KR102598633B1 - Composition for controlling plant disease comprising chrysoeriol 7 or cochlioquinone 9 compound as effective component and uses thereof - Google Patents

Abstract

The present invention relates to chrysoeriol 7 or cochlioquinone 9; Or, it relates to a composition for controlling plant diseases containing agrochemically acceptable salts thereof as an active ingredient, and a method for controlling plant diseases comprising the step of treating plants with the composition.

Description

Composition for controlling plant disease comprising chrysoeriol 7 or cochlioquinone 9 compound as effective component and uses thereof}

The present invention relates to a composition for controlling plant diseases containing chrysoeriol 7 or cochlioquinone 9 as an active ingredient and its use.

Rice ( Oryza sativa L.) is the primary food crop for more than 50% of the world's population and is one of the most important crops in the world. According to a report on agricultural outlook by the OECD, FAO and other international organizations, the world's population will reach about 9.6 billion by 2050 and food demand will increase by more than 60%. Significant increases in rice production are needed over the next few decades to meet the needs of a rapidly growing population.

However, in the natural environment, rice is susceptible to attack by pathogens such as pests, nematodes, viruses, bacteria, and mold. These attacks cause disease in rice and cause significant yield losses worldwide, threatening rice supplies and global food security. Migratory insects also cause major problems in Asia, especially the small brown planthopper (SBPH; Laodelphax striatellus Fallen), Nilaparvata lugens (BPH), and white-backed planthopper (WBPH; Sogatella furcifera Horvath). It is well known for causing serious damage in Southeast Asia.

Some plant extract-derived compounds have been recognized as pesticide substitutes in environmentally friendly agriculture because they contain bioactive substances and cause little harm to humans. Chrysoeriol, also known as 3'-0-methylluteolin, is a 3'-O-methylated flavonoid. Chrysoeriol is mainly found in the leaves of Eriodictyon glutinosum Bentham, but is also present in a variety of plants, vegetables, fruits, and flowers. Chrysoeriol is of great scientific interest due to its antioxidant, anti-inflammatory, antibacterial, and antiviral properties. Glucosides bound to chrysoeriol are isoflavonoids that play an important defense role against invasion by pathogens and insects. Chrysoeriol has antibacterial activity against various pathogens. In particular, it was found to contain a derivative of artemisinin, a malaria drug caused by Plasmodium falciparum . Chrysoeriol strongly induces the expression of heme oxygenase (HO)-1, an antioxidant enzyme, and at the same time promotes intranuclear movement of the transcription factor Nrf2 , thereby strengthening the antioxidant effect. Additionally, it blocks AP-1 activity and strongly inhibits the induction of nitric oxide synthase.

When insects invade the leaves, fungi grow around the wounds and produce a variety of interesting plant toxins. Fungi produce plant toxins that cause plant stress, but they also produce substances to protect plants from insect attack. Cochlioquinone is a fungal flavonoid and antibacterial compound transformed by Clusia spp. WBPH induces the fungus into the leaves, which produces coclioquinone A. The parasitic fungus Cochliobolus miyabeanus produces the yellow pigments Cochlioquinone A and B as small metabolites. Their mixed biosynthesis occurs by introducing a farnesyl unit into an aromatic precursor, in which a secondary methyl group is derived from methionine. Cochlioquinone is a biologically active substance and is sometimes used as a nematicide that competes for specific 3H ivermectin binding sites. Cochlioquinone A1, isolated from Bipolaris zeicola , is used as a powerful angiogenesis inhibitor.

Meanwhile, Korean Patent Publication No. 2017-0095970 discloses an 'antimicrobial composition' containing one or more bioflavonoids, and Korean Patent Publication No. 2016-0114424 discloses 'Chrysoeriol from rice inoculated with white-backed planthoppers'. Although the 'method of separation' is disclosed, there is no description regarding the 'composition for controlling plant diseases containing chrysoeriol 7 or cochlioquinone 9 compound as an active ingredient and its use' of the present invention.

The present invention was derived from the above needs, and the present inventor extracted two flavonoid compounds known as whitebacked planthopper ( Sogatella furcifera ) resistant substances from rice plants, and tested the antimicrobial activity of the compounds against various pathogenic bacteria. and fungi, the chrysoeriol 7 compound was found to be against Fusarium graminearum and Pythium graminicola, and cochlioquinone 9 was against Fusarium graminearum and Pythium graminicola . The compound is effective against Cladosporium herbarum , C. cladosporioides , Gibberella zeae , Fusarium graminearum and Pycium graminicola. The present invention was completed by confirming that it has excellent antifungal activity.

