KR100459066B1 - Method for the biological control of plant diseases using chaetomium globosum f0142 and chaetoviridins a and b produced by same - Google Patents

Abstract

The present invention relates to a method for biological control using ketomolium globosum F0142 (KCTC 0957BP) or ketobiridine A and B of formulas 1 and 2 produced thereby, which is effective for biological control of plant diseases. As a result, the culture of the ketomoglobosum F0142 of the present invention and ketobiridine A and B can be effectively used for biological control of plant diseases such as rice fever, tomato blight, wheat red rust, pepper blight and the like.

Description

METHOD FOR THE BIOLOGICAL CONTROL OF PLANT DISEASES USING CHAETOMIUM GLOBOSUM F0142 AND CHAETOVIRIDINS A AND B PRODUCED BY SAME}

The present invention relates to a method for biological control of plant diseases using Chaetomium globosum F0142 (KCTC 0957BP) and ketobiridine A and B produced thereby.

Although pesticide use is inevitable to improve agricultural productivity, the misuse of synthetic pesticides, which make up many of the pesticides in use, has caused many problems such as soil, water and agricultural pollution, toxicity, and ecosystem disturbances. There is an active research to develop environmentally friendly pesticides. The Korean government also plans to reduce the usage of synthetic pesticides by 50% by 2004, and it is required to develop biopesticides as a representative technology to replace synthetic pesticides. Biopesticides refer to plants, microorganisms, natural enemies and naturally occurring bioactive substances used to effectively protect crops from weeds, pests and pathogenic microorganisms.

According to The Biopesticide Manual, published by The British Crop Protection Council in 1998, those classified as biopesticides and currently commercially available are listed in Table 1. same.

Classification Pesticide Count Natural material 30 Pheromones 45 Biological system 60 Natural enemies 40 Etc 13

Particularly, as a biopesticide using mold, verticillium lecani (Verticillium lecanii,Pesticide), Gliocladium viens (Gliocladium virens,Fungicides), Pheniophora zygantia (Peniophora gigantea,Fungicides), Trichoderma spp.Trichodermaspp., fungicides), choletotricom gloosporioides (Colletotrichum gloeosporioides,Herbicides) and phytoprovoraPhytophthora palmivora,6 paper products such as herbicides).

Ketomium globosum has been reported to have an antagonistic effect on several soil and plant infectious plant diseases (Di Pietro et al.Phytopathology,82, 131-135, 1992; Boudreau and Andrews,Phytopathology,77, 1470-1475, 1987; Di Pietro et al.,J. Plant Dis. & Protection,98, 565-573, 1991; Hubbard, etc.Can. J. Micorbiol.,29, 431-437, 1982; Kommedahl and Chang Mew,Phytopathology,65, 296-300, 1975; Vannacci and Harman,Can. J. Microbiol.,33850-856, 1987; Walter and Gindrat,Can. J. Microbiol.,34, 631-637, 1988), which has not yet been developed as a biopesticide. The fungus is known to live in soil and grow in plants, especially in areas rich in cellulose and lignin, and also known as rots of wood and natural fibers (Von Arx, etc.).Supplements,84, 1-162, 1986). As one of the materials of biological pesticides for controlling plant diseases, the fungus of the present invention has been studied by several researchers. Banach and Halman said that the bacteriumAlternaria raphani) And Alternaria Brassica (Alternaria brassicicolaHas been reported to be effective in controlling seed infectious diseases (Vannacci and Harman,Can. J. Microbiol.,33, 850-856, 1987), Di Pietro et al., Ketomium Globosoom Pisium Ultimum (Pythium ultimumHas been reported to be able to control mosslock disease caused by sugar beet (Di Pietro et al.Phytopathology,82, 131-135, 1992). Ketium globosum is also known as the Venturi inner equalizer that causes apple tree black star blight.Venturia inaequalisHas been reported to have antagonistic effects (Boudreau and Andrews,Phytopathology,77, 1470-1475, 1987).

