KR101891296B1 - Composition for controlling plant diseases including Chitinophaga sp. HK235 strain and its culture broth, its fraction as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for controlling plant diseases, comprising a strain of the genus HK235 belonging to the genus Kirtiona, a culture of the genus HK235, and a fraction thereof as an active ingredient. The strain of the genus HK235 belonging to the genus Kitinopa, the culture thereof, It exhibits a controlling effect on plant diseases important in the production of crops such as blast foliage, sheath blight, gray mold, plague, powdery mildew, red rust and anthracnose, so that it can be usefully used for a plant disease control composition.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for controlling plant diseases, comprising a strain of the genus HK235 belonging to the genus Chitinosa, a culture thereof, and a fraction of the culture medium as an active ingredient. HK235 strain and its culture broth, its fraction as an active ingredient}

The present invention relates to a composition for controlling botanical disease comprising a strain of the genus HK235 belonging to the genus Chitinosa, a culture thereof, and a fraction of the culture medium as an active ingredient.

In the production of crops, various plant pathogens, insects and weeds inhibit the growth of crops and cause a decrease in yield, so their control is an important part of agriculture. In modern agriculture, most of the control chemicals use chemically synthesized medicines. The use of synthetic pesticides greatly increases the productivity of the crops, but the use of synthetic pesticides over many years has increased the frequency of drug-resistant pests , And the concern about human and animal toxicity, environmental pollution and ecosystem disturbance of residual pesticides is increasing. Therefore, policy regulations on the use of synthetic pesticides have been strengthened, especially in OECD countries including Korea. Cluster analysis of millions of compounds registered in the American Chemical Society's Chemical Information Database (CAS) can be categorized into only a few hundred leading substances. If regulations are tightened on the basis of leading substances, synthetic pesticides The number of people will be reduced. As described above, there is a demand for eco-active substances that can substitute synthetic materials due to the environment-friendly aspects of synthetic pesticides, safety concerns, and resistance to pest resistance. Also. In addition to the eco-friendly agriculture policy to reduce the use of synthetic pesticides, there is a growing demand for new environmentally friendly control measures.

Biopesticide, unlike synthetic pesticides, is an eco-friendly crop protection agent with little impact on humans, animals and ecosystems. It can be used to produce high value-added agricultural products such as organic agricultural products. Can be effectively used for controlling. Biological pesticides include microbial pesticides that utilize the functions of microorganisms and biochemical pesticides that utilize microbial or plant-derived metabolites. The development of environmentally friendly natural pesticides has been attracting attention since the Rio Conventions on Environmental Conventions, which will replace 20% of the world's pesticides with bio-pesticides. According to the Global Biopesticide Market (2016-2021), the global biopharmaceutical market, which was $ 3.7 billion in 2015, is expected to grow by 14.1% a year and reach $ 7.7 billion by 2021, As of March 2016, a total of 427 pesticides have been announced as pesticide-controlling pesticides. Of these, 204 are plant extracts, 10 are microbial extracts, and 62 are microbial agents.

Kitty and old woman (Chitinophaga) in strains containing the strain was classified in the past Plexiglas bakteo (Flexibacter), Flavobacterium (Flavobacterium), Saito Parker (Cytophaga) a Gram-negative soil bacteria with chitin resolution of the type history, Various functional peptides derived from a strain of Kittenopha have been reported. Korean Patent No. 10-173956 discloses a cosmetic using a novel strain having an antiseptic, wrinkle-improving and whitening effect as well as an antiseptic efficacy, and International Patent Publication No. 2016-045783 discloses an antifungal activity derived from chitinopa ≪ / RTI >

Accordingly, the present inventors have completed a novel plant control composition using a strain of Kittenopea, a culture solution thereof and fractions thereof as an environmentally friendly plant disease control means.

Korean Patent No. 10-173956 International Patent Publication No. 2016-045783

It is an object of the present invention to provide a composition for controlling plant diseases, which comprises a strain of the genus HK235 belonging to the genus Kirtiona, a culture thereof and a fraction thereof, as an active ingredient.

In order to achieve the above object, the present invention provides a strain of Chitinophaga sp. HK235 deposited with Accession No. KACC 81044BP.

In addition, the present invention provides a composition for inhibiting plant diseases, wherein the Kitsch wolfma comprises as an active ingredient at least one selected from the group consisting of a strain HK235, a culture thereof, and a fraction of the culture broth.

