KR101837622B1 - Composition for controlling plant diseases comprising an extract of Maesa japonica, and method for controlling plant diseases using the same - Google Patents

Abstract

The present invention provides a composition for controlling a plant disease comprising a Maesa japonica extract or a fraction thereof as an active ingredient and a method for controlling a plant disease using the same, wherein the composition for controlling a plant disease of the present invention comprises Since it shows harmless and harmless to the human body and exhibits activity against plant fungal diseases such as rice blast, rice sheath blight, wheat red rust, tomato blight or tomato gray fungus disease without inducing environmental pollution, development of environmentally friendly natural material fungicide And can be usefully used for producing high value added organic agricultural products.

Description

[0001] The present invention relates to a composition for controlling a plant disease, which contains an extract of Bile tree, as an active ingredient, and a method for controlling plant diseases using the same,

The present invention relates to an environmentally friendly plant disease controlling composition comprising an extract of Maesa japonica or a fraction thereof as an active ingredient.

In the latter half of the 20th century, American plant pathologist Bollog developed modern farming practices represented by hybrids, synthetic pesticides, and synthetic fungicides. This is called the "Green Revolution," and the use of pesticides in modern agriculture has become essential for preserving yields. In the production of crops, various plant pathogens, pests and weeds inhibit the growth of the crops, and if not properly controlled, yields of 30 ~ 100% will occur depending on the type of crops to be cultivated. Although a variety of synthetic pesticides have been developed and used actively to protect crops, the frequency of occurrence of harmful organisms resistant to medicines has increased due to the use of synthetic pesticides for many years, and the human body due to the residual toxicity of abused pesticides Concerns about health, environmental pollution and ecosystem disturbance are growing, and policy regulations on the use of synthetic pesticides are strengthening, especially in the OECD countries including Korea. Cluster analysis of millions of compounds registered in the American Chemical Society's Chemical Information Database (CAS) can be categorized as a group of only a few hundred leading substances. As regulations are strengthened on the basis of lead compounds, The number of pesticides is bound to decline. In addition to eco - friendly agricultural policies aimed at reducing the use of synthetic pesticides, the demand for new environmental - friendly control measures is increasing, and bio - pesticides are emerging as an alternative to synthetic pesticides.

Bio-pesticides are eco-friendly crop protection agents that have little effect on human life and ecosystem, unlike synthetic pesticides. They can be used to produce agricultural products with high added value such as organic agricultural products and can be effectively used to control pests that are difficult to control with synthetic pesticides . Biological pesticides include microbial pesticides that utilize the functions of microorganisms and biochemical pesticides that utilize metabolites derived from plants or microorganisms. Since the Rio Conventions on Environmental Conventions to replace 20% of the world's pesticides with biopesticides, the development of pesticides using environmentally friendly natural pesticides, especially plant extracts, has attracted attention. The bio-pesticide market, which was $ 1.3 billion in 2011, is expected to reach $ 3.2 billion by 2017, growing at an average annual rate of 16%. The Korean government is also concentrating on eco-friendly agricultural policies, and the public awareness of eco-friendly agricultural products is rising, and research on bio-pesticides, which are now being carried out separately at government-funded research institutes, universities and some small and medium-sized venture businesses, do. An analysis of pesticide-related papers 3236 (1996-2013) obtained from Elsevier's database showed that interest in bio-pesticides was steadily increasing due to an increase in the number of papers with an average annual growth rate of 12%.

In Korea, the RDA administers the registration and management of pesticides for pest control used in environmentally friendly agriculture, and provides a list of products that can be used for environmentally friendly organic agriculture by publishing a list of 'environmentally friendly organic materials'. As of March 2016, a total of 427 pesticides were declared as pesticides for the control of pests, of which 204 were plant extracts, 10 were microbial extracts, and 62 were microbials. About 48% The extract was used.

Maesa japonica (Thunb) Morizi & Zoll. Is an evergreen bush bush that belongs to the primevalle Maesaceae taxonomically. This species was collected in Jeju Island and published as an unrecorded species in 2006. It is composed of a genus Maesa 1, with a pair of follicles on peduncle, no callus, and a large number of seeds, separated from Myrsinaceae and Primulaceae. The stem grows straight and is 1 to 1.5 m high. The roots come out of the nodes touching the ground and touching the ground. Leaves are opposite, oval or oblong, 5 to 17 cm long, 2 to 5 cm wide. The back side of the leaf is green. Flowers bloom from April to May, hanging from the axilla of leaves. Calyx is divided into 5 branches, sepals are dull triangles. There are 5 stamens and 1 pistil. The fruit is round with bowels. Not only in Korea, but also in Taiwan, Vietnam, Japan, and China.

In Chinese traditional medicine, leaves and roots are known to treat common cold symptoms, and other species belonging to the genus Mesa ( Maesa spp.) Have been used in folk remedies in Africa and Asia. Maesa lanceolata was used in traditional Rwandan medicine in Africa to treat hepatitis, dysentery, dermatosis, and neuropathy, which was used in folk remedies to prevent cholera in East Africa. Maesa balansae has been used in traditional Vietnamese medicine for the treatment of allergies, sprains, parasitic infections, skin ulcers, hangovers and headaches. Maesasaponins isolated from the leaves of Maesa lanceolata showed various physiological activities such as antiviral, anti-molluscic, hemolytic and anti-angiogenic activities, and mesanganin isolated from the fruit of this plant maesanin) and mesesquinone (maesaquinone) showed immunological enhancement and anticancer activity. Mesa Balan aged methoxy bowl fluoride (maesabalides) isolated from leaves of (Maesa balansae) exhibited hangri Schumann parasite (antileishmania) activity. Maesaquinone and acetylmaesaquinone were isolated from bark, root and fruit of Maesa japonica and maejaposides, a kind of maesasaponins, were isolated from leaves. . The cytotoxicity and antimutagenic activity of MRC-5 in the n-butanol fraction of leaf extract of Maesa japonica have been reported, and the leaf, stem and flower extracts of bilem trees have been effective in preventing skin aging It has been registered as a US patent (US 8709507 B2) as a raw material. However, there has been no report on the activity of plant extracts for controlling plant diseases.

