KR20060016851A - Streptomyces koyangensis sp. nov. vk-a60 strain effective against plant pathogens and fungicides produced from it - Google Patents

Abstract

The present invention is a novel Streptomyces koyangensis sp.nov.VK -A60 strain isolated from soil and antibiotic 4-phenyl-3-butenoic acid, which is isolated and extracted from the strain culture medium. A method for producing (4-Phenyl-3-butenoic acid) and a fungicide for controlling plant diseases comprising the antibiotic as an active ingredient. According to the present invention of the above configuration, it can provide an environmentally friendly pollution-free fungicide that is low toxicity and does not cause adverse effects on the environment, while having a high control effect of the organic synthetic pesticide level, which can be used for controlling plant diseases such as cucumber anthrax and rice fever. have.

Phytopathogenic bacteria, Streptomyces, fungicides, cucumber anthrax, rice fever

Description

Novel Streptomyces goyangensis VK-A60 strain having antimicrobial activity against phytopathogens and fungicides produced from the strain {Streptomyces koyangensis sp. nov. VK-A60 strain effective against plant pathogens and fungicides produced from it}

1 is a scanning electron micrograph of the Streptomyces Goyangensis VK-A60 strain according to the present invention.

Figure 2 is a schematic diagram based on the 16S rDNA nucleotide sequence of 30 Streptomyces.

Figure 3 is a graph showing the inhibitory effect on phytopathogens of Streptomyces Goyangensis VK-A60 strain according to the present invention according to the culture medium and incubation time.

Figure 4 is a process showing the process of purifying 4-phenyl-3-butenoic acid in the culture filtrate of Streptomyces Goyangensis VK-A60 strain according to the present invention.

Figure 5 shows the results of comparing the control effect of antibiotics against rice fever and cucumber anthrax according to the present invention in pot experiments of greenhouse conditions with tricyclazole and chlorothalonil as a commercial disinfectant graph.

The present invention relates to a novel Streptomyces koyangensis sp. Nov. Strain having antimicrobial activity against phytopathogenic fungi, and to fungicides using antibiotics produced therefrom. More specifically, antifungal by producing and secreting 4-phenyl-3-butenoic acid, an antibiotic substance having an antimicrobial effect against phytopathogenic fungi such as cucumber anthrax and rice fever. A novel Streptomyces koyangensis sp.nov.VK -A60 strain having sexual activity, and a fungicide using the antibiotic 4-phenyl-3-butenoic acid produced therefrom.

In general, cucumber anthrax, rice fever, etc. are economically important plant diseases in Korea, which will be described in detail as follows.

Anthrax of gourds and vegetable crops occurs in the upper part of plants such as leaves, stems and fruits when the temperature and humidity are high. If it is not controlled at the early stage of development, it is very difficult to control crops at the late stage of crop growth and harvesting is impossible. Often they end up ruining farming.

The anthrax disease occurs frequently in high temperature, but rarely occurs in house cultivation where muddy water does not adhere to the leaves, and occurs on leaves, stems, and fruits. Leaves make dark-brown lesions in circular to irregular shapes, and stems are fusiform and stems form centrally depressed lesions. As the disease progresses further, the lesions are torn and open. Lesions appear long along the vine, except in the duct. Afterwards, many small sunspots attach to the lesions. The onset of the fruit is pitted with dark spores on the spots.

Pathogens, such as cucumber anthrax, belong to incomplete fungi and form conidia. The conidia itself is sticky and sticky to the viscous material and unlike the germs, it is difficult to be scattered by the wind and mainly spreads by water. Because of this, a lot of rainfall occurs a lot, and in the non-rain house cultivation is less likely to occur.

The pathogens are prone to mycelial growth, mainly living in the mycelium form along with the damaged foliage, which is likely to occur in the field. Therefore, it is dangerous to continuously cultivate not only watermelon but also watermelon and crops, such as cucumber or melon, which are susceptible to this disease. When the soil and the air are humid and continue, or when the muddy water attaches to the vines or leaves during rainfall, the germs can attach to the leaves or vines and winterize in the soil, causing the next year. In addition, it may be infected by attaching to the surface of a seed, farm equipment or land. The growth temperature of pathogens is 22-28 ℃ and breeds in the range of 6-30 ℃. Since this disease is a disease that causes a great deal of damage, it is difficult to control when the disease starts, so care must be taken not to cause it.