The present invention also relates to a method for treating a plant disease, comprising the step of treating the plant, its seed or its habitat, wherein the kittenofa is selected from the group consisting of strain HK235, a culture thereof, and a fraction of the culture broth. Provides a control method.

The HK235 strain of the genus Ketinopa according to the present invention, the culture medium thereof, and the fraction of the culture medium have a controlling effect against plant diseases important for crop production such as blast foliage, sheath blight, gray mold, plague, powdery mildew, red rust and anthracnose And thus it can be usefully used for a composition for controlling plant diseases, which contains it as an active ingredient.

Fig. 1 shows the result of testing soil bacteria showing a bactericidal effect against a gray mold germ ( Botrytis cinerea ).
FIG. 2 is a micrograph of the strain HK235 ( Chitinophaga sp. HK235, Accession No .: KACC 81044BP) cultured on an agar medium (A: Strain, B: Scanning electron microscope photograph).
Fig. 3 shows the result of confluent cultivation showing the antagonistic action of the strain HK235 on a plant pathogenic fungus.
FIG. 4 is a flow diagram showing a result of a comparison analysis of Kittenoa according to the present invention with species having a base sequence of 16S rRNA gene of strain HK235.
Fig. 5 shows the result of comparing the growth conditions of the strain HK235.
A: Comparison of growth by medium (TSB: Tryptic soy broth medium, LB: Luria broth medium, MB: malt extract broth medium, NB: nutrient broth medium)
· B: Comparison of growth by culture temperature
FIG. 6 shows the results of inhibition of Botrytis cinerea against tomato gray mold fungi by solvent fraction of cultured strain HK235 according to the present invention.
A: Negative control (Aq: water fraction, BuOH: n - butanol fraction, EA: ethyl acetate fraction, Hex: n - hexane fraction)
B: Comparison of fractions by media (TSB: Tryptic soy broth medium, LB: Luria broth medium, MB: Malt extract broth medium, NB: Nutrient broth medium)
FIG. 7 shows the results of the test for the control of the cultivation of the strain HK235, the culture filtrate, and the cell suspension of Tomato gray mold fungus according to the present invention. (Culture broth: Culture solution, Culture filtrate: Culture filtrate, Cell: Cell suspension)
FIG. 8 shows the results of investigating the activity of controlling the tomato gray mold of butanol fraction and HP-20 column fraction according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides the strain HK235 (Chitinophaga sp. HK235) deposited with Accession No. KACC 81044BP.

In addition, the present invention provides a composition for controlling plant diseases, wherein the chitinopa deposited with Accession No. KACC 81044BP comprises at least one selected from the group consisting of strain HK235, a culture thereof, and a fraction of the culture solution as an active ingredient.

The culture solution is a culture solution in which a strain has been cultured, or the culture solution is filtered by a physical method.

The fraction of the culture broth is fractionated using a column using fractionation using an adsorbent or at least one selected from the group consisting of hexane, ethyl acetate, butanol and water.

The adsorbent that may be used in the column may be selected from the group comprising styrene-divinylbenzene, silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin.

The plant disease is caused by Magnaporthe oryzae , Rhizoctonia solani , Botrytis cinerea , Phytophthora infestans , Blumeria graminis, Puccinia, recondita ) and anthracnose ( Colletotrichum coccodes ).

Wherein the plant is at least one selected from the group consisting of rice, tomato, barley, wheat, radish, red pepper and tomato.

The composition for controlling plant diseases according to the present invention may be a simple mixture of kithinopa strain HK235, a culture thereof, an extract of the culture solution or an active substance isolated from the extract. Alternatively, the composition for controlling plant diseases may be prepared by mixing the active substance isolated from the culture medium, the extract of the culture medium or the extract, and the inert carrier with each other, and then mixing the emulsion, the emulsion, the fluidizing agent, the wettable powder, the granulated wettable powder, By adding a surfactant and other necessary auxiliaries to the mixture so that it can be formulated into granules, granules, and the like. The above-mentioned plant disease controlling composition can be used as seed treatment agent by itself or by adding another inert ingredient.

