JP6635524B2 - Plant disease control agent - Google Patents

Description

  The present invention relates to a plant disease controlling agent comprising proanthocyanidin as an active ingredient, and a method for controlling a plant disease comprising applying the controlling agent to a plant.

  If plant disease caused by fungal disease, bacterial disease, plant virus disease, etc., causes loss of world agricultural production by as much as 15%, of which crop production loss due to plant virus disease exceeds 6 trillion yen annually Is expected. Plant virus diseases cause a variety of abnormalities in crops, severely affecting yield and quality.

  As a plant virus control agent, lentemin is registered as an agricultural chemical in Japan, but there is no chemical pesticide that is a specific medicine (lentemin is a registered trademark of Noda Shokubai Kogyo Co., Ltd. 17774, 19439 and 19440). Therefore, it is a fact that the time-consuming and time-consuming cultivated pest control, such as vector control and attenuated virus inoculation, is carried out to reduce the damage of plant virus diseases.

  For this reason, development of a controlling agent exhibiting an excellent effect on plant virus diseases is eagerly desired.

  As a control agent for plant virus diseases, for example, there are reports that amino acid fermentation by-products, dehydroascorbic acid, ascorbic acid (vitamin C), lignin extract, and the like are effective (Patent Documents 1 to 4).

WO 2013/065439 JP 2014-5232 A Patent No. 5574266 International Publication No.2017 / 125993

  The present invention has been made in view of such circumstances, and an object of the present invention is to identify a molecule having an excellent control effect on a plant disease, and to control a plant disease control agent and a control method using the molecule. Is to provide.

  As a result of intensive studies to solve the above problems, the present inventors have found that a specific extract fraction from a plant has an excellent control effect on various plant viruses and plant pathogens. The present inventor further tried to identify the active ingredient contained in the extracted fraction, and finally found out that proanthocyanidin was the active ingredient, thereby completing the present invention.

  That is, the present invention relates to a plant disease controlling agent comprising a purified plant product as an active ingredient, and a method for controlling a plant disease including applying the controlling agent to a plant, and more specifically, the following inventions are provided. It is.

  (1) An agent for controlling plant diseases, including a purified plant product obtained by purification without going through a fermentation process.

  (2) The control agent according to (1), wherein the purification comprises extraction with ethanol or elution with acetonitrile.

  (3) The control agent according to (1) or (2), wherein the purification comprises fractionation at a molecular weight of at least 3500.

  (4) A plant disease controlling agent comprising proanthocyanidin as an active ingredient.

  (5) The control agent according to (4), comprising a purified plant product containing proanthocyanidin.

  (6) The control agent according to (4) or (5), which contains 1% or more by weight of proanthocyanidin on a dry basis.

  (7) The control agent according to any one of (1) to (6), wherein the plant disease is a plant virus disease, a plant fungal disease, or a plant bacterial disease.

  (8) The control agent according to (7), wherein the plant disease is mosaic disease.

  (9) The control agent according to (7), wherein the plant disease is anthrax or black spot bacterial disease.

  (10) A method for controlling plant diseases, which comprises applying the control agent according to any one of (1) to (9) to plants.

  The use of the control agent of the present invention makes it possible to safely control plant diseases without causing phytotoxicity such as leaf atrophy and spots due to components contained in plants. Since the controlling effect of the controlling agent of the present invention was observed not only on the leaves directly sprayed but also on the upper leaves thereof, it is considered that the controlling effect of the controlling agent causes an improvement in plant immunity (resistance to pathogens). Therefore, according to the controlling agent of the present invention, a controlling effect can be provided very effectively.