On the other hand, rice fever occurs worldwide and is one of the most important diseases for rice. In particular, irrigation or cultivation in places with high rainfall and where a large amount of nitrogen fertilizer is the most important disease. To date, there have been several outbreaks of blast disease in many parts of the world, in some regions 50-90% of the average annual crop has been tolerated. The plague invades the leaves, in which the inner part of the fusiform plaque is grayish white, the outer part is dark green to dark brown, and a yellow flock is formed on the boundary with the healthy part. Sometimes these lesions enlarge and fuse to kill the entire leaf. In addition, if it occurs in the vinegar not only leaves but also connected to the leaves, stem stems or nodes occur between the white water comes out in the extractor or the stem is broken.

Bacteria overwinter with mycelia or conidia on diseased rice straw or seeds or weed hosts. Blast bacteria form and scatter conidia during high relative humidity. Mature conidia are contagious in air. Conidia, which have settled in rice, cling to sticky viscous secretions at their ends. When rice leaves or stems are wet, conidia spores germinate, and germination tubes form adherents, through which they penetrate the surface of the plant or enter the pores. Seedlings and young leaves and stems are more susceptible than adult plants and mature tissues. It takes 4-5 days from invasion to lesion formation at optimal temperature conditions. In humid weather or high relative humidity, the disease develops within a few hours of the disease.

Since such rice fever and cucumber anthrax cause severe yield reduction in the entire crop harvest, cultivation of resistant varieties is currently carried out for its control, but until now, the most effective control method has been chemical control using a fungicide. It is known. However, due to the increasing use of organic synthetic pesticides, the adverse effects on the environment and human beings have reached a serious level, causing social problems.

In view of this, it is urgently required to develop a low-toxic, pollution-free fungicide that can be used for controlling cucumber anthrax and rice fever. In particular, the use of secondary metabolites secreted from microorganisms as a low-toxic pollution-free fungicide is proposed, because the microbial secondary metabolite has a number of useful properties as a fungicide for controlling plant diseases. In other words, since antibiotics derived from microorganisms are biosynthesized in nature, they are rapidly decomposed in the natural environment and are less damaged by soil residual toxicity. It has the advantage of reducing side effects, including appearance, residual in soil, and damage to livestock.

Therefore, in recent years, the development of new pesticides, taking advantage of these microbial secondary metabolites, do not focus only on the efficacy of the drug like conventional pesticides, low toxicity to human beings, high activity against pests and weeds, Research is focused on the development of lead compounds derived from natural products with high levels of safety, high selectivity, low toxicity and environmental stability.

On the other hand, such microorganism-derived antimicrobial material is used as such, but is also widely used as a precursor for the development of new, more powerful and safe fungicides. For example, β-methoxyacrylates azoxystrobin and kresoxim-methyl are Venturia inaequalis and Cercospora arachidicola. ), Was developed from strobilurin, which inhibits Plasmopara viticola and Phytophthora infestans , corn fusarium graminearum and wheat galacchia sival Feniclonil and fludioxonil , which have antimicrobial effects on Gerlachia civalis, are agricultural fungicides developed from pyrrolnitrin.

Microorganisms derived from microorganisms that have been on the market for the past 30 years are blasticidin S, polyoxin, kasugamycin, validamycin and midiomycin. have. The first material developed as an agricultural antibiotic was blasticidine S, which showed excellent control against rice fever. Polyoxin D is used for pathogenic fungi of fruit trees, vegetables, and flowers. Group Estonia solani (Rhizoctonia solani) is for causing rice sheath blight control is sold as Zn-salt field name, car number azithromycin may utilize rice blast and bacterial Pseudomonas (Pseudomonas) prevention, wheat audio azithromycin is different It is used to control powdery mildew in crops.

Meanwhile, a large number of known antibiotics have been isolated from actinomycetes. Among them, actinomycetes in Streptomyces include aminoglycosides, anthracyclines, glycopeptides, and beta-lactams. It is well known for producing antibiotics with various chemical structures such as β-lactams, macrolides, nucleic acids, peptides, and polyenes. This is the fungus being tried.