In addition, a lot of reports have been reported on the mechanism of antagonistic effects of ketomoglobosum on plant pathogenic fungi. In case of replacement with Rhizoctonia solani or Alternaria brassicicola fungus, ketomyum globosum is a hyphal coiling coiling the mycelia of these plant pathogenic fungi. As phenomena have been shown, ketomim globosum is known to have an antagonistic effect by overlapping parasites of these plant pathogens (Vannacci and Harman, Can. J. Microbiol. , 33 , 850-856, 1987; Walter and Gindrat, Can.J. Microbiol. , 34 , 631-637, 1988). On the other hand, the hyphae coiling phenomenon was not observed for P. ultimum and Venturi inner equals (Hubbard et al . , Can. J. Microbiol. , 28 , 431-437, 1982; Walter and Gindrat, Can. J. Microbiol. , 34 , 631-637, 1988). Baudrio and Andrew did not identify antibiotics produced by the ketomolium globosum strain, which had an antagonistic effect against Venturia innerequal bacteria, but found that antagonistic effects were also observed when treated with the bacterial culture. It has been reported that antibiotics are deeply involved in this activity (Boudreau and Andrews, Phytopathology , 77 , 1470-1475, 1987). Di Pietro et al. Have isolated and identified two antimicrobial actives from the ketomime globosum strain, which is very effective against beetroot's mozzarlock disease, and the two substances are 2- (buta-1,3-dienyl) -3-hydroxy. 4- (penta-1,3-dienyl) -tetrahydrofuran [2- (buta-1,3-dienyl) -3-hydroxy-4- (penta-1,3-dienyl) -tetrahydrofuran] (BHT ) And ketomin, especially in liquid culture, have been shown to be highly correlated with the control activity of beetroot's mozzarella disease (Di Pietro et al., Phytopathology , 82 , 131-135, 1992).

On the other hand, a substance called ketobiridine produced from the ketomolium globosum strain of the present invention is ketomium globosum flaboviridae (Chaetomium globosumvar.flavo-viridaeHas been reported as a pigment produced by the present invention, and so far, ketobiridine A, B, C, and D are known as four ketobiridine derivatives. Among them, ketobiridine A weakly inhibits an enzyme called monoamine oxidase, and also in vitro (in vitro) From rice fever (Magnaporthe griseaHas been reported to inhibit growth (Takahashi et al.,Chem Pharm. Bull.,38, 625-628, 1990), in vitro for other plant pathogens (in vitro) Antimicrobial activity and in vivo (in vivoPlant disease control activity in) has not been reported for any plant disease, including rice fever. On the other hand, monascorubrin and monascoflavin, which are structurally similar to ketobiridine, are harmless to human beings as they are used as food and alcohol pigments in Asian countries.

Accordingly, the inventors of the present invention are ketodium globosum F0142, a plant endogenous fungus isolated from a plant inhabiting Gangwon-do, and the ketobiridine A and B produced by the same against plant diseases such as rice fever, tomato late blight and wheat red rust. After confirming that there is an antimicrobial activity, the treatment of the liquid culture, vesicle spores or solid culture of ketodium globosum F0142, and ketobiridine A and B, respectively, revealed that it is effective in the control of the plant disease and completed the present invention. It was.

It is an object of the present invention to provide a novel ketomium globosum strain effective for the control of plant diseases.

Another object of the present invention is to provide a method for separating ketobiridine A and B from Ketodium globosum F0142.

It is still another object of the present invention to provide a method for controlling rice fever, tomato late blight and wheat red rust using Ketomium globosum F0142 or ketobiridine A and B produced thereby.

Figure 1 shows the germination of terminal hair, vesicles, vesicles and vesicles of Ketomium Globosum F0142 (KCTC 0957BP),

Figure 2 shows the hydrogen nuclear magnetic resonance spectrum of ketobiridine A,

Figure 3 shows the carbon nuclear magnetic resonance spectrum of ketobiridine A,

4 shows a hydrogen nuclear magnetic resonance spectrum of ketobiridine B,

Figure 5 shows the carbon nuclear magnetic resonance spectrum of ketovirindine B.

