KR20050072341A - Streptomyces roseoflavus ls-a24 strain effective against plant pathogenic fungi and fungicides produced from it - Google Patents

Abstract

The present invention relates to a novel Streptomyces roseoflavus LS-A24 strain isolated from soil , and staurosporine, which is an antibacterial substance extracted and extracted from the strain culture medium, and this antibiotic. It relates to a fungicide for plant disease control to be used.

The novel strains of the present invention and the antimicrobial substances isolated therefrom are Botrytis cinerea , Botantis cinerea , Collectotrichum orbiculare , Cladosporium cucumerinum ), Didymella bryoniae , Fujium oxysporum f.sp. Lycopene has an anti-bacterial effects, etc. When Percy (Fusarium oxysporum f.sp. Lycopersici), Rhizoctonia solani (Rhizoctonia solani), and between the pie Saratov seashell kaepsi (Phytophthora capsici).

Description

Streptomyces roseoflavus LS-A24 strain effective against plant pathogenic fungi and fungicides produced from it with antibacterial activity against phytopathogenic fungi

The present invention relates to a Streptomyces roseoflavus strain having a novel antifungal activity, and to a fungicide using antibiotics produced therefrom, and specifically, to a plant hospital such as red pepper bacterium and gray mold fungi. Streptomyces roseoflavus LS-A24 strain having a novel antifungal activity by producing and secreting an antibiotic, staurosporine, which has an antimicrobial effect against fungi, and produced therefrom It relates to a fungicide using the antibiotic staurosporin.

Generally, pepper blight and gray mold are economically important plant diseases in Korea.

Cayenne pepper plague occurs mainly in the roots and stems from the seedling stage to the entire growing season.However, pathogens splash into the rainwater and occur on the ground, such as leaves, fruits, and branches. In the middle of growth or in later stages, the diseased tree is wilted at first and later turns yellow to dry.

Phytophthora capsici , a causative agent of cayenne pepper, is a water-loving hemi-aquatic bacterium that easily forms a zygote capsule in water or on an incubator, where the spilled swab swims in 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, most infectious agents are difficult to control because it is introduced from the soil.

Gray mold is a disease that occurs in the fruits, stems, and branches of horticultural crops such as red pepper, cucumber, citrus, leek, tomatoes, potatoes, and flower crops. It is a disease that causes rotting inwards and a lot of gray mold on the infected area.

Pathogens overwinter in the form of fungal nuclei or conidia in the diseased area and become infectious agents. In plant cultivation, many occur when the temperature is high around 20 degrees, and during the rainy season during the summer rainy season.

Such pepper blight and gray mold disease cause severe yield reduction of more than 20% in the whole crop harvest. Therefore, cultivation and crop rotation of resistant varieties are currently being carried out for its control, but so far the most effective Silver is known to be a chemical control using a fungicide. Looking at the scale of such chemical control, even in the case of pepper blight, the amount of fungicide used for red pepper cultivation soil is about 1,000 M / T (1993) in the packaging of 83,000 information. The adverse effects on the human beings have reached a serious level, causing social problems.

From this point of view, there is an urgent need to develop a low-toxic, pollution-free fungicide that can be used for the control of pepper blight, gray mold, and the like.

The use of secondary metabolites secreted from microorganisms has emerged as a low-toxic, pollution-free bactericide because microbial secondary metabolites have various useful properties as fungicides for controlling plant diseases. It is rapidly decomposed in the environment, so there is little damage due to soil residual toxicity, and the chemical structure and biological effects are varied, and it has side effects including the emergence of pathogenic strains resistant to organic synthetic pesticides, residues in soil, and damage to livestock. It has the advantage of being reduced.

In recent years, the development of new pesticides, taking advantage of these microbial secondary metabolites, does not focus only on medicinal effects like conventional pesticides, has low toxicity to livestock, high activity against pests and weeds, and is safe for environmental and natural ecosystems. Research is focused on developing lead compounds derived from natural products with high selectivity, low toxicity and environmental stability.

On the other hand, although such microorganism-derived antimicrobials are used as such, they are also widely used as precursors for the development of new and more powerful fungicides. For example, β-methoxyacrylate azoxystrobin (β- The methoxyacrylates azoxystrobin and kresoxim-methyl are Venturia inaequalis , Cercospora arachidicola , Plasmopara viticola , and phytoprosolain . Fenyllonil, which was developed from strobilurin, which inhibits Phytophthora infestancs , and has antimicrobial effects on corn Fusarium graminearum and Gerlachia civalis . Fludioxonil is an agricultural fungicide developed from pyrrolnitrin.