In the study related to the present invention, the actinomycetes of Koyangensis sp. Nov. , A new species of Streptomyces spp., Were isolated during the screening process, and the bioactive substances produced by the Streptomyces goyangensis species were isolated. The activity as an agricultural antibiotic was evaluated.

The present invention has been made to solve the problems and demands of the prior art as described above, the object of the present invention is a low-toxic and environmentally friendly disinfectant that can be used for controlling plant diseases such as rice fever and cucumber anthrax. It is to provide.

In order to achieve the above object, the present invention provides a novel Streptomyces koyangensis sp.nov.VK -A60 strain isolated from soil and an antibiotic substance isolated and extracted from the strain culture solution. It provides a method for producing phenyl-3-butenoic acid and a fungicide for controlling plant diseases using the antibiotic.

The present inventors have been conducting researches to develop antifungal antibiotics from soil microorganisms since 1990 in the Department of Agricultural Biology, Korea University, and have discovered a number of antagonistic fungi that produce antifungal antibiotics showing excellent antimicrobial activity against phytopathogenic fungi. I've done it.

The present inventors separated about 1,000 actinomycetes from soil samples in various regions of Korea, and tested the control effect on plants under agar medium and greenhouse conditions. As a result, the antimicrobial effect was prevented against plant diseases including cucumber anthrax and rice fever bacteria. Excellent novel actinomycetes strain VK-A60 strain was selected.

Actinomycetes strain VK-A60 of the present invention was identified as a new species through physiological, biochemical characteristics, and 16S rDNA sequence analysis, and Streptomyces koyangensis sp. Nov. VK-A60 (type species VK-A60 ^T )) and then deposited [Microbial Accession Number: KCCM 10555].

Subsequent studies were conducted to screen the drug by separating and purifying antibiotics having antimicrobial activity from the culture filtrate of the Streptomyces koyangensis sp.nov . VK-A60 strain. As a result, it was confirmed that one of them has an excellent antimicrobial activity against phytopathogenic fungi, and the molecular structure of the antibiotic was identified, 4-phenyl-3-butenoic acid (4-Phenyl-3- butenoic acid) is to complete the present invention.

Looking at the process of obtaining the 4-phenyl-3-butenoic acid in more detail, the Streptomyces koyangensis sp. Nov. VK-A60 strain is glyceroldextrin medium (GDB) The culture filtrate was adsorbed onto HP-20 resin and developed with methanol. Then, the fraction having an antimicrobial activity was extracted with ethyl acetate and concentrated to perform silica gel column chromatography. And, collecting the fractions showing the antimicrobial activity C ₁₈ resin reverse phase flash column chromatography (C ₁₈ resin reversed-phase flash column chromatography) to performed, and by collecting the fractions showing the antifungal activity Sephadex LH-20 column chromatography (Sephadex LH again -20 column chromatograhy) to obtain 4-phenyl-3-butenoic acid according to the present invention as a secondary metabolite in the antimicrobial activity fraction 65-189.

The 4-phenyl-3-butenoic acid separated and purified from the actinomycetes strain Streptomyces Goyangenssis VK-A60 strain by the above method has the following molecular structure.

To date, 4-phenyl-3-butenoic acid was first reported in 1973 by Khristov as an antibacterial substance against Trichomonas, and subsequently by Trifnov et al. It turns out that there is. In addition, it has been reported to act as a modulator of glutamic acid transpeptidase and to inhibit peptidyglycine amidating monooxygenase, but it is known about plant disease antifungal activity. It is a barless substance.

The antibiotic 4-phenyl-3-butenoic acid isolated and purified from the actinomycetes strain Streptomyces Goyangensis VK-A60 strain of the present invention as described above is in vitro in vitro Oythrax aneurysm ( Colletotrichum orbiculare ), rice fever ( Magnaporthe grisea ) and Pythium ultimum were found to have specific antimicrobial activity (see Table 1). In addition, the antibiotic 4-phenyl-3-butenoic acid according to the present invention was found to have the same level of control effect against rice fever and cucumber anthrax in comparison with the control group, which is an organic synthetic fungicide, in greenhouse pot tests. (See FIG. 5).