In order to solve the above problem, the present invention is chrysoeriol 7 (chrysoeriol 7) or cochlioquinone 9 (cochlioquinone 9); Alternatively, a composition for controlling plant diseases containing agrochemically acceptable salts thereof as an active ingredient is provided.

Additionally, the present invention provides a method for controlling plant diseases comprising treating plants with the composition.

The composition of the present invention contains chrysoeriol 7 or cochlioquinone 9 compounds as active ingredients, which have excellent growth inhibition effects against fungal pathogens, and can be usefully used to improve the productivity of plants. It is believed that it can be done.

Figure 1 shows LC/MS results of chrysoeriol 7 (C7) and coclioquinone 9 (C9) compounds. The flow rate was consistently 50 μl/min with a capillary temperature of 320°C and a capillary voltage of 49V. (A) Chrysoeriol 7. Mass: 267.36 and 381.37 m/z, 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one, C ₁₆ H ₁₂ O ₆ , molecular weight = 300.26 g/mol. (B) Cochlioquinone 9. Mass: 381.38 and 480.40 m/z, (3R)-9-[(1S,2R,3S)-2-acetyloxy-1,3-dimethylpentyl]-1,2,3,4aβ ,5,6,6a,12,12aβ,12b-decahydro-12β-hydroxy-3α-(1-hydroxy-1-methylethyl)-6aα,12bα-dimethylpyrano[3,2-a]xanthene-8,11-dione , C ₃₀ H ₄₄ O ₈ , molecular weight = 532.7 g/mol.
Figure 2 shows the antimicrobial activity results of compounds C7 (left) and C9 (right).

In order to achieve the object of the present invention, the present invention is chrysoeriol 7 (chrysoeriol 7) or cochlioquinone 9 (cochlioquinone 9); Alternatively, a composition for controlling plant diseases containing agrochemically acceptable salts thereof as an active ingredient is provided.

In the composition for controlling plant diseases of the present invention, chrysoeriol 7 or cochlioquinone 9 is a flavonoid compound. Chrysoeriol 7 (5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)chromen-4-one) is a compound represented by the following formula 1, and coclioquinone 9((3R)-9- [(1S,2R,3S)-2-acetyloxy-1,3-dimethylpentyl]-1,2,3,4aβ,5,6,6a,12,12aβ,12b-decahydro-12β-hydroxy-3α-(1 -hydroxy-1-methylethyl)-6aα,12bα-dimethylpyrano[3,2-a]xanthene-8,11-dione) is a compound represented by the following formula (2).

As used herein, the term "agrochemically acceptable salt" may include, for example, metal salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, etc. there is. Suitable metal salts include, for example, alkali metal salts such as sodium salts, potassium salts, etc.; Alkaline earth metal salts such as calcium salts, magnesium salts, barium salts, etc.; Aluminum salts, etc. may be included. Salts with organic bases are, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N Salts with , N-dibenzylethylenediamine, etc. may be included. Salts with inorganic acids may include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with organic acids may include, for example, salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. . Salts with basic amino acids may include, for example, salts with arginine, lysine, ornithine, etc. Salts with acidic amino acids may include, for example, salts with aspartic acid, glutamic acid, etc.

In the composition for controlling plant diseases according to the present invention, the plant disease may be a disease caused by a fungus, and the fungus includes Fusarium graminearum , Pythium graminicola , and Clado. It may be one or more selected from the group consisting of Cladosporium herbarum, C. cladosporioides , and Gibberella zeae , but is not limited thereto.

In one embodiment of the present invention, the chrysoeriol 7 compound is cochlioquinone 9 against Fusarium graminearum and Pythium graminicola. The compound is effective against Cladosporium herbarum , C. cladosporioides , Gibberella zeae , Fusarium graminearum and Pycium graminicola. It showed excellent antifungal activity.

The composition for controlling plant diseases of the present invention may further include agrochemically acceptable carriers and/or excipients. Excipients included in the composition of the present invention are commonly used in the agricultural field, for example, oxides such as diatomaceous earth and slaked lime, phosphates such as apatite, sulfates such as gypsum, clay, kaolin, bentonite, acid clay, quartz, etc. It may include additional solid carriers such as mineral powders such as silica, fillers, anti-coagulants, surfactants, emulsifiers, preservatives, etc. In addition, the composition of the present invention can be formulated using methods known in the art to treat plants to allow rapid, sustained, or delayed release of the active ingredient. For formulation, commonly used additives such as surfactants, diluents, dispersants, and auxiliaries are mixed with the active ingredients to create hydrating agents, suspensions, emulsions, emulsions, emulsifiers, liquids, dispersible liquids, granular hydrates, granules, powders, etc. It can be formulated and used in various forms such as liquid moisturizer, sleep aid, tablet, etc.

