KR101166853B1 - Compositions for protecting Plant diseases - Google Patents

Abstract

The present invention relates to a composition for controlling fungal plant diseases, comprising a liquid culture product of lichen-forming mold and / or eucalyptus plant extracts, and a method for controlling fungal plant diseases using the same. The control composition according to the present invention is derived from natural products and is harmless to the human body and exhibits high control activity against plant diseases without causing environmental pollution. Can be used.

Lichen forming fungus, eukaryptus, usnic acid, gallic acid, plant disease

Description

Compositions for protecting plant diseases

The present invention relates to a composition for controlling fungal plant diseases, comprising a liquid culture product of lichen-forming mold and / or a plant extract of Eucalyptus genus, and a method for controlling plant diseases using the same.

Plant diseases include 1) fungal diseases, 2) prokaryotic diseases, 3) parasitic diseases, 4) viruses and viroids, 5) nematodes, 6 A) due to protozoa, and 7) other abiotic causes. Among the biological factors, plant disease caused by fungi is the most serious. These fungi include: 1) large numbers of populations, 2) different types of propagules (hyphae, conidia, streptococcus, spermatozoa, green spores, spores, etc.); , Fungal nuclei, etc.), 4) small genome size, resulting in mutations that are difficult to control.

Currently, synthetic pesticides are most widely used to prevent or kill the spread of pests by spraying fungicides directly on plants in order to control disease caused by the above causes. In the case of such fungicides, the pests are directly killed, so that the edible effects appear quickly and are easy to use, but due to continuous overuse, drug resistance of various diseases is induced. In addition, drugs that are highly effective are highly toxic and social anxiety is created by environmental degradation such as soil pollution and water pollution. As a result, eco-friendly agricultural policies are being implemented to reduce the use of synthetic pesticides by 40%, especially among OECD member countries including Korea. As part of this policy, technology that emerges as an alternative to synthetic pesticides is biopesticides.

Bio pesticides include microbial pesticides and biochemical pesticides, which are environmentally friendly crop protection agents. Unlike synthetic pesticides, biopesticides can produce high value-added agricultural products because they are safe for human beings and ecosystems, and effectively control pests that are difficult to control with synthetic pesticides. Increase the production of safe crops and at the same time solve the problems caused by misuse of synthetic pesticides.

Milsana ™ is the most typical disinfectant product that has been successfully commercialized using plant extracts worldwide. This product is an eco-friendly disinfectant developed using extracts of Reynouria sachalinensis. It has been shown to exert resistance to plant diseases by inducing resistance to plants rather than directly killing plant pathogens. In addition, cinnamaldehyde, which is registered as a natural fungicide, is an oil extracted from seeds of Cassia tora, as well as insecticidal activity, Verticillium genus, Rhizoctonia genus, Pythium It is reported to have bactericidal activity against phytopathogens such as genus and Fusarium moniliforme var.subglutinans (canker disease). In addition, jojoba oil extracted from jojoba seeds has bactericidal activity against powdery mildew as well as insecticidal activity. Macleaya genus extract is Macleaya cordata extract and is effective in powdery mildew, Alternaria leaf spots and Septoria leaf spots. In addition, fatty acids based on oleic acid are also effective in powdery mildew.

Therefore, the present inventors have conducted research to develop a natural fungicide that is harmless to the human body by using natural products. As a result, the liquid culture product of lichen-forming fungus and the plant extract of Eucalyptus plant have excellent control against fungal plant diseases. The present invention was completed by confirming that it can be used as a natural fungicide for controlling plant diseases.

One object of the present invention is to provide a composition for controlling fungal plant diseases comprising extracts of liquid culture products of lichen forming fungus genus Ussnea.

Another object of the present invention is an extract of a liquid culture product of the lichen forming fungus genus Ussnea, a substance of any one or more of usnic acid and its agriculturally acceptable salts, and eucalyptus plant extracts, gallic acid and agriculturally It is to provide a fungal plant disease control composition comprising any one or more of the acceptable salts as an active ingredient.

Another object of the present invention to provide a method for controlling fungal plant diseases using the composition.

In one aspect, the present invention relates to a composition for controlling fungal plant diseases comprising extracts of liquid culture products of lichen forming fungus of the genus Usnea.