As described above, Streptomyces koyangensis sp.nov.VK -A60 strain of the present invention, the culture filtrate of the strain, and the antimicrobial substance 4-phenyl-3-bute purified from the strain Norick acid is a substance of natural origin, and is a substance that can be easily degraded by microorganisms in a natural environment. Therefore, the culture filtrate of the strain according to the present invention and the antimicrobial material may be prepared as a pollution-free, environmentally friendly fungicide composition by mixing a filler or the like commonly used in the art to which the present invention belongs, very useful for plant disease control It is expected to be used.

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 invention, it is apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Selection of Antagonistic Actinomycetes

In order to select antagonists, soil 10 cm deep from topsoil was taken from vegetable soils in Goyang, mixed 5 g of sample soil with 50 ml of sterile water, and shaken for 30 minutes with a rotary shaker (150 rpm). Prepared. The prepared suspension was filtered through Whatman 1 filter paper and diluted with sterile water in a 1: 100 (v: v) ratio. Humic acid vitamin agar: 1.0 g humic acid, 0.5 g Na ₂ HPO ₄ , 1.71 g KCl, 0.05 g MgSO ₄ · 7H ₂ O, 0.01 g FeSO ₄ 7H ₂ O, 0.02 g CaCO ₃ , 0.5 mg thiamine-HCl, 0.5 mg riboflavin, 0.5 mg niacin, 0.5 mg pyridoxin-HCl, 0.5 mg inositol, 0.5 mg Ca-pantothenate, 0.5 mg p-aminobenzoic acid, 0.25 mg biotin , 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 floras thus produced, the floras that appeared to be actinomycetes were selected. Here, the bacterium that appears to be actinomycetes refers to a flora that forms a rigid mycelium on an agar plate by forming a substrate mycelia unlike ordinary bacteria.

Antibacterial activity against cucumber anthrax bacterium, red pepper blight, gray fungus, apple anthrax, rice leaf blight and rice fever was tested on these selected floras. Specifically, the selected actinomycetes are streaked at 30 mm away from the plate of V8 juice agar (V8A) and incubated at 27 ° C. for 36 hours, and then placed in the center of the medium. Mycelial disks (mycelial disk, 7 mm in diameter) removed from the culture of phytopathogenic fungi, such as growing cucumber anthrax, were 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.

Example 2 Identification of Actinomycetes Species Using Physiological, Biochemical Properties, and 16S rDNA Sequence Analysis of Antagonistic Actinomycetes VK-A60

The strain VK-A60 of the present invention isolated and selected in Example 1 generates substrate hyphae on the medium, and the diaminopimelic acid (DAP) of the cell wall is L-form (electron injection). As a result of observing the spores under a microscope, it was confirmed that the spreptomyces was deceased by the fact that it was a reflexible (rectiflexible) type and had a smooth spore surface (see 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 VK-A60 showed 67.8% G + C content, which is important for the identification of fungi, and showed different characteristics from other actinomycetes, and formed melanin pigment in ISP 7 medium, elastin (Elastin), and arbutin (Arbutin). ), Starch, tyrosine, tyrosine, aesculin, xanthine, casein, and gelatin, but not cellulose.

L-Arabinose, D-fructose, Mannitol, Xylose could be used as carbon sources, but Adonitol, Dextran (Dextran), meso-inositol, Melezitose, D-Melibiose, Raffinose, Rhamnose, Sucrose, Xylitol (Xylitol) was not available.

L-cysteine, L-valine, L-phenylalanine and L-histidine were used as the nitrogen sources, but amino-n-butyric acid and L-hydroxyl were used. Proline (Hydroxyproline) was 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 actinomycetes in GenBank, and 16S rDNA of Streptomyces sp. VK-A60 strain according to the present invention and other actinomycetes were shown in FIG. 2 by a cluster method in a DNAstar program.

Through the above physiological and biochemical tests and 16S rDNA sequencing, the actinomycetes strain of the present invention was identified as Streptomyces koyangensis sp.nov ., And Streptomyces koyangensis VK-A60 ( Streptomyces koyangensis). sp.nov.VK-A60).

Example 3 Culture Conditions of Antagonistic Actinomycetes VK-A60

Since the substance secreted by the microorganism varies 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 VK-A60 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 medium (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 inoculation, the culture filtrate was recovered and concentrated from each medium at 2 days intervals from 2 days to 14 days, and the diameter (mm) of low support was measured by a paper disk method to compare the antibacterial activity against phytopathogens (see FIG. 3).