The microbial fungicides on the market for the past 30 years include blasticidin S, polyoxin, kasugamycin, validamycin and mildiomycin. Among the first agricultural antibiotics, blasticidin S has been shown to have excellent control against rice fever. , polyoxin D is used for pathogenic fungi of fruit trees, vegetables, and flowers, polyoxin D is sold under the name Zn-salt for the control of rice leaf blight caused by Rhizoctonia solani , and kasugamycin is used for the control of rice fever and bacterial Pseudomonas. Mildiomycin is used to control powdery mildew in many crops.

On the other hand, were a large number of antibiotics already known in conventional isolated from Streptomyces, among others Streptomyces (Streptomyces) the genus Streptomyces is the aminoglycoside (aminoglycosides), not bitter cyclin (anthracyclines), glycopeptides (glycopeptides), beta-lactams ( It is well known for producing antibiotics with various chemical structures such as β-lactams, macrolides, nucleic acids, peptides, and polyenes. It's a species being tried.

However, with this active navigation, and even less, Streptomyces (Streptomyces) of Rose O 'Rab's (roseoflavus) from the genus Streptomyces species of Streptomyces is Agricultural up despite the possibility is high to generate a variety of physiologically active substances and yet Antibiotics Research There is not much research on this.

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 disinfectant that can be used in the control of plant diseases such as pepper blight, gray mold, etc. It is.

To this end, the present invention, the production of a novel Streptomyces roseoflavus LS-A24 strain isolated from the soil and antibiotics staurosporine, which is isolated and extracted from the strain culture solution It provides a method and a fungicide for controlling plant diseases using the antibiotic.

The present inventors separated about 1,000 actinomycetes from the mud flat soil of the west coast of Korea, and assayed the titer under agar medium and greenhouse conditions. Actinomycete strain LS-A24 producing (staurosporine) was selected and identified.

Actinomycetes strain LS-A24 of the present invention was identified as Streptomyces roseoflavus through physiological, biochemical properties, and 16S rDNA sequence analysis.

Specifically, the present inventors have been conducting research to develop antifungal antibiotics from soil microorganisms since 1990 at the Department of Agricultural Biology, Korea University. Many antagonists generate antifungal antibiotics showing excellent antimicrobial activity against phytopathogenic fungi. Actinomycetes were discovered, and the screening effects of several antibiotics separated and purified from the antagonistic actinomycetes as a single component confirmed that one of the antibiotics had an excellent antimicrobial activity against phytopathogenic fungi. As a result of identifying the molecular structure of the antibiotic, the present invention was completed by identifying staurosporin.

That is, the present inventors separated about 1,000 actinomycetes from soil samples of various tidal flat areas in Korea, and tested the control effect on plants under agar medium and greenhouse conditions. Details -A24 strains to identify a kind of Streptomyces ROGER O 'Rab's (Streptomyces roseoflavus) and Streptomyces ROGER O'Rab's LS-A24 (Streptomyces roseoflavus LS- A24) termed deposited after [microorganism accession number: KCCM 10505], the present invention was completed, and subsequent studies were performed to isolate and purify the antimicrobial agent staurosporine produced therefrom, to determine its structure, and to find an excellent antifungal effect. Explained as follows.

In order to select antagonistic actinomycetes, soil 10cm deep from the topsoil was collected from the lake soil in the Sunghwan area, 5g of each sample soil was mixed with 50ml of sterile water, and shaken for 30 minutes in a rotary shaker (150rpm) to prepare a soil suspension. It was. The prepared suspension was filtered through Whatman 1 filter paper and diluted with sterile water in a 1: 100 (v: v) ratio. 1 ml of each diluent was dissolved at 40 ° C. 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 ㆍ H ₂ O, 0.01 g FeSO ₄ H 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 , 50mg cycloheximide, 18g agar, 1 liter H ₂ O, adjusted to pH 7.2; vitamins and cycloheximide were filter-sterilized and malt extract agar: 3g malt extract, 0.5g peptone, 0.5 g glucose, 0.85% NaCl, 18 g agar, 1 liter H ₂ O), and inoculated agar plate medium was incubated at 28 ℃ for 14 days until colonies were generated.

