antibiotic produced by the antagonistic actinomycetes strain Promicromonospora KH-28 of the present invention.

9 shows Fab-mass spectra of antibiotics produced by the present invention antagonistic actinomycetes promicromonospora genus KH-28.

The present invention comprises the steps of selecting the antagonistic actinomycetes from low diseased arable lands of the country; After culturing the selected antagonistic actinomycetes in a chitinase production medium, the antifungal chitinase production was investigated, the activity of chitinase enzymes was measured, and the antifungal antibiotics were produced in the antagonistic actinomycetes producing chitinase. Selecting only strains producing the substance; The antagonistic actinomycetes of the present invention were identified as the result of the identification of the above-described chitinase and antibiotic-producing strains based on the microbial classification method and the general bacteriology method of the genus KH-28 ( Promicromonospora sp. KH-28) (Accession No. KCTC 8946P); Investigating the antimicrobial activity of the antagonistic actinomycetes promicro monospora genus KH-28 in 12 kinds of phytopathogenic fungi; Investigating the optimal culture temperature, pH and incubation time for the production of antibiotics of the antagonistic actinomycetes promicromonospora genus KH-28 using the modified Bennett medium as a base medium; Extracting and purifying antibiotics by extracting the culture filtrate of KH-28 of the genus antagonistic actinomycetes promicromonospora of the present invention with n -butanol and then purifying with silica gel column chromatography and Sephadex column chromatography; Analyzing the extracted and purified antibiotics using an analyzer such as an ultraviolet spectrometer, a TLC, a mass spectrometer, and a nuclear magnetic resonance spectrometer; Planting peppers infected with Fusarium oxysporum pathogen into the soil as a disease-bearing plant and injecting the antagonistic actinomycetes promicromonosporus genus KH-28 of the present invention into the soil and then investigating the growth of plant growth do.

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 of Antagonistic Actinomycetes

To select actinomycetes from low-border arable soils around the country, 1 g of rhizosphere soil samples were collected, heat-treated at 80 ° C for 30 minutes, and then placed in 10 mL of sterile saline solution and stirred for 20 minutes. Diluted at ^-3 to 10 ^-5 times, 0.1mL was separated into starch-nitrate agar medium for separation, followed by incubation at 28 ° C for 2 to 4 weeks to isolate the filamentous colony. . As a result, over 500 strains of the typical actinomycetes observed in colony and optical microscopy of the cultured and isolated strains were firstly selected and stored by lyophilization.

Example 2: Antifungal Chitinase Productivity and Activity of Antagonistic Actinomycetes

In order to investigate whether chitinase is produced in the antagonistic actinomycetes selected in Example 1, chitinase production medium (colloidal chitin 4g, K ₂ HPO ₄ 0.7g, MgSO _{4 · 5H 2 O 0.5g, KH 2} PO 4 0.3g, FeSO 4. · 7H 2 O 0.01g, MnCl 2 1.0 mg, plate (separate to the plate) of _{ZnSO 4 1,0 mg, agar 20g,} pH 7.0) After inoculation of one strain, it was examined whether colloidal chitin was decomposed to form a clear zone. In addition, the culture supernatant was precipitated with ammonium sulfate and dialyzed with membrane color (membrane sack; MW 12,000) to obtain a modified chitinase enzyme. Enzyme activity was measured by adding 2.0 mL of 0.1M citrate buffer (pH 6.0) and 1.0 mL of 0.5% colloidal chitin, adding 2.0 mL of enzyme solution and shaking for 1 hour at 40 ° C. The reaction was stopped by heating at 100 ° C. for 15 minutes. The reaction solution was centrifuged at 5000 rpm to separate it from undecomposed chitin, and the amount of N-acetylglucosamine in the supernatant was confirmed by the method of Reissig et al. It was defined as the amount of enzyme that produced 1 μM of N-acetylglucosamine. 28 ° C in Bennett medium (glucose 10g, peptone 2g, yeast extract 1g, beef extract 1g, pH 7.0) to select strains that produce antifungal antibiotics among the selected actinomycetes producing chitinase The culture filtrate incubated for 5 days was filtered through membrane color (membrane sack; Sigma, Co., MW 12,000) to separate small molecules and polymers, and then to the Fusarium oxysporum , a plant root bacterium. The antimicrobial activity was investigated by paper disk method. As a result, as shown in FIG. 1, the KH-28 strain having the strongest antifungal activity and the extracellular chitinase productivity was selected.