As a result of the test, the antagonistic bacterium VK-A60 according to the present invention was found to have the greatest inhibitory effect against Oythrax aureus and rice fever when used for 14 days in glycerol dextrin medium (GDB). Therefore, it was found that the most amount of antimicrobial material was produced in the culture conditions.

Example 4 Isolation and Purification of Antibiotic 4-phenyl-3-butenoic Acid

In order to isolate and purify the secondary metabolite having antimicrobial activity from the antagonistic Streptomyces Goyangensis VK-A60 strain of the present invention identified in Examples 1 to 3 and established in culture conditions, the procedure shown in FIG. 4 was performed. .

First, the glycerol dextrin medium (GDB), which produces the most antimicrobial substance, is cultured in bulk for 14 days at 28 ° C., and then the culture filtrate from which the mycelium is removed is collected by centrifugation. In the culture filtrate, methanol / water 0: 100, 20:80, 40:60, 60:40, 80:20, 100: 0 (V / V) mixed solvent was used as an eluent in Diaion HP-20 resin. Flash column chromatography was performed.

Of the fractions obtained, the fractions of MeOH 80% and 100% having antimicrobial activity were combined, extracted with ethyl acetate, and the antimicrobial activity was collected from the organic solvent layer. Then, concentrate it and re-use silica gel as eluent with methanol / chloroform 0: 100, 10:90, 80:20, 70:30, 50:50, 30:70, 10:90 (V / V). Flash gel chromatography (Silica gel column flash chromatography) was performed.

90% of the antimicrobial activity was observed with the collection of 100% chloroform fraction (chloroform fraction) standing C ₁₈ resin reverse phase flash column chromatography (C ₁₈ resin reversed-phase flash column chromatography) to perform the to exhibit antimicrobial activity MeOH 60% and 80 Fractions of% were collected. This was again eluted with methanol using Sephadex LH-20 column chromatography and purified to obtain 4-phenyl-3-butenoic acid in the antimicrobial activity fraction 65-189.

The result was analyzed by FAB mass spectrum, and the molecular weight was 162 and the molecular formula was C ₁₀ H ₁₀ O _2. Nuclear magnetic resonance (NMR) analysis showed 4-phenyl-3-butenoic acid. (4-Phenyl-3-butenoic acid).

Example 5 Antimicrobial Activity of 4-Phenyl-3-butenoic Acid Against Phytopathogenic Fungi

In order to measure the antimicrobial activity of 4-phenyl-3-butenoic acid obtained in Example 4, the following phytopathogenic fungi inhibition test was performed.

Colletotrichum orbiculare , Manaporthe grisea , Pythium ultimum , Alternaria mali , Cladosporium cucumerinum ), Botrytis cinerea , Cylindrocarphon destructans , Fujium oxysporm f.sp. When lycopene Percy (Fusarium oxysporum f.sp. lycopersici), pie Saratov kaepsi between Sora (Phytophthora capsici), Rhizoctonia solani (Rhizoctonia solani), Crescent Loti's Nias Klee Tio Rum (Sclerotinia scleotiorum) plant pathogenic fungi such as They were cultured in potato dextrose agar medium.

4-phenyl-3-butenoic acid was mixed with a 1% potato agar liquid medium in a micro-well plate and placed in a series of concentrations (0.1-100 µg / ml), followed by the fungal spores or yeasts. The suspension was placed in each well at a concentration of 10 ⁵ spores / ml.

After incubating the inoculated well plate at 28 ° C. for 2 to 4 days, the lowest concentration among the fungi that did not occur mycelial growth was evaluated as the minimum inhibitory concentration (MIC), and the results are shown in Table 1 below. (Minimum inhibitory concentration of 4-phenyl-3-butenoic acid against various phytopathogenic fungi).