It is a figure which shows the infection suppression effect with respect to ToMV of the moon peach subspecies extract. In addition, in the figure, "RIBS juice" is an extract squeezed in Okayama Prefecture, and "Okinawa juice" is an extract squeezed in Okinawa Prefecture. It is a figure which shows the infection suppression effect with respect to ToMV and TMV of the moon peach extract obtained using the blade of the juice extractor with different raw materials. It is a figure which shows the infection suppression effect with respect to ToMV of each material in the upper leaf of Bensamiana tobacco. It is a figure which shows the infection suppression effect with respect to ToMV of each material in the upper leaf of a tomato. It is a figure which shows the infection suppression effect with respect to the cruciferous vegetable anthrax of the moon peach extract applied to Arabidopsis thaliana and bok choy. It is a figure which shows the infection suppression effect with respect to the black spot bacterial pathogen of the moon peach extract applied to Arabidopsis thaliana. It is a figure which shows the infection suppression effect with respect to the black spot bacterial pathogen of the moon peach extract applied to the bok choy. It is a figure which shows the infection suppression effect with respect to ToMV of each acetonitrile fraction applied to Bensamiana tobacco. It is a figure which shows the dry matter weight of each acetonitrile fraction. It is a figure which shows the molecular weight distribution and the absorption spectrum of the component contained in the acetonitrile fraction which were analyzed by gel permeation chromatography. FIG. 3 is a diagram showing the results of MALDI-TOF mass spectrometry of the molecular weight distribution of components contained in the acetonitrile fraction. It is a figure which shows the result of the infrared absorption (IR) analysis of the partial structure of the component contained in an acetonitrile fraction. FIG. 3 is a diagram showing the results of proton NMR analysis of partial structures of components contained in the acetonitrile fraction. FIG. 4 is a view showing the result of pyrolysis GC-MS analysis of partial structures of components contained in an acetonitrile fraction. It is a figure which shows the result of carbon NMR analysis of the partial structure of the component contained in an acetonitrile fraction.

  The present invention relates to an agent for controlling plant diseases, including a purified plant product obtained by purification without going through a fermentation process.

  In the method described in WO 2017/125993, fermentation is performed in the purification step in order to obtain a lignin hydrolyzate as an active ingredient, but the present invention does not require fermentation in the purification step. Further, in the purification step of the publication, contact with iron ions (use of an iron crusher) is required to obtain a lignin degradation product, but in order to obtain a purified proanthocyanidin in the present invention, iron ion No contact is required, for example, a crusher made of stainless steel may be used.

  The purification step in the present invention can include extraction with ethanol or elution with acetonitrile. When performing extraction with ethanol, the ethanol concentration is usually 40% to 80%, preferably 60% to 80%. On the other hand, when performing elution with acetonitrile, the acetonitrile concentration is usually 5% or more, preferably 5% to 30%, and more preferably 5% to 20%. Acetonitrile at a concentration of 40% or more may be used, but it is conceivable that the proportion of inactive ingredients in the extract will increase.

  Further, for example, fractionation at a molecular weight of 3500 (eg, fractionation at a molecular weight of 4000 or more, 5000 or more, 6000 or more, 7000 or more, 8000 or more, 9000 or more, 10,000 or more) can be included. This makes it possible to remove low molecular weight components that do not contribute to the activity of controlling plant diseases.

  In addition, in the present invention, other purification steps may be combined as necessary.

  In the present invention, it has been found that proanthocyanidins are the active ingredient (or one of them). Therefore, the present invention provides a plant disease controlling agent comprising proanthocyanidin as an active ingredient.

  In the present invention, “proanthocyanidins” are compounds in which a plurality of flavan-3-ols such as catechin and epicatechin are bonded. The proanthocyanidin used in the control agent of the present invention is not particularly limited as long as it has a plant disease control effect, and may be a naturally-derived purified product or an artificially synthesized compound. The purified product may be a completely purified product or a crude product.

  The preferred form of proanthocyanidins in the present invention is a purified plant. A purified product of proanthocyanidin (extract) is usually 0.1 mg / ml or more, preferably 0.3 mg / ml or more, more preferably 0.5 mg / ml or more (for example, 1 mg / ml or more, 5 mg / ml or more). , 10 mg / ml or more, 20 mg / ml or more). In order not to cause adverse effects such as phytotoxicity, it is usually considered that the dose is preferably 50 mg / ml or less. Further, the purified proanthocyanidin usually contains proanthocyanidin in a dry weight of 1% or more, preferably 5% or more, more preferably 10% (eg, 20% or more, 30% or more, 50% or more). Accordingly, the present invention provides a plant disease controlling agent comprising proanthocyanidin at the above-mentioned concentration or ratio. Proanthocyanidins can be used alone or in combination of two or more.