Plant extracts have shown various physiological activities and have been used in various fields through traditional medicine and folk remedies. Based on this, the present inventors tried to use plant extract as an environmentally friendly plant disease control means.

It is an object of the present invention to provide a method for preventing and treating harmful insects such as Magnaporthe oryzae , Rhizoctonia solani , Botrytis cinerea , Phytophthora infestans , Rust ( Puccinia recondita ) and pepper anthracnose ( Colletotrichum The present invention also provides a composition for controlling plant diseases, which comprises Maesa japonica extract or a fraction thereof as an active ingredient, which exhibits an excellent controlling effect on plant diseases important for the production of crops,

In order to solve the above-mentioned object, the present invention provides a plant disease controlling composition comprising Maesa japonica extract or a fraction thereof as an active ingredient.

The present invention relates to a composition for the control of plant diseases, which contains Maesa japonica extract as an active ingredient. The composition for controlling plant diseases according to the present invention is a natural product harmless to the human body, ( Rhizoctonia solani ), Botrytis cinerea , Phytophthora infestans , Puccinia recondita , and Colletotrichum anthracnose ( Colletotrichum anthracnose) are the most common species of the genus , such as Magnaporthe oryzae , Rhizoctonia solani , coccodes and the like, it can be developed as an environmentally friendly biological pesticide and can be usefully used in the production of high value added organic agricultural products.

Fig. 1 shows the results of controlling the ethanol extract of Maesa japonica (3,000 ppm) against rice blast.
Left untreated control; The middle is the bille tree extract; And on the right were treated with synthetic antimicrobial tricyclazole (10 ppm).
Fig. 2 shows the in vitro antimicrobial activity of Magnaporthe oryzae of ethyl acetate extract (900 ㎍) and n-butanol extract (900 쨉 g) of Maesa japonica by the paper disk agar diffusion method to be.
Fig. 3 is a graph showing the chromatogram of the fraction E222241 isolated from the ethyl acetate layer of the leaf extract of Maesa japonica at 280 nm wavelength using high performance liquid chromatography and the UV absorption spectrum of the analyzed peak and the antimicrobial activity Respectively.
FIG. 4 is a graph showing the chromatogram of the fraction B944 isolated from the n-butanol layer of the leaves and stem methanol extract of Maesa japonica at 230 nm wavelength using high performance liquid chromatography and the UV absorption spectrum of the analyzed peak, Activity.
Figure 5 shows the chromatogram of the fraction B9453 isolated from the n-butanol layer of the leaves and stem methanol extract of Maesa japonica at a wavelength of 210 nm using high performance liquid chromatography and the UV absorption spectrum of the analyzed peak, Activity.

In the present specification, the term " control " is used to mean prevention and avoidance of diseases and insects as well as removal and death. However, the control effect of plant disease control by preventive treatment is the main control mechanism. Therefore, the control effect of plant extracts and fractions on the plant diseases was tested by prophylactic treatment.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for controlling a plant disease comprising an extract of Maesa japonica or a fraction thereof as an active ingredient.

It is preferable, but not limited, that the bille-tree extract or the fraction thereof is produced by a manufacturing method comprising the following steps:

1) Extracting the extract with an extraction solvent;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and then drying to prepare an extract of Bile wood; And

4) a step of extracting the extract of the bilemen of the step 3) with an organic solvent to prepare the bilemen fractions.

In the above method, the tree of step 1) can be used without limitation such as cultivated or marketed. The above-mentioned tree can be used as leaves, stalks or roots, but is not limited thereto. The harvesting time of the tree is preferably September, but is not limited thereto.

In the above method, it is preferable to use water, an alcohol, n-hexane, methylene chloride, ethyl acetate, acetone or a mixture thereof in the extraction solvent in step 1).

As the alcohol, a C ₁ to C ₄ lower alcohol is preferably used, and as the lower alcohol, ethanol, methanol, propanol or butanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. The extraction solvent is preferably added by 1 to 10 times, more preferably 2 to 3 times, to the amount of dried bamboo. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. In addition, the extraction number is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In the above method, the organic solvent in step 4) is preferably, but not limited to, n-hexane, chloroform, ethyl acetate, n-butanol or water. The fractions can be obtained by suspending the extracts of Bilequinia species in water and then fractionating the fractions in the order of n-hexane, chloroform, ethyl acetate, n-butanol and water in the order of n-hexane fraction, chloroform fraction, ethyl acetate fraction, n- Water fraction, and more preferably an ethyl acetate fraction or an n-butanol fraction. However, the present invention is not limited thereto. The fractions can be obtained by repeating the fractionation process from the above-mentioned Bile tree extract for 1 to 5 times, preferably 3 times, followed by fractionation and concentration under reduced pressure, but the present invention is not limited thereto.

The plant disease is preferably one or more selected from the group consisting of rice blast, rice sheath blight, tomato gray mold, tomato blight, wheat blight and anthracnose.

The composition may include Botrytis < RTI ID = 0.0 > cinerea , Alternaria brassicicola , Colletotrichum fructicola , Fusarium < RTI ID = 0.0 > oxysporum , Magnaporthe oryzae , Phytophthora ( Phytophthora) infestans , Rhizoctonia solani , and Sclerotinia sclerotiorum . It is preferred that Agrobacterium tumefaciens , Burkholderia sp., and the like are used for inhibiting the growth of at least one phytopathogenic fungus mycelial growth selected from the group consisting of Agrobacterium tumefaciens , Burkholderia glumae) and Peck tobak Te Karo Solarium Sat boreom (Pectobacterium carotovorum subsp . carotovorum ), but the present invention is not limited thereto.

The extract or the fraction thereof is preferably contained at a concentration of 100 to 3,000 占 퐂 / ml in the composition for controlling a plant disease, but is not limited thereto. It is more preferable that the extract or the fraction thereof is contained at a concentration of 1,000 to 3,000 占 퐂 / ml.

In the concrete examples of the present invention, the extracts obtained from the extracts obtained with the 70% ethanol aqueous solution at a concentration of 3,000 ㎍ / The control efficacy against the remaining six plant diseases was shown. In particular, the control activity against rice blast, wheat red rust and red pepper anthracnose showed strong control activity of 80% or more (see Table 1 and FIG. 1).

