Method for controlling plant diseases

Abstract

A method for controlling plant diseases comprising the steps of: (1) preparing plasma water by dispersing and / or dissolving, in water, an air plasma-activated gas obtained by subjecting air to an atmospheric pressure plasma treatment; and (2) applying the plasma water to soil.

Description

Methods for controlling plant diseases

[0001] This invention relates to a method for controlling plant diseases.

[0002] Soil disinfection is widely practiced to suppress pathogenic microorganisms in the soil. Known methods of soil disinfection include soil sterilization using pesticides (such as chloropicrin) and soil reductive disinfection using rice bran or wheat bran. For example, Patent Document 1 discloses technology for chloropicrin emulsion, and Patent Document 2 discloses technology for soil reductive disinfection using wheat bran.

[0003] Japanese Patent Publication No. 2013-135693 Japanese Patent Publication No. 2006-61003

[0004] However, the methods described above all suppress disease outbreaks by reducing the concentration of pathogenic microorganisms in the soil before planting, and there was room for improvement regarding the occurrence of diseases caused by pathogenic microorganisms that increased after planting.

[0005] The present invention aims to provide a method for controlling plant diseases that is effective in suppressing pathogenic microorganisms in the soil, not only before planting but also after planting.

[0006] In order to solve the above problems, the inventors conducted extensive research and found that by preparing plasma water by dispersing and / or dissolving air plasma-activated gas, obtained by treating air with atmospheric pressure plasma, in water, and applying this plasma water to the soil, it is effective in suppressing pathogenic microorganisms in the soil not only before planting but also after planting.

[0007] The present invention was completed based on these findings and includes the following broad embodiments: [Claim 1] A method for controlling plant diseases, comprising the steps of: (1) preparing plasma water by dispersing and / or dissolving an air plasma-activated gas obtained by plasma treatment of air at atmospheric pressure in water; and (2) applying the plasma water to soil. [Claim 2] The method according to Claim 1, wherein step (2) includes irrigating soil in which a plant is growing with plasma water. [Claim 3] The method according to Claim 1 or 2, wherein step (2) applies the plasma water to the soil to reduce the causative fungus of the plant disease by changing the amount of soil components that affect the growth of the causative fungus of the plant disease. [Claim 4] The method according to any one of Claims 1 to 3, wherein the causative fungus of the plant disease decreases two days or more after application of plasma water to the soil. [Claim 5] The method according to any one of Claims 1 to 4, wherein the plant disease is caused by a Fusarium fungus. [Item 6] A method for sterilizing soil, comprising the steps of: (1) preparing plasma water by dispersing and / or dissolving an air plasma-activated gas obtained by treating air with atmospheric pressure plasma in water; and (2) applying the plasma water to soil. [Item 7] A plant disease control agent comprising plasma water, wherein the plasma water is obtained by dispersing and / or dissolving an air plasma-activated gas obtained by treating air with atmospheric pressure plasma in water.

[0008] According to the present invention, it is possible to provide a method for controlling plant diseases that is effective in suppressing pathogenic microorganisms in the soil not only before planting but also after planting.

[0009] This document outlines the plasma water generator used in Manufacturing Example 1. It describes the procedure for a sterilization test of Fusarium-contaminated soil using plasma water in Example 1. It shows the results of a sterilization effect test on Fusarium by plasma water treatment in Example 1. It shows the effect of plasma-treated water on the growth of Eustoma in Example 2. Eustoma was treated with 600 ml of plasma water once a week for 2 months. Distilled water was treated similarly as a negative control. Chloropicrin was also treated according to a predetermined method. The document describes the procedure for a sterilization test of Fusarium-contaminated soil by periodic irrigation with plasma water in Example 3. It shows the results of measuring Fusarium concentration using plasma water in Example 3. (A) Photograph of Fusarium concentration in plasma-treated soil after plate dilution culture using Fo-G2 selective medium. (B) Bacterial concentration calculated from the number of colonies formed on the medium. (C) Bacterial concentration calculated from the number of colonies formed on the medium. Example 3 demonstrates the suppression of lisianthus wilt disease by plasma water. Six plants were used in both the distilled water treatment group and the plasma water treatment group. In the distilled water treatment group, wilting of the plant body and yellowing of the leaves, which are early symptoms of wilt disease, were observed, but in the plasma water treatment group, the early symptoms of wilt disease were not observed. An overview of the experiment in Example 4 is shown. The results of the fungal concentration in Example 4 are shown. (A) Results in the topmost soil section. (B) Results in the third section from the top soil section. (C) Results in the seventh section from the top soil section (approximately 20 cm deep from the top). An overview of the experiment in Example 5 is shown. The principal component analysis results of the metabolome analysis data in Example 6 are shown. (A) An overview of each culture medium in Example 7 is shown. (B) The state of the culture medium on day 5 of cultivation in Example 7 is shown. (C) The results on day 5 of cultivation in Example 7 are shown.

[0010] In this specification, singular nouns (a, an, the, etc.) include both singular and plural forms unless otherwise explicitly stated or the context clearly contradicts them. In this specification, "comprise" also includes "consist essentially of" and "consist of."

[0011] In this specification, "plant disease" refers to an abnormality in a plant caused by a series of actions by a certain cause, and when the cause of the disease is a microorganism (filamentous fungi, bacteria, viruses, viroids, phytoplasmas, nematodes), it is specifically called an infectious disease.

[0012] In this specification, "control" means preventing, suppressing, or eliminating plant diseases, preferably soil-borne plant diseases.

