KR20210019716A - Composition for controlling plant diseases including Platycladus orientalis extract or fractions of thereof as an active ingredient and method of controlling plant diseases using the same - Google Patents

Abstract

The present invention relates to a composition for controlling plant diseases, comprising a Platycladus orientalis extract or a fraction thereof as an active ingredient, and to a method for controlling plant diseases using the composition. The Platycladus orientalis extract or fraction thereof is harmless to the human body as a natural product. In addition, since it is biodegradable in nature and does not cause environmental pollution, it has an effect of controlling plant diseases, thereby being usefully used as the composition for controlling plant diseases.

Description

Composition for controlling plant diseases including Platycladus orientalis extract or fractions of thereof as an active ingredient and method of controlling plant diseases using the composition for controlling plant diseases containing the extract or a fraction thereof as an active ingredient using the same}

The present invention relates to a composition for controlling plant diseases and a method for controlling plant diseases using the composition, and more particularly, to a composition for controlling plant diseases containing an extract or a fraction thereof as an active ingredient.

In crop production, plant diseases are one of the harmful factors that inhibit the healthy growth of plants and are caused by microbial factors such as fungi, bacteria, mycoplasma, viruses, and nematodes. When plant diseases occur in large-scale crop cultivation areas, not only economic losses due to significant decrease in yield, but also disruption in stable production of main grains can be a serious social problem in terms of food security.

Synthetic pesticides have been used as the main means of controlling plant diseases in modern agriculture. However, the existing synthetic pesticides have been used in large quantities for many years, accelerating the emergence of drug-resistant pathogens, increasing the control costs every year, and there are constant concerns of civil society and academia about the problem of human toxicity of pesticide residues and disturbance of the ecosystem. Comprehensive pest control, which focuses on the use of biological pesticides in reducing the use and spraying frequency of synthetic pesticides while solving the drug resistance problem, can be a new alternative to conventional agricultural methods dependent on synthetic pesticides.

Biological pesticides are developed based on natural substances derived from biological materials such as plant extracts or using live microorganisms such as fungi, bacteria, and viruses to control pests. Therefore, biological pesticides are natural products derived from nature and have nature-friendly characteristics, and have the advantage of being weak or harmless in toxicity compared to synthetic pesticides.

Accordingly, policy restrictions on the use of synthetic pesticides are being strengthened in OECD countries, including Korea. When a group analysis of millions of compounds registered in the American Chemical Association's Chemical Information Database (CAS) is classified as a group of hundreds of leading substances, the types of synthetic pesticides that can be used when the regulation of the lead substance standard is enforced. And the number is bound to drop sharply. Therefore, it is necessary to develop new eco-friendly plant disease control measures to replace the existing synthetic pesticides.

Plants are used to adapt to various habitats, such as alkaloids, terpenoids, flavonoids, glycosides, quinones, coumarins, phenolic, and essential oils. (essential oil), phytoalexin, etc. have evolved to biosynthesize various substances, and from these substances, secondary metabolites that function to inhibit the invasion of plant pathogens and the feeding of pests are biosynthesized and accumulated in plants. As such, plant extracts are widely used as materials for biological pesticides.

Plant extracts and essential oils derived from neem, canola, pyrethrin, limonene, tea tree, and rotenone are used as biological pesticides. In addition, Rheum rhabarbarum Nakai root extract can be used for barley powdery mildew ( Blumeria graminis). f. sp. hordei ) and wheat red rust ( Puccinia) triticina) showed excellent effects on prevention, gamguk (Chrysanthemum indicum L.) Oil Tobacco Station Essen Sean pathogens (Phytophthora nicotianae ) showed activity to inhibit the growth of alfalfa ( Medicago sativa ) and Trevesia palmata (Trevesia palmata A number of research results supporting the use of plant extracts, such as that the saponin compound derived from L.) exhibits bactericidal activity against several plant pathogens including Magnaporthe oryzae, have been reported. Has been done. In addition, Japanese Patent Application Laid-Open No. 2012-102023 discloses a cypress ( Chamecyparis obtusa) has disclosed an antimicrobial agent for controlling plant diseases and a method for producing the same, which contains the leaf extract as an active ingredient.

On the other hand, cypress ( Platycladus orientalis (L.) Franco) is taxonomically one of the conifers and is the only species of Platycladus. It is native to northern China and grows wild in Asia, including Korea, Japan, India, and Iran. It has been reported that the cypress tree extract exhibits various pharmacological activities such as anti-inflammatory, antioxidant, antimicrobial, and anticancer, and terpene compounds having pharmacological actions such as antifibrosis and inflammation have been reported from the cypress extract.

Terpene is a generic term for natural organic compounds containing isoprene (C ₅ H ₈ ) as a constituent unit, and has (C ₅ H ₈ )n as a basic skeleton, and is also called a terpenoid. Terpene is the main component of plant essential oil, and it is produced in the early stages of biosynthesis, in which acetic acid becomes steroids (sitosterol and stigmasterol) through squalene under the action of enzymes in plants. Terpenes are classified according to the number of isoprene units (C ₅ H ₈ )n, and isoprene itself is a hemeterpene, a carbon number of 10 is a monoterpene, a carbon number of 15 is a sesquiterpene, Those with 20 carbons are classified as diterpenes. In addition, terpenes are classified into chain terpenes, monocyclic, monocyclic, and tricyclic terpenes, and compounds having the same carbon skeleton and alcohol, aldehyde, ketone, and carboxylic acid are also included in each category. Terpene has the function of preventing oxidation of the fruit bark, leaves and stems of plants from ultraviolet rays, the function of preventing other plants from growing around, the function of preventing the invasion of phytopathogenic fungi or bacteria and directly attacking them.

Japanese Patent Application Publication 2012-102023

It is an object of the present invention to provide a composition for controlling plant diseases that is harmless to the human body and exhibits excellent control effects against plant diseases without causing environmental pollution.

Another object of the present invention is to provide a method for controlling plant diseases that is harmless to the human body and exhibits an excellent control effect against plant diseases without causing environmental pollution.

To achieve the above object,

The first aspect of the present invention is a cypress ( Platycladus Orientalis ) It provides a composition for controlling plant diseases containing as an active ingredient at least one selected from the group consisting of extracts and fractions thereof.

A second aspect of the present invention provides a method for controlling plant diseases comprising the step of treating a plant, a seed thereof, or a habitat thereof with the composition for controlling plant diseases.

The white white tree of the present invention ( Platycladus orientalis ) extract or a fraction thereof is a natural product, harmless to the human body, biodegradable in nature, does not cause environmental pollution, and has the effect of controlling plant diseases, so it can be usefully used as a composition for controlling plant diseases.

FIG. 1 is a synthetic pesticide disinfectant tricyclazole 10 μg/ml(a) and 3000 μg/ml of a cypress extract in order to evaluate the control effect of a cypress extract or an organic solvent fraction on rice blast disease. (b), 2000 μg/ml (c) of the hexane fraction and 2000 μg/ml (d) of the ethyl acetate fraction were sprayed, respectively, and the rice blast bacteria were inoculated, and then the photograph of the rice and the untreated area (e) were inoculated with the rice blast bacteria. It is a picture of rice after one.

Hereinafter, the present invention will be described in detail.

The present inventors have platycladus while trying to investigate the control effects on plant diseases of various plant extracts to develop eco-friendly plant disease control agents (Platycladus orientalis (L.) Franco) extract from rice blast disease (cause: Magnaporthe oryzae ) and wheat red rust (cause: Puccinia triticina ) was found to have excellent control effects.

In the present specification, the term "control" shall be used in the sense of not only preventing and avoiding diseases or pests, but also removing and killing. However, in controlling plant diseases, the control effect of the preventive treatment is the main control mechanism, so the investigation of the control effect of the extracts and fractions of Platycladus orientalis on plant diseases was tested as a preventive treatment.

One aspect of the present invention is a cypress ( Platycladus Orientalis ) It provides a composition for controlling plant diseases containing as an active ingredient at least one selected from the group consisting of extracts and fractions thereof.

Here, the extract may _{be extracted using any one selected from the group consisting of water, C 1} to C ₄ lower alcohol, hexane, and ethyl acetate as a solvent, but is not limited thereto.

The C ₁ to C ₄ lower alcohol may be methanol, ethanol or butanol, preferably methanol or ethanol, and more preferably methanol.

In addition, the fraction of the extract may be fractionated using any one selected from the group consisting of hexane and ethyl acetate as a solvent, but is not limited thereto.

The plant disease may be one or more plant diseases selected from the group consisting of rice blast disease, wheat red rust, pepper anthrax, phalaenopsis bacterial brown spot disease, crop soft rot and kiwi ulcer disease, but is not limited thereto. The crop may be Chinese cabbage, radish, tomato, sweet potato, potato, onion, carrot, etc., but is not limited thereto.

The composition for controlling plant diseases is Magnaporthe oryzae ), Puccinia triticina triticina ) and Colletotrichum coccodes may inhibit the growth of one or more phytopathogenic fungi selected from the group consisting of, but is not limited thereto.

The composition for controlling plant diseases is Exidovorax Abene subsp. Cattrayer ( Acidovorax avenae subsp. cattleyae ), Dickeya chrysanthemi , Pseudomonas syringe pv. Actinidie ( Pseudomonas syringae pv. actinidiae ) may inhibit the growth of one or more phytopathogenic bacteria selected from the group consisting of, but is not limited thereto. The Dikkeya chrysanthemai is a crop soft rot, and the crop may be cabbage, radish, tomato, sweet potato, potato, onion, carrot, etc., but is not limited thereto.

The cypress ( Platycladus orientalis ) extract or a fraction thereof may contain one or more compounds represented by any one of the following Formulas 1 to 8:

[Formula 1]

;

;

[Formula 2]

;

;

[Formula 3]

;

;

[Formula 4]

;

;

[Formula 5]

;

;

[Formula 6]

;

;

[Formula 7]

; 및

; And

[Formula 8]

.

.

The active ingredient may be included in a concentration of 1 to 100000 μg/ml in the composition for controlling plant diseases, may be included in a concentration of 10 to 10000 μg/ml, and may be included in a concentration of 100 to 5000 μg/ml, preferably It may be included in a concentration of 250 to 3,000 μg / ml. The concentration represents the concentration contained in the composition when the plant disease has occurred, its seeds or its habitat, and if the concentration is lower than the lower limit of the above range, the concentration of the active ingredient is too low to control plant diseases. There is a problem of falling, and if the concentration is higher than the upper limit of the above range, the concentration of the active ingredient is too high than necessary, which is uneconomical and may cause a negative impact on the environment. However, the amount of the composition for controlling plant diseases is not limited to the concentration range, and may be appropriately adjusted in consideration of the type, degree of occurrence, and environment of phytopathogenic fungi or phytopathogenic bacteria.