The composition for controlling plant diseases according to the present invention is prepared, for example, in the form of directly sprayable solutions, powders and suspensions or as highly concentrated aqueous, oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, scattering substances or granules. It can be done, but is not limited to this. Additionally, the composition for controlling plant diseases can be used by spraying, spraying, spraying, scattering or pouring. The form of use depends on the intended purpose; in all cases it should be ensured that the distribution of the composition according to the invention is as fine and uniform as possible.

The composition for controlling plant diseases of the present invention can be formulated in various forms. The formulation can be prepared, for example, by adding a solvent and/or carrier. Often, inert additives and surface-active substances, such as emulsifiers or dispersants, are mixed into the formulation. Suitable surface-active substances include aromatic sulfonic acids (e.g. lignosulfonic acid, phenol-sulfonic acid, naphthalene- and dibutylnaphthalenesulfonic acid), fatty acids, alkyl- and alkylarylsulfonates, alkyl lauryl ethers, alkalis of fatty alcohol sulfates. Metals, alkaline earth metals, ammonium salts, sulfated hexa-, hepta- and octadecanols, salts of fatty alcohol glycol ethers, sulfonate naphthalene and its derivatives, condensates of formaldehyde, naphthalene or naphthalenesulfonic acid, phenol and formaldehyde. Condensates, polyoxyethyleneoctyl phenol ethers, ethoxylated isooctyl-, octyl- or nonylphenol, alkylphenyl or tributylphenyl polyglycol ethers, alkylarylpolyether alcohols, isotridecyl alcohol, fatty alcohol/ethylene oxide condensates. , ethoxylated castor oil, polyoxyethylene alkyl ether or polyoxypropylene, lauryl alcohol polyglycol ether acetate, sorbitol ester, lignin-sulfite waste liquid, or methylcellulose, but is not limited thereto.

Suitable solid carrier materials are, in principle, all porous and agriculturally acceptable carriers, for example mineral earths (e.g. silica, silica gel, silicates, talc, kaolin, limestone, lime, chalk, bowl, red clay, clay, Dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetics), fertilizers (e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea), vegetable products (e.g. grain flour, bark meal, wood meal) and nut shell powder) or cellulose powder, but is not limited thereto. Additionally, the above solid carrier may be used alone or in a mixture of two or more types.

The composition for controlling plant diseases of the present invention can be used for irrigation, dipping, foliar spraying, seed disinfection, or agricultural equipment disinfection, but is not limited thereto.

The composition for controlling plant diseases of the present invention can use chrysoeriol 7 or cochlioquinone 9 as an active ingredient alone or in combination with two or more other antibacterial/antifungal substances.

The composition for controlling plant diseases of the present invention can be mixed with dispersing agents, penetrants, or surfactants to improve crop absorption and effectiveness.

The present invention also provides a method for controlling plant diseases, comprising treating plants with the composition for controlling plant diseases of the present invention.

The composition for controlling plant diseases of the present invention is a composition containing chrysoeriol 7 or cochlioquinone 9 as an active ingredient.

In the method for controlling plant diseases according to the present invention, the plant diseases are the same as described above.

The composition of the present invention can be treated to plants by conventional methods. When applied to plants, the treatment can be sprayed or applied directly to the leaves, stems, branches, roots, and seeds of plants, mixed with general cultivation soil such as rice fields or fields, or seedling media or medium, or applied to plants grown in water. In some cases, it can be treated on the surface of the water to control diseases. Specific application methods are application treatment, dipping treatment, fumigation treatment or spray treatment, for example, the composition can be sprayed on soil, plant leaves, stems, seeds, flowers or fruits. In order to apply the composition of the present invention to plants, it can be diluted in water or an appropriate medium.