Lichen-forming fungi of Usnea in the present invention is a fungus belonging to lichenized fungi streptococci, and the fungus of Usnea is, for example, Usnea longgishima ( Usnea longissima , Usnea barbata , Usnea florida , Usnea hirta , Usnea rubicunda , Usnea rubiginea , Usnea rubiginea , Usnea subfloridana .

In the present invention, the "liquid culture product" refers to the culture product of the lichen-forming fungus of the genus Usnea generated while culturing the lichen-forming fungus of the genus Usna. The lichen forming fungus of Ussnea of the present invention, unlike other lichen forming fungi, the liquid culture product has antibacterial activity.

In the present invention, the "extract of the liquid culture product" refers to a substance having antifungal activity isolated by extracting the liquid culture product of the lichen-forming fungus of the genus Usnea with an organic solvent. In addition, in the present invention, "extract" is used to include not only the extract fraction, but also its dry powder or any form formulated using the same. As the extraction organic solvent, C ₁ -C ₄ alcohols such as methanol, ethanol, propanol, butanol or an aqueous solution thereof, hexane, ethyl acetate, acetone, methylene chloride, etc. may be used alone or in combination.

For example, the lichen forming fungus in Usnea is incubated in a liquid culture solution for a certain period of time, and then the culture filtrate is recovered, which is sequentially fractionated with an organic solvent, and then concentrated under reduced pressure. At this time, the fractionation process may be carried out by a process commonly used in the art. The usable solvents include hexane, ethyl acetate, methylene chloride, chloroform, acetone, ethyl ether; Or C ₁ -C ₄ alcohols such as methanol, ethanol, propanol, butanol, and the like.

In one specific embodiment, the liquid culture product of lichen forming fungus of the genus Usnea, unlike the liquid culture product of lichen forming fungus of the genus other, exhibits distinct antimicrobial activity against fungal plant pathogens and can be used for useful plant disease control. .

In another aspect, the present invention provides an extract of the liquid culture product of the lichen forming fungus genus Ussnea, substances of any one or more of usnic acid and its agriculturally acceptable salts, and eucalyptus plant extracts, gallic acid and agricultural It relates to a fungal plant disease control composition comprising any one or more substances of the salt acceptable as an active ingredient.

Usnic acid of the present invention refers to a compound having a structure such as the formula (1).

In the present invention, the Usnic acid of Formula 1 may be present in the form of a salt. The salt is an agrochemically acceptable salt of an inorganic acid such as hydrochloric acid or sulfuric acid, or an organic acid such as p-toluenesulfonic acid.

Usonic acid of Formula 1 or an agriculturally acceptable salt thereof is formulated from metabolism and culture products of microorganisms, preferably lichen-forming molds, more preferably Ussenia lichen-forming molds, preferably liquid cultures. Can be separated. In one specific implementation, it was isolated from the liquid culture product of Usnea longissima.

In one specific implementation, it was confirmed that the Usnic acid of the formula (1) is excellent in the activity of inhibiting the mycelial growth of phytopathogens can be used to control fungal plant diseases.

Eucalyptus plants of the present invention include, for example, Eucalyptus darlympleana , Eucalyptus globules , Eucalyptus unigera , etc. Preferably it is Eucalyptus darlympleana .

"Eucalyptus genus plant extract" of the present invention is meant to include crude extracts and organic solvent soluble extracts of eucalyptus plants, the "crude extracts" refers to water, lower alcohols, such as methanol, ethanol , Preferably produced by extraction with methanol or a mixture thereof. The "organic solvent soluble extract" is prepared by extracting the above crude extract with an organic solvent, for example, hexane, butanol, acetone, ethyl acetate, chloroform, dichloroform or dichloromethane, preferably butanol, ethyl acetate or dichloromethane. Say what you did.

In one specific implementation, Eucalyptus darlympleana ) to about 400 g to 600 ml of C ₁ -C ₄ alcohol was added to 100 g of leaves and evaporated under reduced pressure at 55 ° C or less, preferably 20 to 55 ° C to obtain a crude extract, which was further extracted with another organic solvent. An organic solvent extract was obtained. In this case, the organic solvent usable may include hexane, butanol, acetone, ethyl acetate, chloroform, dichloroform or dichloromethane. In the present invention, it is also possible to perform the fractionation process commonly used in the art.