Example 3: Taxonomic Identification of the Antagonists of the Invention

The antagonistic actinomycetes KH-28 selected in Example 2 were identified taxonomically in accordance with Bergy's manual of systematic bacteriology and Manual of methods for general bacteriology, especially for actinomycetes. The Streptomyces Project (ISP), Numerical Taxonomy Method was identified. Morphological characteristics were observed by light microscopy and electron microscopy after incubation in International Streptomyces Product (ISP) medium, and biochemical properties were analyzed by diaminopimelic acid in cell wall, glucose analysis in cell, etc. It was confirmed. In addition, the physiological characteristics of carbon and nitrogen sources, antimicrobial activity test and substrate degradation were examined. The results of 41 unit trait experiments were stochastically identified by Streptomyces sp and species by using the TAXON program, a computer program developed by Ward et al. As a result, as shown in Table 1, growth was generally good in 12 medium, but poor in ISP6 and ISP7 medium. The aerial mycelium was grey, and the pigment in the medium was generally brown, and yellow in ISP1 and ISP5 medium. Morphological characteristics of the spores were observed on an electron microscope (SEM) using colonies grown in ISP4 medium. As shown in FIG. 2, the spores form a rugose and the chains of the spores are spiraled. (Table 2). As a result of confirming the composition of LL, meso and 3-OH diaminopimelic acid (DAP), which are cell wall components in actinomycetes, it is composed of diaminopimelic acid (diaminopimelic acid) as shown in FIG. It could be confirmed that it is not. In addition, the total sugar analysis in the cell could not identify any sugar as shown in FIG. Therefore, the antagonistic actinomycetes KH-28 of the present invention showed different results from those containing LL-DAP, which is a characteristic of Streptomyces genus, and was confirmed by the microorganism classification volume 4 (volumn 4 of the Bergy's manual). The genus Promicromonospora has been shown to be most suitable. In addition, the optimum growth temperature was 30 ℃, the growth was possible at 45 ℃ and also at pH 10, but did not grow at 10 ℃ and pH 4.3 as a result of confirming the culture, physiological characteristics, etc. of the main strain (Table 3). Proteases such as lecitinase and lipolytic enzymes were not produced, but the degradability of chitin and pectin was strong. Inhibitors such as antibiotics neomycin, rifampicin and NaCl, sodium azide and phenol were not resistant. Hydrogen sulfide production and xanthine degradation were positive, but nitrate production and allantoin and arbutin degradation were negative. Antibacterial activity was shown against Bacillus subtillis and Psudomonas fluoresence strains, but not against Aspergillus niger . In addition, sugar utilization was generally good, but D-fructose, adonitol, and cellobiose showed weak growth. Nitrogen availability was low in L-histidine. As a result of the analysis it was confirmed that the genus Promicromonospora ( Promicromonospora ). Therefore, the antagonistic actinomycetes of the present invention was named Promicromonospora sp.KH -28 ( Promicromonospora sp. KH-28) and was deposited as KCTC 8946P on June 14, 1999 at the Gene Bank in the Biotechnology Research Institute.

Culture Characteristics of Antagonistic Actinomycetes KH-28 Badge growth A mycelium Water soluble pigment Tryptone Yeast Extract Broth (ISP1) M Light yellow Light yellow Yeast malt extract agar (ISP2) G grey Brown Oatmeal Baby (ISP3) G grey Brown Inorganic salt starch agar (ISP4) G grey Light brown Glycerol Asparagine Agar (ISP5) G Grayish white yellow Peptone-Yeast Extract Iron Agar (ISP6) P Light yellow Light brown Tyrosine Baby (ISP7) P grey No color Starch baby G grey Yellowish light brown Nutrition baby M White Light brown Potato Dextrose Baby G grey Brown Glucose peptone agar M Yellowish white Light brown Zapek Baby G grey Pinkish brown [Note] G: Good, M: Normal, P: Poor

Morphological Characteristics of Antagonistic Actinomycetes KH-28 formation Morphological characteristics Colony surface Powder Spore chain gyre Spore surface decoration Pleated Colony size - Other water soluble pigments none Backside color Light yellowish brown Aerial mass color grey Melanoid color none

