WO2012046758A1 - Gramineous plant disease resistance enhancer and gramineous plant disease prevention method using same - Google Patents

Gramineous plant disease resistance enhancer and gramineous plant disease prevention method using same Download PDF

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WO2012046758A1
WO2012046758A1 PCT/JP2011/072955 JP2011072955W WO2012046758A1 WO 2012046758 A1 WO2012046758 A1 WO 2012046758A1 JP 2011072955 W JP2011072955 W JP 2011072955W WO 2012046758 A1 WO2012046758 A1 WO 2012046758A1
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disease resistance
disease
amino acid
rice
plant
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PCT/JP2011/072955
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French (fr)
Japanese (ja)
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直樹 角谷
武田　泰斗
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味の素株式会社
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof

Definitions

  • the present invention relates to a disease resistance enhancer and a disease control method for gramineous plants that contain an amino acid as an active ingredient and have a low environmental load and are safe for users and consumers.
  • Static resistance means resistance due to the physical and chemical properties of a plant before being infected with pathogenic bacteria, such as plant cell wall hardness and thickness, and accumulation of polyphenols.
  • dynamic resistance means a series of defense responses in which plant cells or tissues are activated in response to an enemy attack and signs.
  • the dynamic resistance reaction of higher plants includes the generation of active oxygen and associated hypersensitive cell death, the formation of papillas, and the genes that encode phytoalexins synthase enzymes that have antibacterial activity against pathogens.
  • PR Pieris-related
  • induction resistance Resistance based on physical and chemical barriers, which is constructed in plant tissues by artificially treating plants with pathogens that have no affinity with plants or elicitors, is called induction resistance. This phenomenon can be regarded as a result of the dynamic resistance inherent in the plant being derived by pretreating the plant with a specific substance called a resistance inducer.
  • the response is weak compared to the induction resistance, but when a pathogen is invaded, the phenomenon that a dynamic resistance can be induced quickly and strongly is created in the plant cell is called priming.
  • Dynamic resistance is classified into Local Acquired Resistance (LAR) and Systemic Acquired Resistance (SAR) based on spatial differences.
  • SAR is basically a phenomenon in which when chemical, physical or biological stress is applied to a part of a plant, the information is transmitted to the whole body and a new resistance to the stress is induced in the whole body.
  • LAR is distinguished from SAR.
  • SAR is induced systemically, SAR is thought to be SAR, but SAR is thought to be transmitted through multiple signals, and both are qualitatively identical from the expression pattern of disease-responsive genes. It is thought that it is not (for example, nonpatent literature 1).
  • SA Salicylic acid
  • Non-Patent Document 2 In rice, it is shown that the downstream of SA branches into an NPR1 route and a WRKY45 route (Non-Patent Document 2). WRKY45 encodes a transcription factor and controls the expression of many genes involved in disease resistance such as PR protein gene. A strong expression strain of the WRKY45 gene exhibits strong resistance to rice blast fungus (Non-patent Document 2).
  • the method of fertilizing silicon is widely known as a method for increasing static resistance. Particularly in rice, it is considered that treatment with silicon physically strengthens the cell wall and suppresses infection of pathogenic bacteria such as rice blast fungus.
  • Non-Patent Document 3 As a method of suppressing disease by treating rice with silicon, a method of fertilizing silica gel is known (Non-Patent Document 3). In addition, a method has been reported in which histidine, lysine, glutamine, glycine, imidazole, and sodium silicate are applied to rice to promote uptake of silicic acid (Non-patent Document 4).
  • a drug using dynamic resistance is called a plant resistance inducer.
  • a pesticide that directly acts on a plant pathogen such as a bactericide
  • Plant resistance inducers do not act directly on pathogenic bacteria, but control disease infection by inducing plant resistance. No cases have been accepted.
  • resistance-inducing pesticides are considered to have a relatively low environmental impact, including organisms other than plants, because they have little bactericidal action on organisms.
  • probenazole (trade name: Orizemate, Meiji Pharmaceutical Co., Ltd.), benzothiazole (BTH) acibenzoral S-methyl (ASM, trade name: Vion. Syngenta Sakai Japan ( Co., Ltd.), thiadiazole carboxamide-based thianidyl (trade name: Vuget. Nippon Agricultural Chemicals Co., Ltd.) is sold.