In one specific embodiment, the plant extract of the genus Eukaryptus of the present invention showed antimicrobial activity against various phytopathogens, in particular excellent antibacterial activity against pepper anthrax. Therefore, the eucalyptus plant extract of the present invention can be used for the purpose of controlling natural plant diseases.

Gallic acid of the present invention is a compound having a structure as shown in the following formula (2), the compound name is 3,4,5-trihydroxybenzoic acid (3,4,5-trihydroxybenzoic acid).

In the present invention, the gallic acid of Formula 2 may be present in the form of a salt. The salt is an agrochemically acceptable salt of an inorganic acid such as hydrochloric acid or sulfuric acid, or an organic acid such as p-toluenesulfonic acid.

The gallic acid of Formula 2 or an agrochemically acceptable salt thereof may be isolated or chemically synthesized from a plant extract, and in one specific embodiment, a plant extract of the genus Eukaryptus, or Eucapritus Darlimpreana ( Eucalyptus darlympleana ) can be obtained by fractionation from the extract.

In one specific embodiment, the gallic acid of the formula (2) was confirmed to inhibit the mycelial growth of plant pathogens, the production of conidia spore germination and the formation of spores of the spores.

Extracts of the liquid culture product of Ussenia lichen forming fungus of the present invention, Usnic acid or agriculturally acceptable salts thereof, Eucalyptus plant extracts, Gallic acid or agriculturally acceptable salts thereof are plant pathogens, preferably fungi Sex plant pathogens, more preferably pepper anthrax, rice blast, rice leaf blight, tomato ash fungus, tomato blight, wheat red rust, barley powdery mildew, pisium mozambique, fungus rot fungus and alter It has antimicrobial activity against narcosis bacteria, even more preferably pepper anthrax. In addition, the extract of the liquid culture product of Usnea lichen-forming fungus of the present invention, Usonic acid or its agriculturally acceptable salt, Eucalyptus plant extract, Gallic acid or its agriculturally acceptable salt are both spores and mycelium It can be controlled, sprayed at high concentration, no harm, no harm to human body or plant.

In particular, an extract of the liquid culture product of the genus Lichen forming fungus of Ussenia, any one or more substances of Usnic acid and its agriculturally acceptable salts, and any of Eucalyptus plant extracts, gallic acid and its agriculturally acceptable salts When the above substances are mixed and treated with plant pathogens, the antimicrobial activity is remarkably superior to those when the respective substances are treated. In one specific embodiment, the treatment of the mixture showed about 3.3 to 10.6% improved antimicrobial activity than the treatment of each substance, and nearly the same as the treatment of the chemical control agent propineb hydrate. It was.

The control composition of the present invention may further include a substance contained in a conventionally used fungicide, and in addition to the active ingredient, an agriculturally acceptable solid carrier, liquid carrier, liquid diluent, liquefied gas diluent, solid diluent as excipients Or other suitable auxiliaries such as emulsifiers, dispersants or foaming agents. The composition for controlling the active ingredient and the excipients may be formulated into various formulations known in the pesticide field, and any formulation method commonly used in the pesticide field may be used for formulation.

The control composition of the present invention may preferably be formulated in a hydrated, granulated, powdered, emulsified, sprayed, smokescreen, capsule and gelled formulation and contacted through a formulation such as a donut for buoyancy of the formulation. Preferably provided as.

It may be formulated by mixing with the present agriculturally acceptable carrier material or diluent. In addition, these compositions may be used in admixture with fertilizers or surfactants or known plant disease control agents as any additional component. As used herein, the term "diluent" means an agriculturally acceptable liquid or solid added to the composition of the present invention for ease of use or for diluting the composition of the present invention to the desired active concentration. Talc, kaolin, zeolite, xylene, diatomaceous earth, water and the like.

The control composition of the present invention is a fungal plant, such as pepper anthrax, rice blast, rice leaf blight, tomato gray mold, tomato blight, wheat red rust, barley powdery mildew, pisium mozzarella, fusidia rot or alternaria disease. In order to effectively control pathogens, the liquid culture product of Ussenia fungus is used at a concentration of 500 to 3,000 µg / ml, or the extract of the liquid culture product of Ussenia fungus or Usnic acid or an agriculturally acceptable salt thereof is 100 Preference is given to using a concentration of from 100 to 3,000 µg / ml, and a plant extract of the genus Eukaryptus or gallic acid or an agriculturally acceptable salt thereof at a concentration of 100 to 3,000 µg / ml. However, the amount of use can be appropriately adjusted in consideration of the type of crop, growth degree, cropland environment, the extent of the development of plant diseases.