  The control agent of the present invention may contain other active ingredients as long as the control effect on plant diseases is not inhibited. For example, it can be used in combination with a known drug such as lentemin in order to further enhance the control effect against plant diseases or to widen the range of plant diseases to be applied. In addition, plant viruses can be used in combination with known insecticides, acaricides, antibacterial agents, and the like, because there are cases in which plant viruses are transmitted by insect vectors.

  The control agent of the present invention may contain other optional components as long as the effect of controlling plant diseases is not inhibited. Other optional components include, for example, spreading agents, fillers, extenders, binders, humectants, disintegrants, lubricants, diluents, excipients, and the like.

  The method for applying the control agent of the present invention to plants is not particularly limited, and examples thereof include spraying, coating, and dipping. When plant tissue culture is performed, addition to the culture medium may be considered. Since a very high infection control effect is observed in the area where the control agent of the present invention is applied, application methods include application to whole plants and leaves by spraying or application, and application to seeds and bulbs. Immersion is preferred. The control agent of the present invention is advantageous in that it can exhibit a control effect on plant diseases even in a site surrounding the applied site.

  The dosage form of the control agent of the present invention can take various forms depending on the application method and the like. Examples include liquid preparations such as emulsions, oils, aerosols and flowables, as well as wettable powders, aqueous solvents, powders, granules and tablets. When applied to a plant by spraying or the like, a liquid formulation or a dosage form that can be made liquid at the time of application is preferable.

  The application rate is not particularly limited as long as it has an effect of controlling plant diseases, and can be appropriately adjusted by those skilled in the art.

  The application time of the control agent of the present invention to plants is not particularly limited, but preventive control is most effective. Specifically, application from the seedling raising stage to before harvest is effective. There is no particular limitation on the number of times the pesticide is applied.

  Plant virus diseases to which the control agent of the present invention is applied include, for example, those caused by infection with a tobamovirus, a potexvirus, a carlavirus, a cucumovirus, a carmovirus, or a begomovirus. Illness.

  Examples of tobamoviruses include tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), cucumber green mottle mosaic virus (KGMMV), capsicum mild mottle virus (PMMoV), watermelon green mottle mosaic virus (CGMMV), and the like. Examples of the virus of the genus Potex virus include psyllium mosaic virus (P1AMV) and potato X virus (PVX). Examples of the virus of the genus Carlavirus include, for example, potato mosaic virus (PVM), Examples of the cucumber virus include, for example, cucumber mosaic virus (CMV), and examples of the carmovirus include, for example, melon necrotic spot virus (MNSV), and examples of the begomovirus include, for example, Yellow tomato Such as wound Virus (TYLCV) include, but are not limited thereto.

  The plant virus disease targeted by the control agent of the present invention is preferably a mosaic disease caused by infection with various mosaic viruses or potato X virus.

  Examples of the filamentous fungi to which the control agent of the present invention is applied include powdery mildew (Erysiphe spp., Sphaerotheca spp., Leveillula spp.), Botrytis spp., Fulvia fulva spp., Corynespora. ), Genus Albugo, downy mildew (Pseudoperonospora, Peronospora, Plasmopara, Bremia), pyricularia (Pyricularia), blast (Pyricularia) grisea), sesame leaf blight (Cochliobolus miyabeanus), circospora related fungi (Cercospora, Cercosporella, Pseudocercospora, Paracercospora Mycovellosiella), anthrax (Colletotrichum), Glomer , Rust fungus (Puccinia genus), Alternaria (Alternaria) genus, Sclerotinia (Sclerotinia) genus, soil-borne fungal root-knot fungus (Plasmodiophora brassicae), Phytophthora (Phytophthora) genus, Fusarium (Fusarium) genus, Verticillium (Pythium) genus, Rhizoctonia (Rhizoctonia) genus. Preferably, it is an anthrax fungus.