  • Patent Document 1 a polysaccharide degradation product
  • Patent Document 2 cerebrosides
  • Patent Document 3 jasmonic acid
  • Non-Patent Document 5 chitin oligosaccharide
  • Non-Patent Document 6 ⁇ -1,3- and ⁇ -1,6-glucan oligosaccharide
  • Patent Document 4 bile acid
  • Non-Patent Document 7 Lipopolysaccharide
  • Non-Patent Document 1 Non-Patent Document 1
  • Non-patent Document 10 Non-patent Document 10
  • a priming effect substance refers to a substance that, by pre-treating a plant with this, significantly enhances the cellular response to elicitors and pathogens and creates a situation in plant cells that can induce dynamic resistance quickly and powerfully.
  • ⁇ -aminobutyric acid BABA
  • BABA ⁇ -aminobutyric acid
  • Non-patent Document 9 Non-patent Document 9 controls or suppresses the disease of pearl millet.
  • sulfur-containing amino acids Patent Documents 7 and 8
  • aminobutyric acid Patent Document 7
  • glycine Patent Document 9
  • Patent Documents 10, 11, and 12 mixed fertilizers containing amino acids
  • Patent Document 5 amino acid fermentation broth
  • Patent Document 6 amino acid fermentation broth
  • Patent Document 6 amino acid fermentation broth
  • Patent Document 6 an extract obtained by heat-treating microbial cells in an acidic solution
  • Patent Document 13 plant disease control by an amino acid mixture (proline, methionine)
  • Non-patent Document 14 Although there has been a report on disease control by fermentation liquid of lysine or glutamic acid (Patent Document 14), no data is described, the active ingredient is unknown, and it is not clear whether it is due to disease resistance induction. . Furthermore, it has been reported that the treatment of sugarcane leaves with DL-phenylalanine promotes the synthesis of phenolic substances and inhibits the spore germination and germ tube elongation of rice sesame leaf blight fungi (Non-patent Documents) 11) It is not described whether phenylalanine treatment actually suppresses invasion or infection of pathogenic bacteria.
  • JP-A-5-331016 Japanese Patent No. 2846610 Japanese Patent Laid-Open No. 11-29412 JP 2006-219372 JP Japanese Patent Laid-Open No. 6-80530 International Publication No. WO / 2009/088074 JP2003-34607 Patent No. 4287515
  • Chinese Patent Publication No. 1640233A Chinese Patent Publication No. 1316893C
  • Chinese Patent Publication No. 1328769A China Patent Publication No. 101182270A China Patent Publication No. 101142925A
  • the object of the present invention is to provide a disease resistance enhancer for gramineous plants that is highly safe and inexpensive for consumers and users, and a disease control method for gramineous plants using the same.
  • the present inventors searched for a substance that induces disease resistance of rice and found that L- or D-amino acids have high disease resistance-inducing activity.
  • L- or D-amino acids have high disease resistance-inducing activity.
  • glutamic acid, asparagine, aspartic acid, threonine, valine, arginine, and alanine to rice, dynamic resistance to disease is systemically induced.
  • these amino acid processing showed the high control effect with respect to diseases, such as a rice blast fungus.
  • diseases such as a rice blast fungus.
  • it has been clarified that the same phenomenon is caused in other gramineous plants.
  • a disease resistance enhancer for gramineous plants comprising an amino acid selected from the group consisting of glutamic acid, asparagine, aspartic acid, threonine, alanine, valine, arginine, and serine.
  • the disease resistance enhancer which is applied to gramineous plants by rhizosphere application or foliar application.
  • the disease resistance enhancer, wherein the gramineous plant is rice, shiba or corn.
  • the disease resistance enhancer, wherein the disease is Gramineae blast or rice leaf blight.
  • the disease resistance enhancer comprising the amino acid in a total amount of 0.2 to 200 mM.
  • the disease resistance enhancer wherein the amino acid is L-form.
  • the disease resistance enhancer wherein the amino acid is D-form.
  • the disease resistance enhancer further comprising a disease resistance inducer other than amino acids.
  • the disease resistance enhancer comprising glutamic acid.
  • fungicides that act directly against phytopathogenic fungi such as fungicides often show resistance mutants to drugs with continued use, but plant resistance inducers are resistant mutants of drugs. It is known that is difficult to appear and can be used for a long time.