As a result of controlling the inhibitory activity against 7 plant diseases, the extracts of leaves, stem extracts and root extracts of bilem trees were treated at a concentration of 3,000 ㎍ / ㎖, and the rice blast fungus, rice sheath blight, The control effect against wheat red rust showed a high control effect of over 73%. The leaf and stem extract showed 50% control against pepper anthracnose. At the concentration of 1,000 ㎍ / ㎖, the two kinds of extracts showed 80% higher control value against rice blast, and 50 ~ 60% control value against rice sheath blight and wheat red rust (Table 2) ).

In addition, when the extracts of Pinus sylvestris extracts were treated at a concentration of 3,000 ㎍ / ㎖, the control activity against rice blast fungus was 98% regardless of harvesting time. In September, Herbicide blight, tomato gray mold, and red pepper anthracnose were shown to have a somewhat higher control effect (see Table 3).

In addition, the inhibition of mycelial growth inhibition on 8 plant pathogenic microbial strains of Lilium leaves and stem extracts revealed that when the leaves and stem extracts of Liliaceae were treated at a concentration of 333 ㎍ / ㎖ or more, the mycelial growth of Magnaporthe oryzae (72.1%), Alteraria brassicola (53.4%), Sclerotinia sclerotiorum (40.7%), and Staphylococcus aureus were found to be 100% Cholate tricum fructicola (37.8%) in order of inhibition rate of mycelial growth. When treated at a concentration of 1,000 ㎍ / ㎖, mycelial growth inhibition rate against the P. aeruginosa pitoflora infestans increased to 69.3%. However, the rate of inhibition of mycelial growth against the P. aeruginosa fungus, P. vorotis cinerea, The inhibition rate of mycelial growth was low at 20.2 ~ 35.7%, and the mycelial growth inhibition activity of the extracts of Lilium leaves and stem extracts against rice blast fungi was highest (see Table 4).

In addition, the bacterial growth inhibition activity of nine phytopathogenic bacterium of Lilium leaf and stem extracts showed that the growth of Agrobacterium tumefaciens decreased to 52.1% And 111 ㎍ / ㎖ inhibited the growth of Burkholderia glume by 40%, but the growth inhibitory activity did not increase with increasing treatment concentration. When treated at a concentration of 1,000 μg / ml, the effect of inhibiting the growth of Pectobacterium carotovorum was inhibited up to 17.3% (see Table 5).

In addition, the inhibitory activity against the seven plant diseases of the extract of methanol extract of bichescholesterol showed that the ethyl acetate fraction and the n - butanol fraction had a control value of over 90% against rice blast fungus at the concentration of 1,000 ㎍ / ㎖, 67% of the red rust showed a control value. However, the aqueous solution layer showed no controlling activity. Therefore, the antimicrobial active substance of bile extract was not a water-soluble substance but a non-polar substance (see Table 6).

The ethyl acetate fraction and the n - butanol fraction inhibited mycelial growth of the rice blast fungus by the methanol extract of Bilkeo extract. Therefore, it was confirmed that a substance exhibiting antimicrobial activity under in vitro conditions is an active ingredient showing a controlling activity against rice blast (see FIG. 2).

Therefore, the extract of the invention and its fractions are natural products harmless to the human body, biodegraded in the natural environment and have a small environmental load, and thus can be used for producing Magnaporthe oryzae , Rhizoctonia solani ), tomato gray mold ( Botrytis cinerea ), tomato blight ( Phytophthora infestans ), wheat red rust ( Puccinia recondita ) and pepper anthracnose ( Colletotrichum coccodes and the like. Therefore, it can be developed as an environmentally friendly composition for controlling plant diseases or as a biochemical pesticide, and can be usefully used in the production of high value-added organic agricultural products.

The composition for controlling plant diseases according to the present invention may further contain a surfactant and a fermented alcohol, and the amount of the surfactant further included is 1 to 10 parts by weight based on 100 parts by weight of the total composition, and the amount of the fermented alcohol May be 1 to 20 parts by weight based on 100 parts by weight of the total composition.

The surfactant may be at least one selected from the group consisting of polyoxyethylene sorbitan lauryl, sodium dodecylbenzene sisphonate, and silicone polyether copolymer, but is not limited thereto. The fermentation alcohol means a commonly used fermentation alcohol, preferably 95% alcohol. In addition, the composition of the present invention may further include a known plant extract having a sterilizing effect for enhancing the sterilizing effect or expanding the sterilization range.

The present invention provides a plant disease control method comprising treating a plant or a soil with a composition for controlling plant disease, the treatment method is not particularly limited, and examples thereof include a method of adding to water or fertilizer added to plants or soil .

More specifically, the fruit and vegetables include branches, peppers, strawberries, watermelons, cucumbers, melons, tomatoes, bell peppers, amber and the like, and more preferably, Leafy vegetables include cabbage, leek, lettuce, and vegetable vegetables, and root vegetables may contain nothing.

The plant disease controlling composition may be diluted with an appropriate amount and may be used in an amount of not more than 500 times (for example, 10 to 500 times), preferably not more than 300 times (for example, 10 to 300 times) May be diluted to a dilution magnification of 200 times or less (for example, 10 to 200 times).

No particular limitation is imposed on the diluent used for diluting the control composition of the present invention, and a diluent commonly used in the related art can be used. For example, water can be used.

The controlling method of the present invention is preferably applied to a plant or soil, wherein the composition for controlling a plant disease is sprayed on the foliage of a plant every 2 to 3 days at intervals of 3 to 5 days.

The main component of the composition for controlling plant diseases according to the present invention is a plant secondary metabolite, which is suitable for environmentally friendly agriculture in which it is impossible to dispense organic synthetic fungicide in principle and there is no harmfulness and residual toxicity to human and livestock, So it can be processed even during the harvesting period of horticultural plants.

It is a safe control material which can substitute organic synthetic fungicide safely in plants when spraying these compositions. Also, even when mixed with chemical pesticides or sprayed in the middle of treatment, it is possible to control the plant diseases, which can contribute to a reduction in the number of spraying of chemical pesticides.