The control composition of the present invention formulated as described above is treated to plants or areas in need of control, pepper anthrax, rice blast, rice leaf blight, tomato ash fungus, tomato blight, wheat rust, wheat flour, barium You can control Mozolox disease, Fusidium wilting disease or Alternaria disease. The preferred method is to apply the control composition so as to be in direct contact with the plant pathogens to control, the most preferred is a method of mixing the soil or spraying directly to the crop.

As described above, the control composition of the present invention is derived from lichen-forming fungi or natural products of plants, so it is harmless to the human body and shows high control activity against fungal plant pathogens without causing environmental pollution. It can be usefully used for the development of fungicides and production of high value-added organic agricultural products.

The invention will be explained in more detail through the following examples. These examples are presented by way of example only, so that the present invention may be more understood, these examples should not limit the scope of the invention.

Example 1 Investigation of Antimicrobial Activity against Culture Products of Lichen Formation Mold

Fourteen kinds of lichen-forming fungal strains showing excellent antimicrobial activity against the red pepper anthrax bacterium Colletotrichum accutatum were selected (see Table 1) to search for antimicrobial active substances in culture products. Specifically, lichen-forming molds cultured in MY medium for 2 months were immersed under sterile conditions (maceration), and then inoculated with MY liquid medium (2L), and the bottom aeration culture was performed at 18 ° C. for 2 months (see FIG. 1). After completion of the culture, the culture filtrate was recovered, and the culture product was sequentially fractionated by hexane (n-hexane), ethyl acetate, and water fractions, concentrated under reduced pressure, and redissolved in 5% DMSO to inhibit the antimicrobial activity against agar well. It was investigated using the well method.

Antimicrobial Activity of Sequential Fractions of Lichen-forming Mold Culture Filtrate Against Pepper Anthrax Bacteria Isolation number Lichen-forming fungi n-hexane ethyl acetate water 030397 Cetrelia japonica - - - 050657 Lecanora argentata - - - CH050099 Ramalina complanata - - - 041183 Parmelia sp. - - - 040810 Parmelia adaugescens - - - 041466 Cladonia metacorallifera - - - 040040 Umbilicaria esculenta - - - 050319 Ramalina litoralis - - - CH050316 Usnea longissima ++ + - H06020 Evernia prunastri - - - H06049-1 Cladonia coicraea - - - H06099 Lasallia pustulata - - - H060125 Hypogymnia physodes - - - 030730 Myelochroa irrugans - - -

As can be seen in Table 1, the antimicrobial activity of the sequential fraction of the culture filtrate showed a distinct antimicrobial activity only in Usnea longissima , unlike solid culture (see Figure 2). This indicates that the growth of lichen-forming fungi and the production of secondary metabolites are very different depending on the culture conditions, which is consistent with the report that the production of secondary metabolites is more generated under stress conditions than the culture conditions most favorable for fungal growth. Could know.

Example 2 Isolation of Antimicrobial Active Substances from Culture Products of Lichen Forming Mold and Evaluation of Antimicrobial Activity of the Separated Substances

In order to separate the antimicrobial active material from the culture filtrate of lichen-forming mold, a process as described in FIG. 3 was performed. The resulting fraction was confirmed to be Usnic acid by using the standard of Usnic acid on TLC (see Fig. 4), and the final result was Usnic acid using HLPC (Yamamoto Professor's Laboratory, Akita University, Japan). It was confirmed (see FIG. 5).

As a result of investigating the antimicrobial effect against red pepper anthrax bacteria using the isolated usnic acid, it was confirmed that mycelial growth is completely inhibited at a concentration of 2,000 ppm (see FIG. 6).

Example 3 Antimicrobial Activity of Eucalyptus Plants

The leaves of Eucalyptus darlympleana plants of ages 5 to 7 were collected, dried naturally, and dried in a 65 ° C. dryer for 3 days. The dried leaves were then milled and stored at 4 ° C. for experiment.