  Examples of bacteria to which the control agent of the present invention is applied include, for example, Agrobacterium (Agrobacterium), Clavibacter (Clavibacter), Erwinia (Erwinia), Pseudomonas (Pseudomonas), Ralstonia (Ralstonia), Corynebacterium Examples include bacteria belonging to the genus (Corynebacterium), the genus Curtobacterium, the genus Burkholderia, the genus Xanthomonas, the genus Rhizobacter, and the genus Clostridium. Preferred is Pseudomonas syringae pv. Maculicola, which causes black spot bacterial disease.

  Plants to which the control agent of the present invention is applied are not particularly limited as long as the above-mentioned pathogens are infected.For example, tobacco, tomato, capsicum, cucumber, melon, watermelon, apple, leek, onion, leek, pepper, Potato, rice, barley, pumpkin, Chinese cabbage, cabbage, bok choy, lettuce, strawberry, flowers (chrysanthemum, rose, carnation, tulip, cattleya, cymbidium, eustoma, starches, gerbera, etc.). As described above, the control agent of the present invention can be applied to a wide range of plants such as Solanaceous plants, Cucurbitaceae plants, Brassicaceae plants, Rosaceae, and Poaceae plants. For the relationship between plant viruses and host plants, refer to the Japanese Plant Disease Database (Agricultural Biological Resources Genebank).

  Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

[Example 1] Purification of moon peach extract without fermentation process As a herbaceous case, the foliage portion of moon peach (getto (Sannin)) of the genus Ganaceae is added to a juicer and water is added. Without extracting the liquid extracted from the plant, the contaminants were removed by a centrifuge (6000 rpm, 5 minutes), and the supernatant was filtered with a filter paper and a filter having a pore size of 0.22 μm (manufactured by TPP).

  In the method described in WO 2017/125993, in the purification process, fermentation of the squeezed liquid is performed, but the method according to the present example does not perform fermentation, and thus is included in the obtained extract. Components are considered to be different.

[Example 2] Infection-suppressing effect of Toro peach subspecies extract on ToMV The plant extract prepared in Example 1 was evaluated using a ToMV-Bensamiana tobacco evaluation system. That is, a plant extract solution (diluted [10%, 20%, 50%, undiluted]) containing 0.01% final concentration of mylino was spray-treated on Bensamiana tobacco leaves. The virus was inoculated. The virus was inoculated by transcribing and synthesizing RNA using 2 μg of ToMV-GFP (virus to which green fluorescent protein GFP was added) plasmid (pTLBN.G3) using AmpliCap-Max ^™ T7 High Yield Message Maker Kits. Was diluted 40-fold, and 10 μl was applied to two places of Bensamiana tobacco leaves using carborundum. Three days after the inoculation of the virus, fluorescent spots of GFP (corresponding to the infection site of ToMV and the growth site) were counted, and the rate of inhibition of virus infection was evaluated to evaluate the plant extract. As a result, a strong virus infection inhibitory effect was observed at all dilution ratios (FIG. 1).

The infection control rate was calculated from the following equation.
Infection control rate (control value)% = 100-{(average number of fluorescent spots of treated plants) / (average number of fluorescent spots of untreated plants)} x 100
[Example 3] Infection suppression effect on ToMV and TMV of moon peach extract obtained using blades of juice extractors with different raw materials As an example of a herbaceous plant, moon peach (Ganto (Sannin)) of the genus Gingeraceae The stem and leaves are put into a juicer equipped with an iron or stainless steel blade, and the liquid extracted from the plant is collected without adding water, and the contaminants are removed by a centrifuge (6000 rpm, 5 minutes). After removal, the supernatant was filtered through filter paper and a filter having a pore size of 0.22 μm (manufactured by TPP).

  As for ToMV, a 10% solution containing 0.01% mylino was sprayed, and after 3 days, 2 μg of the plasmid was transcribed. Then, 40 μl of diluted ToMV-GFP (a virus to which green fluorescent protein GFP was added) was added to each plant in an amount of 10 μl. Each third leaf was inoculated with carborundum, and three days later, GFP fluorescence spots on the inoculated leaves were measured. The infection suppression rate for the control was calculated, and the average value was shown. For each treatment, three individuals of Bensamiana tobacco were used.