[0013] In one embodiment, the present invention provides a method for controlling plant diseases. The method of the present invention includes the following steps (1) and (2): (1) A step of preparing plasma water by dispersing and / or dissolving an air plasma activated gas obtained by treating air with atmospheric pressure plasma in water. (2) A step of applying the plasma water to soil.

[0014] In step (1), an air plasma activated gas obtained by treating air with atmospheric pressure plasma is dispersed and / or dissolved in water to prepare a dispersed solution of air plasma activated gas (hereinafter also referred to as plasma water).

[0015] Air-atmospheric pressure plasma treatment includes methods such as dielectric barrier discharge, which uses a dielectric material to cover the electrode surface with an insulator (dielectric) and drive the discharge by applying a low-frequency AC voltage to the electrode, and a method that uses a high-voltage pulsed power supply to prevent overheating by switching off the power before the plasma heats up. Here, dielectric barrier discharge has the property that when a voltage is applied, charge accumulates on the dielectric surface, creating an electric field opposite to that on the electrode surface, and self-terminating the discharge. As a result, the time scale of the discharge is limited to a scale shorter than the relaxation time of the gas temperature, and it is preferable because it is possible to generate atmospheric pressure-non-equilibrium plasma with a low gas temperature.

[0016] In one embodiment, a preferred apparatus for atmospheric pressure plasma processing is one in which, for example, an AC voltage of a predetermined frequency is applied between electrodes to generate a discharge, and then gas (air) is supplied between the electrodes to generate plasma from the molecules in the gas, and a gas stream containing this plasma is emitted. An example of such an apparatus is an atmospheric pressure plasma jet.

[0017] The voltage conditions are preferably 1 to 30 kVp-p, and more preferably 5 to 20 kVp-p. The frequency is preferably 1 to 100 kHz, and more preferably 3 to 10 kHz.

[0018] The air plasma activated gas obtained by the atmospheric pressure plasma treatment of air described above contains various chemical species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). The air plasma activated gas used in the present invention preferably contains ozone (O3) in a concentration of 10 to 50,000 ppm, more preferably 1,000 to 5,000 ppm, nitrogen pentoxide (N2O5) in a concentration of 0.1 to 500 ppm, more preferably 10 to 200 ppm, and nitric acid (HNO3) in a concentration of 0.1 to 500 ppm, more preferably 10 to 200 ppm.

[0019] Dispersion and / or dissolution of air plasma activated gas in water can be achieved, for example, by bubbling or spraying. The water may be, but is not limited to, sterile water, distilled water, purified water, pure water, ultrapure water, RO water, or ion-exchanged water.

[0020] The bubbling method is a method of blowing air plasma activated gas into water. Examples of bubbling methods include (i) directly bubbling a large volume of water, and (ii) using a small volume of water in the bubbling section (dissolution solution generation section), releasing the bubbling liquid to the outside while introducing the same amount of water into the dissolution solution generation section. In the case of method (i), since the air plasma activated gas contains not only gaseous ozone but also highly soluble nitrogen oxides such as nitric acid and nitrous oxide, these nitrogen oxides dissolve in the water, and the pH of the solution decreases in proportion to the bubbling time. Also, the larger the volume of liquid, the longer it takes for the dissolved ozone concentration to rise, so bubbling for a long time is required. On the other hand, in the case of method (ii), since bubbling is continuously performed on a volume of purified water to continuously generate the dissolution solution, the rise in nitric acid concentration in the solution can be reduced and the dissolved ozone concentration can be stabilized quickly, which is preferable.

[0021] In the bubbling method, the amount of gas introduced is preferably 0.1 to 200 slm, and more preferably 1 to 20 slm. Furthermore, the amount of water in the dissolution generation section of (ii) above in the bubbling method is preferably 10 to 20,000 mL, and more preferably 100 to 2,000 mL.

[0022] The spraying method is a method of generating a dissolution solution by spraying water into an activated gas chamber filled with activated gas. By forming the water into tiny droplets, the air plasma activated gas can be dissolved in the water at high speed and with high efficiency.

[0023] The volume of the gas-filled section is preferably 1 to 10,000 L, and more preferably 100 to 1,000 L. The gas introduction rate is preferably 0.1 to 200 slm, and more preferably 1 to 20 slm.

[0024] Examples of spraying methods include atomizers and spray nozzles (single-fluid nozzles, two-fluid nozzles).

[0025] The dissolved ozone concentration in the plasma water is preferably 0.1 to 10,000 μM, and more preferably 1 to 100 μM, immediately after generation.

[0026] Plasma water may contain optional components as long as they do not interfere with the effects of the present invention. Examples of such components include fertilizers, pH adjusters, surfactants, antifreeze agents, and decomposition inhibitors. These components may be contained individually or in combination of two or more types. The amount of these components contained is not particularly limited.

[0027] In step (2), the plasma water prepared in step (1) above is applied to the soil.

[0028] In this invention, soil includes not only cultivated land such as fields where crops are grown, but also soil in planters and flowerpots, as well as growing beds such as rock wool and vermiculite on which crops are grown. In one embodiment, it is preferable that the soil is used for soil cultivation or hydroponic cultivation of plants.

[0029] The method of applying plasma water to soil is not particularly limited, as it varies depending on the cultivation method of the plants using that soil, but examples include spraying and irrigation. Here, spraying can be done by watering the soil, for example. Irrigation can be done by watering the soil surface (ground surface) or by drenching the soil. From the viewpoint of suppressing the reduction of the effects of the present invention due to the diffusion of air plasma activated gas into the atmosphere, it is preferable to apply it to the soil by irrigation.