Among the germs generated in this way, the bacterium that appears to be actinomycetes, that is, unlike ordinary bacteria, selects a bacterium that forms a physiological mycelia and forms a hard mycelium on agar plate. The antimicrobial activity against bacteria and rice fever was tested.

To this end, the selected actinomycetes are streaked at 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 the causative fungal bacterium, incubated for another 5 days and confirmed the formation of low support.

The antagonistic bacteria selected by the production of low support were grown at 0.3% TSB medium, mixed with 15% glycerol, and stored at -70 ° C. Glass test tubes with lids for daily use (10 ×) 28 mm) was used to inoculate 0.3% TSA medium.

This separation, to identify the selected strain as Streptomyces ROGER O 'Rab's (Streptomyces roseoflavus) Streptomyces ROGER O'Rab's LS-A24 (Streptomyces roseoflavus LS- A24) termed, and antimicrobial activity over the test as described below The secondary metabolite having was purified.

Streptomyces roseoflaves LS-A24 strain was cultured in glycerol peptone broth, 28 ° C. for 12 days, and then subjected to flash chromatography with HP-20 resin. The fractions of 40%, 60% and 80% of MeOH with antimicrobial activity were combined and extracted with ethyl acetate to show antimicrobial activity in this organic solvent layer. The layer was concentrated and again subjected to C ₁₈ resin flash chromatography. Collect 60% and 80% MeOH fraction and perform thin layer chromatography, develop chloroform: methanol 9: 1 to collect Rf 0.37 band showing antimicrobial activity with MeOH, and then refine it with Sephadex LH-20 column chromatography 61-157 were obtained and the antimicrobial activity fraction was purified by C ₁₈ reverse phase HPLC (eluent: 30% -1000% acetonitrile linear gradient system) at a rate of 2ml / min. Obtained and identified as staurosporin.

In this manner, the staurosporin isolated and purified from the actinomycetes Streptomyces roseoflavus LS-A24 strain of the present invention has the following molecular structure.

As known to date, staurosporin is a microbial alkaroid-based compound originally isolated from Streptomyces by S. Omura et al. In 1977. After antimicrobial and hypotensive activity was discovered, it has been known as a protein kinase C inhibitor. Since research on kinase inhibitors have anticancer effects, it has received much attention for medical use using its pharmacological effects, but little is known about phytopathogenic antifungal activity.

As described above, it was confirmed that the antibiotic staurosporin isolated and purified from the actinomycetes Streptomyces roseosporin LS-A24 strain of the present invention had the same level of control effect as compared to the conventional organic synthetic fungicide. In particular, by showing the specific antimicrobial activity in vitro , as well as Phytophthora capsici , Magnaporthe grisea , Rhizoctonia solani, Cucumber rot fungus ( Botrytis cinerea ), etc. It is expected to be useful for controlling plant diseases in the future.

Hereinafter, 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 Identification of Actinomycetes Species Using Physiological, Biochemical Properties and 16S rDNA Sequence Analysis of Antagonistic Actinomycetes LS-A24

The strain LS-A24 of the present invention isolated from Sunghwan Lake soil produced substrate hyphae on the medium, diaminopimelic acid (DAP) on the cell wall was L-form, and the spores were observed by electron microscopy. Spiral, spore surface was found to be smooth to see Streptomyces cheat (Fig. 1). Physiological and biochemical tests for species identification were carried out using the methods described in the Manual's manual of systematic bacteriology.

The streptomyces strain LS-A24 did not form melanin pigment in ISP 6, ISP 7 medium, elastin (Elastin), arbutin (Arbutin), starch (Starch), Tyrosine (Tyrosine), Escurin (Esculin) However, Xanthine, Casein and Gelatin were not degraded. Xylose and L-Arabinose could be used as the carbon source, but the strain was sucrose, inositol, mannitol, raffinose, and ramnose. ), Melezitose, Xylitol, and Fructose were not available. L-valine and L-histidine were used as nitrogen sources, but L-cysteine, L-phenylalanine and L-hydroxylproline were not available.

Genomic DNA of the strain was extracted according to Pospiech and Neumann's method, and then the base sequence was analyzed by amplifying 16S rDNA using primers fD1 (AGAGT TTGAT CCTGG CTCAG) and rP2 (ACGGC TACCT TGTTA CGACTT).

The analyzed sequences were compared with those of other staphylococci in GenBank, and the 16S rDNA of the streptomyces sp. LS-A24 of the present invention and other Streptomyces strains were plotted by the cluster method in the DNAstar program and shown in FIG. 2.