  Regarding TMV, a 10% solution containing 0.01% mylinol was sprayed, and 3 days later, 2 μg of the plasmid was transcribed, and 6-fold diluted TMV-GFP (virus to which green fluorescent protein GFP was added) was added to each plant at 10 μl. Each third leaf was inoculated with carborundum, and three days later, GFP fluorescence spots on the inoculated leaves were measured. The infection suppression rate for the control was calculated, and the average value was shown. For each treatment, three individuals of Bensamiana tobacco were used.

  As a result, a strong virus infection-suppressing effect on ToMV and TMV was observed, regardless of whether the juice was extracted using an iron or stainless steel blade equipped with a blade (FIG. 2).

  In the method described in WO 2017/125993, iron and iron ions were considered to be related to the antiviral effect, but in the extract obtained by the purification method of Example 1, the antiviral effect Iron and iron ions were not related to the effect.

[Example 4] Pest control effect on ToMV of ethanol extract of moon peach against ToMV The stem and leaves of moon peach (Gettou (Sannin)) of the genus Gingeraceae are cut with scissors equipped with stainless steel blades, and 20 g of the cut material is cut. On the other hand, it was immersed in 40 ml of a 0-100% ethanol solution. The mixture was reciprocally shaken (180 rpm) at 25 ° C. overnight to collect a liquid extracted from the plant. Next, impurities were removed by a centrifuge (6000 rpm, 5 minutes), and the supernatant was filtered with filter paper.

  A 20% solution of each plant extract to which 0.01% mylinol was added was spray-treated on Bensamiana tobacco and allowed to stand for 3 days. After transferring 2 μg of the plasmid by the method of Example 2, 10 μl of ToMV-GFP diluted 40-fold was inoculated into the third leaf at two locations using carborundum, and the spot of GFP fluorescence of the inoculated leaf was measured three days later. . The infection suppression rate for the control was calculated, and the average value was shown. For each treatment, three individuals of Bensamiana tobacco were used.

  As a result, a strong virus infection-suppressing effect was observed in the extract with the 60-80% ethanol solution (Table 1). In addition, the numerical value in parenthesis in a control value means minus.

[Example 5] Inhibitory effect of viral infection on upper leaves (1) Bensamiana tobacco To the Bensamiana tobacco, the plant extract prepared in Example 1 (10% solution and 0.01% solution containing 0.01% mylino) ) Was sprayed. Ascorbic acid and lentemin were used as positive controls. Three days after the treatment with each material, 2 μg of the plasmid was transcribed by RNA, and 20 μl-diluted ToMV-GFP was inoculated to the third leaf of each plant by 10 μl using a carborundum. Three days later, the amount of ToMV infection was quantified using real-time PCR. The average value and the standard error were determined using three or more Bensamiana tobacco plants per treatment. As a result, even in the upper leaves, a strong virus infection inhibitory effect on ToMV was observed (FIG. 3).

(2) Tomato
A 14-day-old tomato (cultivar Regina) was spray-treated with the plant extract prepared in Example 1 (50% solution containing 0.01% mylino). Ascorbic acid and lentemin were used as positive controls. Three days after each material treatment, 2 μg of plasmid was transcribed with RNA, and ToMV-GFP diluted 5-fold was inoculated to the first leaf of each plant by 10 μl using a carborundum. Six days later, the amount of ToMV infection in the inoculated leaves and upper leaves was quantified using real-time PCR. The average value and the standard error were determined using three or more tomatoes per treatment. As a result, even in the upper leaves, a strong virus infection inhibitory effect on ToMV was observed (FIG. 4).