Growth Characteristics of Antagonistic Actinomycetes KH-28 Property result Optimum growth temperature rangeGrow at 10 ℃ Grow at 45 ℃ No growth at 28 ~ 35 ℃ Optimum pH range Grow at pH 4.3 Grow at pH 10 6.8 ~ 7.0 No growth Rectinase voice Lipolytic enzyme voice Chitin breakdown positivity Pectin degradation positivity Neomycin (50 μg / mL) No growth Rifampicin (50 μg / mL) No growth NaCl (7% w / v) No growth Sodium azide (0.01% w / v) No growth Phenol (0.1% w / v) No growth Nitrate reduction voice Hydrogen sulfide production positivity Xanthine positivity Allantoin voice Arbutin voice Bacillus subtilis Inhibited Pseudomonas Fluorescence Inhibited Aspergillus Niger Do not inhibit

Example 4: Inhibition of growth of various phytopathogens of the present invention antagonistic actinomycetes promicromonospora genus KH-28

Antagonistic actinomycetes Promicro monospora genus KH-28 (Promicromonosporasp. KH-28) cultured filtraten-The inhibitory activity of phytopathogen growth was examined by the paper disk method of the material extracted and concentrated in the butanol layer. Experimental results, as shown in Table 4 Fusarium Solani (F. solani),Fusarium oxysporm (F. oxysporum),Alternaria MarieAlternaria mali),Phytophthora Capsi (Phytophthora capsici),Alterna Kiki (Alternaria kiki) It showed inhibitory effect on the fungus, etc., and choletotricum gloeosporioides (Colletotrichum gloeosporioides),Penicillium Expanium(Penicillum expansum),Fusarium Moni(F. moni),Raizoktonia(Rizoctoniasp.) and other pathogenic fungi did not show inhibition.

Antifungal Spectrum of the Antagonistic Actinomycetes Promicromonosporus KH-28 Against Various Plant Pathogenic Fungi Test mold Antifungal activity Pairing culture Antibiotic Crude Extract ^* (crude antibioic) Pythium ultimum - - Fusarium solani (root rot) + + Fusarium oxysporum + + Rhizoctonia sp. - - Fusarium moni ( Fusarium moni ) - - Alternaria kiki + + Colletotrichum gloeosporioides - - Penicillum expansum - - Stemphylium sp. + + Septoria solani - - Puccinia adenophora - - Phytophthora capsi (Peace) ( Phytophthora capsici ) + + [Note] +: Inhibition,-: No inhibition *: Antibiotic crude extract was extracted with n -butanol from the culture filtrate, concentrated 100-fold and verified by paperdist method.

Example 5 Optimal Conditions by Antibiotic Production of the Agonist Actinomycetes Promicromonospora Species KH-28

In order to investigate the optimal incubation time for the antibiotic production of the antifungal antagonistic actinomycetes selected in Example 2, inoculated with 1% of the starter cultures spawned in each antibiotic production medium to incubate at 30 ° C. The growth and bacterial antagonism of F. oxysporum in the culture supernatants was investigated by the respective analytical methods. In order to investigate the effects of optimal culture temperature and pH on the production of antibiotics of selected antifungal antagonistic actinomycetes, strains were added to antibiotic production medium (glucose 10g, peptone 2g, yeast extract 1g, broth 1g, pH 7.0). Inoculation and incubation at each culture temperature were analyzed for the production of antifungal antibiotics. In addition, the antibiotic production rate was analyzed by adjusting the initial pH of the antibiotic production medium to 2-11. At this time, the basic medium was Bennett medium, and the culture filtrate was extracted with n -butanol, concentrated 100 times, and the average inhibition distance was determined by checking the distance of the inhibition zone 3 times by the paper disk method. Investigate. As a result, antibiotic production according to pH was the most productive at pH 7.0 as shown in Figure 5 and relatively good within the pH range of 5-9. In addition, in order to investigate the effect on the production of antibiotics by the growth temperature of the antagonistic actinomycetes promic monospora KH-28, the production of antibiotics from 15 ℃ to 45 ℃ as shown in Figure 6 30 When incubated at ℃ was the highest productivity and was relatively good within the range of 26 ~ 40 ℃. In order to confirm the antibiotic productivity by incubation time, after 7 days of incubation for each incubation time, as shown in FIG. 7, the highest productivity was obtained when incubated for 5 days and there was no change in inhibition after 5 days. After days, the inhibition was slightly decreased.

Example 6: Extraction and Purification of Antibiotics from the Culture of KH-28 Genus Antagonistic Actinomycetes Promicromonospora

The present invention Antagonistic Actinomycetes main Pro micro mono spokes la in KH-28 The production of the 5-day medium 10L for separating and purifying the antibiotics that was filtered, after which the culture filtrate cultured n is an organic solvent-extracted with n- butanol The butanol layer was concentrated, dissolved in distilled water, stored at 4 ° C. for 1 day, and the centrifuged precipitate was dissolved in methanol to obtain methanol: H ₂ O in silica gel column chromatography (70-230 mesh, Merck, 4 × 20 cm). It was developed at a ratio of 3: 3. It was also eluted with methanol using Sephadex LH-20 column chromatography (Merck, 4 x 20 cm).