The composition for controlling plant diseases is a cypress ( Platycladus orientalis ) extract or a simple mixture of fractions thereof. Alternatively, the composition for controlling plant diseases is formulated with the extract or fraction of the extract and an inert carrier, and the mixture is formulated as an emulsion, emulsion, fluidizing agent, wettable powder, granulated wettable powder, powder, granule, etc. It is prepared by adding surfactants and other necessary auxiliaries to the mixture so that it can be made. The above-mentioned composition for controlling plant diseases can be used as a seed treatment agent in the present invention by itself or by adding other inactive ingredients.

Examples of liquid carriers that can be used in the formulation include 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 diisopropylether and dioxane; Acid amides such as N , N -dimethyl formamide 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, attapulgite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and talcite; Natural organic substances such as corn leaf stalk powder and walnut husk 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, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalene sulfonate form. Aldehyde polycondensate; And nonionic surfactants such as polyoxyethylene alkyl aryl ethers, polyoxyethylene alkylpolyoxypropylene block copolymers and sorbitan fatty acid esters and cationic surfactants such as alkyltrimethylammonium salts.

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

In one embodiment of the present invention, the cypress ( Platycladus orientalis ) extract or a fraction thereof was confirmed to exhibit a growth inhibitory effect on phytopathogenic fungi or phytopathogenic bacteria (see Examples 1 to 4), from which the composition for controlling plant diseases containing the extract or fraction as an active ingredient It can be seen that the plant disease control effect.

The cypress ( Platycladus orientalis ) extract or fraction may be prepared by a manufacturing method comprising the following steps, but is not limited thereto:

1) Cypress tree ( Platycladus orientalis ) extracting by adding an extraction solvent;

2) filtering the extract of step 1);

3) preparing an extract of Cypress (Platycladus orientalis ) by concentrating the filtered extract of step 2) under reduced pressure and drying it; And

4) Step 3) of Platycladus extracts orientalis) to extract additional organic solvent platycladus (Platycladus orientalis ) preparing a fraction.

In the above method, the cypress of step 1) ( Platycladus orientalis ) can be used without limitation, such as grown or commercially available. The cypress ( Platycladus orientalis ) leaves, stems, or roots are all available, but are not limited thereto.

The extraction solvent of step 1) may _{be any one selected from the group consisting of water, C 1} to C ₄ lower alcohol, hexane and ethyl acetate, or a mixed solution thereof, but is not limited thereto.

The C ₁ to C ₄ lower alcohol may be methanol, ethanol or butanol, preferably methanol or ethanol, and more preferably methanol.

As the extraction method, shaking extraction, Soxhlet extraction, or reflux extraction may be used, but the present invention is not limited thereto. The extraction solvent may be extracted by adding 1 to 10 times to the amount of dried cypress (Platycladus orientalis ), and it may be extracted by adding 2 to 3 times. The extraction temperature may be 20°C to 100°C, 20°C to 40°C, and room temperature, but is not limited thereto. In addition, the extraction time may be 10 to 48 hours, may be 15 to 30 hours, may be 24 hours, but is not limited thereto. Further, the number of extractions may be 1 to 5 times, may be repeatedly extracted 3 to 4 times, and may be 3 times, but is not limited thereto.

In the above method, the vacuum concentration in step 3) may be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying may be vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

In the above method, the organic solvent in step 4) may be any one selected from the group consisting of hexane and ethyl acetate, but is not limited thereto. The fraction is cypress ( Platycladus orientalis ) may be any one of the solvent fractions obtained by suspending the extract in water and then fractionating with hexane and ethyl acetate, but is not limited thereto. The fraction is the cypress ( Platycladus orientalis ) from the extract can be obtained by repeating the fractionation process 1 to 5 times, can be obtained by repeating 3 times, and concentrated under reduced pressure after fractionation, but is not limited thereto.

In addition, another aspect of the present invention is a cypress ( Platycladus orientalis ) provides a plant disease control method comprising the step of treating a plant, a seed thereof, or a habitat thereof, a composition for controlling plant diseases containing as an active ingredient at least one selected from the group consisting of extracts and fractions thereof.

The plant may be one or more plants selected from the group consisting of rice, wheat, pepper, phalaenopsis, radish, cabbage, tomato, sweet potato, potato, onion, carrot, and kiwi, but is not limited thereto.

The plant disease may be one or more plant diseases selected from the group consisting of rice blast disease, wheat red rust, pepper anthrax, phalaenopsis bacterial brown spot disease, crop soft rot and kiwi ulcer disease, but is not limited thereto. The crop may be Chinese cabbage, radish, tomato, sweet potato, potato, onion, carrot, etc., but is not limited thereto.

The treatment may be an indirect spraying of spraying the composition directly on the plant, spraying it on the soil in which the plant is growing, or spraying the cultivation medium of the plant.

The control method of the present invention includes treatment of stems and leaves of plants, treatment of places where plants grow (eg soil), treatment of seeds such as seed sterilization/seed coating, and treatment of roots.

Treatment of the stems and leaves in the control method of the present invention may include, in particular, application on the plant surface, such as spraying the stems and leaves. Treatment of the soil in the control method of the present invention may include, for example, spraying onto the soil, mixing with the soil, and spraying the liquid treatment agent into the soil (irrigation of the liquid treatment agent, injection into the soil, dropping of the liquid treatment agent). Examples of places to be treated are planting holes, furrows, around replanting blood, around planting furrows, the entire surface of the growth area, between soil and plants, between roots, and under the stem of the plant. , Main furrow, growing soil, pond. It includes a seedling box, a seedling tray, and a seedling. The treatment may be performed before spraying, during spraying, immediately after spraying, during a simulated cultivation period, before cultivation settling, cultivation settling, and at a growing period after cultivation settling. In the above-mentioned soil treatment, the active ingredient may be applied to the plants at the same time, or a solid fertilizer such as a paste fertilizer containing the active ingredient may be applied to the soil. The active ingredient can be mixed in an irrigation liquid, for example injected into an irrigation system (irrigation tube, irrigation pipe, sprinkler, etc.), mixed in a liquid overflowing between furrows, or mixed in a water culture medium. I can. Alternatively, the irrigation liquid and active ingredients can be premixed and used for treatment by suitable irrigation methods, including, for example, irrigation methods mentioned above and other methods such as sprinkling and flooding.

The volatilization treatment method according to the control method of the present invention, for example, the volatilization of the sprayed composition by spraying treatment on media such as soil for cultivating plants with the composition for controlling plant diseases of the present invention, hydroponic medium for culturing plants, and bed It is a method of protecting the plant from pests and diseases, and in addition, the composition may be placed around the plant to expose the plant to the volatilized gaseous composition.

The seed treatment method in the control method of the present invention is, for example, a method of treating seeds to be protected from pests with the composition for controlling plant diseases of the present invention, a specific example of which is to atomize the suspension of the composition for controlling plant diseases of the present invention And spray treatment method of spraying on the seed surface; Spray treatment method of applying the wettable powder, emulsion, fluidizing agent, etc. of the composition for controlling plant diseases of the present invention on the seed surface by itself or by adding a small amount of water; An impregnation treatment method of impregnating seeds into a solution of the composition for controlling plant diseases of the present invention for a specific period of time; It includes a film coating treatment method and a pellet coating treatment method.

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

Emulsions, wettable powders, fluidizing agents, etc. 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, etc. are usually used for treatment without dilution.

The control method of the present invention can be used in arable or uncultivated areas such as rice fields.

In one embodiment of the present invention, the cypress ( Platycladus orientalis ) extract or a fraction thereof was confirmed to exhibit a growth inhibitory effect on phytopathogenic fungi or phytopathogenic bacteria (see Examples 1 to 4), from which the composition for controlling plant diseases containing the extract or fraction as an active ingredient It can be seen that can be usefully used in a plant disease control method.

Further, another aspect of the present invention provides a use of a composition for controlling plant diseases, containing an extract of Platycladus orientalis and a fraction thereof as an active ingredient in controlling plant diseases.

In addition, another aspect of the present invention,

It provides a composition for controlling plant diseases containing as an active ingredient at least one compound selected from the group consisting of compounds represented by the following formulas A and B, stereoisomers thereof, or agrochemically acceptable salts thereof:

[Formula A]

(In Formula A,

R ¹ is CH ₃ or H, and

,

,

또는

이다); 및R ² is

,

,

or

to be); And

[Formula B]

(In Formula B,

은 단일결합 또는 이중결합이고,

Is a single bond or a double bond,

R ¹ is OH or H,

R ² is CH ₃ or CH ₃ OH, and

R ³ is absent or OH).

Another aspect of the present invention provides a composition for controlling plant diseases comprising as an active ingredient at least one compound selected from the group consisting of compounds represented by the following Formulas 1 to 8:

[Formula 1]

;

;

[Formula 2]

;

;

[Formula 3]

;

;

[Formula 4]

;

;

[Formula 5]

;

;

[Formula 6]

;

;

[Formula 7]

; 및

; And

[Formula 8]

.

.

The compound is a cypress ( Platycladus orientalis ) may be derived from an extract. However, it is not limited thereto.

Specifically, the cypress ( Platycladus orientalis ) from the extract, a solvent fraction is prepared, and the fraction is fractionated by liquid chromatography to separate the compound.

The liquid chromatography technique refers to a chromatography technique in which the mobile phase is a liquid, and is performed in a column filled with a stationary phase or a plane to which the stationary phase is attached. Cypress ( Platycladus orientalis ) fraction is not limited as long as it is a solvent fraction, but may be an ethyl acetate fraction or a hexane fraction, but is not limited thereto. As the mobile phase, organic solvents such as water, hexane, methanol, ethanol, acetonitrile, acetone, chloroform, dichloromethane, and ethyl acetate may be used alone or in combination, and as a fixed phase, silica gel, Diaion HP-20 , RP-18 or Sephadex LH-20 may be used, but is not limited thereto.

The plant disease may be one or more plant diseases selected from the group consisting of rice blast disease, potato blight, tomato blight, wheat red rust, phalaenopsis bacterial brown spot, crop soft rot, and kiwi ulcer disease, but is not limited thereto. The crop may be Chinese cabbage, radish, tomato, sweet potato, potato, onion, carrot, etc., but is not limited thereto.

The composition for controlling plant diseases is Magnaporthe oryzae ), Phytophthora infestans infestans ) and Puccinia triticina (Puccinia triticina) may inhibit the growth of one or more phytopathogenic fungi selected from the group consisting of, but is not limited thereto.