Example 6 Inhibitory Effect on Infection with Pathogenic Bacteria (1) Anthrax Bacteria Using the plant extract derived from moon peach prepared in Example 1, the effect of inhibiting infection with cruciferous vegetables on anthrax was evaluated by the following method. A 50% plant extract (with 0.01% myrine) was sprayed on foliage of Arabidopsis thaliana. Two days after the application of the plant extract, Arabidopsis was spray-inoculated with a cruciferous vegetable anthrax (5 × 10 ⁵ spores / ml). Five days after the inoculation, the inoculated leaves were sampled, and the amount of infection of Arabidopsis thaliana by Brassica anthracnose was determined by qRT-PCR. As a control, the same quantification was performed by spraying only a spreading agent containing no plant extract (0.01% mylinol added).

  As a result, a significant effect of suppressing infection was observed in the plant extract as compared with the control (FIG. 5).

In addition, using the plant extract derived from moon peach prepared in Example 1, the effect of inhibiting infection with strawberry anthrax (Colletotrichum gloeosporioides) was evaluated by the following method. A 50% plant extract (with 0.01% myrinoux) was sprayed onto foliage strawberry. Three days after the application of the plant extract, strawberries were inoculated by spraying with strawberry anthrax (5 × 10 ⁵ spores / ml). Six days after the inoculation, the amount of strawberry anthracnose infecting strawberry was evaluated by examining the symptoms. As a control, the same evaluation was performed by spraying only a spreading agent containing no plant extract (0.01% mylinol added). The severity is expressed by the following equation.
Severity = ｛(1n ₁ + 2n ₂ + 3n ₃ + 4n ₄ + 5n ₅ ) / (5 × number of surveys)｝ × 100
The disease incidence was surveyed by dividing the disease severity into the following five categories.
0: No symptom, 1: Microscopic spot, 2: Lesion is found in area of 25% or less of leaf 3: Lesion is found in area of 25 to 50% of leaf, 4: 50% of leaf Lesion is observed in the above area or petiole is broken. 5: Withering
n ₅ from n ₁ is the number of individuals controlling value is expressed by the following scheme.
Control value = {1-degree of disease in treated area / degree of disease in untreated area) x 100
As a result, a significant effect of suppressing infection was observed in the plant extract as compared with the control (Table 2).

(2) Black Spot Bacterial Bacteria Using the plant extract derived from moon peach prepared in Example 1, the effect of suppressing infection against black spot bacilli was evaluated by the following method. A 50% plant extract (with 0.01% myrineau) was sprayed onto foliage of Arabidopsis thaliana and bok choy (variety: Shapao). Two days after the application of the plant extract, Arabidopsis thaliana and bok choy were each spray-inoculated with black spot bacterial pathogen (1 × 10 ⁸ cfu / ml). Three days after inoculation, the inoculated leaves were sampled, and the amount of infection of Arabidopsis thaliana and bok choy by the bacterial spot of black spot was quantified by qRT-PCR. As a control, the same quantification was performed by spraying only a spreading agent containing no plant extract (0.01% mylinol added).

  As a result, a significant infection-suppressing effect was observed in the plant extract as compared with the control (FIGS. 6 and 7).

[Example 7] Control effect of acetonitrile extract of moon peach on ToMV Okinawa peach squeezed solution was applied to a reverse phase carrier (Sep-Pak C18), washed with water, and then 5%, 10%, 15%, Stepwise eluted fractions were obtained with 20% and 40% acetonitrile. Acetonitrile contained in the obtained fraction was distilled off by an evaporator, and the remaining solution was freeze-dried. The freeze-dried product of each of the obtained fractions was dissolved in water, and the effect of suppressing virus infection was evaluated.

  In the evaluation of the virus infection-suppressing effect, the virus was inoculated (recruited inoculation) 3 days after treating the Bensamiana tobacco with the moon peach extract. In this evaluation, a virus in which a protein GFP gene that emits fluorescence was introduced into ToMV, and which emitted fluorescence upon infection was used.

  As a result, a virus infection inhibitory effect was observed in the fractions eluted with 5%, 10%, 15%, and 20% acetonitrile (FIG. 8).