Phytopathogenic fungi Minimum Inhibition Concentration (μg / mL) Alternaria Mali 30 Botrytis Cinema > 100 Cladosporium Kucumeninum 30 Colletotricum Obicularie 3 Cylindro carpon destructurals 30 Fujium Ogiforum f.sp Lycopisissi > 100 Magnaporte Gracia 10 Phytopsora Capshisai 30 PC Ultimate 0.5 Raiatonia Solani 30 Screrotine Sclethiorum > 100

As a result of the test, 4-phenyl-3-butenoic acid isolated from the antagonistic Streptomyces Goyangenssis VK-A60 strain of the present invention was collected from C. orbiculare ( C. orbiculare ), Magnaforte lycia ( M. grisea ) and P. ultimum showed antimicrobial activity at very low concentrations of 10 μg / ml or less. Also, A. mali , C. cucumerinum , B. cinerea , C. destructans , Fusarium Oxysporm f.sp. Lycopene in Percy city (F. oxysporum f.sp. lycopersici), pie Saratov kaepsi between Sora (P. capsici), Rhizoctonia solani (R. solani), Crescent Loti's Nias Klee Tio Rum (S. scleotiorum) Showed antimicrobial activity at low concentrations of 100 μg / ml or less.

Example 6 Comparison of the Control Effects of 4-phenyl-3-butenoic Acid on the Control Effects of Rice Fever by the Fungicide Tricyclazole and the Control of Otathrax by the Fungicide Chlorotalonyl

To compare the effectiveness of rice febrile control of 4-phenyl-3-butenoic acid produced from Tricyclazole, a commercial fungicide for rice fever, in greenhouse greenhouse rice seedlings, and VK-A60 strain of antagonist Streptomyces spp. Investigate. Two drugs dissolved in methanol and water were treated by spraying 8-leaf rice seedlings at concentrations of 0, 1, 10, 50, 100 and 500 μg / ml, respectively. After 1 day, the spore suspension of rice blast bacteria was inoculated by spraying rice leaves, and then wet-treated for 24 hours.

Five days after inoculation, the number of lesions of rice leaves was counted to evaluate the progress of the disease. Typical lesions of rice febrile disease began to appear after 4 days of inoculation. 10 µg / ml of 4-phenyl-3-butenoic acid and tricyclazole began to inhibit the lesions in rice leaves, and as the concentration increased, the progress of blast disease in rice leaves was suppressed. Although 4-phenyl-3-butenoic acid had a slightly lower inhibitory effect on rice fever compared to tricyclazole, treatment with 500 µg / ml showed a similar inhibitory effect to tricyclazole (part A of FIG. 5). Reference).

In addition, chlorotalonyl, a commercial fungicide for cucumber anthrax, and 4-phenyl-3-butenoic acid produced from the antagonist Streptomyces Goyangenssis VK-A60 strain of the present invention in three-leaf cucumber plants in greenhouse conditions The control effect was compared and investigated.

Chlorotalonyl and 4-phenyl-3-butenoic acid diluted from 0 (no treatment) to 10, 50, 100 and 500 μg / ml, respectively, were sprayed on the leaves of the three-leaf cucumber plant. After 1 day, the spore suspension of cucumber anthrax was diluted to an appropriate concentration and uniformly sprayed on the leaves of cucumber plants, and then wet-treated for one day. Six days after the inoculation, the progress of anthrax was evaluated by counting the number of lesions on the secondary leaves of cucumber plants.

When 10 µg / ml 4-phenyl-3-butenoic acid and chlorothalonil were treated with cucumber plants in the third leaf phase, anthrax was not suppressed, but 4-phenyl-3-butenoic acid was 100 µg / ml. Treatment of anthrax was greatly inhibited, and at 500 μg / ml, almost no lesion was seen in 4-phenyl-3-butenoic acid and chlorotalonyl (see part B of FIG. 5).

From these test results, it can be seen that the soil actinomycetes derived from 4-phenyl-3-butenoic acid used in the present invention have almost the same level of control effect as the organic synthetic fungicide active ingredient.

As described above, the Streptomyces goyangensis VK-A60 strain having an antimicrobial activity as described above is isolated and identified, and the secondary metabolite 4-phenyl-3-butenoic acid is isolated and purified and the antimicrobial activity is confirmed. , Streptomyces koyangensis sp.nov.VK -A60 strain or a fungicide for controlling plant diseases using the culture filtrate of the strain, and containing purified 4-phenyl-3-butenoic acid Fungicides for controlling plant diseases can be provided.

In addition, the Streptomyces koyangensis sp.nov.VK -A60 strain of the present invention, the culture filtrate of the strain, and the antimicrobial substance 4-phenyl-3-butenoic acid purified from the strain are All of them are naturally derived materials, which can be easily decomposed by microorganisms in a natural environment, and thus can be used to prepare a pollution-free, eco-friendly fungicide composition alone or by mixing fillers commonly used in the art to which the present invention pertains. It can be said.