The composition for controlling plant diseases is Exidovorax Abene subsp. Cattrayer ( Acidovorax avenae subsp. cattleyae ), Dickeya chrysanthemi , Fectobacterium carotoborum subsp. Carotovorum ( Pectobacterium carotovorum subsp. carotovorum ) and Pseudomonas syringe pv. Actinidie ( Pseudomonas syringae pv. actinidiae ) can inhibit the growth of one or more phytopathogenic bacteria selected from the group consisting of, but is not limited thereto. The Dikkeya chrysanthemai and Fectobacterium carotoborum subsp. Carotoborum is a crop soft rot, and the crop may be cabbage, radish, tomato, sweet potato, potato, onion, carrot, etc., but is not limited thereto.

The compound may have one or more selected from the group of compounds represented by the following Formula 1, Formula 2 and Formula 7 as an active ingredient, and the plant disease is from the group consisting of rice blast, potato blight, tomato blight, and wheat red rust. It may be one or more plant diseases of choice:

[Formula 1]

;

;

[Formula 2]

; 및

; And

[Formula 7]

.

.

The active ingredient may be included in a concentration of 1 to 100000 μg/ml in the composition for controlling plant diseases, may be included in a concentration of 10 to 10000 μg/ml, and may be included in a concentration of 50 to 5000 μg/ml, preferably It may be included in a concentration of 100 to 2000 μg / ml. The concentration represents the concentration contained in the composition when the plant disease has occurred, its seeds or its habitat, and if the concentration is lower than the lower limit of the above range, the concentration of the active ingredient is too low to control plant diseases. There is a problem of falling, and if the concentration is higher than the upper limit of the above range, the concentration of the active ingredient is too high than necessary, which is uneconomical and may cause a negative impact on the environment. However, the amount of the composition for controlling plant diseases is not limited to the concentration range, and may be appropriately adjusted in consideration of the type, degree of occurrence, and environment of phytopathogenic fungi or phytopathogenic bacteria.

The compound represented by Formulas A to B, the labdane diterpnoid compound represented by Formulas 1 to 4, and the pimarane diterpenoid compound represented by Formulas 5 to 8 are pesticides It can be used in the form of a scientifically acceptable salt, and as a salt, an acid addition salt formed by agrochemically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, etc., acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Examples of such agrochemically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, i. Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube Rate, sebacate, fumarate, maleate, butine-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β -hydroxybutyrate, Glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

The acid addition salt can be prepared by a conventional method, for example, a labdane diterpnoid compound and a pimarane diterpenoid compound in methanol, ethanol, acetone, methylene chloride, The precipitate produced by dissolving in an organic solvent such as acetonitrile and adding an organic or inorganic acid may be filtered and dried, or may be prepared by distilling a solvent and an excess of acid under reduced pressure and drying to crystallize under an organic solvent.

In addition, bases can be used to make agrochemically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

Further, the compounds of Formulas A to B, labdane diterpnoid compounds of Formulas 1 to 4, and pimarane diterpenoid compounds of Formulas 5 to 8 are agrochemically acceptable It may be used in the form of not only salts, but also solvates, stereoisomers, and hydrates that may be prepared therefrom.

Plant disease control containing the compounds of Formulas A to B, labdane diterpnoid compounds of Formulas 1 to 4, and pimarane diterpenoid compounds of Formulas 5 to 8 as active ingredients The composition is a mixture of a compound of Formulas A to B, a labdane diterpnoid compound of Formulas 1 to 4, and a pimarane diterpenoid compound of Formulas 5 to 8 and an inert carrier, It is prepared by adding a surfactant and other necessary auxiliary agents to the mixture so that the mixture can be formulated as an emulsion, emulsion, fluidizing agent, wettable powder, granulated wettable powder, powder, granule, and the like. The above-mentioned composition for controlling plant diseases can be used as a seed treatment agent in the present invention by itself or by adding other inactive ingredients.

Examples of liquid carriers that can be used in the formulation include 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 diisopropylether and dioxane; Acid amides such as N , N -dimethyl formamide 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, attapulgite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and talcite; Natural organic substances such as corn leaf stalk powder and walnut husk 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, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalene sulfonate form. Aldehyde polycondensate; And nonionic surfactants such as polyoxyethylene alkyl aryl ethers, polyoxyethylene alkylpolyoxypropylene block copolymers and sorbitan fatty acid esters and cationic surfactants such as alkyltrimethylammonium salts.

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

In one embodiment of the present invention, the labdane diterpnoid compound of Formulas 1 to 4 and the pimarane diterpenoid compound of Formulas 5 to 8 are phytopathogenic fungi or phytopathogenic bacteria It was confirmed that it exhibits a growth inhibitory effect on (see Examples 8 to 10), from which the labdane diterpnoid compounds of Formulas 1 to 4 and the pimarane diterpnoids of Formulas 5 to 8 It can be seen that the composition for controlling plant diseases containing the (pimarane diterpenoid) compound as an active ingredient has a plant disease control effect.

Further, another aspect of the present invention is a compound of Formulas A and B, a labdane diterpnoid compound of Formulas 1 to 4, and a pimarane diterpenoid compound of Formulas 5 to 8 It provides a plant disease control method comprising the step of treating a plant, a seed thereof, or a habitat thereof, a composition for controlling plant diseases containing as an active ingredient.

The plant may be one or more plants selected from the group consisting of rice, potato, tomato, wheat, phalaenopsis, radish, cabbage, sweet potato, onion, carrot, and kiwi, but is not limited thereto.

The plant disease may be one or more plant diseases selected from the group consisting of rice blast disease, potato blight, tomato blight, wheat red rust, phalaenopsis bacterial brown spot, crop soft rot, and kiwi ulcer disease, but is not limited thereto. The crop may be Chinese cabbage, radish, tomato, sweet potato, potato, onion, carrot, etc., but is not limited thereto.

The treatment may be an indirect spraying of spraying the composition directly on the plant, spraying it on the soil in which the plant is growing, or spraying the cultivation medium of the plant.

The control method of the present invention includes treatment of stems and leaves of plants, treatment of places where plants grow (eg soil), treatment of seeds such as seed sterilization/seed coating, and treatment of roots.

Treatment of the stems and leaves in the control method of the present invention may include, in particular, application on the plant surface, such as spraying the stems and leaves. Treatment of the soil by the control method of the present invention may include, for example, spraying onto the soil, mixing with the soil, and spraying the liquid treatment agent into the soil (irrigation of the liquid treatment agent, injection into the soil, dropping of the liquid treatment agent). Examples of places to be treated are planting holes, furrows, around replanting blood, around planting furrows, the entire surface of the growth area, between soil and plants, between roots, and under the stem of the plant. , Main furrow, growing soil, pond. It includes a seedling box, a seedling tray, and seedlings. Treatment may be carried out before spraying, during spraying, immediately after spraying, during a simulated cultivation period, before cultivation settling, cultivation settling, and at a growth period after cultivation settling. In the above-mentioned soil treatment, the active ingredient may be applied to the plants at the same time, or a solid fertilizer such as a paste fertilizer containing the active ingredient may be applied to the soil. The active ingredient can be mixed in an irrigation liquid, for example injected into an irrigation system (irrigation tube, irrigation pipe, sprinkler, etc.), mixed in a liquid overflowing between furrows, or mixed in a water culture medium. I can. Alternatively, the irrigation liquid and active ingredients can be premixed and used for treatment by suitable irrigation methods, including, for example, irrigation methods mentioned above and other methods such as sprinkling and flooding.

The volatilization treatment method according to the control method of the present invention, for example, the volatilization of the sprayed composition by spraying treatment on media such as soil for cultivating plants with the composition for controlling plant diseases of the present invention, hydroponic medium for culturing plants, and bed It is a method of protecting the plant from pests and diseases, and in addition, the composition may be placed around the plant to expose the plant to the volatilized gaseous composition.

The seed treatment method in the control method of the present invention is, for example, a method of treating seeds to be protected from pests with the composition for controlling plant diseases of the present invention, a specific example of which is to atomize the suspension of the composition for controlling plant diseases of the present invention And spray treatment method of spraying on the seed surface; Spray treatment method of applying the wettable powder, emulsion, fluidizing agent, etc. of the composition for controlling plant diseases of the present invention on the seed surface by itself or by adding a small amount of water; An impregnation treatment method of impregnating seeds into a solution of the composition for controlling plant diseases of the present invention for a specific period of time; It includes a film coating treatment method and a pellet coating treatment method.

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

Emulsions, wettable powders, fluidizing agents, etc. 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, etc. are usually used for treatment without dilution.

The control method of the present invention can be used in arable or uncultivated areas such as rice fields.

In an embodiment of the present invention, it is shown that the labdane diterpnoid compounds of Formulas 1 to 4 and the pimarane diterpenoid compounds of Formulas 5 to 8 exhibit a control effect against plant diseases. It was confirmed (see Examples 8 to 10), from which it can be seen that the composition for controlling plant diseases containing the terpenoid compounds represented by Formulas 1 to 8 as an active ingredient can be usefully used in a method for controlling plant diseases. .

In addition, another aspect of the present invention is a compound of Formulas A to B, a labdane diterpnoid compound of Formulas 1 to 4, and a pimaran dieterfe of Formulas 5 to 8 in controlling plant diseases. It provides the use of a composition for controlling plant diseases containing a noid (pimarane diterpenoid) compound as an active ingredient.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Manufacturing example 1> Containing cypress extract as an active ingredient Plant disease Preparation of control composition

Step 1: Preparation of cypress tree extract

Cypress ( Platycladus) orientalis ) to prepare a composition for controlling plant diseases containing the extract as an active ingredient, Platycladus orientalis ) extract was prepared. Specifically, the leaf and stem samples of the dried cypress ( Platycladus orientalis ) were cut finely, methanol was added, extracted for 24 hours at room temperature, filtered through a filter paper, and the filtrate was concentrated under reduced pressure to 100 g of cypress ( Platycladus orientalis ) extract was obtained.

Step 2: Preparation of a composition for controlling plant diseases

2 ml of a sample in which the extract prepared in step 1 was dissolved in methanol at a concentration of 60 mg/ml was diluted with 38 ml of distilled water. A composition for controlling plant diseases containing an extract of Platycladus orientalis at a concentration of 3000 μg/ml by adding Tween 20 as a surfactant to the diluent to a concentration of 0.025% (w/v) Prepared 40 ml of the solution.