  • the L- or D-amino acid that is an active ingredient of the disease resistance enhancer of the present invention is not a direct bactericidal action against pathogenic bacteria, but prevents disease infection by inducing disease resistance. Mutant strains are unlikely to appear, and it can be expected that they can be used over a long period of time, which is extremely useful industrially.
  • D-amino acids are considered less susceptible to degradation by microorganisms in the environment than L-amino acids. In one embodiment of the present invention, it is expected that a stable disease control effect can be realized by using a D-amino acid.
  • an existing plant disease resistance inducer for example, a plant disease resistance inducer registered with an agrochemical and an amino acid
  • the control effect is increased or the amount of the plant disease resistance inducer used is reduced. be able to.
  • the figure which shows the rice blast control effect by the rhizosphere application of an amino acid The figure which shows the rice blast control effect by the foliar application of an amino acid.
  • PBZ represents probenazole (hereinafter the same).
  • cht3 represents the chitinase 3 gene
  • OsNPR1 represents the NPR1 gene in the rice genome.
  • ORZ represents oryzate (agrochemicals containing probenazole as an active ingredient).
  • the figure which shows the response with respect to the amino acid of the rice cultivated aseptically on the artificial culture medium. con represents a negative control (hereinafter the same).
  • the expression level of the disease response gene in the root tissue is shown.
  • the expression level of the disease response gene in the root tissue is shown.
  • control represents a control.
  • the expression level of the disease response gene in the 4th leaf is shown.
  • L-Glu represents L-glutamic acid, and con represents a control.
  • L-Glu represents L-glutamic acid, and con represents a control.
  • the disease resistance enhancer for gramineous plants of the present invention contains an amino acid selected from the group consisting of glutamic acid, asparagine, aspartic acid, threonine, alanine, valine, arginine, and serine as an active ingredient. Of these amino acids, glutamic acid, asparagine, aspartic acid, threonine, and alanine are preferred.
  • the disease resistance enhancer may contain each amino acid alone, or may contain any two or more amino acids. When a plurality of amino acids are included, one is preferably glutamic acid. Moreover, the disease resistance enhancer may contain amino acids other than these amino acids.
  • the amino acids may be either L-form or D-form, and may be a mixture containing L-form and D-form at an arbitrary ratio.
  • serine is preferably D-form.
  • the amino acid may be a free form or a salt such as ammonium salt, sodium salt or potassium salt.
  • the form of the amino acid is not particularly limited as long as it contains the amino acid, and may be a generally sold reagent, a refined or crude product produced by a fermentation method, a by-product generated in a purification process, and a marine product. It may be a composition containing the amino acid, such as an extract from or a protein hydrolyzate.
  • disease resistance inducers other than amino acids in addition to the amino acids.
  • the disease resistance inducer other than amino acids is not particularly limited as long as it is a compound other than amino acids and can induce dynamic resistance or priming to gramineous plants.
  • Probenazole (trade name: oryzate) Meiji Seiyaku Co., Ltd.), benzothiazole (BTH) acibenzoral S-methyl (ASM, trade name: Vion. Syngenta Sakai Japan Co., Ltd.), thiadiazole carboxamide-based thianidyl (trade name: Vuget. Nippon Agricultural Chemicals Co., Ltd.) And other commercially available drugs.
  • the terms “disease resistance enhancer” and “disease resistance inducer” refer to the disease resistance enhancer of the present invention and substances having a disease resistance inducing action other than amino acids for convenience. It is to distinguish, not to distinguish their actions.
  • the disease resistance enhancer of the present invention has a disease resistance inducing action. Therefore, the disease resistance enhancer of the present invention can also be called a disease resistance inducer.
  • a preferred amino acid is glutamic acid.
  • the gramineous plant disease resistance enhancer may contain any component in addition to the amino acid and the disease resistance inducer other than amino acids.
  • Such components include solvents, carriers, pH adjusters, spreading agents for increasing the spreading power to plants, surfactants for increasing the permeability to plants, etc., for enhancing the fertilization effect.
  • Fertilizer components such as minerals, agricultural chemical components, binders, extenders and the like. As these components, components usually used for agricultural chemicals, fertilizers and the like can be used as long as the effects of the present invention are not impaired.
  • the dosage form of the gramineous plant disease resistance enhancer according to the present invention may be any use form such as solution, powder, granule, emulsion, wettable powder, oil, aerosol, flowable, etc., and the form of the drug for application
  • the use form and application method are not particularly limited.