In accordance with the above object, in the present invention, ketomy globosum F0142 (KCTC 0957BP), a new strain showing control activity against various plant diseases, and the ketobiri of the above-mentioned strains or Formulas 1 and 2 produced by the same Biological control methods using chaetoviridin A and B are provided:

Formula 1

Formula 2

Treatment of ketomim globosum F0142 and ketobiridine A and B of the present invention can effectively control plant diseases such as rice fever, tomato blight, wheat red rust, and pepper blight.

Ketomium globosum F0142 (KCTC 0957BP) of the present invention is when the bacteria are incubated in potato agar medium (potato 200 g, dextrose 20 g, agar 15 g, distilled water 1 l), the bacterial tip is white but the middle part is yellow. Old hyphae shows black color.

In order to obtain ketobiridine A and B, which exhibit antimicrobial activity against plant diseases, from the ketium globosum F0142, the bacterium was transferred to a liquid medium, for example potato juice medium (potato juice 200 g, dextrose 20 g, distilled water 1 L) After incubation at 25 ℃ to 28 ℃ for 7 to 21 days, preferably 14 days, the culture is centrifuged. The supernatant was extracted with ethyl acetate and then concentrated under reduced pressure. The ethyl acetate extract was added to a silica gel column and eluted with diethyl ether and dichloromethane in order.

The eluted fractions were mixed and dissolved with chloroform: methanol (9: 1, v / v) and then separated by silica gel column chromatography using the same solvent as eluent. The eluate is separated by thin layer chromatography (TLC) using a developing solvent, and the antimicrobial activity of each fraction is assayed to select samples showing high activity. Ketobiridine A and B can be separated from this sample by silica gel column chromatography using a 2: 1 to 1: 5 (v / v) mixed solvent of hexane: ethyl acetate as the eluent.

Ketobiridine A is produced in an amount of 35 mg / l and ketoviridin B in an amount of 80 mg / l from 1 L of the fermentation broth in which the ketodium globosum F0142 of the present invention is cultured in potato juice medium at 25 ° C. for 14 days .

Ketomium Globosum F0142 can be sprayed in the form of liquid or solid cultures, mycelium, or follicular spores to control plant diseases, and can also be applied directly to the plants pure purified ketovirindin A and B from the strain. .

Plant diseases in the present invention may include rice fever, rice leaf blight blight, tomato ash fungus, tomato blight, wheat red rust and barley flour, etc., Ketomium globosum F0142 is particularly rice blight, tomato blight and wheat Excellent biological control against rust.

In order to show the biological control effect, the spray treatment of the mycelia or spores of ketium globosum F0142 to the plant may vary depending on the type and incidence of the disease, but based on the liquid culture cultured for 14 days 1 It can be treated in the amount of / 3 diluent to 1/27 diluent, and especially when treated in the amount of 1/3 dilution, it shows an excellent control effect.

In the case of treating ketovirindin A and B, it can be treated in an amount of 62.5 μg / ml to 250 μg / ml by foliar spraying.

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Selection of Strains

Five pines, two firs, three ginkgos, three samples, three acacias, four plies, two larch, three oaks, and autumn leaves in Gangwon-do. Whole branches and stems of 31 plants were collected, including one tree sample, two pine tree samples, and three blood samples. These plant samples were cut to 1 cm in size, immersed in 96% ethanol for 1 minute, 3.25% sodium hypochlorite (NaOCl) for 3 minutes, and then surface sterilized with 96% ethanol for 30 seconds, and then alcohol. Flame sterilization was done using a lamp. The sterilized sample was washed in corn-malt-yeast extract agar medium (17 g of corn agar, 20 g of malt extract, 2 g of yeast extract, 1 l of distilled water) to which 50 mg / l chloramphenicol was added. A total of 187 bacteria were isolated by culturing for 10 days at. 187 species of bacteria were isolated and cultured in potato agar medium (potato 200 g, dextrose 20 g, agar 15 g, distilled water 1 L) to isolate single mycelia.