Example 7: Instrumental analysis of antibiotics produced by the antagonistic actinomycetes of the present invention

The antibiotics extracted and purified in Example 6 were analyzed using an ultraviolet spectrometer (UV), thin film chromatography (TLC), mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), etc. The analysis was carried out. As a result, as shown in Table 5, the antibiotics were composed of yellow pigment, and the characteristics of the purified antibiotics were found to be soluble in solvents such as n -butanol, methanol, and ethanol, and dissolved in chloroform, toluene, n -hexane, etc. It wasn't. Various biochemical reactions showed positive reactions to 2: 4-dinitrohydrazine (2: 4-Dinitrohydrazine) and showed fluorescent color upon UV irradiation. However, it was negative in the amino acid component identification reaction such as ninhydrin reaction and negative in the non-reducing sugar test results by DNS method. The developing solvent was developed on silica gel TLC with ethanol: ammonia: water = 8: 1: 1 to confirm Rf value of 0.87. UV absorbance spectra showed absorbance at 260 and 440 nm. The ¹ H-NMR and Fab mass spectra shown in FIGS. 8 and 9 show that the pyramidine skeleton has a structure at peaks of 7.713, 7.627, and 4.275 ppm, and peaks of 2.149, 1.884, 1.345, 0.972, and 0.867 ppm. The hydrogen group of the long aliphatic chain was found in. It also showed a molecular weight of 503 dalton.

Analysis of antibiotics produced by KH-28 in the antagonistic actinomycetes promicromonospora KH-28 Exterior Orange-brown Color Response Positive Voice 2: 4-dinitrohydrazine, I ₂ , H ₂ SO ₄ ninhydrin, DNS Soluble Water Soluble Insoluble n -butanol, methanol, ethanol, chloroform, ethyl acetate, acetone, H ₂ O, ethyl ester, n -hexane TLC, RF Ethanol: Ammonia: H ₂ O (8: 1: 1) Methanol: H ₂ O (7: 3) 0.870.74 UV λ ^MeOH nm 240,440

Example 8 Investigation of Biocontrol Activity by Plant Experiment of Inventive Antagonistic Actinomycetes Promicromonospora Genus KH-28

In the present embodiment, to determine whether the antagonistic actinomycetes strain of the present invention, Promicromonospora genus KH-28 inhibits phytopathogens, the pepper as a pathogen plant in a petri dish at 28 ℃ constant temperature room After two sheets of filter paper, the water was soaked properly and incubated for 3 days, the seeds were selected to germinate. When transplanted into a 24-neck plastic pot containing sterilized bermuculite and grown into three germ layers, it was transplanted into a 24-neck plastic pot mixed with other sterilized vermuculites, pathogens and antagonistic actinomycetes. Growth was observed. Experimental results showed that B group mixed with pathogens and antagonists was better than A group inoculated with pathogens and control group C, which was not inoculated with antagonists and pathogens, and especially B group mixed with pathogens and antagonists than control group C. The growth of host plants was found to be good.

As described above, the antagonistic actinomycete strain Promicromonosporus genus KH-28 (Accession No. KCTC8946P) of the present invention selected from the soil of low disease farmland and identified as promicromonospora is a plant. It has an excellent effect of strongly inhibiting the pathogenic fungi Fusarium solanie, Fusarium oxysporum, Alternaria Marie, Phytopopera capsi and Alternaria Kiki. In addition, the antagonistic actinomycete of the present invention is a very useful invention for the biopesticide industry because it has an excellent effect of producing a large amount of antibiotics composed of alicyclic derivatives having a molecular weight of 503dalton when incubated at pH 7.0 and a temperature of 30 ° C for 5 days.

Claims (2)

KH-28, a genus of antagonistic actinomycetes promicromonospora, which produces antifungal antibiotics and chitinases (Accession No. KCTC 8946P). The antagonistic actinomycetes Promicromonospora genus KH-28 (Accession No. KCTC 8946P) described in claim 1 was inoculated into antibiotic production medium (glucose 10g, peptone 2g, yeast extract 1g, broth 1g) to pH 5-9 , Production method of antifungal antibiotics comprising culturing at a temperature of 26 ~ 40 ℃.