  • the carrier component examples include vermiculite, talc, diatomaceous earth, kaolin, calcium carbonate, clay, calcium hydroxide, clay when the gramineous plant disease resistance enhancer of the present invention is a bottom floor additive or a solid agent.
  • Inorganic substances such as silica gel and solid carriers such as wheat flour and starch can be used.
  • the gramineous plant disease resistance enhancer is a liquid, water, aromatic hydrocarbons such as xylene, alcohols such as ethanol and ethylene glycol, ketones such as acetone, ethers such as dioxane and tetrahydrofuran , Liquid carriers such as dimethylformamide, dimethyl sulfoxide, acetonitrile and the like can be used.
  • a solid or powdery gramineous plant disease resistance enhancer may be dissolved or dispersed in a solvent such as water or alcohol. Further, the liquid gramineous plant disease resistance enhancer may be diluted with a solvent such as water or alcohol.
  • the gramineous plant disease resistance enhancer is used as a disease control agent.
  • a method for controlling a gramineous plant disease which comprises applying the gramineous plant disease resistance enhancer according to the present invention to a gramineous plant.
  • the content of amino acids in the gramineous plant disease resistance enhancer is not particularly limited, and can be appropriately set according to the application rate described later.
  • the content of amino acids in the gramineous plant disease resistance enhancer is not particularly limited as long as an effective amount for enhancing disease resistance can be applied, but when it contains an amino acid alone, for example, usually 0.2 to 200 mM, Preferably it is 1 to 100 mM. In the case of containing a plurality of amino acids, the total content is 0.2 to 200 mM, preferably 1 to 100 mM.
  • concentration is a density
  • concentration is a density
  • the content is not particularly limited, but is usually 0.1 mM to 200 mM, preferably 1 mM to 50 mM, for example.
  • the content is not particularly limited, but is usually 0.1 mM to 200 mM, preferably 1 mM to 50 mM, for example.
  • disease resistance inducers that are registered as agricultural chemicals, it depends on the recommended amount.
  • the amount ratio of the amino acid and the disease resistance inducer other than the amino acid is, for example, usually 1: 1/10000 to 1: 1000, preferably 1: 1/200 to 1:20.
  • Plants targeted by the disease resistance enhancer of the present invention are plants belonging to the family Gramineae, and include rice (Oryza sativa), shiba (Zoysia genus plant), wheat (Triticum genus plant), maize (Zea mays), barley ( Hordeum vulgare), rye (Secale cereale), sugarcane (Saccharum officinarum) and the like. Examples of varieties include, but are not limited to:
  • Shiba varieties include Himeko-ryashiba, Noshiba, Centipedegrass, Bermudagrass, Tifton Shiva, Riviera, Sun Devil II, Tiffway, Bahiagrass, St. Augustine Singrass and the like.
  • ⁇ Barley varieties include Nijo barley, Shijo barley, Rojo barley, bare barley, and Sachiho golden.
  • Rye varieties include Haruka, Haruichi, King Rye, Ryotaro, Petkus, etc.
  • sugarcane varieties include agricultural forest 8, agricultural forest 9, agricultural forest 10, agricultural forest 11, agricultural forest 13, agricultural forest 15, agricultural forest 19, agricultural forest 20, F161, KY96-189, KY96T-537, etc. .
  • the expression of disease-responsive genes in rice was significantly increased by amino acid application, and the disease resistance by amino acid application is not caused by other substances such as silicic acid. It was shown to be due to the induction of mechanical resistance, particularly systemic acquired resistance. Since the dynamic resistance of plants is non-specific to pathogenic bacteria, the target disease is not particularly limited. For example, plant diseases caused by filamentous fungi, bacteria and viruses are included.
  • Examples of typical rice diseases are rice leaf blight, rice anthracnose, rice coat blight, rice seedling blight, rice leaf sheath browning, rice scab, rice stripe disease, and rice yellowing atrophy Etc.
  • wrinkles examples include wrinkle dwarf disease, wrinkle mosaic disease, wrinkle dwarf disease, buckwheat horn disease, wrinkle white leaf disease, wrinkle rust disease, wrinkle blast disease, and wrinkle blight.