Example 2: In vitro of strains for plant diseases in vivo Antimicrobial Activity

The tip of each of the 187 strains cultured in potato agar medium was cut into 8 mm diameter discs and inoculated into 4 sterile 200 ml potato juice medium (potato 200 g, dextrose 20 g, distilled water 1 l). , Shaking culture at 25 ℃ for 10 days with stirring at 150 rpm. The supernatant obtained by centrifuging the culture solution at 10,000 rpm for 15 minutes was used as a sample.

Processed cultures of 187 plant endogenous fungi to select strains with antimicrobial activity against six plant diseases including rice fever, rice leaf blight, tomato ash fungus, tomato blight, wheat red rust, and wheat flour. In vivo antimicrobial activity was assayed. The mixed solution obtained by mixing 250 μg / ml Tween 20 with 30 ml of each plant endogenous fungal sample prepared above was sprayed on rice, tomatoes, barley and wheat, and then air-dried for one day and then inoculated with each pathogen.

Rice pots, tomatoes, barley, and wheat were sown in 70 cm diameter plastic pots, and seedlings were grown for 1 to 3 weeks in a greenhouse of 25 ± 5 ° C. Rice febrile disease is a two-leaf seedling.Magnaporthe grisea; Source: Spore Suspension of Korea Research Institute of Chemical Technology (5 × 10)⁵Spores / ml) were inoculated by spraying and treated for one day on a 25 ° C. wet room to induce onset, and then placed in a constant temperature room at 25 ° C. for 5 days. Paddy leaf blight blight is in three leafy seedlings.Thanatephorus cucumeris; Obtained from Korea Chemical Research Institute, inoculated with 7 days of culture medium (90 g of wheat bran, 15 g of chaff, 100 ml of distilled water) and treated for 4 days in a humidified environment at 25 ° C. Incubated for days. Tomato blight is a late blight in two-leaf tomato seedlings.Phytophthora infestans; Source: Gangneung National University Yuzu sleeping bag (10⁵Inoculation was carried out by spraying the influx of the influenza suspension extracted from the saccharin sac / ml) and then treated for 4 days in a wet room to induce the onset. Tomato gray mold disease is caused by tomato fungusBotrytis cinerea; Obtained from: Korea Research Institute of Chemical Technology⁶Spores / ml) were treated after 3 days on a 20 ° C humid. Wheat red rust is a foliar seedling of foliar seedlings.Puccinia recondita; Obtained from: University of Incheon) spores were suspended in 250 μg / ml Tween 20 solution in an amount of 0.67 g spores / l and sprayed with a spore suspension to induce the onset in a 20 ° C. humidification chamber at 20 ° C. Transfer to incubation and incubated for 7 days. Barley powdery mildew disease is a type of powdery mildew bacterium that was subcultured to host plants during the first stage of barley seedlings.Blumeria graminisf. sp.hordei; Obtained from: Korea Chemical Research Institute) was inoculated by dropping an appropriate amount of spores and developed in a constant temperature room at 20 ℃. As a control, 30 ml of Tween 20 solution (250 µg / ml) containing 1% DMSO was used.

Lesion area ratios were investigated after 7 days for rice fever, wheat rust, and barley powdery disease after 8 days, and after 3 days and 4 days for tomato ash and tomato blight, respectively. Among them, F0142 strain having the strongest antibacterial activity was selected. Control effect of the F0142 strain on the plant disease is shown in Table 2.

Control Effects of F0142 on Plant Disease Plant disease Rice fever Rice leaf pattern Tomato gray mold Tomato plague Wheat Red Rust Wheat flour Control price (%) 93 0 14 99 98 0 Control Value (%) = (Non-Treatment Control Incidence-Tr.