100 g of the pulverized eucalyptus leaves were extracted three times with 500 ml of methanol, and then filtered, and the resultant was evaporated under reduced pressure at a temperature of 55 ° C. or lower. Thereafter, the concentrate was re-dissolved in 30 ml of methanol, and then the inoculation of 500 μl of the concentrate and methanol (control) were measured for antimicrobial activity against C. coccodes, C. gloeosporioides, and C. acutatum by the Agwell method. The results are shown in Fig.

As shown in FIG. 7, the eucharyptus extract exhibited antimicrobial activity, in particular, excellent antimicrobial activity against C. acutatum .

Example 4 Isolation of Antimicrobial Activity from Eucalyptus Plant Extracts and Investigation of Antimicrobial Activity

The methanol extract of the plant of the genus Eukaryptus of Example 3 was sequentially partitioned into CH ₂ Cl ₂ , ethyl acetate, butanol and water, and the antimicrobial activity of each of these fractions against C. coccodes, C. gloeosporioides, and C. acutatum . Was investigated by the Agwell method. As a result, the ethyl acetate and butanol fractions were identified as antimicrobial activity (not shown), and purified using silica gel and Sephadex LH-20 column chromatography to identify compounds (Park et al ., Inhibitory effects of methanol extract, phenolic acids and flavonoids from the leaves of Eucalyptus darlympleana against 1,1-Diphenyl-2-picrylhydrazyl radical. Natural Product Sciences 10: 244-247).

The identified compound was confirmed to be gallic acid of Chemical Formula 2, and the treatment of garlic acid by the concentration of pepper anthrax C. acutatum inhibited the mycelial growth of C. acutatum , and inhibited 60% mycelial growth at 2,000 ppm. (See Figure 8). In addition, C. acutatum condensed spore germination was suppressed at about 70% at a concentration of 125 ppm, and in the case of some normally germinated spores at this concentration, adhesion formation was inhibited (see FIGS. 9 and 10).

Example  5: lichen Culture products Eucalyptus  Biology Using Genus Plant Extracts Control  Manufacture and effect

Lichen liquor is very non-polar and hardly soluble in water. Eucalyptus extract also has low solubility in water, so these natural raw materials were completely dissolved using DMSO and used as a raw material for liquid hydrating agents. At this time, the amount of DMSO used was adjusted to the final concentration of 5%, and when only DMSO was sprayed on the red pepper seedlings, it was confirmed that no effect was observed. Fully inhibited mycelial growth by Usnic acid was investigated at 2,000ppm, and about 200% of mycelial growth was detected at 2,000ppm of gallic acid isolated from eucharyptus, and spore germination and adhesion formation of anthrax were completely inhibited at 500ppm. In consideration of the fact that the final spray concentration for the actual package verification was prepared to adjust to 500ppm for lichen extracts to 500ppm for the eucalyptus extract. Water was diluted in the raw material to the above concentration for the actual package spraying, and tween 20 (0.1%) was added as an electrodeposition agent and sprayed evenly to flow through the pepper plant. As a result, the mature pepper plant was 250 to 300 ml per week. Natural pesticides were sprayed.

The test was carried out in the 3rd repetition of the ingot method on the plastic house packaging of Sunchon National University affiliated farm, which was the same as the open field red pepper cultivation test package. Unlike the open-air pavement, where natural infections occur well, pepper anthrax does not occur in the isolated plastic house. Therefore, in order to induce the occurrence of red-rop anthrax, the on-site pavement was infected with pepper anthrax on October 8 and October 15 The diseased fruits from which the conidia were flowed out from the spore layer were hung on the ceiling of the plastic house at each treatment section, and each night, the sprinkling of conidia was sprayed by spraying clean water with a sprayer at 6 o'clock to inoculate red pepper seedlings. In order to test the control effect on pepper anthrax, the lichen extract to be tested and the extract of eukaryptus were treated alone or mixed three times at intervals of about 10 days from October 9, 2008. Was sprayed with a propineb hydrating agent used as a control agent. An outbreak investigation was conducted on October 30 to test the control effect of red pepper anthrax in each treatment group, and the outbreak rate was calculated and evaluated in the same manner as the field cultivation field test.