  • the diseases of wheat include wheat dwarf disease, wheat red mold disease, wheat red rust disease, wheat horn horn disease, wheat leaf blight, wheat spot disease, wheat blast disease, wheat horn spot disease, wheat black spot disease, wheat black rust disease, wheat Examples include macular disease, wheat anthrax, wheat blight, and wheat powdery mildew.
  • Maize diseases include corn streak dwarf disease, corn yellowing disease, corn mosaic disease, corn leaf blight, corn sesame leaf blight, corn leaf blight, corn leaf blight, corn blast, corn brown blight, corn Examples include blight, maize seedling blight, corn southern rust, and corn rust.
  • Barley diseases include barley powdery mildew, barley ring rot, barley streak, barley blight, barley soot, barley cloud, barley dwarf, barley black spot, barley black spot, etc. Can be mentioned.
  • diseases of rye include rye yellow leaf disease, rye ergot, rye red rust, rye spot disease, rye powdery mildew, rye black spot disease, rye red mold disease, and rye streak bacterial disease.
  • Sugarcane diseases include sugarcane mosaic disease, sugarcane red stripe disease, sugarcane red rot, sugarcane peel disease, sugarcane white stripe disease, sugarcane downy mildew, sugarcane dwarf disease, sugarcane eye spot disease, sugarcane pseudo red stripe disease, Examples include sugarcane brown stripe disease, sugarcane leaf blight, sugarcane smut, sugarcane ring spot disease, and sugarcane rust disease.
  • blast fungus Magnaporthe oryzae
  • the plant disease control method of the present invention has a main purpose of preventing disease, and is preferably used prior to the time when the disease occurs. However, even after the occurrence of a disease, an effect of suppressing the expansion or attenuating the disease can be expected.
  • a disease resistance enhancer By applying a disease resistance enhancer to a gramineous plant, resistance to the above diseases can be enhanced.
  • the method of application is not particularly limited, and can be used by a method according to the dosage form, the state of paddy field or field, and the situation of the farmer. For example, it can be sprayed, dripped or applied as a liquid or an emulsion to not only the plant growth point but also a part or the whole of the plant body including stems and leaves.
  • application to the rhizosphere for example, surface application to the soil, irrigation, mixing into the soil, or immersion treatment in the root can be mentioned. Of these, rhizosphere application is preferred.
  • the gramineous plant disease resistance enhancer of the present invention is applied to the rhizosphere, systemic acquired resistance or priming is induced in the above-ground part.
  • this Gramineae plant disease resistance enhancer to gramineous plants
  • the frequency varies depending on the purpose of administration, the growth stage of gramineous plants, and the like. For example, if it is before rice planting, the disease of a young disease can be suppressed by applying to a nursery.
  • the application amount of the gramineous plant disease resistance enhancer may vary depending on the concentration of the active ingredient, the application time, the frequency of application, the type of plant, the cultivation density, the growth stage, the application method, and the like.
  • the application rate is usually, for example, 0.2 to 200 mM at 100 to 5000 L / ha, preferably 2 to 100 mM at 500 to 1000 L / ha, as the amount of amino acid.
  • the total amount is usually 0.2 to 200 mM at 100 to 5000 L / ha, preferably 2 to 100 mM at 500 to 1000 L / ha.
  • the application amount is the same as that in the foliar application.
  • the disease resistance enhancer containing an amino acid and the disease resistance inducer other than the amino acid may be separately applied to the grass family plant.
  • Example 1 Effect of amino acid on control of rice blast (1) Cultivation and application method of plants Rice (cultivar "Nihonbare") was cultivated as follows. Putting soil mixed with cultivated soil for horticulture (Powersoil; Kureha Chemical Co., Ltd.) and vermiculite (S.K. The sowed rice was sown and cultivated for 14 days. The incubator was cultivated at a temperature of 23 to 25 ° C., a daily period of 14 hours light, and a light intensity of about 100 ⁇ mol m ⁇ 2 s ⁇ 1 .
  • the rhizosphere application was performed by immersing the pot in an amino acid solution as described above, and the foliar application was performed by direct spraying. Inoculation of rice blast fungus was performed by spraying the conidia suspension (1 ⁇ 10 5 conidia / mL) on the leaf surface. After spray inoculation, the plant was infected with rice blast fungus by leaving it in a dark place for 24 hours. Evaluation was carried out by measuring the number of diseased lesions occurring on each treated leaf 4 days after the inoculation. Instead of the amino acid solution, water was used in the negative control group, and 0.5 mM probenazole was used as a positive control in the foliar spray test. The control value was calculated according to the following formula. The experiment was repeated 3 to 4 times, and the average control value in each experiment is shown in FIG. 1 and FIG.