Examples of liquid carriers which can be used in the formulations are water; Alcohols such as methanol and ethanol; Ketones, such as acetone and methyl ethyl ketone; Aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene and methylnaphthalene; Aliphatic hydrocarbons such as hexane, cyclohexane, kerosene and light oil; Esters such as ethyl acetate and butyl acetate; Nitriles, such as acetonitrile and isobutyrnitrile; Ethers such as diisopropyl ether and dioxane; Acid amides such as N, N-dimethylformamide and N, N-dimethylacetamide; Halogenated hydrocarbons such as dichloromethane, trichloroethane and carbon tetrachloride; Dimethyl sulfoxide; And vegetable oils such as soybean oil and cottonseed oil.

Examples of solid carriers that can be used in the formulation include fine powders or granules such as minerals such as kaolin clay, at perpulite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and talcite; Natural organic materials such as corn leaf powder and walnut shell powder; Synthetic organic materials such as urea; Salts such as calcium carbonate and ammonium sulfate; Synthetic inorganic materials such as synthetic hydrated silicon oxide; As the liquid carrier, aromatic hydrocarbons such as xylene, alkylbenzene and methylnaphthalene; Alcohols such as 2-propanol, ethylene glycol, propylene glycol and ethylene glycol monoethyl ether; Ketones such as acetone, cyclohexanone and isophorone; Vegetable oils such as soybean oil and cottonseed oil; Petroleum aliphatic hydrocarbons, esters, dimethylsulfoxide, acetonitrile and water.

Examples of surfactants include anionic surfactants such as alkyl sulfate ester salts, alkyl aryl sulfonate salts, dialkyl sulfosuccinate salts, polyoxyethylene alkyl aryl ether phosphate ester salts, lignosulfonate salts and naphthalenesulfonate forms Aldehyde polycondensates; And nonionic surfactants such as polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl polyoxypropylene block copolymers and sorbitan fatty acid esters and cationic surfactants such as alkyl trimethyl ammonium salts.

Examples of other formulation auxiliaries are water-soluble polymers such as polyvinyl alcohol and polyvinylpyrrolidone, polysaccharides such as arabic gum, alginic acid and its salts, CMC (carboxymethyl-cellulose), xanthan gum, inorganic materials such as aluminum magnesium silicate and alumina sol alumina sol, preservatives, colorants and stabilizers such as PAP (acid phosphate isopropyl) and BHT (butylhydroxytoluene).

In a specific example of the present invention, the strain HK235 (Fig. 1, Fig. 2 and Fig. 4), which is a novel strain identified through screening of strains having a controlling effect against a plant pathogenic bacterium, And have inhibitory activity against plant pathogenic bacteria (Tables 1 and 2, 3 and 6 to 8).

Therefore, the strain of the genus HK235 belonging to the genus Chitinosa according to the present invention, the culture thereof, and the fraction of the culture can be useful for controlling plant diseases.

The present invention also includes a step of treating any one or more selected from the group consisting of the strain HK235, the culture thereof, and the fraction of the culture broth deposited with the accession number KACC 81044BP to the plant, its seed or its habitat To provide a plant disease control method.

The treatment may be an indirect application in which the composition is sprayed directly on the plant, on the soil in which the plant grows, or on the medium for cultivation of the plant.

The controlling method of the present invention includes treatment of stem and leaves of a plant, treatment of the place where the plant grows (for example, soil), treatment of seeds such as seed sterilization / seed coating and treatment of roots.

The treatment of the stems and leaves with the control method of the present invention may include, in particular, application on plant surfaces such as spraying on stems and leaves. As the treatment of the soil with the controlling method of the present invention, for example, spraying on the soil, mixing with the soil, application of the liquid treating agent into the soil (irrigation of the liquid treating agent, injection into the soil, Examples of sites to be treated include planting holes, furrows, planting furrows, peripheries of the growing area, soil-to-plant parts, root-to-root parts, , Main furrows, growing soil, marmots. A box for cultivation, a tray for cultivation, and a plate. The treatment may be carried out before, during, immediately after the spraying, during the simulated growth period, before the settlement, during the settlement, and after the settlement. In the above-mentioned soil treatment, the active ingredient may be applied to the plant at the same time, or a solid fertilizer such as paste fertilizer containing the active ingredient may be applied to the soil. The active ingredient may be mixed in an irrigation liquid and may be introduced into an irrigation facility (an irrigation tube, an irrigation pipe, a sprinkler, etc.), mixed in a liquid that floods between troughs, or mixed in a water culture medium . Alternatively, the irrigation liquid and the active ingredient may be premixed and used for treatment by suitable irrigation methods, including, for example, the irrigation methods mentioned above and other methods such as spraying and flooding.