The present invention provides a plant disease controlling agent (product) comprising a composition for controlling a plant disease. The herbicide may be formulated as a concentrate and may be diluted to an appropriate dilution by the end user.

The controlling agent according to the present invention is a flowable formulation, suspension, microsuspension, emulsion (e. G., Suspension emulsion, microemulsion), powder (e. G., Wettable powder) , Granular concentrates, pastes, and the like.

In addition, the plant disease control agent of the present invention may contain other components such as insecticides, other fungicides, herbicides, plant growth regulators, fertilizers and minerals as necessary as an effective ingredient.

The plant disease control agent may contain 0.1 to 99 parts by weight, preferably 10 to 90 parts by weight, more preferably 50 to 90 parts by weight, based on the weight of the active ingredient, of the composition for controlling plant diseases, One or more surfactants and / or any other inert ingredients known in the art, such as protective colloids, adhesives, thickeners, thixotropic agents, penetrants, preservatives, stabilizers, antifoaming agents, cryoprotectants, , Dyes, pigments, colorants and polymers.

Examples of the penetrating agent include fatty alcohol alkoxylates, mineral oils, vegetable oils, mineral oils, and esters of vegetable oils.

Examples of the stabilizer include epoxidized plant and animal oils, nonionic polyoxyethylene surfactants, anionic polyoxyethylene surfactants, polyhydric alcohols and basic substances.

Examples of the defoaming agent include trade name Anti-mousse (manufactured by BELCHIM CROP PROTECTION).

Examples of the cryoprotectant include ethylene glycol, propylene glycol, and the like.

Examples of the carrier include a solid carrier and a liquid carrier. Examples of the solid carrier include starch, sugar, cellulose powder, cyclodextrin, activated carbon, soybean powder, wheat flour, rice hull powder, wood flour, fish meal, The same vegetable powder; Mineral powders such as talc, kaolin, bentonite, organic bentonite, calcium carbonate, calcium sulfate, sodium bicarbonate, zeolite, diatomaceous earth, white carbon, clay, alumina, silica, sulfur powder and slaked lime. Examples of the liquid carrier include water; Vegetable oils such as soybean oil and cottonseed oil; Animal oil such as bovine fat, light oil; Alcohols such as ethyl alcohol and ethylene glycol; Ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone and isophorone; Ethers such as dioxane and tetrahydrofuran; Aliphatic hydrocarbons such as kerosine, kerosene, liquid paraffin, and cyclohexane; Aromatic hydrocarbons such as toluene, xylene, trimethylbenzene, tetramethylbenzene, and solvent naphtha; Halogenated hydrocarbons such as chloroform and chlorobenzene; Acid amides such as N, N-dimethylformamide; Esters such as acetic acid ethyl ester and glycerin of fatty acid; Nitriles such as acetonitrile; Sulfur-containing compounds such as dimethyl sulfoxide, and N-methyl-2-pyrrolidone.

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

However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Tree ( Maesa  japonica ) 7 kinds of extract In botanical bottles  Confirmation of control activity against

<1-1> Tree  Preparation of ethanol extract

In order to prepare the extract of bamboo tree, the extracts of Maesa japonica were cut into small pieces and 70% ethanol aqueous solution was added thereto. The extract was extracted at room temperature for 24 hours and filtered. The filtrate was concentrated under reduced pressure to give 120 mg An extract was obtained.

For the evaluation of the phytotoxic effect of the extracts, 2 ㎖ of the plant extract solution dissolved in methanol at a concentration of 60 ㎎ / ㎖ was diluted with 38 ㎖ of distilled water, and the concentration of the tween 20 (tween 20) was 250 ㎍ / ㎖ To prepare 40 ml of an extract solution having a concentration of 3,000 占 퐂 / ml. Here, distilled water containing 5% of methanol and 250 占 퐂 / ml of Tween 20 was used as the control.

<1-2> Tree  Identification of the control activity of ethanol extracts

The inventors of the present invention found that Magnaporthe oryzae , Rhizoctonia spp., And Rhizoctonia spp. solani ), tomato gray mold ( Botrytis cinerea ), tomato blight ( Phytophthora infestans), barley powdery mildew (Blumeria graminis f. sp. hordei ), wheat leaf rust (Puccinia recondita ) and anthracnose ( Colletotrichum coccodes ) were investigated as follows.

Specifically, two pots were used per plant, and the extract solution was sprayed on the leaf surface, then air-dried for 24 hours, and each plant pathogen was inoculated. The rice, tomato, barley and wheat plants used in the experiment were filled with plastic pots with a diameter of 4.5 ㎝ of about 70% of the ground or horticultural soil, seeded and cultivated in a greenhouse at 25 賊 5 캜 for 1 to 4 weeks .

The rice blast fungus was sprayed with a spore suspension (5 × 10 ⁵ spores / ㎖) of Magnaporthe oryzae (Korea Research Institute of Chemical Technology), which is a causative organism of blast fungus , And then cultured in a constant temperature room at 25 DEG C for 4 days to induce the onset.

Rhizoctonia solani ( Rhizoctonia solani , Korea Research Institute of Chemical Technology), which is a causative agent of sheath blight, was cultivated for 7 days in medium (90 g of bran, 15 g of rice hull and 100 ml of distilled water) Seedlings were inoculated, treated for 4 days in a wet condition at 25 ° C, and cultured in a constant temperature room at 25 ° C for 4 days to induce the onset.

Tomato blight was sprayed on tomato seedlings from 3 to 4 leafy tomatoes by inoculation with a suspension of the extruded juveniles from Phytophthora infestans (Kangnung National University) (10 ⁵ sporangia / ㎖) Actually, the cells were treated for 2 days and cultured in a constant temperature and humidity room at 25 ° C for 1 day to induce the onset.

Tomato gray mold was detected in 3 ~ 4 seedlings of tomatoes by Botrytis , a causative organism of gray mold spore suspension of the cinerea, Korea Research Institute of Chemical Technology) (after treatment with ^{5 × 10 5 spores / ㎖)} , it was induced by the onset for 3 days in a moist substance of 20 ℃.