< Manufacturing example 2> of cypress extract Fractions Contained as an active ingredient Plant disease Preparation of control composition

Step 1: Preparation of a fraction of the cypress tree extract

After dissolving 10 g of the extract of white cypress prepared in step 1 of Preparation Example 1 with 500 ml of distilled water, extraction was performed twice with 500 ml of hexane to obtain a hexane layer and an aqueous solution layer. The aqueous solution layer was again extracted twice with 500 ml of ethyl acetate and butanol to obtain an ethyl acetate layer, a butanol layer, and an aqueous solution layer. Then, these were concentrated under reduced pressure to obtain a dried hexane fraction (3.5 g), ethyl acetate fraction (2.7 g), butanol fraction (2.2 g) and water fraction (1.1 g).

Step 2: Preparation of a composition for controlling plant diseases

The hexane fraction and ethyl acetate fraction prepared in step 1 were dissolved in methanol at a concentration of 20 mg/ml, and 4 ml of each sample was diluted with 36 ml of distilled water. For the butanol fraction and the water fraction, 2 ml of each sample dissolved in methanol at a concentration of 40 mg/ml was diluted with 38 ml of distilled water. Tween 20, a surfactant, was added to the diluent to a concentration of 0.025% (w/v), and the concentration of 2000 μg/ml was used as Platycladus orientalis ) 40 ml of a solution of a composition for controlling plant diseases containing a fraction of the extract as an active ingredient was prepared.

< Example 1> Containing cypress extract as an active ingredient Plant disease Control composition Plant disease Control effect evaluation

In order to confirm whether the composition for controlling plant diseases containing the extract of C. as an active ingredient according to the present invention exhibits the effect of controlling plant diseases, an experiment was performed according to the following procedure, and the results are shown in Table 1.

Specifically, the composition for controlling plant diseases prepared in Preparation Example 1 is a rice blast disease (cause: Magnaporthe oryzae ), rice leaf sheath blight (cause: Rhizoctonia solani ), tomato gray mold disease (cause: Botrytis cinerea ), tomato blight (cause: Phytophthora infestans ), barley powdery mildew (cause: Blumeria graminis f. sp. hordei ), wheat red rust (cause: Puccinia triticina ) and pepper anthrax ( Cause : Colletotrichum coccodes), in order to check whether there is a control effect, the following experiment was performed in greenhouse conditions. At this time, the untreated group was treated with distilled water containing 5% (v/v) of methanol and 0.025% (w/v) of Tween 20, and tricycla, a synthetic pesticide disinfectant as a control agent, was used to evaluate the control effect against rice blast disease. Treated with 10 μg/ml of sol (tricyclazole).

Plants used in the experiment were rice ( Oryza sativa L.), tomato ( Solanum lycopersicum L.), wheat ( Triticum aestivum L.) barley ( Hordeum sativum Jessen) and red pepper ( Capsicum annuum L.). Each plant was cultivated for 1 to 4 weeks in a greenhouse at a temperature of 20 to 30°C by sowing seeds after filling up to 70% of the water supply or horticultural soil in a plastic pot having a diameter of 4.5 cm. After spray-spraying the solution of the control composition prepared in Step 2 of Preparation Example 1 on the cultivated plants and air-drying for 24 hours, the causative bacteria of the plant diseases were respectively inoculated, and three pots were used for each phytopathogenic fungus. .

Rice blast is the causative agent of seedlings in the 3rd to 4th leaf stage, Magnaporthe oryzae, Korea spore suspension of the Research Institute of Chemical ^{Technology) (5 × 10 5 spores /} ml) of the spray vaccination, and were treated for one day in a moist fact the temperature is 25 ℃, the constant temperature hangseupsil temperature is 25 ℃, with relative humidity of 80% It was cultivated for 4 days and induced the onset.

Rice leaf sheath blight is the causative agent of Rhizoctonia solani (Rhizoctonia). solani , Korea Research Institute of Chemical Technology) was cultured in a medium containing 90 g of bran, 15 g of rice husk and 100 ml of distilled water for 7 days, and the obtained culture was inoculated into 5-leaf seedlings and treated in a humid chamber at 25°C for 4 days, Onset was induced by cultivation for 4 days in a constant temperature room with a temperature of 25°C.

Tomato pestilence is caused by spray inoculation of the suspension of the sporangia ^{(5×10 4} sporangia/ml) of Phytophthora infestans (Gangneung National University), which is the causative agent, on tomato seedlings of 3-4 leaf stage, and then the temperature is 20. Onset was induced by treatment for 2 days in a humid room at °C and incubation for 2 days in a constant temperature and humidity room at a temperature of 20 °C.

^{Tomato gray mold disease is treated with a spore suspension (5×10 5} spores/ml) of Botrytis cinerea (Korea Research Institute of Chemical Technology), which is a causative agent in tomato seedlings of 3-4 leaf stage, and then the temperature is 20℃. In fact, it was cultured for 3 days to induce onset.

Wheat red rust is Puccinia triticina (Puccinia) , a causative agent in single-leaf seedlings. triticina , Korea Research Institute of Chemical Technology) was spray-treated by suspending the spores in 0.025% (w/v) Tween 20 solution in an amount of 0.67 g spores/ml, and treated in a humid room with a temperature of 20°C for a day, and then the temperature was increased. The onset was induced by incubating for 6 days by transferring to a thermostat at 20°C.

Barley powdery mildew is a causative agent that is subcultured in a host plant, on one leaf seedling of barley. graminis f. sp. hordei (Korea Research Institute of Chemical Technology) spores were shaken and inoculated, and incubated for 7 days in a constant temperature room at a temperature of 20℃ to induce onset.

Pepper anthrax is spray-inoculated with a spore suspension (4 × 10 ⁵ spores/ml) of Colletotrichum coccodes (Korea University), which is the causative agent, on 3 to 4 leaf pepper seedlings, and in a humid room with a temperature of 25°C. After 2 days of wet room treatment, the onset was induced by incubating for 1 day in a constant temperature and humidity room with a temperature of 25°C and a relative humidity of 80%.

Tomato gray mold disease and pepper anthrax 3 days after inoculation, tomato blight 4 days after inoculation, rice blast disease 5 days after inoculation, wheat red rust and barley powdery mildew 7 days after inoculation, rice leaf sheath blight 8 days after inoculation The lesion area ratio (%) was investigated. The control agent (%) was calculated according to Equation 1 below using the lesion area ratio (%) obtained from the above investigation, and the results are shown in Table 1.

[Equation 1]

sample density
(μg/ml) Control (%) RCB RSB TGM TLB WLR BPM PAN Cypress tree extract 3000 90 25 21 0 90 0 40

(RCB: rice blast, RSB: rice sheath blight, TGM: tomato gray mold, TLB: tomato blight, WLR: wheat red rust, BPM: barley powdery mildew, PAN: pepper anthrax)

When the composition for controlling plant diseases containing cypress extract as an active ingredient was treated at a concentration of 3000 μg/ml, it exhibited excellent control of 90% against rice blast disease and wheat red rust (see FIG. 1), and pepper anthrax A 40% control was shown for (see Table 1). Through the above results, it can be seen that the cypress tree extract has a plant disease control effect, from which it can be seen that the cypress tree extract can be usefully used as a composition for controlling plant diseases.

< Example 2> of cypress extract Fractions Contained as an active ingredient Plant disease Control composition Plant disease Control effect evaluation

In order to confirm whether the composition for controlling plant diseases containing the fractions of the extract of cypressaceae according to the present invention as an active ingredient exhibits the effect of controlling plant diseases, the following experiment was performed, and the results are shown in Table 2.

Specifically, the composition for controlling plant diseases prepared in Preparation Example 2 was rice blast disease, which is a plant disease (cause: Magnaporthe oryzae ), rice leaf sheath blight (cause: Rhizoctonia solani ), tomato gray mold (causing bacteria: Botrytis cinerea ), tomato blight (cause: Phytophthora infestans ), barley powdery mildew (cause: Blumeria graminis f. sp. hordei ), wheat red rust (cause: Puccinia triticina ) and pepper anthrax (cause of: Colletotrichum coccodes ), except that the composition for controlling plant diseases prepared in Preparation Example 2 was treated, and the experiment was conducted in the same manner as in Example 1. Performed. The experimental results are shown in Table 2.

sample density
(μg/ml) Control (%) RCB RSB TGM TLB WLR BPM PAN Hexane fraction 2000 88 15 14 0 80 0 30 Ethyl acetate fraction 2000 75 0 21 0 67 0 0 Butanol fraction 2000 0 5 14 0 20 0 0 Water fraction 2000 0 0 0 0 0 0 0

(RCB: rice blast, RSB: rice sheath blight, TGM: tomato gray mold, TLB: tomato blight, WLR: wheat red rust, BPM: barley powdery mildew, PAN: pepper anthrax)

When the composition for controlling plant diseases containing the hexane fraction as an active ingredient is treated at a concentration of 2000 μg/ml, 88% of control against rice blast disease, 80% of wheat red rust and 30 against red pepper anthrax % Control.

When the composition for controlling plant diseases containing the ethyl acetate fraction as an active ingredient was treated at a concentration of 2,000 μg/ml, it showed 75% control against rice blast disease and 67% against wheat red rust (Fig. 1). Reference).

The composition for controlling plant diseases containing a butanol fraction or a water fraction as an active ingredient did not show a significant control effect (see Table 2).

Through the above results, it can be seen that the fraction of the cypress tree extract, particularly the hexane fraction or the ethyl acetate fraction, has a plant disease control effect, and can be usefully used as a composition for controlling plant diseases.

On the other hand, based on the fact that the hexane fraction and ethyl acetate fraction showed a high control effect against rice blast and wheat red rust, Cypress (Platycladus orientalis ) The bactericidal active substance of the extract was not a water-soluble substance, but a non-polar substance, and it was found that the hexane fraction and ethyl acetate fraction can be used for separation and purification of the bactericidal active substance.

< Example 3> Cypress tree extract or its Fraction Plant pathogenicity Inhibiting the growth of mold Check the effect

In order to confirm whether the composition for controlling plant diseases containing the extract or fractions thereof according to the present invention as an active ingredient exhibits an effect of inhibiting the growth of phytopathogenic fungi, an experiment was performed according to the following procedure, and the results are shown in Table 3 Shown in. Specifically, the extract of cypress trees prepared in step 1 of Preparation Example 1 or the hexane fraction, ethyl acetate fraction, butanol fraction, or water fraction prepared in step 1 of Preparation Example 2 are rice blast bacteria Magnaporthe oryzae ) to confirm whether or not there is a growth inhibitory effect, in vitro analysis (assay) was performed.

A minimum inhibitory concentration (MIC) value was calculated by a broth micro-dilution method using a 96-well plate, and the effect of inhibiting the growth of phytopathogenic fungi was evaluated.