The volatilization treatment method according to the present invention can be applied to a plant disease control composition of the present invention, for example, by volatilization of a composition sprayed on a medium such as a hydroponic medium or a mat for cultivation of soil or plants in which the plant is cultivated To protect the plant from pests and diseases. In addition, the composition may be placed around the plant to expose the plant to the volatile gaseous composition.

The seed treatment method according to the present invention is, for example, a method for treating seeds to be protected from pests and insects by a plant disease control composition of the present invention. Specific examples thereof include microparticles of a suspension of the plant disease controlling composition of the present invention And spraying on the seed surface; A spraying treatment method in which a wettable powder, an emulsion, a fluidizing agent or the like of the plant disease control composition of the present invention is applied on its own surface or on a seed surface by adding a small amount of water; Impregnation treatment in which the seed is impregnated in a solution of the plant disease controlling composition of the present invention for a specific period of time; A film coating treatment method and a pellet coating treatment method.

When a plant or soil for plant growth is treated with a compound according to the present invention, the throughput may vary depending on the type of plant to be treated, the type and frequency of occurrence of the insect to be controlled, the formulation type, the treatment period, the climatic conditions and the like.

Milk, wettable powders, fluidizing agents and the like are usually diluted with water and then sprayed for treatment. In this case, the concentration of the active ingredient is usually in the range of 0.0001 to 3% by weight, preferably 0.0005 to 1% by weight. Powders, granules and the like are usually used for treatment without dilution.

The controlling method of the present invention can be used in cultivated land such as paddy field or non-cultivated land.

The plant disease is caused by Magnaporthe oryzae , Rhizoctonia solani , Botrytis cinerea , Phytophthora infestans , Blumeria graminis, Puccinia, recondita ) and anthracnose ( Colletotrichum coccodes ).

The plant is at least one selected from the group consisting of rice, tomato, barley, wheat, radish, red pepper and tomato.

In a specific example of the present invention, the strain HK235 (Fig. 1, Fig. 2 and Fig. 4), which is a novel strain identified through screening of strains having a controlling effect against a plant pathogenic bacterium, And have inhibitory activity against plant pathogenic bacteria (Tables 1 and 2, 3 and 6 to 8).

Therefore, the method of treating the strain of the genus HK235 belonging to the genus Chitinosa according to the present invention, the culture solution thereof, the fraction of the culture solution to the plant, its seed or its habitat can be useful for controlling plant diseases.

Hereinafter, the present invention will be described in detail with reference to examples.

EXAMPLES The following Examples and Experiments are for the purpose of illustrating the present invention, but the present invention is not limited by the following Examples and Experimental Examples.

Example  1. Selection of antagonistic microorganisms

Microorganisms useful for plant disease control were selected from the soil microbial culture filtrate library of the Eco - friendly New Materials Research Center, Korea Research Institute of Chemical Technology. 5, 10, and 20% (v / v) concentrations of the soil microbial culture filtrate were added to 100 μl of a PDB (potato dextrose broth; BD Biosciences) medium in a 96 well plate, and spores of Botrytis cinerea Was inoculated at a concentration of 10 ^-5 spores / mL and cultured at 20 ° C for 3 days.

As a result, as shown in FIG. 1, it was confirmed that culture filtrate of HK235 strain among seven Kittenopa accelerating strains completely inhibited the growth of gray mold fungus from 5% concentration, and the strain was observed after pure culture.

Specifically, the cells were inoculated on a medium of 3% TSA (tryptic soy agar; BD Biosciences) and used as a cylindrical type (average diameter: 0.4 쨉 m, average length: 4.1 쨉 m) . For long-term storage, the strain was cultured in TSB (trptic soy broth; BD Biosciences) medium for 2 days, mixed with 40% glycerol aqueous solution at 1: 1 and stored at 80 ° C.