Wheat red rust is caused by Puccinia , a causative organ of rust, Recondita , Incheon University) was suspended in 250 .mu.g / ml Tween 20 solution in an amount of 0.67 g spores / l, treated for 20 days at 20 ° C in a humidified chamber, transferred to a constant temperature chamber at 20 ° C, To induce the onset.

Barley powdery mildew is the cause of a subculture in the host plant, the first leaf stage seedlings of barley powdery mildew Blue Almeria Grammy Nice Pomeranian seupesal lease Hall Day (Blumeria graminis f. sp. hordei , Korea Research Institute of Chemical Technology) were inoculated and incubated for 7 days in a constant temperature chamber at 20 ° C to induce the onset. Tomato gray mold and tomato blight were examined 3 days after inoculation, 5 days after inoculation with rice blast, 7 days after inoculation with wheat red rust, barley powdery mildew, and 8 days after inoculation with rice sheath blight.

In order to test the antifungal activity against pepper anthracnose, a plastic pot having a diameter of 7.0 cm was filled with about 70% of the soil for horticultural use, and then the best pepper seeds were sown. The seeds were raised in greenhouse to 3-4 weeks, 1-1> were sprayed on the leaf surface. After spraying the sample, it was dried at room temperature for 24 hours and then colletotrichum (4 × 10 ⁵ spores / ㎖) of the spore suspension of the coccodes (Korea University). (25 ℃, relative humidity 80%) for 1 day after 2 days of damp treatment at 25 ℃ on the wet field. The percentage of lesion area (%) was examined 3 days after inoculation.

The% control was calculated according to the following equation using the percent area percentage of the lesion obtained from the above.

[Equation 1]

As a result, as shown in Table 1, when the extract obtained with a 70% ethanol aqueous solution was treated at a concentration of 3,000 μg / ml in the bark beetle outbreak, it was confirmed that it exhibited the controlling activity against the remaining six plant diseases except the barley powdery mildew (Table 1 and Fig. 1). Especially, rice flour disease, wheat red rust and red pepper anthracnose showed strong control activity of 80% or more.

sample Concentration (/ / ml) Control (%) RCB RSB TGM TLB WLR BPM PAN Bilye tree seed extract 3,000 94 55 43 94 100 0 79 RCB: rice blast; RSB; Rice sheath blight; TGM: Tomato gray mold disease; TLB: tomato blight; WLR: wheat red rust; BPM: barley powdery mildew; PAN: pepper anthracnose

Tree ( Maesa  japonica ) 7 kinds of extracts by site In botanical bottles  Confirmation of control activity against

<2-1> Tree  Preparation of extracts by site

The present inventors finely cut 500 g of dried bamboo leaves and stem water samples and 10 L of 70% aqueous methanol solution was added thereto, and the methanol extract obtained by extraction at room temperature for 24 hours was filtered using a filter paper. The filtrate was concentrated under reduced pressure to obtain 78.3 g Leaf and stem extracts were obtained.

In the same manner, 300 g of root drying sample was finely cut, and 3 L of a 70% methanol aqueous solution was added. The mixture was extracted at room temperature for 24 hours and concentrated under reduced pressure to obtain 34.8 g of root extract.

2 ml of each sample obtained by dissolving the above extract in methanol at two concentrations of 20 mg / ml and 60 mg / ml was diluted in 38 ml of distilled water, and the concentration of the electrodeposited tween 20 (tween 20) was adjusted to 250 μg / ml To prepare 40 ml of the extract solution of 1,000 μg / ml and 3,000 μg / ml, respectively. Here, distilled water containing 5% of methanol and 250 占 퐂 / ml of Tween 20 was used as the control.

<2-2> Tree  Identification of control efficacy of extracts by site

The present inventors evaluated the plant disease control effect in the same manner as in Example <1-2> in order to confirm the controlling activity of the extract of the bamboo plant extract obtained in Example <2-1> above.

As a result, as shown in Table 2, when treated with a leaf extract, stem extract or root extract solution at a concentration of 3,000 μg / ml, the control against rice blast, rice sheath blight, and wheat rust was more than 73% The extracts of leaf and stem extracts showed 50% inhibitory activity against pepper anthracnose. At the concentration of 1,000 ㎍ / ㎖, the two kinds of extracts showed more than 80% control effect against rice blast, and 50 ~ 60% of control against rice blast fungus and wheat red rust (Table 2). The extracts of each part of the bilet trees showed little difference in plant disease control activity.

sample Concentration (/ / ml) Control (%) RCB RSB TGM TLB WLR BPM PAN Leaf tree stem extract 3,000 90 75 14 7 83 0 50 1,000 87 50 0 0 60 0 20 Bile root extract 3,000 90 100 14 7 73 0 30 1,000 80 60 0 7 53 0 20 RCB: rice blast; RSB; Rice sheath blight; TGM: Tomato gray mold disease; TLB: tomato blight; WLR: wheat red rust; BPM: barley powdery mildew; PAN: pepper anthracnose

Tree ( Maesa  japonica ) Seven kinds of extracts In botanical bottles  Confirmation of control activity against

<3-1> Of the tree  Preparation of Extract by Collection Period

The present inventors cut out the outbreaks of the bille trees collected in the period (January, June, September, December) by chopping and methanol for 24 hours at room temperature, filtered using a filter paper, To obtain a dried extract.

2 ㎖ of the bilayers outpost specimen of each sampling period dissolved in methanol at a concentration of 60 ㎎ / ㎖ was diluted with 38 ㎖ of distilled water and added to the concentration of 250 ㎍ / ㎖ of the tween 20 of the electrodeposition agent. 40 ml of the extract solution of each period was prepared. Here, distilled water containing 5% of methanol and 250 占 퐂 / ml of Tween 20 was used as the control.

<3-2> Tree  Identification of control efficacy of extracts by collection period

The present inventors evaluated the plant disease control effect in the same manner as in Example <1-2> in order to confirm the controlling activity of the extracts obtained by the extracting time of the bale trees obtained in the above Example <3-1>.