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. White-backed planthopper breeding and plant materials

Fifty male and female white-backed planthoppers (WBPH) were obtained from the National Institute of Agricultural Science, Rural Development Administration. The aviary was made of acrylic board (50 x 50 x 40 cm), and a 100 ㎛ mesh net was used on the back for ventilation and water supply. The insect farm was maintained at a temperature of 27 ± 1°C, humidity of 60-70%, and light intensity of 2000 lux over a 16-hour period. Chucheong rice is one of the most preferred rice varieties by WBPH and was used for food by WBPH. 20-25 g of seeds were sterilized using Sportak pesticide, then soaked in water and placed in an incubator at 33°C for 3 days, and washed with clean water every morning. After sowing in 21 The leaves were provided as food to the dogs. Food was changed weekly to help sustain the next generation of WBPH. When WBPH was in the 2nd or 3rd instar, it was inoculated into plant materials such as Cheongcheong rice, Nakdong rice, and TN1 rice.

Cheongcheong, Nakdong, and TN1 were harvested at the Kyungpook National University experimental site located in Gunwi in 2018. Cheongcheong is a highly resistant cultivar, while Nakdong and TN1 are cultivars sensitive to WBPH. 60 g of seeds were sterilized in 1 L of water at 33°C for 3 days and then washed with clean water every morning. After sowing in plastic containers (21 Fifty 2nd or 3rd instar WBPH were inoculated in Chungcheong, Nakdong, and TN1 for one week.

2. Bacterial and fungal pathogens

Two types of bacteria and 11 types of fungi that cause disease in rice were obtained from the National Institute of Agricultural Science, Rural Development Administration (Table 1). The freeze-dried bacteria were suspended in 5 mL of Luria-Bertani (LB) medium and cultured for 3 days at 30°C with a stirring speed of 130 rpm. The freeze-dried mold was suspended by adding 0.3 to 0.5 mL of sterilized distilled water, and then 0.1 mL was inoculated onto potato dextrose agar (PDA) solid medium using a pipette and cultured at 25°C for 1 week. After culturing, growth and contamination of the strain were confirmed, and pure culture strains were used in the experiment.

3. Extraction and separation of compounds

After cutting the leaves of Cheongcheong, Nakdong, and TN1 inoculated with WBPH, liquid nitrogen was added and ground in a mortar and pestle. 35g of each leaf was used. Then, 35 g of plant sample and 350 mL of 70% methanol were added to a 1000 mL Erlenmeyer flask and reacted overnight at 20°C while stirring at a speed of 130 rpm. A filter paper (Whatman Grade 2 filter paper diameter: 15 cm, pore size: 8 uL) was fixed to the funnel, the sample was poured onto the filter paper, and this filtration process was repeated twice.

Samples were washed using an equal volume of n-hexane. Equal amounts of sample and n-hexane were added to the separation glass, shaken for a few minutes and left until the layers were completely separated before collecting the bottom layer of the solution. Samples were concentrated using a rotary evaporator. Concentration was performed under the following conditions: water bath temperature of 30℃, cooling solution of -3~5℃, and speed of 6~8. The sample was concentrated until the volume was approximately 10 mL and then concentrated further using a heating block. The sample in each tube was heated at 60°C until it reached approximately 0.3 mL.

125 g of Silica Gel 60 was poured into a glass column (10 x 250 mm diameter) using a funnel. After washing the silica gel with 20% methanol, the concentrated sample was added until the silica gel was submerged. Pour 20 mL solution into the column and wait until the solution falls. The elution gradient was 4% methanol and 20% acetone. After collecting the solution in the tube until all samples had fallen, the eluate was dried with a heating block at 50°C. After drying, the compound was confirmed using TLC silica gel 60F ₂₅₄ play with a mobile phase of chloroform:methanol:1-butanol:water = 4:5:6:4.

The silica matrix containing the compound was cut to separate C7 and C9. Each compound was then separated from the silica matrix using a small column and eluted with 20 mL of 4% methanol (in 100% acetone) to obtain purified compounds. The purity of the compound was confirmed by TLC silica gel 60F ₂₅₄ plate.

4. LC/MS

The present inventors analyzed the compounds using MSQ Plus Single Quadrupole mass spectrometer (Termo Fisher Scientific, USA). The injection concentration was a 1:1000 diluted sample using 50% methanol (in 0.1% formic acid), and the flow rate was 50 L/min.

5. Testing the antimicrobial activity of compounds

The separated compounds were dried using a heating block, dissolved in distilled water at three concentrations (100 ppm, 500 ppm, and 1,000 ppm), and then 0.1 mL was added to the medium. In the case of bacteria, the liquid LB medium was suspended in distilled water to adjust the OD value to 1.0 at 600 nm, and then 0.1 mL of bacterial culture was dropped into the LB medium using a pipette and cultured at 30°C in dark conditions. In the case of fungi, a portion (0.5 x 0.5 cm) of the cultured mycelium was transferred to PDA medium and cultured under dark conditions at 25°C. The growth rate was checked for 1 to 2 weeks after inoculation, and the diameter of the colony was measured. The following formula was used to calculate the growth inhibition rate (%) by the compound.