[0030] One method of irrigating the ground surface is to lay permeable pipes (sprinkler tubes) on top of the soil and flow plasma water through them. While simply flowing water over the soil surface is sufficient for irrigation, it is preferable to cover the soil surface with a substantially airtight sheet (mulching) and lay permeable pipes inside to irrigate, as this requires less water. Examples of substantially airtight sheets include polyethylene film and polyvinyl chloride film or sheets.

[0031] One method of irrigating the soil is to flow plasma water through permeable pipes buried in the soil. The irrigation depth can be adjusted as appropriate depending on the depth at which the target organisms live and the type of plants. It is also possible to supply water from pipes buried at multiple depths or to use it in combination with surface irrigation. For example, pipes can be buried in the soil at a depth of 10 to 30 cm, preferably 20 cm, for irrigation.

[0032] Examples of permeable pipes include permeable unglazed ceramic pipes, porous rubber pipes with fine interconnected pores, and PVC pipes with numerous holes. In the case of pipes with numerous holes, the hole size is preferably 1-3 mm, and the holes should be 5-10 cm apart. 2 It is preferable to have one or more of these. Furthermore, it is preferable to install one pipe in the center of each furrow.

[0033] The amount of irrigation water varies depending on the concentration of chemical species such as ozone in the plasma water, the irrigation method, the timing of irrigation, the type and composition of the soil being irrigated, and the organisms to be eradicated. However, in one embodiment, 3 L / day to 7 L / day per meter of furrow is preferred, and 5 L / day per meter of furrow is more preferred. It is preferable to use an amount of plasma water that is completely absorbed into the soil and does not flow over the surface.

[0034] The number of times plasma water is applied to the soil can be appropriately determined depending on the target plant species, the type of fungus to be controlled, etc.

[0035] Plasma water should preferably be applied to the soil at a frequency of, for example, once to several times a week.

[0036] There are no particular restrictions on the timing of applying plasma water to the soil; it can be applied at any time, such as before planting, during planting, or after planting. Since the plasma water used in this invention does not have a negative effect on plant growth, it can also be applied to soil while plants are growing. In one embodiment, step (2) above includes irrigating soil where plants are growing with plasma water. More preferably, it includes irrigating soil before plant growth with plasma water, and irrigating soil where plants are growing with plasma water.

[0037] From the perspective of the stability of plasma water, the time from preparation of plasma water to application to soil is preferably within 30 minutes after preparation of plasma water. In a preferred embodiment, the application to soil in step (2) is performed immediately after the plasma water is prepared in step (1).

[0038] In one embodiment, in the step (2), it is preferable to reduce the causative bacteria of plant diseases by changing the amount of a soil component that affects the growth of said causative bacteria through applying the plasma water to soil.

[0039] Examples of soil components that affect the growth of the above-mentioned plant diseases include p-benzenesulfonic acid, nicotinamide mononucleotide, N-acetylglucosamine-1-phosphate, deoxyadenosine, glucose-1-phosphate, 2'-deoxycytidine, phosphorylcholine, 3-methoxy-4-hydroxyphenylethylene glycol, 2'-deoxyguanosine, xanthopterin, gluconic acid, gluconolactone, N-acetylglucosylamine, uridine diphosphate-N-acetylglucosamine, uridine diphosphate-N-acetylgalactosamine, saccharopin, γ-glutamic acid-methionine, 3-phenylpropionic acid, trimethyllysine, lysine, N-lactoylphenylalanine, and N6-methyllysine. Asymmetric dimethylarginine (ADMA), proline, symmetric dimethylarginine (SDMA), Nw-methylarginine, homoarginine, arginine, nicotinamide riboside, 3',5'-cyclic cytidine monophosphate, 2',3'-cyclic cytidine monophosphate, N6,N6-dimethyllysine, 3-sulfopropanediol, ornithine, N-(o-toluyl)glycine, phenaceturic acid, N-formylphenylalanine, ibuprofen, choline, gibberellic acid, N-acetylputrescine, cysteic acid, tryptophan, 1-methyladenine, citrulline, xanthurenic acid, proline-hydroxyproline, γ-glutamic acid-arginine, p-hydroxyphenylacetylglycine, alanine, N,N-dimethylhistidine, mimosine, S-methylcysteine, γ-aminobutyric acid, prostaglandin F2α, betainaldehyde, morpholine, tropin, 2-oxoglutaric acid, 3-methylcytidine, 2'-O-methylcytidine, ferulic acid, hercinin, tyrosine, serotonin, glycine, prostaglandin Examples include E2, histidine, nicotinic acid, pipecolic acid, cyclohexylamine, tetrahydrouridine, valine, trimethylamine-N-oxide, hypotaurine, N-methylserotonin, 4-(β-acetylaminoethyl)imidazole, isonicotinamide, nicotinamide, 2-amino-2-methyl-1-propanol, 3-aminoisobutyric acid, leucine, phenylalanine, alanine-hydroxyproline, hexamine, threonine, ectoin, N1-methylguanosine, alanine-alanine, acetyl-L-carnitine, isoleucine, methionine sulfoxide, kynurenine trimethyllysine, N-lactoyl, asparagine, aspartic acid, glutamine, glutamic acid, methionine, serine, cysteine, etc.