After inoculation of 5 hyphae discs of Magnaporthe oryzae , a rice blast bacteria in 100 ml PDB medium (potato dextrose broth medium; 0.4% potato starch, 2% dextrose) in an Erlenmeyer flask, 7 to 10 days 2 ml of the hyphae suspension prepared by grinding the culture solution shaken at 25° C. at 150 rpm with a blade mixer was mixed well with the inoculated 100 ml PDB medium, and each 50 μl was dispensed into wells.

Finally, in 100 μl well, the cypress tree prepared in step 1 of Preparation Example 1 The extract and the hexane fraction, ethyl acetate fraction, and butanol fraction prepared in step 1 of Preparation Example 2 were included in concentrations of 250, 500, and 1,000 μg/ml, respectively, and the methanol content was 5% (v/v). Did not exceed. In the untreated group, only 5% (v/v) of methanol was added. Thereafter, the culture was performed at 25° C. for 2 to 3 days, and the concentration at which the growth of rice blast bacteria was completely inhibited was set as the minimum inhibitory concentration.

sample Minimum inhibitory concentration (μg/ml) Cypress tree extract 250 Hexane fraction of cypress extract 250 Ethyl acetate fraction of cypress extract 250 Butanol fraction of cypress extract 500 Water fraction of cypress extract >1,000

Cypress extract, its hexane fraction and ethyl acetate fraction completely inhibited the growth of rice blast bacteria at a concentration of 250 μg/ml, respectively, and the butanol fraction completely inhibited the growth of rice blast bacteria at a concentration of 500 μg/ml (Table 3). Reference). Through the above results, it can be seen that the cypress extract or its fraction, particularly the hexane fraction or the ethyl acetate fraction, has an effect of inhibiting the growth of phytopathogenic fungi, and can be usefully used as a composition for controlling plant diseases.

< Example 4> Cypress tree extract or its Fraction Plant pathogenicity Inhibition of growth of bacteria Check the effect

In order to confirm whether the composition for controlling plant diseases containing the extract or a fraction thereof according to the present invention as an active ingredient exhibits an effect of inhibiting the growth of phytopathogenic bacteria, an experiment was performed according to the following procedure, and the results are shown in Table 4 Shown in.

Specifically, the cypress extract prepared in step 1 of Preparation Example 1 or the hexane fraction or ethyl acetate fraction prepared in step 1 of Preparation Example 2 is a phalaenopsis bacterial brown spot blotch, Exidoborax abene subsp. Cut tray (Acidovorax avenae subsp. cattleyae ), Agrobacterium tumefaciens (Agrobacterium tumefaciens), a bacterial root lump disease, Burkholderia glumae (Birkholderia glumae), a fungus of red pepper ulcer, Clavibacter miciganensis subsp. Mishiganensis ( Clavibacter michiganensis subsp. michiganensis ), Dickeya chrysanthemi (Dickeya chrysanthemi ), which is a crop soft rot, and Fectobacterium carotoborum subsp. Carotovorum ( Pectobacterium carotovorum subsp. carotovorum ), Kiwi ulcer pathogen Pseudomonas syringe pv. Actinidiae ( Pseudomonas syringae pv. actinidiae ), Pseudomonas syringae pv. Pseudomonas syringae pv. lachrymans ), Ralstonia solanacearum (Ralstonia solanacearum), a green blight of eggplant crops, and Xanthomonas arborichola pv. In order to confirm whether there is a growth inhibitory effect on pruni (Xanthomonas arboricola pv. pruni), an in vitro assay was performed.

A minimum inhibitory concentration (MIC) value was calculated by a broth micro-dilution method using a 96-well plate, and the effect of inhibiting the growth of phytopathogenic bacteria was evaluated.

The 10 phytopathogenic bacteria were inoculated in TSB medium (tryptic soy broth medium: 1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.25% K ₂ HPO ₄ , 0.5% NaCl), and kiwi ulcer pathogen Pseudomonas Syringe pv. Actinidia and Xanthomonas arborcola pv. Fruny was incubated with shaking at 150 rpm for 2 days at 25°C, and the remaining bacteria were incubated with shaking at 150 rpm for 2 days at 30°C. Thereafter, it was diluted with TSB medium so that the bacterial concentration was about 1×10 ⁷ CFU/ml. The diluted bacterial culture solution was inoculated, and finally about 1 × 10 ⁵ CFU/ml.

After dissolving the cypress extract prepared in step 1 of Preparation Example 1 and the hexane fraction and ethyl acetate fraction prepared in Step 1 of Preparation Example 2 in methanol at a concentration of 100 mg/ml, respectively, methanol was diluted by a double dilution method. It was diluted to prepare a stock solution for each concentration. Using a 96-well plate, in 100 ml of TSB medium per well, the cypress extract prepared in step 1 of Preparation Example 1 and the hexane fraction and ethyl acetate fraction prepared in step 1 of Preparation Example 2 were each finally 1.6 , 3.1, 6.3, 12.5, 25, 50, 100, 200, 400 μg / ml of the stock solution was dispensed for each concentration to be included in the concentration, at this time, the content of methanol did not exceed 1%. The experiment was repeated three times for each concentration, and only 1% of methanol was added to the untreated group. Thereafter, culture was performed for 72 hours, and the concentration at which the growth of phytopathogenic bacteria was completely inhibited was set as the minimum inhibitory concentration.

sample Minimum inhibitory concentration (μg/ml) Aa ^a At Bg Cm Dc PC Psa Psl Rs Xa Cypress tree extract 400 - - - 400 - 400 - - - Hexane fraction 400 - - - 400 - 400 - - - Ethyl acetate fraction 400 - - - 400 - 400 - - -

( ^a Aa, Exidovorax Abene subsp.Cattray( Acidovorax avenae subsp. cattleyae ); At, Agrobacterium tumefaciens ( Agrobacterium tumefaciens ); Bg, Burkholderia Glume glumae ); Cm, Clavibacter michiganensis subsp. michiganensis ); Dc, Dickeya chrysanthemi ; Pc, Fectobacterium carotoborum subsp. Carotovorum ( Pectobacterium carotovorum subsp. carotovorum ); Psa, Pseudomonas Syringe pv. Actinidia ( Pseudomonas syringae pv. actinidiae ); Psl, Pseudomonas Syringe pv. Pseudomonas syringae pv. lachrymans ); Rs, Ralstonia solanacearum ; Xa, Xantomonas Arboricola pv. Pruni ( Xanthomonas arboricola pv. pruni ).)

(If the value is not indicated in the table above, the minimum inhibitory concentration is> 400 μg/ml)

The cypress extract prepared in step 1 of Preparation Example 1, and the hexane fraction and ethyl acetate fraction prepared in Step 1 of Preparation Example 2 were respectively 400 μg/ml of Phalaenopsis phalaenopsis brown spot blotch Excidoborax Abene subsp. Cut tray and Dickeya chrysanthemai, a crop soft rot, and Pseudomonas syringe, a kiwi ulcer disease pv. The growth of actinidie was completely inhibited (see Table 4). Through the above results, it can be seen that the cypress extract or its fraction, particularly the hexane fraction or the ethyl acetate fraction, has an effect of inhibiting the growth of phytopathogenic bacteria, and thus can be usefully used as a composition for controlling plant diseases.

< Example 5> Separation of bactericidal active substance from extract of cypress tree

Separation was performed using various chromatography techniques to separate the bactericidal active substance from the Platycladus orientalis extract, and specifically, the following procedure was followed.

In the process as shown in FIG. 1, Compounds 1 to 9 were isolated from 100 g of a cypress extract prepared in step 1 of Preparation Example 1. First, 100 g of cypress extract was suspended in 5 L of distilled water, extracted twice with 5 L of hexane, and extracted twice with ethyl acetate, and the hexane layer and ethyl acetate layer were combined and concentrated under reduced pressure. 46 g of a mixed fraction of dried hexane and ethyl acetate was prepared. After adsorbing 46 g of the mixed fraction of hexane and ethyl acetate on a silica gel (Kiesel gel 60, 70-230 mesh, 500 g; Merck) column, an aqueous methanol solution of chloroform and methanol in a volume ratio of 95:5 to 0:100 It was sequentially eluted and divided into 12 fractions (E1 to E12).

To investigate the chemical characteristics of the fractions, a thin layer chromatography (TLC) technique was used, and after the sample was adsorbed on a silica gel stationary phase, methanol: ethyl acetate (95:5, v/v), methylene chloride: ethyl acetate (95: 5 v / v) or hexane: ethyl acetate (8: 2 v / v) and developed with, p - by coloring with anise aldehyde reagent (p -anisaldehyde reagent) was performed by TLC analysis pattern. Among them, 6.4 g of the E2 fraction was added to a column filled with silica gel, and then a solution obtained by mixing hexane and dichloromethane in a volume ratio of 8:2 to 0:10 was sequentially eluted and divided into 9 fractions (E21 to E29). .

E26 fraction (730 mg) was added to a C18 reverse phase column (Phenomenex Capcell Pak C18 UG, 250 mm × 20 mm, 5 μm) connected to a high-pressure liquid chromatography (HPLC) system, and 85 as a concentration gradient condition. To 100% methanol aqueous solution was eluted at a flow rate of 5 ml per minute, to separate 44.7 mg of compound 3 and 95.1 mg of compound 4.

432 mg of the E27 fraction was dissolved in 1 ml of petroleum ether, and crystallized at -4°C for 4 hours to purify pure compound 9.

2.9 g of the E3 fraction was added to a normal phase column (Biotage SNAP KP-Sil) connected to an MPLC (Medium pressure liquid chromatography) system, and hexane and dichloromethane were added as a concentration gradient of 9:1 to 0:10. The solution mixed in a volume ratio was eluted at a flow rate of 5 ml per minute, and 487.8 mg of compound 5 and 206.8 mg of compound 6 were separated.

3.2 g of the E5 fraction was added to a normal column (Biotage SNAP KP-Sil) connected to the MPLC system, and a solution obtained by mixing dichloromethane and ethyl acetate in a volume ratio of 10:0 to 9:1 under a concentration gradient condition was added to 5 ml per minute. Elute it at the flow rate. Divided into 5 fractions (E51 to E55), 815.4 mg of compound 1 was isolated from the E52 fraction.

393.3 mg of the E54 fraction was added to a normal column (Biotage SNAP KP-Sil) connected to the MPLC system, and a solution obtained by mixing hexane and ethyl acetate in a volume ratio of 93:7 to 8:2 under a concentration gradient condition at a flow rate of 3 ml per minute. By elution, 43.3 mg of compound 7 and 40.2 mg of compound 8 were separated.

2.4 g of the E7 fraction was added to the Biotage ZIP Sphere connected to the MPLC system, and a solution obtained by mixing hexane and dichloromethane in a volume ratio of 97:3 to 88:12 under a concentration gradient condition was eluted at a flow rate of 5 ml per minute. Then, 449.5 mg of compound 2 was isolated.