Example 3: Identification of strain F0142

The F0142 strain was cultured in potato agar medium, but the tip of the fungus was white but the middle was yellow and the old mycelia were black. And when the bacteria grew, a part where many white aerial hyphae were formed was formed. F0142 strains were malt extract agar medium (maltose 12.75 g, dextrin 2.75 g, glycerol 2.35 g, peptone 0.78 g, agar 15 g, distilled water 1 L) according to the method of Domsch et al. (Domsch et al., Compendium of Soil Fungi, 1980). The germination of the virgin, follicular, follicular spores and germinated spores formed by culturing for 14 days at 25 ° C. were observed under an optical microscope and shown in FIG. In addition, the observed morphological properties are shown in Table 3 in comparison with the properties of ketome globosum (Domsch et al., Compendium of Soil Fungi, 1980).

F0142 was identified as Chaetomium globosum according to the observation results, and Ketoum Globosum F0142 was deposited with KCTC 0957BP, deposited on February 12, 2001, at the Korea Biotechnology Research Institute Gene Bank.

Example 4 Control Activity of Liquid Medium Culture of Ketodium Globosum F0142

(1) other control effects during the incubation period

200 ml of potato juice medium was inoculated into a 500 ml Erlenmeyer flask and inoculated with Ketodium globosum F0142, followed by shaking culture at 25 ° C. for 3 days, 7 days or 14 days with stirring at 150 rpm. Samples were prepared by grinding the cultures at each time period, diluting with 400 ml of distilled water, and then adding Tween 20 at a concentration of 250 μg / ml.

Using these samples, the control effect of the plant disease of the ketome globosum F0142 culture according to the culture period was investigated in the same manner as in Example 2. The results are shown in Table 4.

Control Effects of Ketomium Globosum F0142 on Plant Diseases of Plant Cultures with Different Culture Periods Incubation period Control price (%) Rice fever Rice leaf pattern Tomato gray mold Tomato plague Wheat Red Rust Wheat flour 3 days 5 0 6 0 0 0 7 days 73 15 6 98 100 0 14 days 93 55 63 99 100 0

As a result, the three-day culture showed little activity against six plant diseases, but the seven-day culture and the 14-day culture showed control effects against six kinds of plant diseases, especially for rice fever, tomato late blight and wheat red rust. Excellent control effect was shown.

(2) Control effect by treatment concentration

In addition, the 14-day culture sample with the highest antimicrobial activity was treated by various concentrations to investigate the control effect on plant diseases, the results are shown in Table 5.

Control Effect of Ketomium Globosum F0142 on Plant Diseases according to Treatment Concentration Treatment concentration of the culture (diluent) Control is (%) Rice fever Rice leaf pattern Tomato gray mold Tomato plague Wheat Red Rust Wheat flour 1/3 93 55 66 99 100 0 1/9 20 50 0 75 98 0 1/27 30 5 8 38 90 0

As a result, rice fever rapidly decreased when diluting the liquid culture of Ketodium globosum F0142 by 1/9, while tomato blight showed relatively good activity even with 1/9 dilution. Wheat red rust showed a very high control effect of 90% or more even when treated with 1/27 diluent, and it was found that it was particularly effective in controlling wheat red rust.

(3) Control effect on adult plants

Ketomium globosum F0142 liquid culture of the above showed excellent control effect against rice fever, tomato blight and wheat red rust. Among them, an experiment was conducted on plant disease control activity using adult, not seedling, against tomato late blight. Ketomium globosum F0142 was shaken for 14 days in potato juice medium as in Example 2 and the culture was obtained. The ground culture was diluted to 1/5 and 1/10 with distilled water, and then sprayed at an amount of 50 ml per plant to the adult where eggplants were powdered and tomato fruit was opened. 24 hours after the sample was processed,Phytophthorainfestans)of Sponge Suspension Suspension (10⁵After inoculation of the influenza suspension extracted from the saccharin capsules (ml / ml), the incidence was induced in a humidified environment at 20 ° C., and the incidence of the disease was examined 4 days later. As a control, one treated with cymoxanil (eastern Hannong), known as an aliphatic nitrogen fungicide, was treated with 83.3 μg / ml or 250 μg / ml. The results are shown in Table 6.