6. Statistical analysis

All experiments were repeated at least three times, and statistical analyzes were performed using the SPSS program (IMSPSS Statistics version 22, IBMSPSS Statistics version 22; Redmond, USA).

Example 1. Compound extraction efficiency according to variety

Chrysoeriol 7 (C7) and cochlioquinone 9 (C9) were isolated from rice plants inoculated with white-backed planthoppers. For C7, 1.7 mg, 3.6 mg, and 12.1 mg were isolated from Cheongcheong, Nakdong, and TN1 rice, respectively, and for C9, 9.0 mg, 4.1 mg, and 14.0 mg were isolated from rice, respectively. For both C7 and C9, the highest yields were obtained in the TN1 variety.

Example 2. LC/MS data

LC/MS analysis was performed to identify the compounds. The LC/MS chromatograms of C7 and C9 are shown in Figure 1. Positive and negative LC/MS data revealed that C7 had molecular weights of 267.36 and 381.37 m/z, and C9 had molecular weights of 381.38 and 480.40 m/z.

Example 3. Antimicrobial activity using compounds

In the antibacterial activity test, chrysoleriol 7 (C7) treatment concentration-dependently inhibited the growth of Fusarium graminearum and Pythium graminicola (p < 0.05) (Figure 2) ). The inhibition rate of F. graminearum one week after inoculation was 10.9%, 26.6%, and 28.7% at C7 treatment concentrations of 100 ppm, 500 ppm, and 1,000 ppm, respectively. In addition, the inhibition rate of P. graminicola one week after inoculation was confirmed to be 22.6%, 48.54%, and 62.27% at C7 treatment concentrations of 100 ppm, 500 ppm, and 1,000 ppm, respectively (Tables 2 and 3).

In the antibacterial activity test of cochlioquinone 9 (C9), Cladosporium herbarum , C. cladosporioides , Gibberella zeae , and Fusarium gras Growth inhibition of F. graminearum and P. graminicola was confirmed (p < 0.05). One week after inoculation, the inhibition rate of Cladosporium herbarum ( C. herbarum ) was 5.0%, 9.9%, and 10.1% at C9 treatment concentrations of 100 ppm, 500 ppm, and 1,000 ppm, respectively. The inhibition rates of Cladosporium cladosporioides ( C. cladosporioides ) were found to be 12.4%, 14.3%, and 15.5%, respectively, and the inhibition rates of Gibberella zeae ( G. zeae ) were 7.6%, 8.1%, and 24.6%, respectively. In percentage terms, it was found that the inhibition rate was significantly high at the C9 treatment concentration of 1,000 ppm. The inhibition rates of F. graminearum were 9.7%, 11.3%, and 20.7%, respectively, and the inhibition rates of P. graminicola were 26.4%, 27.3%, and 36.2%, respectively, which were relatively high. showed (Table 4 and Table 5).

It was observed that the growth of Fusarium graminearum ( F. graminearum ) and Pyceum graminicola ( P. graminicola ) was inhibited by both C7 and C9 compounds. Antifungal activity against Fusarium graminearum was slightly higher in C7, and antifungal activity against Pycium graminicola was higher in C7. However, both C7 and C9 compounds failed to show antibacterial activity against bacteria. The above results suggested that chrysoeriol 7 or cochlioquinone 9 compounds could be useful as ingredients for controlling plant diseases caused by fungal pathogens.

Claims (7)

chrysoeriol 7 or cochlioquinone 9; Or a composition for controlling plant diseases caused by mold containing agrochemically acceptable salts thereof as active ingredients,
The fungus is a group consisting of Pythium graminicola , Cladosporium herbarum , C. cladosporioides and Gibberella zeae A composition for controlling plant diseases caused by fungi, characterized in that it contains at least one selected from. delete delete The composition for controlling plant diseases caused by fungi according to claim 1, further comprising an agrochemically acceptable carrier or excipient. A method for controlling plant diseases caused by fungi, comprising the step of treating plants with the composition of claim 1 or 4,
The fungus is a group consisting of Pythium graminicola , Cladosporium herbarum , C. cladosporioides and Gibberella zeae A method for controlling plant diseases caused by fungi, characterized in that it is one or more selected from the following. delete delete