As a result, as shown in Table 3, when the seedling extract of Bilye tree collected at different time points was treated at a concentration of 3,000 ㎍ / ㎖, the control effect against rice blast fungus was as high as 98% regardless of harvesting time. In September, the herbicidal activity against rice sheath blight, tomato gray mold, and red pepper anthracnose was somewhat higher than other control (Table 3). Overall, however, there was little difference in the effectiveness of plant disease control over harvesting time.

sample When to collect Concentration (/ / ml) Control (%) RCB RSB TGM TLB WLR BPM PAN Bilye tree seed extract January 3,000 98 10 44 0 47 0 42 June 3,000 98 45 65 0 67 0 33 September 3,000 98 45 70 4 58 0 60 December 3,000 98 23 61 0 47 0 38 RCB: rice blast; RSB; Rice sheath blight; TGM: Tomato gray mold disease; TLB: tomato blight; WLR: wheat red rust; BPM: barley powdery mildew; PAN: pepper anthracnose

Tree ( Maesa  japonica ) 8 kinds of leaves and stem extracts On plant pathogens  Identification of mycelial growth inhibitory activity

The present inventors evaluated the efficacy of inhibiting the mycelial growth of phytopathogenic fungi using a sample obtained by dissolving the bilayre leaf stem extract prepared in Example <2-1> in methanol at a concentration of 100 mg / ml.

Specifically, the leaf and stem extracts dissolved in methanol were mixed in a PDA (potato agar medium, BD Biosciences) at a concentration of 111, 333, and 1,000 μg / ml to a 6-well plate, In the middle, 3 mm diameter fungal hypha disks were inoculated. Eight species of fungi were tested in 3 replicates per concentration. Botrytis cinerea and Phytophthora ( Phytophthora) infestans ) were cultured at 20 ° C, and the remaining Alternaria brassicycola brassicicola , Colletotrichum &lt; RTI ID = 0.0 &gt; fructicola , Fusarium oxysporum , Magnaporthe &lt; RTI ID = 0.0 &gt; oryzae , Rhizoctonia solani , Sclerotinia sclerotiorum were cultured at 25 ° C. The mycelial growth diameter of untreated and treated mycelium was measured when the mold of the non-treatment area was irradiated with radial growth of 70 to 80% of the diameter of the well, which was cultured in a potato agar medium containing 1% methanol. Mycelial growth diameter values were obtained by using mean values of the major axis and minor axis length minus the inoculated mycelial disk diameter of 3 mm.

The mycelial growth inhibition rate (%) was calculated according to the following equation using the mycelial growth diameter value obtained from the above.

&Quot; (2) &quot;

As a result, as shown in Table 4, it was confirmed that mycelial growth of Magnaporthe oryzae, a causative agent of rice blast fungus, was inhibited by 100% when treated with a concentration of 333 ㎍ / ㎖ or higher. When treated at the concentration of 111 ㎍ / ㎖, Magnaporthe lucerne (72.1%), Alteraria brassicycola (53.4%), Sclerotinia sclerotiorum (40.7%), Cola (37.8%), and the inhibition rate was increased with increasing treatment concentration. When treated at a concentration of 1,000 ㎍ / ㎖, mycelial growth inhibition rate against the P. aeruginosa pitoflora infestans increased to 69.3%. However, the rate of inhibition of mycelial growth against the P. aeruginosa fungus, P. vorotis cinerea, Mycelial growth inhibition rate was 20.2 ~ 35.7% (Table 4). The highest inhibitory activity of Mycobacterium tuberosum L. and Stem Extracts against rice blast fungus was observed in the above Example 2 in that the extracts of Lilium japonica leaf and stem extract (1,000 μg / ml) ), Respectively.

Plant pathogenic fungi Concentration (/ / ml) Mycelial growth inhibition rate (%) ± standard deviation Magnaporthe oryzae 111 72.1 ± 0.7 333 100 1,000 100 Alternaria brassicicola 111 53.4 ± 1.9 333 58.4 ± 1.4 1,000 61.4 ± 1.2 Sclerotinia sclerotiorum 111 40.7 ± 0.9 333 44.5 ± 1.3 1,000 44.9 ± 2.5 Colletotrichum fructicola 111 37.8 ± 3.7 333 38.2 ± 1.7 1,000 46.1 ± 2.2 Phytophthora infestans 111 15.6 ± 1.8 333 31.3 ± 3.4 1,000 69.3 ± 3.4 Rhizoctonia solani 111 22.5 ± 0.9 333 30.8 ± 2.4 1,000 35.7 ± 1.6 Botrytis cinerea 111 19.4 ± 1.1 333 21.1 ± 3.7 1,000 32.2 ± 5.9 Fusarium oxysporum 111 9.8 ± 0.7 333 13.1 ± 3.0 1,000 20.2 ± 0.8

Tree ( Maesa  japonica 9 Plant Pathogens of Leaf and Stem Extracts Bacterial growth inhibitory activity against bacteria  Confirm

The present inventors evaluated the activity of inhibiting the growth of phytopathogenic bacteria by using the extract of Lilium japonica leaf and stem extract prepared in Example <2-1> (a sample dissolved in methanol at a concentration of 100 mg / ml).

Specifically, a broth microdilution method was used in a 96-well plate, and in a 100 μl TSB (tryptic soybean broth, BD Biosciences) medium per well, , 333, 1,000 占 퐂 / ml. Nine species of phytopathogenic bacteria, Acidovorax avenae subsp. cattleyae), Agrobacterium tumefaciens (Agrobacterium tumefaciens), Burke holde Ria glue methoxy (Burkholderia glumae , &lt; RTI ID = 0.0 &gt; Clavibacter & lt ; / RTI &gt; michiganensis subsp . michiganensis , Dickeya chrysanthemi , Pectobacterium carotovorum subsp. carotovorum , Pseudomonas sp. syringae pv . lachrymans , Ralstonia &lt; RTI ID = 0.0 &gt; solanacearum), Pseudomonas janto ahreubo Ricola (Xanthomonas arboricola pv . pruni ) was repeated three times for each concentration. Each bacterium was cultured in TSB medium at 30 ° C for 2 days with shaking at 150 rpm, diluted with TSB medium, and measured with a spectrophotometer. The OD ₆₀₀ (optical density measured at 600 nm) was 0.1, 5.0 to 8.0 x 10 &lt; ⁷ &gt; CFU / ml. Of diluted bacterial TSB culture solution was inoculated by 1 ㎕ per well, was adjusted to finally about ^{5.0 ~ 8.0 × 10 5 CFU /} ㎖. After incubation at 30 ° C for 24 hours, the cells were cultured in TSB medium supplemented with 1% methanol and used as an untreated control. Exodoborax, Agrobacterium, Burkholderia, Claviabacter, and Pectobacterium were incubated for 24 hours at OD ₆₀₀ values were measured. Dickey, Pseudomonas, Ralstonia, and Zanthomonas were incubated for 48 hours to measure an OD ₆₀₀ value.