In order to confirm whether the HK235 strain has an antagonistic action against other plant pathogens, a plant pathogen, Alternaria (BD), was added to a PDA (potato dextrose agar; BD Biosciences) brassicicola , Alternaria panax , Botrytis Cinerea ), Colletotrichum coccodes , Fusarium oxysporum , Magnaporthe (mycelial disk, diameter 5 mm) removed from the fungus of P. oryzae , Phytophthora infestans and Rhizoctonia solani , and a strain HK235 And incubated for 7 days.

As a result, as shown in Fig. 3, the strain HK235 exhibited antagonistic activity strongly inhibiting mycelial growth of all eight plant pathogenic bacteria used in the test.

Example  2. Identification of HK235 strain and comparison of bactericidal activity with same strain

DNA of HK235 strain (100 ng /)) was obtained by DNA extraction using CTAB buffer. A 16S rRNA gene fragment was obtained by PCR using a pair of primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3 ') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') as a template.

Using the 16S rRNA sequence of HK235 strain, a systematic analysis was conducted. As a result, it was confirmed that Kitinopa was selected from Chitinophaga jiangningensis ) JN53 (= DSM27406). In the phylogenetic analysis, Kititrona was close to Chitinophaga eiseniae YC6729 (= DSM22224), but the bootstrap value (%) associated with accuracy in the phylogenetic tree was less than 50%. Therefore, it was confirmed that the strain HK235 belongs to the genus Kittenopha, and the other known Kittenopsis has different phylogenetic characteristics from the strain.

In order to compare the bactericidal activity of the strains of Kititnopa strain and HK235 strain, twelve species of Kitinopa strains were distributed from KACC, and the bactericidal activity of the culture filtrate was examined by a paper disk agar diffusion method.

Specifically, each strain was inoculated into TSB medium, cultured at 30 ° C for 2 days with shaking, and centrifuged (10,000 × g, 15 min) to prepare a culture filtrate. 2 ml of the culture filtrate was concentrated with a vacuum concentrator and the paper disk containing the concentrate was placed on hardened agar medium at a concentration of 10 ^-5 spores / mL in a Botrytis cinerea for 3 days at 20 ° C One inhibitory zone was observed. The antifungal activities of the accelerated Kittenoa strains identified through inhibition ring formation were as shown in Table 1 below.

Strain name KACC deposit number Antifungal activity Chitinophaga sp . HK235 81044BP +++ Chitinophaga rupee 14521 - Chitinophaga niastensis 12954 - Chitinophaga polysaccharea 17184 - Chitinophaga cymbidii 17363 - Chitinophaga japonensis 12057 - Chitinophaga arvensicola 17191 - Chitinophaga sancti 13046 - Chitinophaga eiseniae 13774 + Chitinophaga barathri 18797 - Chitinophaga pinensis 12763 - Chitinophaga  terrae 12755 - Chitinophaga costaii 18713 -

 (+++: inhibitory ring> 25 mm, ++: inhibitory ring = 18 to 25 mm, +: inhibitory ring = 10 to 17 mm

 ±: inhibition ring <10 mm, -: no sterilization activity)

As a result, it was confirmed that only the closely related Kititnopa, Chitinophaga eiseniae KACC 13774 (= YC6729; DSM 22224) exhibited bactericidal activity as in strain HK235 as shown in Table 1. In addition, as compared with KACC 12763 strain known to produce a fungicidally active peptide in the past, the fungicidal activity against tomato gray mold fungus was clearly shown, and the strain HK235 of the present invention was found to have an antagonistic bacterial effect against plant pathogenic fungi The strain was confirmed to be superior to the strain. Thus, strain HK235 of the present invention was deposited and received a deposit number as KACC81044BP.

Example  3. Determine optimal culture conditions

In order to confirm the optimum culture condition of HK235 strain, the growth was compared by changing the medium and temperature.

(LB), 5 g yeast extract, 10 g sodium chloride, and pH 7.0) were used as the nutrient broth (NB) Medium, MEB (6 g malt extract, 1.8 g maltose, 6 g dextrose, 1.2 g yeast extract, pH 4.7) medium, TSB (17 g tryptone, 3 g soytone, 2.5 g dextrose, 5 g sodium chloride, 2.5 g dipotassium phosphate, Was inoculated with HK235 strain and cultured at 25 DEG C and 150 rpm for 3 days with shaking.