[0040] In one embodiment, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen types of compounds selected from the group consisting of p-benzenesulfonic acid, nicotinamide mononucleotide, N-acetylglucosamine-1-phosphate, deoxyadenosine, glucose 1-phosphate, 2'-deoxycytidine, phosphorylcholine, 3-methoxy-4-hydroxyphenylethylene glycol, 2'-deoxyguanosine, xanthopterin, gluconic acid, gluconolactone, N-acetylglucosylamine, uridine diphosphate-N-acetylgalactosamine, saccharopine, γ-glutamic acid-methionine, and 3-phenylpropionic acid are preferably increased in amount in soil. In particular, it is preferable that the amount of p-benzenesulfonic acid in soil is increased. In one embodiment, one week after three irrigation treatments with plasma water at a frequency of once a week, the increase in the amount of the compound in soil is preferably 1.2 times or more, more preferably 1.5 times or more, still more preferably 2 times or more, and particularly preferably 3 times or more, as compared with the case where plasma water is not applied to the soil (that is, the case where mere water is applied for irrigation, etc.).

[0041] In one embodiment, trimethyllysine, lysine, N-lactoylphenylalanine, N6-methyllysine, asymmetric dimethylarginine (ADMA), proline, symmetric dimethylarginine (SDMA), Nw-methylarginine, homoarginine, arginine, nicotinamide riboside, 3',5'-cyclic cytidine monophosphate, 2',3'-cyclic cytidine monophosphate, N6,N6-dimethyllysine, 3-sulfopropanediol, ornithine, N-(o-toluyl) Glycine, phenaceturic acid, N-formylphenylalanine, ibuprofen, choline, gibberellic acid, N-acetylputrescine, cysteic acid, tryptophan, 1-methyladenine, citrulline, xanthurene acid, proline-hydroxyproline, gamma-glutamic acid-arginine, p-hydroxyphenylacetylglycine, alanine, N,N-dimethylhistidine, mimosine, S-methylcysteine, gamma-aminobutyric acid, prostaglandin F2α, betainaldehyde, morpholine, tropin, 2-oxoglutaric acid, 3-methylcytidine, 2'-O-methylcytidine, ferulic acid, hercinin, tyrosine, serotonin, glycine, prostaglandin E2, histidine, nicotinic acid, pipecolic acid, cyclohexylamine, tetrahydrouridine, valine, trimethylamine-N-oxide, hypotaurine, N-methylserotonin, 4-(β-acetylaminoethyl)imidazole, isonicotinamide / It consists of nicotinamide, 2-amino-2-methyl-1-propanol, 3-aminoisobutyric acid, leucine, phenylalanine, alanine-hydroxyproline, hexamine, threonine, ectoin, N1-methylguanosine, alanine-alanine, acetyl-L-carnitine, isoleucine, methionine sulfoxide, kynurenine trimethyllysine, N-lactoyl, asparagine, aspartic acid, glutamine, glutamic acid, methionine, serine, and cysteine. It is preferable that the amount of one or more compounds selected from the group in the soil decreases. In particular, it is preferable that the amount of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fourteen or more, fourteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, or nineteen or more compounds selected from the group consisting of lysine, proline, arginine, tryptophan, alanine, tyrosine, glycine, histidine, valine, leucine, phenylalanine, threonine, isoleucine, asparagine, aspartic acid, glutamine, glutamic acid, methionine, and serine in the soil is reduced. In one embodiment, after irrigating with plasma water once a week for three times, the decrease in the amount of compound in the soil one week later is preferably 0.8 times or less, more preferably 0.5 times or less, even more preferably 0.3 times or less, and particularly preferably 0.1 times or less, compared to the case where plasma water was not applied to the soil (i.e., when only water was used for irrigation).

[0042] In the method of the present invention, the reduction in the causative agents of plant diseases varies depending on the type and composition of the soil, the species of bacteria, etc., but for example, it occurs 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days after the application of plasma water to the soil (first application). The reduction in the causative agents of plant diseases does not occur immediately after or 1 day after the application of plasma water to the soil. This is thought to be because, upon application of plasma water to the soil, the components in the plasma water (such as ozone) do not directly act on the causative agents and kill them immediately, but rather the plasma water undergoes a chemical change with substances contained in the soil, causing the changes described in (i) and / or (ii) above, which leads to a reduction in the causative agents. Here, the reduction in causative bacteria is preferably 10% or more, preferably 15% or more, more preferably 20% or more, even more preferably 25% or more, particularly preferably 30% or more, and most preferably 40% or more, compared to the case where plasma water was not applied to the soil (i.e., when ordinary water was used for irrigation, etc.).

[0043] In one embodiment, it is preferable that the amount of plasma water decreases by 10% or more (preferably 20% or more) three days after a single application to the soil. In one embodiment, it is preferable that the amount decreases by 20% or more (preferably 30% or more) seven days after a single application to the soil. In one embodiment, it is preferable that the amount decreases by 40% or more (preferably 50% or more) fourteen days after a single application to the soil.

[0044] The method of the present invention can be used in combination with other chemical and biological control methods, but since the method of the present invention alone provides sufficient control before and after planting, it is preferable not to use it in combination with other control methods. In one embodiment, the method of the present invention does not use any pesticides other than plasma water, such as chloropicrin. In another preferred embodiment, the method of the present invention does not use any pesticides other than plasma water.