Using the OD ₆₀₀ value obtained from the above, the inhibition rate (%) of bacterial growth was calculated according to the following equation.

&Quot; (3) &quot;

As a result, as shown in Table 5, it was confirmed that the effect of suppressing the growth of Agrobacterium tumefaciens to 52.1% was increased as the treatment concentration of the leaf extract and stem extract of bilemens increased. 111 ㎍ / ㎖ inhibited the growth of Burkholderia glume by 40%, but the growth inhibitory activity did not increase with increasing treatment concentration. In addition, it was confirmed that when treated at a concentration of 1,000 占 퐂 / ml, the effect of inhibiting the growth of Pectobacterium carotovorum was inhibited up to 17.3% (Table 5). Overall, the growth inhibitory activity of phytophthora leaves and stem extracts against phytopathogenic bacteria was low.

Phytopathogenic bacteria Concentration (/ / ml) Bacterial growth inhibition rate (%) Acidovorax avenae subsp. cattleyae 111 0 333 8.1 1,000 0 Agrobacterium tumefaciens 111 16 333 32.5 1,000 52.1 Burkholderia glumae 111 40 333 41.1 1,000 37.9 Clavibacter michiganensis subsp. michiganensis 111 6 333 0 1,000 7.6 Dickeya chrysanthemi 111 0 333 0 1,000 0 Pectobacterium carotovarum subsp. carotovarum 111 4.2 333 3.8 1,000 17.3 Pseudomonas syringae pv. lachrymans 111 0 333 5.1 1,000 0 Ralstonia solanacearum 111 5.8 333 0 1,000 0 Xanthomonas arboricola pv. pruni 111 0 333 0 1,000 0

Tree ( Maesa  japonica ) 7 kinds of methanol extract solvent fraction In botanical bottles  Confirmation of control activity against

<6-1> Tree  Solvent of methanol extract Fraction  Produce

To obtain the basic properties of the antimicrobial active substance, the fractions of the extracts of bamboo leaves and stem methanol prepared in Example <2-1> were prepared.

Specifically, 78.3 g of methanol extract of Lilium leaves and stem were dissolved in 3 L of distilled water and extracted twice with an equal volume of ethyl acetate solution. The ethyl acetate layer and the aqueous layer were concentrated under reduced pressure. The concentrated aqueous solution layer was dissolved again with 3 L of distilled water and extracted twice with the same amount of n-butanol. The organic solvent extract and the aqueous solution layer were concentrated under reduced pressure to obtain a dried solvent fraction. 2 ml of each fraction sample dissolved in methanol at a concentration of 20 mg / ml was diluted with 38 ml of distilled water and tween 20 (Tween 20) was added thereto to a concentration of 250 占 퐂 / ml to prepare a fraction solution having a concentration of 1,000 占 퐂 / Ml. Here, distilled water containing 5% of methanol and 250 占 퐂 / ml of Tween 20 was used as the control.

<6-2> Tree  Methanol extract solvent Fraction  Check control activity

The present inventors evaluated the plant disease control effect in the same manner as in Example <1-2> in order to confirm the control activity of the solvent fraction of the methanol extract of B. bistilis obtained in Example <6-1> above.

As a result, as shown in Table 6, the ethyl acetate fraction and the n-butanol fraction showed a control value of over 90% for rice blast fungus at a concentration of 1,000 ㎍ / ㎖, and a control value of 67% against wheat rust Respectively. However, it was confirmed that the aqueous solution layer did not exhibit the controlling activity (Table 6). Therefore, the antimicrobial active substance of the extract of Bilye tree was identified as a nonpolar material rather than a water soluble substance.

sample Concentration (/ / ml) Control (%) RCB RSB TGM TLB WLR BPM PAN Ethyl acetate layer 1,000 90 21 21 8 67 0 25 n-butanol layer 1,000 97 7 7 0 67 0 17 Aqueous solution layer 1,000 17 0 0 0 0 0 8 RCB: rice blast; RSB; Rice sheath blight; TGM: Tomato gray mold disease; TLB: tomato blight; WLR: wheat red rust; BPM: barley powdery mildew; PAN: pepper anthracnose

Tree ( Maesa  japonica ) Methanol extract The rice fraction of the solvent fraction Blast fungus  Identification of Mycelial Growth Inhibitory Activity

In order to understand the basic properties of the antimicrobial active substances of Liliaceae, the in vitro antibacterial activities of the ethyl acetate fraction and the n-butanol fraction prepared in Example <6-1> were examined.

Specifically, it was evaluated by a paper disk agar diffusion method. Mycelial disks of Magnaporthe oryzae of rice blast fungus Magnesium stearate 5 disks were inoculated into 100 ml PDB (potato) medium containing Erlenmeyer flasks and shaken at 150 rpm at 25 ° C for 7 to 10 days. The mixture was ground with a blender mixer One milliliter of the prepared mycelial suspension was mixed and hardened in a 100 ml potato agar medium cooled to 40 DEG C or less. Then, a paper disk (diameter: 8 mm) containing 900 g each of ethyl acetate fraction and n-butanol fraction was placed, For 2 days.

As a result, it was confirmed that the ethyl acetate fraction and the n-butanol fraction inhibited mycelial growth of rice blast fungus bacteria as shown in FIG. 2 (FIG. 2). Therefore, it was confirmed that the fractions of Bilye tree fractions exhibiting antimicrobial activity under in vitro conditions are effective ingredients for control of rice blast.