Control Effect of Ketomium Globosum F0142 on the Plague of Liquid Culture in Tomato Adults sample Treatment concentration of the culture Control is (%) F0142 1/5 dilution 98 1/10 dilution 77 Mountain 250 μg / ml 100 83.3 μg / ml 98

As a result, as in the seedlings, it was found that the liquid culture of ketome globosum F0142 also showed high activity in adults. In the case of 1/5 dilution, the control effect was similar to that of 83.3 ㎍ / ml of simonate.

Example 5 Control Activity of Aspergillus Spores and Solid Medium Cultures

In addition to the liquid culture of Ketodium globosum F0142, control activities by asymptomatic spores and mycelium formed in asymptomatic spores and rice solid medium were investigated. The strains were inoculated in malt extract agar medium and then incubated at 25 ° C. for 14 days, and then scraped by adding sterile water to the medium, followed by filtration with four layers of gauze. Aspergillus spore fluid obtained by filtration was counted under the microscope to adjust the concentration of vesicle spores under a microscope to a concentration of 6.7 × 10 ⁵ follicle spores / ml to 2 × 10 ⁶ follicle spores / ml. Meanwhile, a rice solid medium was prepared by putting 200 g of rice in a 1 L flask, adding 120 ml of distilled water thereto, and then sterilizing twice at 121 ° C. for 15 minutes. Ketomium globosum F0142 was inoculated on the rice solid medium and incubated at 25 ° C. for 7 days. Aspergillus spores and mycelium produced in the solid medium was harvested by adding distilled water to a level of 0.07 g rice medium / ml to 0.2 g rice medium / ml and filtered through a layer of gauze.

The control effect on the six plant diseases was investigated as in Example 2 using the porcine spore sample and the rice solid medium sample obtained by the above method. The results are shown in Table 7.

Control Effects of Cytoplasmic Spores and Rice Solid Cultures of Botome Globosum F0142 on Plant Disease sample Treatment method density Control is (%) Rice fever Rice leaf pattern Tomato gray mold Tomato plague Wheat Red Rust Wheat flour Follicle spores 3 DBI 2 × 10 ⁶ follicle spores / ml 90 0 0 0 90 0 6.7 × 10 ⁵ follicle spores / ml 65 0 0 0 60 0 1 DBI 2 × 10 ⁶ follicle spores / ml 95 20 0 0 97 0 6.7 × 10 ⁵ follicle spores / ml 95 10 0 0 93 0 Rice solid medium 3 DBI 0.2 g rice medium / ml 80 10 0 0 77 17 0.07 g rice medium / ml 83 0 0 0 53 17 1 DBI 0.2 g rice medium / ml 93 0 0 0 100 33 0.07 g rice medium / ml 90 15 0 0 98 0 ^* DBI = Days Before Inoculation

Treatment of asymptomatic spores had no effect on tomato late blight, but was effective on rice fever and wheat red rust. When treated one day before the inoculation, when treated at a concentration of 6.7 × 10 ⁵ follicle spores / ml or more, more than 90% of the two plant diseases were controlled. In addition, even when treated three days before the inoculation showed that more than 60% of the control effect on the two plant diseases, the sperm spores of Ketomium Globosum F0142 showed some degree of sustained effect on these plant diseases.

In the case of rice solid medium incubated for 7 days, it was treated with the amount of 6.7 × 10 ⁵ asymply spores / ml even after harvesting and treating the spores and mycelium formed in the rice solid medium at the ratio of 0.07g rice solid medium / ml distilled water. Similar to that, rice fever and wheat red rust were controlled. Obtaining asymptomatic spores and mycelia using a solid rice medium is a much more economical and convenient method than obtaining a liquid culture or obtaining asymptomatic spores using a solid medium.