In order to determine optimal culture temperature for optimal culture conditions, growth was compared at 20, 25, 30, and 37 ℃. After inoculation on TSB medium, the absorbance (OD ₆₀₀ ) of the culture broth was measured every day, 2, and 3 days by shaking flask culture.

As a result, as shown in FIG. 5, the growth of HK235 strain was the most active when cultured in the TSB medium under the conditions according to the medium, and the growth rate was the fastest at 25 ° C and 30 ° C according to the temperature, The difference in temperature was not significant. Therefore, it was confirmed that culturing at 1-25 days at 25 ~ 30 ℃ is a favorable condition for culturing the strain.

Example  4. HK235 Culture filtrate By fraction  Comparison of bactericidal activity

To investigate the fungicidal activity, the fungicidal activity of tomato gray mold was evaluated for each fraction by paper disk agar diffusion method.

Specifically, strain HK235 was inoculated into TSB medium, cultured at 30 ° C for 2 days with shaking, centrifuged (10,000 × g, 15 min), and the culture filtrate was mixed with an equal amount of organic solvent and then separated using a separatory funnel The organic solvent was separated into an organic solvent layer and a water layer, and hexane, ethyl acetate and butanol were sequentially used as the organic solvent. 2 ml of each fraction was concentrated with a vacuum concentrator, and the resulting concentrate was applied to a paper disk (diameter: 8 mm), spores of Botrytis cinerea were cultured on a potato agar medium at a concentration of 10 ⁵ spores / The paper disk was placed, and the result was observed by incubating at 20 ° C for 3 days.

In addition, the culture filtrate obtained by centrifuging (10,000 × g, 15 min) for each of the various media of Example 3 was extracted with a liquid funnel and an organic solvent to compare the bactericidal activity against the gray mold fungi according to the culture medium.

As a result, as shown in FIG. 6, the inhibitory ring was formed only in the butanol fraction (BuOH), and the bactericidal activity inhibiting the growth of the gray mold fungus was observed in the butanol fraction regardless of the composition of the culture solution. Were found to be soluble in butanol.

Example  5. Culture medium, Culture filtrate , Of the cell suspension  Control effectiveness comparison

To evaluate the plant disease control effect, the culture medium, culture filtrate and cell suspension were compared.

Specifically, strain HK235 was inoculated into TSB medium by shake flask culture, followed by shake culture at 25 ° C and 150 rpm for 2 days, and then centrifuged (10,000 × g, 15 min) to prepare a culture filtrate. The centrifuged baeterial cells were suspended in distilled water at a concentration of 2 × 10 ⁹ cells / mL. Tween 20, a surfactant, was added as an adjuvant (electrodeposition agent) for enhancing emulsifying property to a concentration of 250 μg / mL in order to evaluate a plant disease control effect. At this time, TSB medium containing 250 占 퐂 / mL Tween 20 was used as a control. The in vivo bactericidal activity against tomato gray mold pathogen (Botrytis cinerea) was investigated. The tomato gray mold was treated with a spore suspension (5 × 10 ⁵ spores / mL) of Botrytis cinerea in a tomato seedling of 3 to 4 and incubated for 3 days in a wet condition at 20 ° C. Respectively. The control value (%) was calculated according to the following equation using the lesion area percentage (%) obtained from the above.

[Equation 1]

(%) = {1- (diseased area ratio of treated area / diseased area ratio of untreated area)} x 100

As shown in FIG. 7, 91% and 69% of the HK235 culture and culture filtrate showed the control, respectively, but the cell suspension treatment showed almost no control of the tomato gray mold disease.

Example  6. Culture medium and Culture filtrate  7 things In botanical bottles  Control activity against

In order to evaluate the plant disease control effect, a strain culture solution of HK235 and a culture filtrate obtained by centrifuging it were prepared by the method of Example 5 above.