Tree ( Maesa  japonica ) Separation of the antimicrobial active substance from the ethyl acetate solvent fraction of the methanol extract

The inventors of the present invention have confirmed that the bichromatic fractions exhibiting antimicrobial activity are active ingredients exhibiting control activity against rice blast fungi. Therefore, experiments for separating and purifying antimicrobial active materials using various chromatographic techniques have been conducted.

Specifically, a silica gel liquid chromatography method was used to separate the antimicrobial active substance from the ethyl acetate fraction (4.45 g) obtained in the above Example <6-1>. The sample was loaded in a small amount of dichloromethane and loaded, and dichloromethane and methanol were then added in a ratio of 99: 1, 97: 3, 95: 5, 70: 30 &lt; / RTI &gt; by volume. The fractions were separated into E1 to E9 according to a thin film chromatography (TLC) pattern.

Among them, fraction E2 (849 ㎎) showed antimicrobial activity against rice blast fungus in the paper disk agar diffusion method. Therefore, fraction E2 was further fractionated by column chromatography on Sephadex LH-20 (GE Healthcare Life Sciences), and fraction E222241 (2.4 mg) showing antimicrobial activity was fractionated by high performance liquid chromatography (HPLC) Respectively. A Waters 515 HPLC system was used. The column was a Luna C18 (2) (5 μm, 4.6 × 250 mm) from Phenomenex Inc. and a 210-250 nm column using a photodiode array detector. An absorption spectrum of 400 nm wavelength was obtained. A linear gradient of 15% aqueous acetonitrile in 80% acetonitrile in 20% aqueous acetonitrile was followed by elution with 80% aqueous acetonitrile. The total analysis time was 30 minutes and the flow rate was 1 ml / min. Peaks detected at a wavelength of 280 nm were aliquoted and the in vitro antimicrobial activity against rice blast was evaluated by paper disk agar diffusion method.

As a result, an antimicrobial active substance (▼) showing a retention time of 15.4 minutes as shown in Fig. 3 was analyzed (Fig. 3). This antimicrobial active substance had a characteristic of exhibiting a maximum absorption wavelength value at 274 nm.

Tree ( Maesa  japonica ) Separation of antimicrobial active substance from methanol extract n-butanol solvent fraction

The present inventors also conducted the following experiment to isolate the antimicrobial active substance from the n-butanol fraction (18.71 g) obtained in the above Example <6-1>.

Specifically, a silica gel liquid chromatography method was used. About 500 g of silica gel was filled in a column (6 cm in diameter × 60 cm in height), and the sample was dissolved in a small amount of dichloromethane and loaded. Dichloromethane and methanol were then added at a ratio of 99.5: 0.5, 99: 1, 95: : 1, 85:15, 80:20, 70:30, 60:40, 50:50 by volume. The fraction B9 (3,433 mg), which exhibited antimicrobial activity against rice blast fungus, was fractionated by Sephadex LH-20 column chromatography in a paper disk agar diffusion method, and the fractions were separated into B1 to B14 according to the thin film chromatography pattern. Respectively. From these, two antimicrobially active fractions B944 (7.6 mg) and B9453 (17.0 mg) were obtained and analyzed by HPLC, respectively, in the same manner as in Example 8 above. Peaks detected at the wavelength of 210 nm or 230 nm were aliquoted and the in vitro antimicrobial activity against rice blast was evaluated by paper disk agar diffusion method.

As a result, as shown in FIG. 4, an antimicrobial active substance (●) exhibiting a residence time of 22.2 minutes was detected by HPLC analysis of the antimicrobial active fraction B944 (7.6 mg). This substance exhibited maximum activity at 222 nm and 273 nm And exhibited absorption wavelength values (FIG. 3). Based on the retention time in the column and the UV absorption spectrum, it was presumed to be a compound of a different kind from the antimicrobial active substance isolated in Example 8 above.

As a result of analysis of another antibacterial active fraction B9453 (17.0 mg) by HPLC, two antimicrobially active substances showing retention times of 11.4 minutes (♦) and 11.7 minutes (★) were detected as shown in FIG. The two antimicrobial active substances exhibited a maximum absorption wavelength value at 216 nm (Fig. 5). Because the residence time in the column is similar to the UV absorption spectrum, it is assumed that the two antimicrobially active substances are similar compounds. In addition, it can be seen that the antimicrobial active material isolated in Example 8 and the antimicrobial active material in FIG. 4 are different from each other.

Claims (10)

A plant disease control selected from the group consisting of rice blast fungus, rice sheath blight disease, tomato gray mold fungus, tomato pest, wheat red rust and red pepper anthracnose containing an extract of Maesa japonica or a fraction thereof as an active ingredient / RTI &gt;
The composition according to claim 1, wherein the extract is extracted with water, a C ₁ to C ₄ lower alcohol, n-hexane, methylene chloride, ethyl acetate, acetone or a mixture thereof as a solvent.
[3] The composition according to claim 2, wherein the lower alcohol is ethanol, methanol, propanol or butanol.
[Claim 2] The composition according to claim 1, wherein the fractions are fractionated using an organic solvent.
5. The composition according to claim 4, wherein the organic solvent is hexane, ethyl acetate or n-butanol.
The method according to claim 1, wherein the fraction is a hexane fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction obtained by phyllotaxis fractionation in the order of hexane, ethyl acetate, n-butanol, Wherein the composition is a fraction.
delete The composition of claim 1, wherein the composition is selected from the group consisting of Botrytis cinerea , Alternaria brassicicola , Colletotrichum fructicola , Fusarium &lt; RTI ID = 0.0 &gt; oxysporum , Magnaporthe oryzae , Phytophthora infestans , Rhizoctonia &lt; RTI ID = 0.0 &gt; solani and Sclerotinia sclerotiorum . The composition according to claim 1, wherein the plant pathogenic fungus is a plant growth regulator.
The method of claim 1, wherein the composition comprises Agrobacterium tyume Pacific yen (Agrobacterium tumefaciens), Burke holde Ria glue methoxy (Burkholderia glumae ) and Pectobacterium carotovorum subsp . carotovorum , which inhibits the growth of any one or more phytopathogenic bacterium.
[Claim 2] The composition according to claim 1, wherein the extract or the fraction thereof is contained in a composition for controlling plant diseases at a concentration of 100 to 3,000 [mu] g / ml.

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