As shown in Table 2, the average incidence of untreated was 32.9%, whereas the incidence of pepper anthrax was 7.6% in the lichen extract alone treatment group, and the incidence of pepper anthrax was 10.0% in lichens and eucharib. The incidence of pepper anthrax was 6.5% in the Tooth extract blend treatment and 6.2% in pepper anthracnose in the propineb hydration treatment.

The control value was calculated based on the rate of incidence of pepper anthrax in each treatment group, and the lichen extract alone treatment was 76.9%, the eucharyptus extract alone treatment was 69.6%, the lichen and eucharyptus extract mixed treatment was 80.2%, and the propineb hydration treatment was 81.1%. Therefore, lichens and eucalyptus extracts alone and their mixtures have a control effect, in particular, lichens and eucalyptus extract mixtures have been found to have a control effect comparable to the chemical chemical propinev hydrate.

Control of Pepper Anthrax in Green House Test drug Incidence and Rate (%) Difference
(DMRT) Control
(%) Ⅰ repeat ⅡRepeat ⅢRepeat Average Lichen extract 7.5 9.6 5.6 7.6 a 76.9 Eucalypt Extract 9.2 7.3 13.5 10.0 a 69.6 Lichen + Eucalyptus Extract 2.4 10.8 6.3 6.5 a 80.2 Propineb Hydrating 6.6 3.9 8.1 6.2 a 81.1 No treatment 32.5 35.9 30.2 32.9   b

(Pharmaceutical treatment; October 9, October 16, October 23, pepper anthrax outbreak investigation: October 30)

1 is a process flow chart for obtaining a sequential fraction through the bottom aeration liquid culture of lichen forming mold.

Figure 2 is an illustration of the antimicrobial activity of the n-hexane fraction of the liquid culture filtrate of Usnea longissima against Colletotrichum accutatum , a pepper anthrax bacterium.

3 is a process flow chart for separating antimicrobial active material from the liquid culture filtrate of Usnea longissima .

Figure 4 is a result of confirming the use of the acidic acid using TLC for the antimicrobial active material isolated from the liquid culture filtrate of Usnea longissima ( Usnea longissima ).

5 is a result of confirming the use of the acidic acid using HPLC for the antimicrobial active material isolated from the liquid culture filtrate of Usnea longissima ( Usnea longissima ).

FIG. 6 shows the results of observing antimicrobial activity against Colletotrichum accutatum , a pepper anthrax bacterium of Usnic acid isolated from the liquid culture filtrate of Usnea longissima .

FIG. 7 is a diagram illustrating the antimicrobial activity of C. coccodes, C. gloeosporioides and C. acutatum in methanol extract of Eucalyptus darlympleana .

FIG. 8 is a diagram showing the mycelial growth inhibitory effect on the pepper anthrax C. acutatum of the gallic acid, a compound isolated from the extract of Eucalyptus darlympleana .

Figure 9 is a diagram showing the effect of inhibiting the conidia spore germination of the pepper anthrax C. acutatum of the gallic acid, a compound isolated from the extract of Eucalyptus darlympleana .

Figure 10 is a diagram showing the inhibitory effect of the adhesion group production on the pepper anthrax C. acutatum of gallic acid, a compound isolated from the extract Eucalyptus darlympleana ( Eucalyptus darlympleana ). A is a control, B is a case of 125ppm gallic acid, C is a case of 500ppm gallic acid. As can be seen in Figure B, spores that germinated normally were found to have suppressed adhering formation.

Claims (8)

Fungi comprising an extract of a liquid culture product of lichen forming fungus of the genus Usnea, and a substance selected from the group consisting of plant extracts of the genus Eucalyptus, agriculturally acceptable salts of gallic acid and gallic acid Composition for controlling sexual plant diseases. The composition for controlling fungal plant diseases according to claim 1, wherein the liquid culture product comprises ausic acid or an agriculturally acceptable salt of ausic acid. delete The plant disease of claim 1, wherein the plant disease consists of pepper anthrax, rice blast, rice leaf blight, tomato ash mold, tomato blight, wheat red rust, barley powdery mildew, piumum mozalococcal disease, fusidium wilting disease, and alternaria disease. Composition for controlling fungal plant diseases, characterized in that at least one from the group. delete delete delete A method for controlling fungal plant diseases, comprising treating a crop or soil with the composition for control of any one of claims 1, 2 or 4.

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