Through the above physiological biochemical test and 16S rDNA sequencing, the actinomycetes strain of the present invention was identified as a type of Streptomyces roseoflavus , and Streptomyces roseoflavus LS-A24 ( Streptomyces roseoflavus LS) -A24).

Example 2 Culture Conditions of Antagonistic Actinomycetes LS-A24

Since the substances secreted by microorganisms vary greatly depending on the culture conditions such as the type of incubator, the culture temperature, and the oxygen supply rate, the medium and culture time for which the antagonistic actinomycetes LS-A24 strain of the present invention can optimally produce antibiotics are determined. For viewing, the following four types of media were selected and tested.

Starch casein medium (SCB, starch casein broth: 20g soluble starch, 0.6g tryptone peptone, 4g K ₂ HPO ₄ , 4g KNO ₃ , 4g NaCl, 1g MgSO ₄ 7H ₂ O, 0.02g FeSO ₄ 7H ₂ O, 0.04 g CaCO ₃ and 1L distilled water)

Starch glucose broth (SGB, starch glucose broth: 20g soluble starch, 10g glucose, 5g yeast extract, 5g casamino acid and 1L distilled water)

Glycerol dextrin broth (GDB, glycerol dextrin broth: 20g glycerol, 20g dextrin, 10g soytone, 3g yeast extract, 2g (NH ₄ ) ₂ SO ₄ , 4g K ₂ HPO ₄ and 1L distilled water)

Glycerol peptone broth (GPB, 20g glycerol, 10g polypeptone, 5g beef extract and 1L distilled water)

After incubation for 2, 4, 6, 8, 10, 12, 14 days, the culture filtrate was concentrated and compared to the antimicrobial activity against plant diseases by the paper disk method is shown in Figure 3 the results.

As a result of the test, the antagonistic bacteria LS-A24 of the present invention had the greatest inhibitory effect against the causative bacterium used in the test when cultured for 12 days in glycerol peptone medium (GPB).

Example 3 Isolation and Purification of the Antibiotic Staurosporin

In order to isolate and purify the secondary metabolite having antimicrobial activity from the antagonistic Streptomyces rosoplavs LS-A24 strain of the present invention identified in Examples 1 and 2 and established in culture conditions, After culturing for 12 days at 28 ° C. using glycerol peptone broth, the resulting glycerol peptone broth was added to methanol / water 0: 100, 20:80, 40:60, 60:40, 80:20 in HP-20 resin. , Flash chromatography was performed using a 100: 0 (V / V) mixed solvent as eluent.

From the fractions obtained, the fraction of MeOH 40%, 60% and 80% with antimicrobial activity and methanol layer with antimicrobial activity extracted from methanol were combined and extracted with ethyl acetate to confirm the antimicrobial activity. Collected, concentrated and concentrated to C ₁₈ resin flash with eluent of methanol / water 0: 100, 20:80, 40:60, 60:40, 80:20, 100: 0 (V / V) Chromatography was performed.

Collect 60% and 80% MeOH fractions, perform thin layer chromatography (TLC) on silica gel plate, expand to chloroform: methanol 9: 1, collect Rf 0.37 bands with antimicrobial activity, collect MeOH, and use Sephadex LH-20 column again. Purification by chromatography gave the antimicrobial activity fraction 61-157, which was purified by C ₁₈ reverse phase HPLC (eluent: 30% -1000% acetonitrile linear gradient system) at a rate of 2ml / min, at 27.37 min. Antibacterial material was obtained.

As a result of analysis by FAB mass spectrum (FAB mass spectrum), the molecular weight was 466, the molecular formula was C ₂₈ H ₂₆ N ₄ O ₃ , nuclear magnetic resonance (NMR) analysis was confirmed as staurosporine (staurosporine).

Example 4 Inhibition Test for Plant Pathogenic Fungi

In order to measure the antibacterial activity of staurosporin obtained in Example 3, the following phytopathogenic fungi inhibition test was performed.

M. grisea , Alternaria mali , Colletotrichum orbiiculare, Botrytis, as well as P. capsici Botrytis cinerea , Cladosporium cucumerinum , Cylindrocarphon destructans , Didymella bryoniae , Fusium oxysporum f.sp. Lycopene Percy when cultured at (Fusarium oxysporum f.sp. lycopercisi), Magna Forte that rayisiah (Mygnaporthe grisea) and Rhizoctonia solani Potato dextrose deck of phytopathogenic fungi such as (Rhizoctonia solani) agar (potato dextrose agar) medium .