By completing the present invention as described above, 4-phenyl which is a novel Streptomyces koyangensis sp.nov.VK -A60 strain isolated from soil and an antimicrobial substance which is separated and extracted from the strain culture medium. 4-buthenic acid (4-Phenyl-3-butenoic acid) and a fungicide for controlling plant diseases using this antibiotic can be provided.

Completion of the present invention, it is possible to provide an environmentally friendly pollution-free fungicide that is low toxicity and does not cause adverse effects on the environment while having a high control effect of the organic synthetic pesticide level, which can be used for controlling plant diseases such as cucumber anthrax and rice fever. .

The fungicide composition provided by the present invention is a major phytopathogen, for example, C. orbiculare , C. orbiculare , M. grisea , P. ultimum , A. mali , C. cucumerinum , B. cinerea , C. destructans , Fusarium oxy Sporm f.sp. When lycopene Percy (F. oxysporum f.sp. lycopersici), pie Saratov kaepsi between Sora (P. capsici), Rhizoctonia solani (R. solani), Crescent Loti's Nias Klee Tio Rum (S. scleotiorum), etc. It can be used for the control of plant diseases.

Claims (8)

Streptomyces koyangensis sp.nov.VK -A60 strain having antifungal activity (Microorganism Accession No .: KCCM 10555). A bactericide for controlling plant diseases, characterized in that it contains a Streptomyces koyangensis sp.nov.VK -A60 strain of claim 1 or a culture filtrate of the strain. The plant disease of claim 2, wherein the plant diseases are phytopathogenic fungi C. orbiculare , M. grisea , P. ultimum , Alternaria A. mali , C. cucumerinum , B. cinerea , C. destructans , Fusarium oxysporum f. sp. When lycopene Percy (F. oxysporum f.sp. lycopersici), pie Saratov kaepsi between Sora (P. capsici), Rhizoctonia solani (R. solani), Crescent Loti's Nias Klee Tio Rum (S. scleotiorum) of A fungicide for plant disease control, characterized in that the plant disease caused by any one. A fungicide for controlling plant diseases comprising 4-phenyl-3-butenoic acid as an active ingredient. The method of claim 4, wherein the 4-phenyl-3-butenoic acid is produced from Streptomyces koyangensis sp. Nov. VK-A60 strain (Microorganism Accession No .: KCCM 10555) Fungicides for controlling plant diseases. According to claim 4 or 5, wherein the plant disease is a phytopathogenic fungi C. orbiculare ( C. orbiculare ), Magna Forte Gracia ( M. grisea ), P. ultimum ), A. mali , C. cucumerinum , B. cinerea , C. destructans , Fujium Oxysporum f.sp. When lycopene Percy (F. oxysporum f.sp. lycopersici), pie Saratov kaepsi between Sora (P. capsici), Rhizoctonia solani (R. solani), Crescent Loti's Nias Klee Tio Rum (S. scleotiorum) of A fungicide for plant disease control, characterized in that the plant disease caused by any one. A Streptomyces koyangensis sp.nov.VK -A60 strain according to claim 1 is cultured in glycerol dextrin medium (GDB), and is obtained by separating and purifying from the culture filtrate. The manufacturing method of 4-phenyl-3- butenoic acid (4-Phenyl-3-butenoic acid) which is used. The method of claim 7, wherein the separation and purification process, Adsorbing the culture filtrate to HP-20 resin and developing with methanol; Extracting the fraction having an antimicrobial activity with ethyl acetate, concentrating, and performing silica gel column chromatography using a methanol / chloroform mixed solvent as an eluent; Collect fractions for antibacterial activity comprising: performing a C ₁₈ reverse-phase resin by flash column chromatography _{(C 18 resin reversed-phase flash} column chromatography), collecting the fractions showing the antifungal activity; Purifying by Sephadex LH-20 column chromatography, characterized in that it comprises the step of collecting 4-phenyl-3-butenoic acid from the antimicrobial activity fraction 65-189 4 -Phenyl-3-butenoic acid (4-Phenyl-3-butenoic acid) manufacturing method.

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