  • Control value (1-average number of lesions per leaf in each ward / average number of lesions per leaf in the negative control ward) x 100
  • Fig. 1 shows the control value by application of amino acid rhizosphere. Compared with the negative control group, it was shown that any amino acid can significantly control the infection of pathogenic bacteria by applying the rhizosphere. In addition, each amino acid other than L-phenylalanine showed a higher sensitivity defense effect than L-phenylalanine.
  • Fig. 2 shows the control value by spraying amino acid leaves. Compared to the negative control group, it was shown that infection with pathogenic bacteria can be significantly controlled by foliar application of amino acids.
  • Example 2 Evaluation of plant disease resistance induction using the increase in the expression of disease response gene as an index (1) Cultivation and application method of plant body The temperature in the incubator is 28-25 ° C and the daily rate is 14 During the light season, the light intensity was cultivated at about 30000 Lx. Nihonbare was used as the rice variety. Rice seeds from which rice husks were removed were germinated at 30 ° C. in the dark for 2 days. Garden soil (power soil; Kureha Chemical Co., Ltd.) and vermiculite (SK Aguri Co., Ltd.) mixed in a ratio of 4: 1 to pot (BEE POT; Caneron Chemical Co., Ltd.) The sown rice was sown and cultivated for 14 days. The rhizosphere application of rice as a test sample was performed by putting 300 ml of the solution in a food pack (Chuo Chemical Co., Ltd., C-AP fruit 200) and immersing the rice in the pot there.
  • a food pack Cho Chemical Co., Ltd.
  • RNA extraction and quantitative RT-PCR Evaluation of disease resistance induction of rice was performed using the root and the fourth leaf.
  • the root or the fourth leaf was sampled from the rice cultivated as described above, immediately frozen in liquid nitrogen, and stored at -80 ° C. It was crushed with a plant crusher (MM300 MIXER MILL GRINDER; Retsch) in a frozen state.
  • RNA extraction using EZ1 RNA Tissue Mini Kit (Qiagen) and Magtration System 12Gc (Qiagen) After RNA extraction using EZ1 RNA Tissue Mini Kit (Qiagen) and Magtration System 12Gc (Qiagen), the chitinase 3 gene, by quantitative PCR using a PCR device (7500 Real Time PCR System; Applied Biosystems) The expression levels of the OsNPR1 gene, the WRKY45 gene, and the PR10 (PBZ1) gene were compared. The eukaryotic transcription elongation factor eEF-1a gene was used as an internal standard.
  • Chitinase gene, NPR1 gene, PBZ1 (PR10) gene plant disease resistance from the molecular level-plant immunity research in the post-genomic era-Isao Shimamoto et al., Shujunsha 2004
  • WRKY45 Plant disease resistance from the molecular level-plant immunity research in the post-genomic era-Isao Shimamoto et al., Shujunsha 2004
  • WRKY45 Shimao Shimamoto et al., Shujunsha 2004
  • Substances that induce the expression of disease response gene expression in the evaluation test include sodium L-glutamate, L-asparagine, sodium L-aspartate, L-threonine, L-valine, L-arginine, L-alanine, L -Phenylalanine or 0.6% oryzate (registered trademark of Meiji Seika Co., Ltd.) was used for comparison.
  • Orizemate is an agrochemical (granule) containing probenazole, a drug that induces SAR, as an active ingredient.
  • FIG. 3 shows the results of applying various amino acid solutions to the rice rhizosphere at a single concentration of 10 mM, respectively, and quantifying the amount of gene transcription in root tissues after 6 hours.
  • FIG. 3 shows the mean and standard error (SE) of three replicate experiments, respectively, and chitinase 3 in root tissue by applying glutamic acid, asparagine, aspartic acid, threonine, valine, arginine, alanine, or phenylalanine, It was revealed that the transcriptional activity of each gene of OsNPR1, WRKY45, PR1, and PR10 (PBZ1) is increased. This indicates that a disease response is occurring in the root tissue. The increase in the transcriptional activity of each gene by each amino acid was more than that of L-phenylalanine.