Staurosporin is mixed with 1% potato agar medium in a micro-well plate and placed in a series of concentrations (0.1 to 100 µg / ml), followed by 10 ⁵ spores of each fungal spore or yeast suspension. Each well was placed at a concentration of / ml.

After incubating the inoculated well plate at 28 ° C. for 2 to 4 days, the lowest concentration among the fungi without mycelial growth was evaluated as the minimum inhibitory concentration, and the results are shown in Table 1 below. It was.

MIC Phytopathogenic fungi Minimum inhibitory concentration (㎍ / mL)  Alternaria Mali Cladosporium Obicularia Botrytis Cineria Cladosporium Cucumerinum Cylindrocarpon Distractance Didimela Brionier Fujium Ogisporum f.sp Lycopissi Magnaporte Gracia Pie Topsora Capshisai Raiztonia Solani > 1001501> 1001050> 10011

As a result of the test, the staurosporin isolated from the antagonistic Streptomyces rosoplavs LS-A24 strain of the present invention is P. capsici , P. capsici , and C. aureus orbiculare ), C. cucumerinum ( C. cucumerinum) and R. solani ( R. solani ) showed high levels of antimicrobial activity, and Botrytis , a cucumber fungus cinerea ) and fujium oxysporum f.sp. Lycopersici ( Fusarium oxysporum f.sp. lycopersici ) and Didymella bryoniae also showed antimicrobial activity at low concentrations such as 50 ㎍ / ㎖ and 10 ㎍ / ㎖. However, it did not show antimicrobial activity against Alternaria Mali and Magna Porte Gracia.

Example 5 Comparison of the Control Effect of Staurosporin and the Fungicide Metalaccil Against Red Pepper Disease

The pesticide control effect of metalaccil, a commercial bactericide for the control of late blight, and staurosporin produced from the antagonistic Streptomyces rosoplavs LS-A24 strain of greenhouse pepper seedlings in greenhouse conditions was investigated.

Staurosporin and metalaxyl were treated at concentrations from 0 (untreated) to 1000 ㎍ / ml and inoculated with late blight after 1 day, and then treated with severity in untreated and untreated treatments. Evaluated.

The degree of disease damage for evaluating the control effect of red pepper disease was evaluated in units of 0 to 5 according to the degree of disease damage in the red pepper seedlings treated based on when the untreated red pepper seedlings were fully diseased and died. When the symptoms of phosphorus disease was not seen at all, and when the pepper seedlings were completely diseased and died, the rating was evaluated as 5 and the results are shown in FIG. 5.

0: no onset

1: lightly wilted and necrotic lesions begin to appear

2: 30-50% of all plants

3: 50-80% of all plants occur

4: 80-90% of all plants occur

5: plant death

As a result, brown plaques began to appear on the stems of untreated red pepper plants from day 2 after the inoculation of the bacterium, whereas no symptoms were observed in the red pepper plants treated with metalaccil and staurosporin. As a result, lesions began to be observed in pepper plants of 1 µg / ml and 10 µg / ml drug treatments, and some withering symptoms were also observed in pepper plants treated with metalaccil 10 µg / ml.

After 5 days of inoculation, pepper plants treated with 100 mg / ml of metalaxyl did not show any symptoms, but antibiotic staurosporin did not develop in 500 µg / ml treated pepper plants.

Since no late blight did not occur in the treatment of more than 500µg / ml of staurosporin treatment, the antifungal substance staurosporin showed the same level of control effect as metalaccil as shown in FIG. Can be. From these test results, it can be seen that the soil actinomycetes-derived staurosporin used in the present invention had almost the same level of control effect as the active ingredient of the organic synthetic fungicide.

As described above, the Streptomyces roseoflavus LS-A24 strain of the present invention having antimicrobial activity is isolated and identified, and the second metabolite, staurosporin, is isolated and purified and the antimicrobial activity is confirmed, thereby streptomycin Plant disease control fungicides using the Streptomyces roseoflavus LS-A24 strain or the culture filtrate of the strain and plant fungicides containing purified staurosporin can be provided. .

In addition, the Streptomyces roseoflavus LS-A24 strain of the present invention, the culture filtrate of this strain, and the antibacterial substance staurosporin purified from the strain are all originally derived from nature, Since the material can be easily decomposed by the microorganisms in the natural environment, it can be said that it can be used to prepare a pollution-free, environmentally friendly fungicide composition alone or by mixing a filler or the like commonly used in the art.