< Example 6> Investigation of the chemical structure of the bactericidal active substance isolated from the extract of Japanese cypress

In order to determine the chemical structure of the bactericidal active material isolated in Example 5, an experiment was performed according to the following procedure, and the results are summarized in Tables 5 to 13.

The sample was dissolved in chloroform-d (CDCl ₃ ^{, 99.96 atom% D, Cambridge Isotope Laboratories) to obtain a 13} C-NMR spectrum in a 500 MHz NMR device (nuclear magnetic resonance, Bruker AMX-500). ^The chemical shift value was expressed as δ ( ppm) based on the 77.0 ppm chloroform solvent peak in the 13 C NMR spectrum. The mass of the separated compound was measured using GC-MS (Gas chromatography-mass spectrometry). ^{The results of the above experiment were compared with the MS and 13} C-NMR data shown in the literature (Chernov et al. 2006), and the chemical structures of the compounds isolated from the extract of cypress trees were identified.

<Example 6-1> Chemical structure of compound 1

As a result of the analysis, Compound 1 was detected as a molecular ion of ^{m/z 346 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 5 below.

¹³ C NMR data were compared with literature (Chernov et al. 2006), and the isolated compound 1 was identified as pinusolide, a labdane diterpnoid compound represented by the following formula (1).

[Formula 1]

location Chemical shift ( δ _c in ppm) ^a
Compound 1 Pinusolide
(Chernov et al. 2006) One 39.2 39.0 2 19.9 19.7 3 38.2 38.0 4 44.3 44.0 5 56.3 56.1 6 26.2 26.0 7 38.7 38.5 8 147.4 147.0 9 55.6 55.5 10 40.3 40.1 11 21.8 21.7 12 24.6 24.5 13 134.8 134.6 14 143.9 143.2 15 70.1 69.5 16 174.4 173.1 17 106.7 106.7 18 177.7 176.6 19 28.8 28.6 20 12.6 12.4 OCH ₃ 51.2 50.7

( ^a Recorded in CDCl ₃ at 125 MHz)

<Example 6-2> Chemical structure of compound 2

As a result of the analysis, Compound 2 was detected as a molecular ion of ^{m/z 362 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 6 below.

¹³ C NMR data were compared with literature (Chien et al. 2004), and the isolated compound 2 was converted to 15-methoxypinusolidic acid, a labdane diterpnoid compound represented by the following formula (2). ).

[Formula 2]

location Chemical shift ( δ _c in ppm)
Compound 2 15-Methoxypinusolidic acid
(Chien et al. 2004) One 39.1 39.0 2 19.8 19.7 3 37.8 37.8 4 44.2 44.0 5 56.2 56.3 6 26.0 25.9 7 38.6 38.4 8 147.3 147.4 9 55.6 55.6 10 40.5 40.4 11 21.7 21.7 12 24.5 24.5 13 139.1 139.1 14 141.6 141.7 15 102.5 102.0 16 171.4 171.0 17 106.8 106.7 18 184.0 183.9 19 29.0 28.8 20 12.7 12.8 OCH ₃ 57.0 57.9

( ^a Recorded in CDCl ₃ at 125 MHz)

<Example 6-3> Chemical structure of compound 3

As a result of the analysis, the compound of Example 3 was detected as a molecular ion of ^{m/z 316 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 7 below.

¹³ C NMR data were compared with literature (Asili et al. 2004), and the isolated compound 3 was identified as lambertianic acid, a labdane diterpenoid compound represented by the following formula (3).

[Formula 3]

location Chemical shift ( δ _c in ppm) ^a
Compound 3 Lambertianic acid
(Asili et al. 2004) One 39.2 39.1 2 20.1 19.9 3 38.2 38.0 4 44.3 44.1 5 56.4 56.2 6 26.2 26.1 7 38.8 38.7 8 148.0 147.8 9 55.4 55.3 10 40.5 40.4 11 23.7 23.6 12 24.4 24.3 13 125.6 125.5 14 111.1 111.0 15 142.8 142.7 16 138.9 138.7 17 106.6 106.5 18 182.2 182.6 19 13.0 12.8 20 29.2 29.0

( ^a Recorded in CDCl ₃ at 125 MHz)

<Example 6-4> Chemical structure of compound 4

As a result of the analysis, Compound 4 was detected as a molecular ion of ^{m/z 302 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 8 below.

¹³ C NMR data the literature (Smith et al 2007.) And to, to the separated compound of formula 4 4 The stage Rab data page cannabinoid (labdane diterpnoid) compound is trans comparison - keomyun acid (trans -communic acid) identified as I did.

[Formula 4]

location Chemical shift (δ _c in ppm) ^a
Compound 4 trans-Communic acid
(Smith et al. 2007) One 39.5 39.4 2 20.2 20.1 3 38.3 38.3 4 44.3 44.4 5 56.3 56.4 6 26.1 26.1 7 38.7 38.6 8 148.3 148.0 9 56.5 56.5 10 40.5 40.2 11 23.4 23.3 12 134.2 133.9 13 133.5 133.4 14 141.7 141.6 15 110.0 109.9 16 11.9 11.1 17 107.6 107.6 18 182.3 177.7 19 29.3 28.9 20 13.0 12.7

( ^a Recorded in CDCl ₃ at 125 MHz)

<Example 6-5> Chemical structure of compound 5

As a result of the analysis, Compound 5 was detected as a molecular ion of ^{m/z 288 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 9 below.

As shown in Table 9 below,

^{In the 13} C NMR spectrum, the 20 carbon signals constituting the diterpene were shown. 1 quarter sp ² carbon ( δ _C 136.6 ppm), 2 methine sp ² carbons ( δ _C 149.0, 128.8 ppm), 1 methylene sp ² carbon ( δ _C 110.0 ppm), 3 ^{quarters in 13 C NMR spectrum} Nuri sp ³ carbons ( δ _C 39.0, 38.6, 37.8 ppm), 3 methine sp ³ carbons ( δ _C 79.6, 54.2, 50.6 ppm), 6 methylene sp ³ carbons ( δ _C 37.9, 36.1, 35.0, 28.4, 22.7 , 18.8 ppm), and 4 methyl carbons ( δ _C 28.5, 26.0, 16.8, 15.3 ppm) signals. Comparing the ¹³ C NMR data with literature (Wong et al. 2010), Compound 5 was converted to 15-sandarakopimaradiene-3 β -ol, which is a pimarane diterpenoid compound of Formula 5 below ( 15-sandaracopimaradien-3 β -ol).

[Formula 5]

location Chemical shift (δ _c in ppm) ^a
Compound 5 8(14),15-Sandaracopimaradien-3 ß -ol
(Wong et al. 2010) One 36.1 36.3 2 28.4 28.1 3 79.6 79.6 4 39.0 39.4 5 54.2 54.6 6 18.8 19.2 7 35.0 34.9 8 136.6 137.0 9 50.6 50.8 10 37.8 37.8 11 22.7 22.6 12 37.9 37.9 13 38.6 38.5 14 128.8 129.3 15 149.0 149.4 16 110.0 110.5 17 26.0 26.4 18 28.5 28.9 19 16.8 16.1 20 15.3 15.4

( ^a Recorded in CDCl ₃ at 125 MHz)

<Example 6-6> Chemical structure of compound 6

As a result of the analysis, Compound 6 was detected as a molecular ion of ^{m/z 288 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 10 below.

^{Comparing the 13} C NMR data with literature (Block et al. 2004), Compound 6 is a pimarane diterpenoid compound of Formula 6, 7,15-isopimaradiene-3 β -ol It was identified as (7,15-isopimaradien-3 β -ol).

[Formula 6]

location Chemical shift (δ _c in ppm) ^a
Compound 6 7,15-Isopimaradien-3ß-ol
(Block et al. 2004) One 38.6 38.6 2 27.4 27.4 3 79.2 79.3 4 37.9 37.8 5 50.0 50.0 6 23.1 23.1 7 121.4 121.4 8 135.4 135.4 9 51.9 51.9 10 37.3 37.3 11 20.1 20.1 12 36.1 36.1 13 35.3 35.3 14 46.0 45.9 15 150.3 150.3 16 109.3 109.2 17 21.5 21.4 18 28.4 28.3 19 15.7 15.6 20 14.9 14.9

( ^a Recorded in CDCl ₃ at 125 MHz)

<Example 6-7> Chemical structure of compound 7

As a result of the analysis, Compound 7 was detected as a molecular ion of ^{m/z 306 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 11 below.

^{Comparing the 13} C NMR data with literature (Kim et al. 2014), Compound 7 is a pimarane diterpenoid compound of the following formula 7 8 ß ,18-dihydroxysandarakopimar-15- It was identified as yen (8 ß ,18-dihydroxysandaracopimar-15-ene).

[Formula 7]

location Chemical shift (δ _c in ppm) ^a
Compound 7 8ß,18-Dihydroxysandaracopimar-15-ene
(Kim et al. 2014) One 39.0 39.2 2 18.0 18.0 3 35.3 35.6 4 37.0 37.3 5 49.4 49.7 6 17.8 17.8 7 43.1 43.4 8 72.6 72.8 9 56.9 57.1 10 37.6 37.9 11 17.0 17.3 12 38.1 38.3 13 36.5 36.7 14 51.5 51.8 15 151.6 151.8 16 108.6 108.8 17 24.2 24.5 18 72.0 72.3 19 17.6 17.7 20 16.1 16.3

( ^a Recorded in CDCl ₃ at 125 MHz)

<Example 6-8> Chemical structure of compound 8

As a result of the analysis, Compound 8 was detected as a molecular ion of ^{m/z 306 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 12 below.

¹³ C NMR data were compared with literature (Asili et al. 2004), and Compound 8 was converted to 15-isopimarene-3 ß ,8 ß -diol, a pimarane diterpenoid compound of Formula 8 below. (15-isopimaren-3 ß ,8 ß- diol).

[Formula 8]

location Chemical shift (δ _c in ppm) ^a
Compound 8 15-Isopimaren-3ß,8ß-diol
(Asili et al. 2004) One 37.7 37.8 2 27.1 27.2 3 79.0 79.0 4 38.9 38.9 5 55.5 55.5 6 17.6 17.6 7 43.5 43.6 8 72.4 72.4 9 56.8 56.8 10 37.0 37.0 11 17.1 17.2 12 38.0 38.0 13 36.5 36.6 14 51.4 51.5 15 151.5 151.5 16 108.6 108.6 17 24.2 24.3 18 28.2 28.2 19 15.5 15.5 20 15.7 15.7

( ^a Recorded in CDCl ₃ at 125 MHz)

<Example 6-9> Chemical structure of compound 9

As a result of the analysis, Compound 9 was detected as a molecular ion of ^{m/z 222 [M + ] in electron impact mass spectroscopy (EIMS), and the 13} C NMR analysis results are shown in Table 13 below.