[0045] The plants targeted by the method of the present invention are not particularly limited, but seed plants are preferred. Examples include cereals, tubers, legumes, vegetables, fruit trees, specialty crops, and flowers.

[0046] Specifically, grains (e.g., rice, wheat, barley, rye, oats, corn, sorghum, millet, foxtail millet, barnyard millet, pearl millet, finger millet, buckwheat), root vegetables (e.g., potatoes, sweet potatoes, taro, yams, konjac), legumes (e.g., soybeans, adzuki beans, kidney beans, peas, broad beans, peanuts, cowpeas, chickpeas, pigeon peas), vegetables (e.g., eggplant, tomato, bell pepper, chili pepper, cucumber) (Examples: melon, watermelon, pumpkin, zucchini, white gourd, bottle gourd, winter melon, bitter melon, cabbage, Chinese cabbage, broccoli, cauliflower, radish, turnip, bok choy, komatsuna, mizuna, green onion, onion, chives, garlic, shallots, asparagus, lettuce, burdock, garland chrysanthemum, butterbur, carrot, mitsuba, parsley, celery, strawberry, spinach, okra, shiso, basil, mint, ginger, myoga), fruit trees (for example) Apples, pears, European pears, quince, Chinese quince, cherries, peaches, plums, Japanese apricots, chestnuts, walnuts, almonds, pecans, grapes, kiwifruit, akebi, persimmons, figs, pomegranates, raspberries, blackberries, blueberries, cranberries, citrus fruits, loquats, olives, bayberries, mangoes, guavas, avocados, dates, coconuts, bananas, pineapples, papayas, passion fruit, acerola), special crops Examples of plants that can be treated include plants (e.g., cotton, flax, rush, rapeseed, sunflower, sesame, oil palm, sugar beet, sugarcane, tea, coffee, cocoa, hops, tobacco), and flowers (e.g., lisianthus, cosmos, morning glory, marigold, impatiens, baby's breath, sweet pea, chrysanthemum, carnation, tulip, lily, daffodil, gladiolus, cyclamen, begonia, water lily, dahlia, rose, cymbidium, cattleya). In the present invention, preferred plants to be treated are lisianthus, tomato, cucumber, watermelon, melon, strawberry, pumpkin, radish, cabbage, turnip, komatsuna, leek, spinach, salad greens, lettuce, chives, sweet potato, celery, and pumpkin, all of which are affected by Fusarium fungi as pathogens.

[0047] The method of the present invention can be effective against plant diseases caused by pathogenic fungi such as filamentous fungi, actinomycetes, and bacteria. Examples of pathogenic bacteria include Fusarium species (e.g., Fusarium avenaceum, Fusarium solani, Fusarium oxysporum, Fusarium Culmorum, Fusarium Moniliforme), Rhizoctonia species (e.g., Rhizoctonia solani), Pythium species (e.g., Pythium hekicoides, Pythium graminicola), Rhizopus species (e.g., Rhizopus chinensis, Rhizopus stolonifer, Rhizopus Delemar), Pyricularia species (e.g., Pyriculariaoryzae Cavara), Cochliobolus species (e.g., Cochliobolus miyabeanus, Cochliobolus sativus), Podosphera species (e.g., Podosphaera leuctricha), and Colletotrichum species (e.g., Colletotrichum orbiculare, Colletotrichum (e.g., gloeosporioides, Colletotrichum circinans, Colletotrichum cucurbitae), Pseudoperonospora species (e.g., Pseudoperonospora subensis, Pseudocercospora fumuli), Venturia species (e.g., Venturia nashicola, Venturiaina equalis), Botrytis species (e.g., Botrytis cinerea, Botrytis elliptica), Puccinia species (e.g., Puccinia tanaceti, Puccinia horiana), Septoria species (e.g., Septoria apii, Septoria lycopersici, Septoria obesa, Septoria lilii), Sclerotinia species (e.g., Sclerotinia sclerotiorum, Sclerotinia intermedia), Sphaerotheca species (e.g., Sphaerotheca pannosa, SphaerothecaExamples include filamentous fungi such as Fusarium fuliginea, Erysiphe species (e.g., Erysiphe cichoracearum, Erysiphe pisi, Erysiphe polygoni), and Sclerotium species (e.g., Sclerotium rolfsii); and bacteria such as Acidovorax species (e.g., Acidovorax avenae, Acidovorax konjaci), Burkholderia species (e.g., Burkholderia glumae, Burkholderia plantarii), and Pseudomonas species (e.g., Pseudomonas fuscovaginae, Pseudomonas syringe, Pseudomonas tolaasii). Among these, the present invention is particularly suitable for plant diseases caused by Fusarium fungi. The method of the present invention can kill these pathogenic fungi and reduce their numbers.

[0048] Furthermore, there are no particular limitations on the diseases that can be controlled using the method of the present invention, and they can be appropriately selected depending on the plants to which the method is applied. For example, damping-off, fusarium wilt, root rot, dry rot, wilt, vine rot, bacterial wilt, bacterial yellow spot, bacterial brown spot, bacterial brown spot, soft rot, bacterial leaf spot, bacterial edge blight, powdery mildew, late blight, fruit rot, brown spot, ring spot leaf blight, sclerotinia rot, black spot, sclerotinia, tarsal fungus, black spot root rot, black spot, white mold, anthracnose, vine blight, seedling blight, gray spot Examples include late blight, gray mold, Verticillium wilt, leaf spot, leaf spot, rose mold, slime mold, downy mildew, Phomopsis root rot, round leaf blight, purple root rot, ring spot, cottony mildew, bacterial black spot, sclerotinia rot, white rust, anthracnose, gray mold, white spot, leaf spot, downy mildew, rhizoctonia, strawberry anthracnose, tomato bacterial leaf spot, and kiwifruit canker. Among these, Fusarium species-induced diseases such as damping-off, yellowing, root rot, dry rot, vine wilt, and wilt are preferred.