By completing the present invention as described above, the novel Streptomyces roseoflavus LS-A24 strain isolated from the soil, and the staurosporine which is an antimicrobial substance separated and extracted from the strain culture medium. ) And fungicides for controlling plant diseases using the antibiotics can be provided.

By the completion of the present invention, it is possible to provide an environmentally friendly pollution-free fungicide that has a high control effect of the organic synthetic pesticides level, which can be used for controlling plant diseases such as red pepper disease, gray mold disease, and the like, which is low toxicity and does not cause adverse effects on the environment. will be.

Fungicide compositions provided by the present invention are major phytopathogens, such as Botrytis cinerea , Cladosporium cucumerinum , Didymella bryoniae , Fuzarium Oxysporum f.sp. Percy lycopene during the (Fusarium oxysporum f.sp. Lycopersici), call tree rekto kyum plant disease caused by a Ob particulate lari (Collectotrichum orbiculare), Rhizoctonia solani (Rhizoctonia solani), between the pie Saratov seashell kaepsi (Phytophthora capsici) It can be used for control.

1 is a scanning electron micrograph of the Streptomyces roseoflavus LS-A24 strain,

Figure 2 is a phylogenetic tree based on the streptomyces 30 species of 16S rDNA nucleotide sequence,

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

Figure 4 is a process chart showing the purification process of staurosporin in the culture filtrate of Streptomyces roseoflavus LS-A24 of the present invention,

5 is a graph comparing the control effect of the antibiotics of the present invention against pepper blight in pot experiments of greenhouse conditions compared with metalaxyl, a commercial fungicide.

Claims (6)

Novel Streptomyces roseoflavus LS-A24 strain with antifungal activity. ( Microbial Accession No .: KCCM 10505 ) It characterized by containing any one of Streptomyces roseoflavus LS-A24 strain of claim 1 or the culture filtrate of the strain, Fungicides for controlling plant diseases. The method of claim 2, Plant diseases include the phytopathogenic fungi Botrytis cinerea , Collectotrichum orbiculare , Cladosporium cucumerinum , Didymella bryoniae ), Fujium oxysporum f.sp. It characterized in that the lycopene during Percy (Fusarium oxysporum f.sp. Lycopersici), Rhizoctonia solani (Rhizoctonia solani) and pie Saratov seashell kaepsi between plant disease caused by any one of (Phytophthora capsici), Fungicides for controlling plant diseases. The Streptomyces roseoflavus LS-A24 strain of claim 1 was incubated at 28 ° C. in glycerol peptone broth for 12 days, and then methanol / water in HP-20 resin. Flash chromatography was performed using 0: 100, 20:80, 40:60, 60:40, 80:20, 100: 0 (V / V) mixed solvent as eluent, From the fractions obtained, the fractions of MeOH 40%, 60% and 80% and the cell mass layers were combined with methanol, and the mixture was extracted with ethyl acetate, collected, concentrated, and methanol / water 0: 100. C ₁₈ resin flash chromatography was carried out using 20:80, 40:60, 60:40, 80:20, 100: 0 (V / V) mixed solvent as eluent. Collect 60% and 80% MeOH fractions, perform thin layer chromatography (TLC) on silica gel plate, develop with chloroform: methanol 9: 1, collect Rf 0.37 band with MeOH, and refine it again with Sephadex LH-20 column chromatography. Fraction 61-157 were obtained, and this antimicrobial activity fraction was purified by C ₁₈ reverse phase HPLC (eluent: 30% -1000% acetonitrile linear gradient system) at a rate of 2ml / min to obtain an antimicrobial substance at 27.37 minutes. Characterized in that, A method for preparing an antimicrobial staurosporine produced from Streptomyces roseoflavus LS-A24 strain having novel antifungal activity. Characterized in that it contains the antimicrobial staurosporine prepared by the method of claim 4, Fungicides for controlling plant diseases. The method of claim 5, Plant diseases include the phytopathogenic fungi Botrytis cinerea , Collectotrichum orbiculare , Cladosporium cucumerinum , Didymella bryoniae ), Fujium oxysporum f.sp. It characterized in that the lycopene during Percy (Fusarium oxysporum f.sp. Lycopersici), Rhizoctonia solani (Rhizoctonia solani) and pie Saratov seashell kaepsi between plant disease caused by any one of (Phytophthora capsici), Fungicides for controlling plant diseases.