^By comparing the 13 C NMR data with the literature (Joseph-Nathan et al. 1984), compound 9 was identified as sedrol (α-cedrol), a sesquiterpene alcohole compound of Formula 9 below.

[Formula 9]

location Chemical shift (δ _c in ppm) ^a
Compound 9 α-Cedrol
(Joseph-Nathan et al. 1984) One 25.4 25.4 2 37.0 37.0 3 41.5 41.5 3a 54.1 54.1 4 31.6 31.6 5 35.4 35.3 6 75.2 75.0 7 61.0 61.0 8 43.4 43.4 8a 56.5 56.6 9 42.0 42.0 10 15.6 15.6 11 30.2 30.2 12 27.6 27.7 13 28.9 28.9

( ^a Recorded in CDCl ₃ at 125 MHz)

< Example 7> of cypress extract Hexane And Ethyl acetate Fraction Analysis of the content of compounds 1 to 9 in

In order to analyze the content (%) of compounds 1 to 9 contained in the mixture fraction of hexane and ethyl acetate of the cypress extract, an experiment was performed according to the following procedure, and the results are shown in Table 14.

After dispensing 100 μg of each of the fractions and compounds 1 to 9 into a vial, 30 μl of a 20 mg/ml methoxyamine hydrochloride pyridine solution was added, and 1% tri Add 50 μl of N,O -bis(triethylsilyl)trifluoroacetamide ( N , O- bis(trimethylsilyl)trifluoroacetamide) solution containing ethylchlorosilane at a time, and 2-chloronaphthalene (2) at a concentration of 250 μg/ml -chloronaphthalene) pyridine solution was added each 10 μl as an internal standard, and reacted at 60° C. for 1 hour to prepare a sample of a derivative compound.

The sample was injected into a DB-5 column (30 m × 0.25 mm, film thickness 0.25 μm, Agilent) connected to a gas chromatography mass-spectroscopy (GC-MS) QP5050, Shimadzu) device. Specifically, the GC inlet temperature was 250°C, and the sample at a concentration of 1 mg/ml was dissolved in chloroform, and 1 μl was injected in a split mode of 1:10. Helium gas was flowed through the mobile phase at a flow rate of 1 ml per minute, the voltage of the detector was 1,588 V, the mass range was 50 to 500 Da, and data were collected in full scan mode. The column oven temperature was increased from 80°C to 5°C per minute to 260°C, and then 3°C per minute to 300°C, and maintained at 300°C for 3 minutes. The peak detected by GC-MS was compared with the mass spectrum of the known material with a retension time (RT) value.

compound content(%) RT (minutes) Pinusolide (compound 1) 8.9 27.3 15-Methoxypinusolidic acid (compound 2) 3.3 28.8 Lambertianic acid (compound 3) 1.5 16.5 trans -Communic acid (Compound 4) 1.2 22.7 8(14),15-Sandaracopimaradien-3 ß -ol (Compound 5) 1.1 22.1 7,15-Isopimaradien-3 ß -ol (Compound 6) 0.5 22.4 8 ß ,18-Dihydroxysandaracopimar-15-ene (Compound 7) 1.4 23.0 15-Isopimaren-3 β ,8 β -diol (Compound 8) 2.4 23.5 α- Cedrol (Compound 9) 5.1 17.0

The analysis results shown in Table 14 were obtained.

The results show that the hexane and ethylacetate fractions of the cypress extract include pinusolide and 15-methoxypinusolidic acid and sesquiterpene alcohol compound α -sedrol as the main components of the Ravdan diterpenoid compound. , Indicates that it contains a large amount of other diterpenoid compounds.

< Example 8> Plant pathogenicity of compounds 1 to 9 Inhibiting the growth of mold Effectiveness evaluation

The labdane diterpenoid compound of Formulas 1 to 4, the pimarane diterpenoid compound of Formulas 5 to 8, and the sesquiterpene alcohol of compound 9 isolated in Examples 5 and 6 In order to confirm whether the (sesquiterpene alcohole) compound exhibits a growth inhibitory effect on phytopathogenic fungi, an experiment was performed according to the following procedure, and the results are shown in Table 15.

Specifically, the compounds are baechugwa crops black pattern germs of Alterna Ria beurasi when coke (Atlternaria brassicicola), crops, gray mold pathogen in beam tri tooth cine LEA (Botrytis cinerea), pepper anthracnose fungus of Collet sat tree glutamicum coco des (Colletotrichum coccodes ), Fusarium oxisporum , a tomato wilt germ f. sp. Growth inhibitory effect on Fusarium oxysporum f. sp. lycopersici , Magnaporthe oryzae , a rice blast bacteria, and Phytophthora infestans , a potato blight and tomato blight. To confirm whether or not, in vitro analysis (assay) was performed.

The effect of inhibiting the growth of phytopathogenic fungi was evaluated by obtaining a minimum inhibitory concentration (MIC) value by a broth micro-dilution method using a 96-well plate.

All strains were spores harvested using PDB medium (potato dextrose broth medium; 0.4% potato starch, 2% dextrose), and a spore suspension was prepared by filtering the harvested spores with 4-layer gauze.

Finally, the spores of phytopathogenic fungi were included in 100 μl wells at ^{a concentration of 1 × 10 4} spores/ml, and the isolated terpenoid compounds (Formulas 1 to 9) were 0.8, 1.6, 3.1, 6.3, 12.5, 25, respectively. , 50, 200 μg / ml was dispensed to be included in the concentration, at this time, the content of methanol did not exceed 1%. The experiment was repeated three times for each concentration, and only 1% of methanol was added to the untreated group. The concentration at which the growth of phytopathogenic fungi is completely inhibited was set as the minimum inhibitory concentration.

compound Minimum inhibitory concentration (μg/ml) Ab ^a Bc Cc Fo Mo Pi Pinusolide (compound 1) - - - - 100 - 15-Methoxypinusolidic acid (compound 2) - - - - 200 100 Lambertianic acid (compound 3) - - - - - - trans -Communic acid (Compound 4) - - - - - - 8(14),15-Sandaracopimaradien-3 ß -ol (Compound 5) - - - - - - 7,15-Isopimaradien-3 ß -ol (Compound 6) - - - - - - 8 ß ,18-Dihydroxysandaracopimar-15-ene (Compound 7) - - - - 100 100 15-isopimaren-3 β ,8 β -diol (Compound 8) - - - - - - α- cedrol (compound 9) - - - - 200 100

( ^a Ab, Alternaria Brassicola brassicicola ); Bc, Botrytis cinerea ; Cc, Colletotrichum coccodes ; Fo, Fusarium Oxysporum f. sp. Fusarium oxysporum f. sp. lycopersici ); Mo, Magnaporthe oryzae ); Pi, Phytophthora infestans ).)

(If the value is not listed in the table above, the minimum inhibitory concentration is> 200 μg/ml)

As shown in Table 15 above,

Pinusolide (Chemical Formula 1), 15-methoxypinusolidic acid (Chemical Formula 2), 8 ß , 18-dihydroxysandarakopimar-15-ene (Chemical Formula 7) and α -sedrol (Chemical Formula 9) are 100 To 200 μg/ml concentration of rice blast bacteria Magnaporthe oryzae completely inhibited the growth, 15-methoxypinusolidic acid (Formula 2), 8 ß , 18-dihydroxysandarakopimar- 15-yen (Chemical Formula 7), α -Sedrol (Chemical Formula 9) at a concentration of 100 μg/ml, Phytophthora infestans, a potato blight and tomato blight infestans ) completely inhibited the growth.

< Example 9> separated Terpenoid Phytopathogenicity of the compound Inhibition of growth of bacteria Effectiveness evaluation

The labdane diterpenoid compound of Formulas 1 to 4, the pimarane diterpenoid compound of Formulas 5 to 8, and the sesquiterpene alcohol of compound 9 isolated in Examples 5 and 6 In order to confirm whether the (sesquiterpene alcohole) compound exhibits a growth inhibitory effect on phytopathogenic bacteria, an experiment was performed according to the following procedure, and the results are shown in Table 16.

Specifically, the compounds are phalaenopsis phalaenopsis brown spot pattern bacteria Exidovorax abene subsp. Cut tray (Acidovorax avenae subsp. cattleyae ), Agrobacterium tumefaciens (Agrobacterium tumefaciens), a bacterial root lump disease, Burkholderia glumae (Birkholderia glumae), a fungus of red pepper ulcer, Clavibacter miciganensis subsp. The system micro-norbornene (Clavibacter michiganensis subsp. Michiganensis), plant soft rot fungus Dick keya My Creations chrysanthemate (Dickeya chrysanthemi ) and Fectobacterium carotovorum subsp. Carotovorum ( Pectobacterium carotovorum subsp. carotovorum ), Kiwi ulcer pathogen Pseudomonas syringe pv. Actinidiae ( Pseudomonas syringae pv. actinidiae ), Pseudomonas syringae pv. Lachrymans ( Pseudomonas syringae pv.lachrymans), Ralstonia solanacearum (Ralstonia solanacearum), an eggplant green blight, and Xanthomonas arborichola pv. In order to confirm whether there is a growth inhibitory effect on pruni (Xanthomonas arboricola pv. pruni), an in vitro assay was performed.

The effect of inhibiting the growth of phytopathogenic bacteria was evaluated by obtaining a minimum inhibitory concentration (MIC) value by a broth micro-dilution method using a 96-well plate.

The 10 phytopathogenic bacteria were inoculated in TSB medium (tryptic soy broth medium: 1.7% tryptone, 0.3% soytone, 0.25% dextrose, 0.25% K ₂ HPO ₄ , 0.5% NaCl), and kiwi ulcer pathogen Pseudomonas Syringe pv. Actinidia and Xanthomonas arborcola pv. Fruny was cultured with shaking at 150 rpm for 2 days at 25°C, and the remaining bacteria were cultured with shaking at 150 rpm for 2 days at 30°C. Thereafter, it was diluted with TSB medium so that the bacterial concentration was about 1 × 10 ⁷ CFU/ml. The diluted bacterial culture solution was inoculated, and finally about 1 × 10 ⁵ CFU/ml.