[0049] In one embodiment, the present invention provides a method for sterilizing soil. The sterilization method of the present invention includes the steps of (1) preparing plasma water by dispersing and / or dissolving air plasma-activated gas obtained by plasma treatment of air at atmospheric pressure in water, and (2) applying the plasma water to soil. Furthermore, in step (2) above, it is preferable to reduce the causative fungi of plant diseases by changing the amount of soil components that affect the growth of the causative fungi by applying the plasma water to the soil.

[0050] The steps (1) and (2) described above are as follows.

[0051] In one embodiment, the present invention provides a plant disease control agent. The control agent of the present invention comprises the plasma water described above.

[0052] The pest control agent of the present invention may contain optional components as long as they do not interfere with the effects of the present invention. Examples of such components include fertilizers, pH adjusters, surfactants, antifreeze agents, and decomposition inhibitors. These components may be contained individually or in combination of two or more types. The amount of these components contained is not particularly limited.

[0053] Furthermore, in one embodiment, a method for controlling plant diseases or sterilizing soil using the above-described air plasma activated gas is provided. This method is carried out by directly applying the air plasma activated gas, obtained by treating air with atmospheric pressure plasma, to the soil.

[0054] The method for applying air plasma activated gas to soil involves burying permeable pipes in the soil and flowing the air plasma activated gas through them during the application period. For example, this can be done by burying pipes at a depth of 20 to 100 cm in the soil. The permeable pipes can be, for example, those with numerous vents.

[0055] The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

[0056] <Manufacturing Example 1: Production of Plasma Water> Air plasma activated gas, which is air containing radicals, active species, and ions discharged after passing through plasma generated by an atmospheric pressure plasma jet, was brought into contact with purified water to produce an air plasma activated gas dispersion solution (hereinafter also referred to as plasma water) containing liquid-phase active species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS). First, air plasma activated gas was generated under atmospheric pressure under the following conditions: ・Introduced gas: Air ・Quartz tube inner diameter: 4 mm ・Discharge power: 0.01 to 50 W ・Total discharge length: 600 mm ・Frequency: 5 to 7 kHz ・Air flow rate: 1 to 4 slm Air plasma activated gas contains various chemical species. The gas-phase active species contained in the plasma activated gas were measured by infrared absorption using a Fourier transform infrared spectrophotometer (FT / IR-6100TUK, JAS.CO, Tokyo, Japan). The presence or absence of CO, CO2, O3, N2O, NO, NO2, N2O4, N2O5, HNO2, HNO3, HNO4, H2O, and H2O2 was checked, and their concentrations were calculated. As a result, ozone (O3) was found at approximately 4000 ppm, nitrous oxide (N2O5) at approximately 20 ppm, and nitric acid (HNO3) at approximately 100 ppm. All other active species were below the detection limit. Subsequently, plasma water was prepared by bubbling air plasma activated gas into ion-exchange purified water (distilled water (DW)) under atmospheric pressure. Specifically, a method was adopted in which the bubbling solution was constantly released to the outside while the same amount of purified water as the amount of released solution was introduced into the solution generation unit (Figure 1). Here, the amount of water in the solution generation unit was approximately 200 mL. The conditions for the air plasma activated gas solution production machine are as follows. - Water introduction rate: 60 mL / min - Air plasma activated gas introduction rate: 1-4 slm Analysis of the ozone concentration and pH of the plasma water immediately after purification revealed that it contained approximately 20 μM of ozone and had a pH of approximately 4. The ozone concentration analysis was performed as follows. Note that this method uses Br in relation to dissolved ozone under low pH conditions. - By adding an aqueous solution, the concentration of Br3 is equal to that of dissolved ozone. -This is based on the formation of an aqueous solution. • Measurement device: UV-Vis spectrophotometer (evolution 220, ThermoFisher Scientific) • Measurement wavelength: 190–450 nm • Measurement method: 2 mL of plasma water was taken and mixed with 200 μL of NaBr solution (a mixture of NaBr 5M and HClO4 1M) to prepare a mixture. Then, 2 mL was taken from the mixture and absorbance was measured to determine Br3 - The absorbance at 266 nm, which is the absorbance peak of the aqueous solution, was measured. Then, Br3 - Molar extinction coefficient of aqueous solution: 40900 M -1 cm -1 Based on this, Br3 from Lambert-Beer's Law - The concentration (i.e., dissolved ozone concentration) was calculated.