Example 6: Isolation and Purification of Antimicrobial Active Compounds

Ketodium globosum liquid culture (8 L) shaken at 25 ° C. for 14 days with stirring at 150 rpm was centrifuged at 10,000 rpm for 15 minutes, then the supernatant was taken out and extracted twice with the same amount of ethyl acetate. In vivo antimicrobial activity was assayed by the method of Example 2 in order to measure the activity of the water layer and ethyl acetate extract obtained by depressurizing and concentrating at 40 ° C. As a result, since ethyl acetate extract (7.3 g) showed antimicrobial activity, flash silica gel column chromatography was performed as follows to separate the active compound therefrom. Ethyl acetate extract was added to a column filled with silica gel (Kiesel gel 60, 70-230 mesh, 200 g; E. Merck) (3.6 x 60 cm) and used as eluent hexane, diethyl ether, dichloromethane, ethyl acetate and methanol 800 ml each was added sequentially and eluted using a vacuum pump. After distilling under reduced pressure and concentration of each eluate, the concentrated solution was treated in an amount of 500 占 // ml, and the antimicrobial activity was assayed in vivo by the method of Example 2, whereby a diethyl ether layer (4.68 g) and a dichloromethane layer (420) were used. Mg) showed activity.

Two fractions showing antimicrobial activity were combined (5.1 g) and dissolved in chloroform: methanol (9: 1, v / v), followed by a column filled with Kaigel gel 60 (70-230 mesh, 200 g) (3.6 × 60 cm). It was added to, followed by elution using the sample dissolving solvent. 10 ml each of the eluate was analyzed by TLC (Kiesel gel 60 F254, 0.25 mm thick, E. Merck, chloroform: methanol (9: 1)). After dividing into eight fractions such as F1, F2, F3, F4, F5, F6, F7 and F8 according to the TLC pattern, each fraction was treated in an amount of 500 ㎍ / ml to assay the in vivo antimicrobial activity as in Example 2. F2 (1.55 g) samples showing high activity against tomato late blight were obtained. F2 (1.55 g) sample was added to a column (3.6 × 60 cm) filled with Kaigel gel 60 (70-230 mesh, 150 g) followed by hexanes: ethyl acetate (2: 1, 1: 1 and 1: 5, v / v) eluting with a solvent to purely separate the two materials. One of the two materials was eluted in hexane: ethyl acetate (1: 1, v / v) and produced 35 mg per liter of fermentation broth which was dark brown and incubated for 14 days. The other was eluted in hexane: ethyl acetate (1: 5), yellow in color, yielding 80 mg per liter of fermentation broth.

Example 7 Structure Determination of Ketobiridine A and B

Two purely purified purified active substances were subjected to mass spectrometry using a mass spectrometer (JEOL JMS-DX303; JEOL Ltd., Tokyo, Japan). As a result, the molecular weights of the two substances were 432 (identified as ketovidin A) and 434 (identified as ketovidin B), and the molecular formulas were estimated to be C ₂₃ H ₂₅ ClO ₆ and C ₂₃ H ₂₇ ClO ₆ . .

Further, were the respective materials 20 mg fully dried placed in a 5 mm NMR tube and dissolved in a 0.5 ㎖ CDCl ₃ NMR analysis (Bruker-AMX 500 (500 MHz )) to determine the structure of the two active substances, ^one H-NMR was measured at 500 MHz and ¹³ C-NMR at 125 MHz. 2, 3, 4 and 5 show the hydrogen and carbon nuclear magnetic resonance spectra of the two active materials. ^{The result} of connecting carbon and hydrogen through the ¹ H- ¹³ C COSY spectrum and the carbon skeleton through the ¹ H- ¹ H COSY spectrum is shown in Table 8. As a result, the two substances were determined as ketobiridine A and B, respectively, and the structural formulas of the two substances were shown in Chemical Formulas 1 and 2, respectively.