  The dry weight of the acetonitrile-eluted fraction is shown in FIG. In addition, all the evaluations of the virus infection suppressing effect in the subsequent experiments were performed using a system using this GFP-inserted ToMV and Bensamiana tobacco.

Example 8 Separation of Active Fraction by Dialysis Membrane The virus infection-suppressing effect of the inner solution of dialysis obtained by dialyzing a squeezed solution of Okinawa Tsukipeach (20 ml) against distilled water was evaluated. Three types (100-500, 1,000, 3,500) having different molecular weight cutoffs were used as dialysis membranes. As a result, the effect of suppressing virus infection was observed in any of the inner dialysis solutions obtained by the three types of dialysis membranes, indicating that the active ingredient had a molecular weight of 3,500 or more.

Example 9 Separation of Active Fraction by Ultrafiltration The above 5%, 10%, 15%, and 20% acetonitrile-eluted fractions were combined and dialyzed against distilled water. A dialysis membrane having a molecular weight cutoff of 3,500 was used. The obtained dialysis solution was freeze-dried and then redissolved in water and subjected to centrifugal ultrafiltration (fraction molecular weight: 10,000). The fractions that passed (that is, molecular weight of 10,000 or less) and those that remained (molecular weight: 10,000 or more) were freeze-dried and evaluated for antiviral activity.

  As a result, it was inferred that the active component had a molecular weight of 10,000 or more, because the activity was present in the remaining fraction of the ultrafiltration.

[Example 10] Analysis of inorganic metal in acetonitrile-eluting fraction In the same manner as in Example 9, a lyophilized product of the acetonitrile-eluting fraction in dialysis solution was obtained. The inorganic metal contained in the obtained freeze-dried product was analyzed by ICP emission.

  As a result, 2.6 mg of magnesium, 2.1 mg of iron, 1.0 mg of calcium, and 1.0 mg of sodium were contained in 1,000 mg of the freeze-dried product.

  When the solubility of the obtained lyophilized product in a solvent was examined, it was soluble in water and dimethylformamide (DMF), but insoluble in tetrahydrofuran (THF) and chloroform.

Example 11 Analysis of Molecular Weight Distribution by Gel Permeation Chromatography In the same manner as in Example 9, a freeze-dried product of the acetonitrile-eluting fraction in the dialyzed solution was obtained. The molecular weight distribution and absorption spectrum of the obtained lyophilized product were examined by gel permeation chromatography using DMF as a solvent.

  As a result, the sample was found to have strong absorption maximums at 283 nm and 263 nm, and its molecular weight was estimated to be 10,000 or more (FIG. 10).

  The weight average molecular weight at the absorption maximum wavelength, which was obtained by using polystyrene having different degrees of polymerization as a calibration curve, is shown in the upper right of the figure.

[Example 12] Analysis of molecular weight distribution by mass spectrometry In the same manner as in Example 9, a lyophilized product of the acetonitrile-eluted fraction in the dialyzed solution was obtained. The molecular weight distribution of the obtained freeze-dried product was estimated by MALDI-TOF mass spectrometry. As a result, it was suggested that the sample was a polymer in which some 288 units of molecular weight were polymerized (FIG. 11). In published literature (Xiaolan Jiang et al., SCIENTIFIC REPORTS 5: 8742, DOI: 10.1038 / srep08742, Sauro Bianchi et al., Phytochemistry 120 (2015) 53-61), molecular weight 288 may be catechin or epicatechin. It has been reported. From this, it was inferred that the active substance was the polymer, proanthocyanidins.

  In addition, when Bensamiana tobacco was sprayed with 500 ppm epicatechin and inoculated with ToMV 2 hours later, a slight infection-suppressing effect was observed.However, when ToMV was inoculated 3 days after spraying, No infection control effect was observed.

Example 13 Estimation of Partial Structure In the same manner as in Example 9, a lyophilized product of the acetonitrile-eluted fraction in the dialysate was obtained. The partial structure of the obtained lyophilized product was estimated by proton NMR and infrared absorption spectrum (FIGS. 12 and 13). As a result, it was speculated that the partial structure contained many aromatic rings, hydroxyl groups, and the like. Further, the partial structure of the obtained freeze-dried product was analyzed by thermal analysis GC-MS, and as a result, it was estimated that a large amount of polyphenols was contained (FIG. 14). The above results further confirmed that the active substance was proanthocyanidins.