[0057] <Example 1: Sterilization of Fusarium-contaminated soil using plasma water> The sterilizing effect of plasma water produced in Production Example 1 on Fusarium avenaceum was investigated. Soil was collected from three locations where damping-off disease occurred in a lisianthus cultivation site and used as contaminated soil. 50 ml of plasma water was gently poured over 50 g of the contaminated soil. As a control, 50 ml of distilled water was gently poured over 50 g of the contaminated soil. After standing at 25°C for 1, 3, 7, and 14 days, the soil was suspended in sterile distilled water, and the serially diluted suspensions were spread on Fo-G2 selective medium, and the bacterial concentration in the contaminated soil was measured by culture (Figure 2). After 1 day of plasma water treatment, no difference in bacterial count was observed between the control (distilled water treatment) group and the plasma water treatment group. Therefore, it was revealed that plasma water does not have an immediate sterilizing effect. On the other hand, in the test plots left for 3, 7, and 14 days, a 23% to 64% reduction in bacterial count was observed in the plasma water treatment plots compared to the control plot (Figure 3). This result suggests that leaving the contaminated soil after plasma water treatment for 3, 7, and 14 days may cause the plasma water to chemically react with substances in the soil, generating substances with bactericidal (or bacteriostatic) activity. This is in contrast to chloropicrin, which is commonly used as a soil disinfectant and does not exhibit such a sustained bactericidal effect.

[0058] <Example 2: Effect of plasma water treatment on the growth of Eustoma> In order to investigate the effect of plasma water treatment on the growth of Eustoma, 600 ml of plasma water was irrigated once a week for 2 months to plants 2 months after sowing. After continuing cultivation, the effect on growth was evaluated. In addition, for Eustoma cultivated by a conventional method, a control plot in which chloropicrin was treated one week before growth evaluation was also set. As shown in Fig. 4, the plasma water-treated plants grew to the same extent as the distilled water-treated plants, and plasma water treatment did not exert a negative effect on the growth of Eustoma. On the other hand, since Eustoma withered in the chloropicrin treatment, it was reconfirmed that chloropicrin cannot be used after planting Eustoma seedlings. From these results, it was revealed that plasma water can be used as a Fusarium control agent that can be treated even after planting of Eustoma seedlings.

[0059] <Example 3: Examination of plasma water treatment conditions> In order to reconfirm that plasma water treatment suppresses the decrease in fungal concentration in Fusarium-contaminated soil and the occurrence of damping-off, plasma water was regularly irrigated, and tests were conducted on "influence on plant growth", "decrease in pathogenic bacteria concentration in soil", and "occurrence of damping-off" (the experimental procedure is shown in Fig. 5). Plants 2 months after sowing were planted in culture soil placed in 500 ml pots, and 600 ml of plasma water was irrigated once a week for 3 weeks. The plants were transplanted together with the culture soil into Fusarium-contaminated soil, and further 600 ml of plasma water was irrigated once a week for 4 weeks (Fig. 5). The experiment was repeated twice, and the inoculum concentration in the first experiment was 1x10 6 bacteria cells / 1 g soil, and the inoculum concentration in the second experiment was 5x10 6The soil was measured using a bacterial count / 1g method. Soil samples were collected at the 7th week of cultivation, and the bacterial concentration was measured using a plate dilution culture method with Fo-G2 selective medium. Simultaneously, the occurrence of damping-off disease was observed. The experimental results are shown in Figure 6. No effect on plant growth was observed due to periodic plasma water treatment. The results of bacterial concentration measurement using the plate dilution culture method with Fo-G2 selective medium (Figure 6A) showed that in the first experiment, plasma water treatment suppressed the Fusarium genus concentration by 97.0%, and in the second experiment, plasma water treatment suppressed the bacterial concentration by 74.4% (Figures 6B, 6C). Furthermore, when the suppression of damping-off disease by plasma water treatment was investigated, wilting and yellowing of leaves, which are early symptoms of damping-off disease, were observed in the distilled water treatment group, but no early symptoms of damping-off disease were observed in the plasma treatment group (Figure 7). Therefore, it was shown that regularly irrigating with plasma water in lisianthus cultivation can reduce the concentration of Fusarium species in the soil and potentially suppress the occurrence of damping-off disease. In addition, the concentration of Fusarium species in the soil of lisianthus cultivation fields was, for example, 10 2 ~10 3 The bacterial count is approximately 1g, which is lower than the bacterial concentration in the above experiment (the bacterial concentration in the first inoculation of the above experiment was 1x10⁻⁶). 6 The bacterial count per 1g of soil, and the bacterial concentration in the second inoculation of the experiment was 5x10. 6 (Bacterial count / 1g soil). Therefore, plasma water can be expected to reduce pathogenic bacteria and suppress the onset of damping-off disease in cultivation sites.

[0060] <Example 4: Treatment of Fusarium-contaminated soil using plasma water> Fusarium-contaminated soil collected from a lisianthus cultivation site was filled into a cylindrical container to a height of 24 cm, and plasma water was irrigated from the top of the cylinder with 50 mL once a week for 4 weeks (Figure 8). Subsequently, as shown in Figure 8, the soil in the cylindrical container was divided into sections every 3 cm from the top of the cylinder, and the bacterial concentration in the soil at the top, the third position from the top, and the seventh position from the top was measured. The results are shown in Figure 9. As shown in Figure 9, the bacterial concentration decreased by approximately 50% at the seventh position from the top (approximately 20 cm from the top), indicating that it had a similar level of pathogen-suppressing effect as the topmost soil.

[0061] <Example 5: Suppression of Fusarium-contaminated soil by treatment with plasma water> We considered that it would be highly likely to maintain a state in which the proliferation of pathogenic bacteria is suppressed by sterilizing the soil with plasma gas before planting lisianthus seedlings and then regularly irrigating with plasma water after planting, and therefore investigated the effect of treating the soil with plasma water on suppressing the proliferation of Fusarium bacteria. Pathogenic bacteria-contaminated soil (initial bacterial concentration 1.6 × 10⁻⁶) 6 After treating the plants with plasma water (12.5 mL once a week for 3 weeks) at a dose of cfu / g, lisianthus seedlings were transplanted, and plasma water treatment was continued (Figure 10). On the 14th day after transplanting, wilting, yellowing, and stunting were observed in the control group that did not receive plasma water treatment, but no such symptoms were observed in the group that received plasma water treatment, indicating that the onset of damping-off disease was suppressed.