Example 8 In vitro of Ketobiridine A and B in vitro Antibacterial activity

In Vitro of Plant Disease of Purely Isolated Ketobiridine A and B In order to measure the antimicrobial activity was carried out in a liquid medium as follows. Ketobiridine A and B, which had been purely separated, were dissolved at a concentration of 10 mg / ml using DMSO, and then diluted by 1 / 3-fold with DMSO. To each well of a 96-well plate, 5 μl of diluted Ketobiridine A and B samples were added to each well.Magnaporthe grisea; Korea Research Institute of Chemical Technology), Fuziium withered fungi (Fusarium oxysporum; Korea Research Institute of Chemical Technology),Red pepper bacterium (Phytophthora capsici; Chungnam National University), Anthrax bacteria (Colletotrichum gloeosporioides; Korea Research Institute of Chemical Technology), Mozolox bacillus (Pythium ultimum; Korea Research Institute of Chemical Technology)Alternaria mali; KyungNong Co., Ltd. and Gray MoldBotrytis cinerea; Potato juice solution inoculated with Korea Research Institute of Chemistry was added to 495 μl per well to treat the ketobiridine A and B concentrations of 100, 33.3, 11.1, 3.70, 1.23 and 0.41 μg / ml, respectively. Mozolox bacillus (Pythium ultimum) Was inoculated with mycelial grinding solution, other bacteria were inoculated with spores, and the control group was treated with 1% DMSO. After incubation for 4 days at 20 ℃ 25 ℃ was observed for the biological control effect on plant diseases. The minimum pharmaceutical concentration that completely inhibits the growth of the strain was determined as the minimum inhibitory concentration, and the results are shown in Table 9.

Minimum Inhibitory Concentration on the Growth of Phytopathogens of Ketobiridine A and B Plant pathogen Ketobiridine A Ketobiridine B Rice fever 1.23 μg / ml 33.3 μg / ml Futurium wilt 33.3 μg / ml > 100 μg / ml Red Pepper Blight 33.3 μg / ml > 100 μg / ml Anthrax 33.3 μg / ml > 100 μg / ml Mozolox bacterium 1.23 μg / ml 33.3 μg / ml Apple tree branch pattern 33.3 μg / ml > 100 μg / ml Gray mold 33.3 μg / ml > 100 μg / ml

As a result, ketobiridine A showed higher antibacterial activity than ketobiridine B, and ketobiridine A and B were more resistant to Magnaporthe grisea and Pythium ultimum than phytopathogens . It was observed to exhibit antimicrobial activity.

Example 9 In vivo of Ketobiridine A and B in vivo Antibacterial activity

In vivo against six plant diseases of ketobiridine A and B The antimicrobial activity was investigated in the same manner as in Example 2. The results are shown in Table 10.

Control Effect of Ketobiridine A and B on Plant Disease sample Concentration (µg / ml) Control is (%) Rice fever Rice leaf pattern Tomato gray mold Tomato plague Wheat Red Rust Wheat flour Ketobiridine A 250 99 20 63 87 87 0 125 94 20 9 50 97 0 62.5 88 0 0 0 83 0 Ketobiridine B 250 96 0 3 0 91 0 125 96 10 20 0 65 0 62.5 94 10 3 0 0 0

Ketoviridin A showed good control against rice fever, tomato blight and wheat red rust, and ketovirid B showed good effect against rice fever and wheat red rust, but weak against tomato blight. .

The present invention relates to a biological control method using the keto irregularities Dean (chaetoviridin) A and B produced by keto hatred Globo breath (Chaetomiumglobosum) is effective for the biological control of plant diseases F0142 and thus, keto hatred Globo breath of the present invention Cultures of F0142 and ketobiridine A and B can be effectively used for the biological control of plant diseases such as rice blast, tomato blight, wheat red rust, pepper blight and the like.

Claims (6)

Ketoium globosum ( Chaetomium globosum ) F0142 (KCTC 0957BP) comprising a tomato plant disease, wheat red rust disease and pepper disease composition comprising a plant disease control composition comprising an active ingredient. Tomato blight, wheat rust and red pepper blight, characterized by spraying a plant, mycelium, spores or ketoviridin A or B isolated therefrom from culture culture of ketomolium globosum F0142 (KCTC 0957BP). Method of controlling plant diseases selected from the group consisting of. delete delete delete delete