Specifically, the culture broth of HK235 strain and culture filtrate of Magnaporthe oryzae , Rhizoctonia spp. solani ), tomato gray mold germ ( Botrytis cinerea , Phytophthora infestans , Blumeria graminis f. sp. hordei ), wheat red rust bacillus ( Puccinia recondita ) and pepper anthracis ( Colletotrichum coccodes ) were investigated in vivo . The culture medium and the culture filtrate were diluted with distilled water at 1, 3, and 10 times, respectively. Tween 20 was added to the mixture to a concentration of 250 μg / mL as an electrodeposition agent. TSB medium containing 250 μg / mL Tween 20 Respectively. Two crops per plant were cultivated in the pot. The extract solution was sprayed on the leaf surface, dried for 24 hours, and inoculated with each plant pathogen. The rice, tomato, barley, and wheat used in the experiment were filled with plastic pots with a diameter of 4.5 cm at a ratio of about 70% for the ground or horticultural soil, seeded and cultivated in a greenhouse at 25 ± 5 ° C for 1 to 4 weeks. The rice blast fungus was sprayed with a spore suspension (5 × 10 ⁵ spores / mL) of Magnaporthe oryzae , which is a causative organism of the blast fungus, in a seedling of 3-4 leaf stage and sprayed in a moist chamber at 25 ° C. for one day After the treatment, the cells were cultured in a constant temperature room at 25 DEG C for 4 days to induce the onset. Rhizoctonia solani , a causative agent of sheath blight, was cultivated for 7 days in medium (90g wheat bran, 15g rice, 100ml distilled water), and the culture was inoculated into 5th seedling seedlings. Treated for 4 days in a wet condition, and cultured in a constant temperature room at 25 ° C for 4 days to induce the onset. In tomato seedlings, 3 ~ 4 tomato seedlings were inoculated with sponge suspension from Phytophthora infestans (10 ⁵ sporangia / mL), which is the causative organ of the disease, Day and incubated for 1 day in a constant temperature and humidity room at 25 ° C. The tomato gray mold was treated with a spore suspension (5 × 10 ⁵ spores / mL) of Botrytis cinerea , which is a causative agent of gray mold, in 3-4 leaf tomato seedlings of tomato, And cultured for 3 days to induce the onset. Wheat red rust is a causative organism of rust in the first seedling seedlings, and Puccinia , an active parasite (parasite) recondita) spores to 250㎍ / mL of Tween 20 and suspended in the solution in an amount of 0.67g spores / ℓ to develop by spraying treatment and after treatment for one day in a constant temperature chamber at 20 ℃ s fact moved six days of culture in 20 ℃ Respectively. Barley powdery mildew was obtained by inoculating a spore of Blumeria graminis f. Sp. Hordei , a causative organism of powdery mildew passaged in host plants, in a first seedling of barley , And the onset was induced by culturing. Tomato gray mold and tomato blight were examined 3 days after inoculation, 5 days after inoculation with rice blast, 7 days after inoculation with wheat red rust, barley powdery mildew, and 8 days after inoculation with rice sheath blight.

In order to test the antifungal activity against pepper anthracnose, a plastic port having a diameter of 7.0 cm was filled with about 70% of the horticultural soil, and the best pepper seed was sowed. The seeds were raised in the greenhouse to the third or fourth leaf stage, . After spraying the sample, it was dried at room temperature for 24 hours and then colletotrichum coccodes ) (4 x 10 ⁵ spores / mL). (25 ℃, relative humidity 80%) for 1 day after 2 days of damp treatment at 25 ℃ on the wet field. After 3 days of inoculation, the area ratio (%) of the lesion was examined and the control value Respectively. The inhibitory activity against the seven plant diseases of the culture solution and culture filtrate of the strain HK235 according to the present invention was as shown in Table 2 below.

Sample
Dilution
factor
Control value (%)
RCB RSB TGM TLB WLR BPM PAN Culture
broth
(Culture solution) One 0 57 ± 15 94 ± 2 29 ± 14 91 ± 4 96 ± 2 77 ± 6 3 0 13 ± 15 91 ± 2 5 ± 8 51 ± 17 79 ± 4 67 ± 12 10 0 0 33 ± 8 0 2 ± 4 44 ± 10 50 ± 15 Culture
filtrate
(Culture filtrate) One 4 ± 7 0 69 ± 4 48 ± 8 91 ± 4 70 ± 6 50 ± 10 3 8 ± 7 0 41 ± 11 19 ± 8 4 ± 4 50 ± 0 23 ± 15 10 4 ± 7 0 14 ± 0 5 ± 8 0 22 ± 10 3 ± 6

TLB: Tomato Plague, WLR: Wheat Red Rust, BPM: Barley Powder, PAN: Anthracnose Anthracnose)

As shown in Table 2, culture and culture filtrate of HK235 strain showed activity against tomato gray mold (TGM), wheat red rust (WLR), barley powdery mildew (BPM), and pepper anthracnose (PAN) control. The inhibitory activity of the culture filtrate on the diluted solution was significantly reduced, whereas the culture medium showed a high inhibitory activity of 91% and 79% in the control of tomato gray mold and barley powdery mildew (BPM) even in the triple dilution.