[0062] <Example 6: Analysis of the mechanism of reduction in Fusarium count due to plasma water treatment> The decrease in Fusarium count in soil treated with plasma water was an unexpected discovery. In the case of plasma water treatment, no decrease in the number of bacteria was observed immediately after treatment, but a decrease in the number of bacteria was observed several days to several weeks after treatment. Therefore, we hypothesized that the interaction between ozone or nitrogen-active species contained in the plasma water and some substance or microorganism in the soil changed the composition of compounds contained in the soil, and that this change in compound composition affected the bacterial concentration. As a first step in verifying this hypothesis, metabolome analysis of soil treated with plasma water and distilled water (control) was performed as follows. (Preparation of treated soil) The soil was treated once a week with plasma water or distilled water (control) (irrigation with 400 ml of plasma water or distilled water per 200 g of soil) for 3 weeks (i.e., 3 treatments), and the soil from the 4th week (i.e., soil one week after the 3rd treatment) was used for analysis. (Pretreatment) To a soil sample treated with plasma water or distilled water, 1500 μL of 50% acetonitrile aqueous solution (v / v) (internal standard concentration: 1 μM) was added to a crushing tube, and the sample was crushed using a crusher under cooling (1100 rpm, 120 seconds x 4 times), and then the same amount of 50% acetonitrile aqueous solution (v / v) was added. After tissue crushing, centrifugation (2,300 × g, 4°C, 5 min) was performed. After centrifugation, the supernatant was transferred to an ultrafiltration tube (Ultrafree MC PLHCC, HMT, centrifugal filter unit 5 kDa). This was centrifuged (9100 × g, 4°C) and ultrafiltration was performed. The filtrate was allowed to dry, dissolved in MilliQ water, and used for measurement. (Candidate metabolite search) Metabolome analysis was performed on the pretreated samples by CE-FTMS. Based on the m / z and MT values ​​of substances registered in the HMT metabolite library, 445 candidate compounds (262 cations, 183 anions) were identified. (Intergroup comparison) For the 445 candidate compound peaks narrowed down through intergroup comparison between the two groups (6 plasma water treatment replicates and 6 distilled water treatment replicates), the relative area value ratios for each group were calculated and Welch's t-test was performed.Principal component analysis of all compounds showing fluctuations between the plasma water-treated and distilled water-treated groups revealed that the dynamics of the compounds were similar in the 6 replicate samples of the plasma water-treated group compared to the distilled water-treated group (Figure 11, contribution rate of the first principal component 32.6%, contribution rate of the second principal component 19.5%). Tables 1-3 show the compounds that showed significant fluctuations due to plasma water treatment. These changes in compound composition are thought to have affected the bacterial concentration. In particular, the increase in p-sulfobenzoic acid was significant. Furthermore, among the compounds that decreased were 13 of the 20 amino acids that make up proteins. In addition, when the relative amounts of other amino acids were examined, all decreased in the plasma water-treated group.

[0063]

[0064]

[0065] <Example 7: Inhibition of Fusarium growth by compounds altered in plasma water-treated soil> Three types of culture media were prepared, and Fusarium bacteria were placed on each medium. The growth of the bacteria was evaluated by measuring the diameter of the colonies on the 5th day of culture (Figure 12). For Fusarium bacteria, they were first cultured on agar medium, and the formed bacterial colony was removed along with the agar medium using a 7 mm diameter cork borer and placed on the following media. (i) Medium to which p-sulfobenzoic acid, which showed the greatest relative increase in plasma water-treated soil, was added (10 mM final concentration added to the basic medium) (ii) Medium from which 20 amino acids, which showed a decrease in relative amount in plasma water-treated soil, were removed (20 amino acids removed from the basic medium) (iii) Results for the basic medium as a control are shown in Figures 12B and 12C. In both cases, the addition of p-sulfobenzoic acid to the basic medium and the removal of the 20 amino acids from the basic medium inhibited bacterial growth by approximately 30%.

Claims

1. A method for controlling plant diseases, comprising: (1) a step of preparing plasma water by dispersing and / or dissolving an air plasma-activated gas obtained by treating air with atmospheric pressure plasma in water; and (2) a step of applying the plasma water to soil.

2. The method according to claim 1, wherein step (2) includes irrigating the soil in which the plant is growing with plasma water.

3. The method according to claim 1, wherein in step (2), the amount of soil components that affect the growth of the plant disease-causing fungi is reduced by applying the plasma water to the soil.

4. The method according to claim 1, wherein the causative fungi of the plant disease decrease two days or more after the application of plasma water to the soil.

5. The method according to claim 1, wherein the plant disease is caused by a Fusarium fungus.

6. A method for sterilizing soil, comprising: (1) a step of preparing plasma water by dispersing and / or dissolving an air plasma-activated gas obtained by plasma treatment of air at atmospheric pressure in water; and (2) a step of applying the plasma water to soil.

7. A plant disease control agent containing plasma water, wherein the plasma water is obtained by dispersing and / or dissolving an air plasma-activated gas, which is obtained by plasma treatment of air at atmospheric pressure, in water.

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