  The freeze-dried product of the dialysis solution obtained in Example 9 was redissolved in distilled water, dialyzed against distilled water using a dialysis membrane having a molecular weight cut-off (10,000), and lyophilized of the dialysis solution. I got something. This was dissolved in acetone and the structure was estimated by C13 carbon NMR (FIG. 15). As a result, it was in good agreement with the C13 carbon NMR data of proanthocyanidin reported in the literature (Thomas L. Eberhardt et al., J. Agric. Food Chem. 42 (1994) 1704-1708). The above results further confirmed that the active substance was proanthocyanidins.

Example 14 Measurement of Proanthocyanidin Concentration The freeze-dried product of the inner dialysis solution (fraction molecular weight: 3,500) obtained in Example 9 was redissolved in distilled water, and a dialysis membrane having a fraction molecular weight (10,000) was used. Then, the solution was dialyzed against distilled water to obtain a lyophilized product of the inner solution of the dialysis. This was dissolved in distilled water to obtain purified anthocyanidins for a calibration curve derived from moon peach.

  For the determination of proanthocyanidins, a method using 4-dimethylaminocinnamaldehyde (DMAC) described in the report by Oki et al. (Tomoyuki Oki et al., Nippon Shkuhin Kagaku Kogaku Kaishi Vol. 60, No. 6, 301-309 (2013)) I almost followed. That is, the proanthocyanidin aqueous solution (10 mg / ml) for a calibration curve was diluted 20-fold with assay solution A (ethanol: methanol: 2-propanol = 90: 5: 5), and further diluted with assay solution B (assay solution A). : Water = 95: 5) to prepare a two-fold dilution series, which was used as a sample for a calibration curve. The moon peach juice was diluted 20-fold with assay solution A (ethanol: methanol: 2-propanol = 90: 5: 5) to obtain a measurement sample. In addition, as a symmetrical sample, a ginger juice and a freeze-dried product of the internal dialysis solution (fraction molecular weight: 3,500) described in Example 9 were also prepared in the same manner as the moon peach juice.

  The DMAC solution was prepared by adding concentrated hydrochloric acid (3 ml) to the above-mentioned assay solution A (27 ml), then adding DMAC (30 mg) to a solution cooled on ice for 15 minutes, dissolving by stirring, and storing on ice until just before use. And a DMAC solution.

  To the wells of a 96-well microplate, dispense 40 μl each of the sample for the calibration curve and the sample for measurement, and add 200 μl of the DMAC solution to the wells to which the sample was added using an 8-well micropipette. A plate seal was attached, and the plate was stirred and left for 20 minutes in a plate reader set at 30 ° C., and the absorbance of each well at 640 nm was measured. A calibration curve was prepared from the absorbance values and concentrations obtained from the calibration curve samples, and proanthocyanidins contained in the peach juice were quantified. As a result, the amount of proanthocyanidins contained in the peach juice was estimated to be in the range of 5 to 20 mg / ml (Table 3).

  In addition, when 10 ml of the moon peach juice 2 shown in Table 2 was freeze-dried, the weight of the obtained dried product was 431.2 mg. When converted from this, the ratio of proanthocyanidins to dry matter is 43.0%. Furthermore, assuming that proanthocyanidin contained in the juice is 5 to 20 mg / ml, the ratio of proanthocyanidin in the dried juice can be estimated to be about 10 to 50%.

  As described above, the control agent of the present invention can exhibit an excellent control effect on plant diseases by utilizing components contained in plants. For this reason, it can greatly contribute to the agricultural field as an agricultural chemical having both safety and effect.

Claims (3)

A plant virus disease controlling agent comprising proanthocyanidin as an active ingredient. Plant virus disease is a mosaic, control agent of claim 1. A method for controlling a plant virus disease , comprising applying the control agent according to claim 1 or 2 to a plant.