Example  7. Culture extracts and Fraction  Test for controlling tomato gray mold

The butanol fraction and the HP-20 column fraction obtained in the above Example were compared for plant disease control activity.

Specifically, strain HK235 was inoculated into TSB medium by shake flask culture, and pre-culture was performed by shaking culture at 25 ° C and 150 rpm for 1 day. 5 mL of the preculture was inoculated into 500 mL of TSB medium The cultures were incubated at 25 ° C and 150 rpm for 3 days. The culture broth was collected by centrifugation (10,000 × g, 15 min) for 5 L culture. The fractions using the column were adsorbed on a Diaion HP-20 column (5 × 30 cm; Mitsubishi chemical), and then fractionated with an aqueous solution of 80% acetone in which fungicidal activity against tomato gray mold was confirmed by paper disk agar diffusion Was adsorbed on a C18 column (10 x 70 cm) and eluted with methanol at a flow rate of 5 mL / min. Butanol fractions were prepared at concentrations of 250, 500, 1000 and 3,000 ㎍ / mL, and HP-20 column fractions were prepared at concentrations of 500, 1000, 2000 and 3,000 ㎍ / mL. As the electrodeposition agent, Tween 20 was added at a concentration of 250 / / mL, and the concentration of methanol used for dissolution was not more than 5%. At this time, a 5% methanol aqueous solution containing 250 / / mL Tween 20 was used as the control. Tomato gray mold germ ( Botrytis cinerea ) was investigated in vivo . The tomato gray mold was treated with a spore suspension (5 × 10 ⁵ spores / mL) of Botrytis cinerea in a tomato seedling of 3 to 4 and incubated for 3 days in a wet condition at 20 ° C. Respectively. (%) Was calculated using the percent area ratio (%) obtained from the above.

As shown in FIG. 8, butanol extract and HP-20 column fractions exhibited 72 to 85% control of tomato gray mold when treated at a concentration of 3,000 ㎍ / mL. In addition, no toxicity to plants was observed at the concentrations used in the test.

Institution name: National Institute of Agricultural Science and Technology Microbiology Bank

Accession number: KACC81044BP

Checked on: 20170117

Claims (9)

Strain HK235 ( Chitinophaga sp. HK235) deposited with Accession No. KACC 81044BP.
( Magnaporthe oryzae ), Rhizoctonia spp. (Including Rhizoctonia spp.) Containing at least one member selected from the group consisting of the strain HK235 belonging to the genus Kittenophorus deposited with Accession No. KACC 81044BP, the culture thereof, The present invention relates to a method for the treatment of fungi such as Solani , Botrytis cinerea , Phytophthora infestans , Blumeria graminis, Puccinia recondita , Colletotrichum coccodes , Alternaria brassicicola , Wherein the composition is derived from at least one bacterium selected from the group consisting of A. panax and Fusarium oxysporum .
[3] The composition according to claim 2, wherein the culture solution is a culture solution in which a strain has been cultured, or filtered by a physical method.
3. The composition according to claim 2, wherein the fraction is fractionated using a solvent selected from the group consisting of hexane, ethyl acetate, butanol and water, or a column using an adsorbent.
delete The composition according to claim 2, wherein the plant is at least one selected from the group consisting of rice, barley, wheat, radish, red pepper and tomato.
Claim, oryzae comprising the step of treating the second composition of claim plant, to its seed or its habitat (Magnaporthe oryzae), sheath blight fungus (Rhizoctonia solani), gray mold pathogen (Botrytis cinerea), reverse pathogens (Phytophthora infestans) Selected from the group consisting of Blumeria graminis, Puccinia recondita , Colletotrichum coccodes , Alternaria brassicicola , A. panax and Fusarium oxysporum . A method for controlling plant diseases derived from any one or more microorganisms.
delete 8. The method according to claim 7, wherein the plant is at least one selected from the group consisting of rice, barley, wheat, radish, red pepper and tomato.

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