The separated compounds were each dissolved in methanol at a concentration of 100 mg/ml, and then diluted in methanol by a double dilution method to prepare a stock solution for each concentration. Using a 96-well plate, each isolated compound is finally stocked at concentrations of 0.8, 1.6, 3.1, 6.3, 12.5, 25, 50, 100, 200 μg/ml in 100 ml of TSB medium per well. The solution was dispensed, and at this time, the content of methanol did not exceed 1%. The experiment was repeated three times for each concentration, and only 1% of methanol was added to the untreated group. Thereafter, culture was performed for 72 hours, and the concentration at which the growth of phytopathogenic bacteria was completely inhibited was set as the minimum inhibitory concentration.

compound Minimum inhibitory concentration (μg/ml) Aa ^a At Bg Cm Dc PC Psa Psl Rs Xa Pinusolide (compound 1) - - - - 100 - 200 - - - 15-Methoxypinusolidic acid (compound 2) - - - - 100 - - - - - Lambertianic acid (compound 3) - - - - 200 - - - - - trans -Communic acid (Compound 4) 200 - - - 200 - - - - - 8(14),15-Sandaracopimaradien-3 ß -ol (Compound 5) 200 - - - 200 - - - - - 7,15-Isopimaradien-3 ß -ol (Compound 6) - - - - 200 - - - - - 8 ß ,18-Dihydroxysandaracopimar-15-ene (Compound 7) - - - - 200 200 - - - - 15-isopimaren-3 β ,8 β -diol (Compound 8) - - - - 200 - - - - - α- cedrol (compound 9) - - - - 200 - - - - -

( ^a Aa, Exidovorax Abene subsp.Cattray( Acidovorax avenae subsp. cattleyae ); At, Agrobacterium tumefaciens ( Agrobacterium tumefaciens ); Bg, Burkholderia Glume glumae ); Cm, Clavibacter michiganensis subsp. michiganensis ); Dc, Dickeya chrysanthemi ; Pc, Fectobacterium carotoborum subsp. Carotovorum ( Pectobacterium carotovorum subsp. carotovorum ); Psa, Pseudomonas Syringe pv. Actinidia ( Pseudomonas syringae pv. actinidiae ); Psl, Pseudomonas Syringe pv. Pseudomonas syringae pv. lachrymans ); Rs, Ralstonia solanacearum ; Xa, Xantomonas Arboricola pv. Pruni ( Xanthomonas arboricola pv. pruni ).)

(If the value is not listed in the table above, the minimum inhibitory concentration is> 200 μg/ml)

As shown in Table 16 above,

Pinusolide (Formula 1) and Methoxypinusolide (Formula 2) at a concentration of 100 μg/ml, Dickeya chrysanthemai (Dickeya chrysanthemi ) completely inhibited the growth, and compounds of Formulas 3 to 8 and Compound 9 completely inhibited the growth of Dickeya chrysanthemai, a crop soft rot, at a concentration of 200 μg/ml. Pinusolide (Chemical Formula 1) is a kiwi ulcer pathogen Pseudomonas syringe pv. Actinidia ( Pseudomonas syringae pv. actinidiae ) completely inhibited the growth, and 8 ß ,18-dihydroxysandarakopimar-15-ene (Chemical Formula 7) was a crop soft rot at a concentration of 200 μg/ml, Pectobacterium carotoborum pv. Carotoborum ( Pectobacterium carotovorum subsp. carotovorum) completely inhibited the growth. trans -Communic acid (Chemical Formula 4) and 8(14),15-Sandarakopimaradiene-3 β -ol (Chemical Formula 5) at a concentration of 200 μg/ml, Phalaenopsis Bacterial Brown Spotted Bacteria Excidovorax Abene subsp . Cut tray (Acidovorax avenae subsp. cattleyae) was completely inhibited.

< Example 10> separated Terpenoid Compound Plant disease Control effect evaluation

In order to confirm whether the terpenoid compound isolated from the extracts of the cypress tree prepared in Examples 5 and 6 exhibited a plant disease control effect, an experiment was performed according to the following procedure, and the results are shown in Table 17.

Specifically, the above compounds, rice blast disease (cause: Magnaporthe oryzae ) or wheat red rust (Cause: Puccinia triticina ) to determine whether there is a control effect, the following experiment was performed in greenhouse conditions.

After dissolving the compounds 1 to 2, compounds 4 to 7 and 9 in methanol, respectively, 0.025% (w/v) of Tween 20 (surfactant) solution was added to obtain a final concentration of 500, 1000, and 2000 μg/ml Was made to be. Compound 4, trans -communic acid, was dissolved in methanol in the same manner as above, except that the final concentrations were 111, 333, and 1,000 μg/ml, and Tween 20 solution was added. The final methanol concentration of all samples was 5% (v/v). At this time, the untreated group was treated with a solution containing 5% (v/v) methanol and 0.025% (w/v) Tween 20. Four pots were used for each plant disease, and the sample was spray-sprayed on the leaf surface of the cultivated plant and air-dried for 24 hours, and then the causative bacteria of the plant diseases were respectively inoculated. The control activity against these rice blast and wheat red rust was investigated according to the method described in Example 1, and the results are shown in Table 17.

compound Concentration (μg/ml) Bottle control (%) Rice blast Wheat red rust Pinusolide (compound 1) 500 75 72 1000 94 80 2000 96 80 15-Methoxypinusolidic acid (compound 2) 500 85 60 1000 91 72 2000 98 92 trans -Communic acid (Compound 4) 111 0 0 333 0 0 1000 19 43 8(14),15-Sandaracopimaradien-3 ß -ol (Compound 5) 500 0 ^-a 1000 0 - 2000 38 - 7,15-Isopimaradien-3 ß -ol (Compound 6) 500 0 - 1000 25 - 2000 56 - 8 ß ,18-Dihydroxysandaracopimar-15-ene (Compound 7) 500 94 - 1000 96 - 2000 96 - α- Cedrol (Compound 9) 500 0 0 1000 0 0 2000 0 0

("-": No data)

As shown in Table 17 above,

Pinusolide (Chemical Formula 1) and 15-methoxypinusolidic acid (Chemical Formula 2) are controlled at a concentration of 500 μg/ml in 75% to 94% against rice blasts and 60% to 72% against wheat red rust. Was shown, and treatment with a higher concentration increased the control effect. 8 ß ,18-dihydroxysandarakopimar-15-ene (Chemical Formula 7) showed 94% control against rice blasts at a concentration of 500 μg/ml, and trans -communic acid (Chemical Formula 4), 8 (14),15-Sandarakopimaradiene-3 ß -ol (Chemical Formula 5) and 7,15-Isopimaradiene-3 ß -ol (Chemical Formula 6) are rice at a concentration of 1000 to 2000 μg/ml It showed 19% to 56% of control against blast disease, and trans -communic acid (Chemical Formula 4) showed 43% control against wheat red rust at a concentration of 1000 μg/ml.

α -Sedrol (Chemical Formula 9) showed the effect of completely inhibiting the growth of Magnaforte Olyse, a rice blast bacteria in Example 8 (in vitro analysis), at a concentration of 200 μg/ml, whereas in Example 10 ( in In vivo analysis) did not show a plant disease control effect even at a concentration of 2,000 μg/ml. Meanwhile, when the above compounds were treated, rice plants ( Oryza sativa L.) or wheat plants ( Triticum aestivum L.) did not show any weakness.

From the above results, the plant disease control effect exhibited by the cypress extract or its fractions is the pinusolide (Chemical Formula 1), 15-methoxypinusolidic acid (Chemical Formula 2), trans -commune. Labdane diterpenoid compound of acid (Chemical Formula 4) or 8(14),15-Sandarakopimaradiene-3 ß -ol (Chemical Formula 5), 7,15-isopimaradiene- It was confirmed that it was caused by a pimarane diterpenoid compound of 3 ß -ol (Chemical Formula 6), 8 ß , 18-dihydroxysandarakopimar-15-ene (Chemical Formula 7).

In addition, based on the results of Examples 1 to 10,

Cypress extract, its organic solvent fraction, isolated labdane diterpenoid compounds of Formulas 1 to 4 or pimarane diterpenoid compounds of Formulas 5 to 8 are non-toxic natural derived from plant extracts. It was confirmed that it could be developed as a fungicide, and the plant disease control activity of the separated labdane diterpenoid compounds of Formulas 1 to 4 and pimarane diterpenoid compounds of Formulas 5 to 8 Was similar to the control spectrum that appeared when treated with the extract of Cyprus chinensis, and showed a higher level of control activity.

Claims (10)

Cypress ( Platycladus) Orientalis ) A composition for controlling plant diseases containing as an active ingredient at least one selected from the group consisting of extracts and fractions thereof.
The method of claim 1,
The extract is a composition for controlling plant diseases, characterized in that the extract is extracted using any one selected from the group consisting of water, C ₁ to C _{4 lower alcohol, hexane and ethyl acetate, or a mixed solution thereof as a solvent.}
The method of claim 1,
The composition for controlling plant diseases, characterized in that the fraction is fractionated using any one selected from the group consisting of hexane and ethyl acetate as a solvent.
The method of claim 1,
The plant disease is a plant disease control composition, characterized in that at least one plant disease selected from the group consisting of rice blast disease, wheat red rust, pepper anthrax, phalaenopsis bacterial brown spot disease, crop soft rot and kiwi ulcer disease.
The method of claim 1,
The composition for controlling plant diseases is Magnaporthe oryzae ), Puccinia triticina triticina ) and Colletotrichum coccodes (Colletotrichum coccodes) A composition for controlling plant diseases, characterized in that it inhibits the growth of one or more phytopathogenic fungi selected from the group consisting of.
The method of claim 1,
The composition for controlling plant diseases is Exidovorax Abene subsp. Cattraye ( Acidovorax avenae subsp. cattleyae ), Dickeya chrysanthemi and Pseudomonas syringe pv. Actinidiee ( Pseudomonas syringae pv. actinidiae ) a composition for controlling plant diseases, characterized in that it inhibits the growth of one or more phytopathogenic bacteria selected from the group consisting of.
The method of claim 1,
The active ingredient is a plant disease control composition, characterized in that contained in a concentration of 250 to 3000 μg / ml in the composition for controlling plant diseases.
The cypress tree of claim 1 (Platycladus orientalis ) Plant disease control method comprising the step of treating a plant, a seed thereof, or a habitat thereof, a composition for controlling plant diseases containing one or more selected from the group consisting of extracts and fractions thereof as an active ingredient.
The method of claim 8,
The plant is a plant disease control method, characterized in that at least one plant selected from the group consisting of rice, wheat, pepper, phalaenopsis, radish, cabbage, tomato, sweet potato, potato, onion, carrot and kiwi.
The method of claim 8,
The plant disease is one or more plant diseases selected from the group consisting of rice blast disease, wheat red rust, pepper anthrax, phalaenopsis bacterial brown spot disease, crop soft rot and kiwi ulcer disease.

Images