  • the response in the root tissue was very stable, and the transcription level of each gene was correlated with the blast control effect. From this, it was considered that the response that occurs in the roots when amino acids were applied in the rhizosphere was the first important step in conferring blast resistance.
  • the transcription amount of the disease response gene in the above-ground part (4th leaf) 48 hours after application of the rhizosphere was measured, a clear increase in the expression of the disease response gene was observed. It has been reported that a high expression strain of WRKY45 shows strong resistance to rice blast (Shimono, M. et. Al., Plant Cell, 2007, 19: 2064-2076). In fact, the application of amino acids in the rhizosphere significantly suppressed the infection of blast fungus, suggesting that SAR was induced.
  • Example 3 Mechanism of disease resistance induction by amino acid application It has been reported that amino acid promotes absorption of silicic acid and, as a result, disease resistance is imparted by physical strengthening of the surface layer of plants (Voleti, SR et al., Crop Protection, 2008, 27: 1398-1402). It has also been reported that silicic acid induces the accumulation of rice antibacterial substance, phytoalexin (Rodorigues, FA, et al., Phytopathology, 2004, 94: 177-183).
  • Example 4 Increase in disease resistance inducing effect by application of a mixture of a known plant disease resistance inducer and an amino acid By using together an amino acid and a known plant disease resistance inducer, the plant disease resistance inducing effect is enhanced. In order to clarify whether this is the case, the following experiment was conducted.
  • rice roots aseptically grown on Murashige-Skoog medium contain 1 mM probenazole, 1 mM sodium L-glutamate (Glu), and both contain 1 mM each. It was immersed in the solution for 6 hours. Further, in order to observe the disease resistance induction in the fourth leaf, rice was grown according to the procedure described in Example 2, the rice root was changed to 0.3% oryzate, 5 mM sodium L-glutamate (Glu), and both Was immersed in the solution containing the above-mentioned concentration for 48 hours. Water was used as a control.
  • FIG. 5 shows changes in the expression level of each gene in the root tissue. It has been clarified that the expression level of each gene in root tissue is significantly increased by using both in combination with probenazole and L-glutamic acid alone.
  • Example 6 Difference in disease resistance-inducing effect between amino acid enantiomers In addition to L-amino acids constituting proteins, there is an effect of inducing disease resistance not only in D-forms that are present in the living body in a small amount. It was evaluated.
  • Example 7 Induction of disease resistance of buckwheat by amino acids Whether disease resistance is induced by amino acids in rice plants other than rice, the following experiment was carried out using Poa pratensis. Chitinase activity was used as an index of disease resistance induction.
  • Chitinase is an enzyme that degrades chitin, which is a cell wall component of filamentous fungi, and is thought to play a part in the defense mechanism of plants. Therefore, the increase in activity is widely recognized as one of the indicators of disease response.
  • Kentucky bluegrass (Takii Tanae Co., Ltd.) was cultivated for 3 weeks under the same conditions as rice, and the rhizosphere was immersed in a 5 mM sodium L-glutamate solution for 24 hours.
  • Chitinase activity was measured by the method by McCreath et al. (McCreath, K. et al., J. Microbiol. Methods 14: 229-237, 1992).
  • the substrate 4MU- (GlcNAc) 3 (4-methylumbelliferyl- ⁇ -dN, N ', N''-triacetylchitobiose; SIGMA M5639) was dissolved in 50% ethanol to a final concentration of 0.4 mM, and -20 Stored at ° C. At the time of use, it was diluted 10 times to obtain a substrate solution. The crude extract fraction was adjusted to 1 ⁇ g / ⁇ L. After 50 ⁇ L of each sample was preincubated on a 96-well plate at 37 ° C. for 10 minutes, 50 ⁇ L of the substrate solution was added and the reaction was started at 37 ° C.
  • Example 8 Induction of maize disease resistance by amino acids In order to further verify whether induction of disease resistance by amino acids is a phenomenon widely observed in gramineous plants, experiments using maize were conducted.
  • Example 6 an increase in chitinase activity was used as an index for resistance induction.
  • a corn variety, Honey Bantam (Sakata Seed Co., Ltd.) was cultivated for 2 weeks after sowing, and the rhizosphere was immersed in a 5 mM sodium L-glutamate solution for 24 hours. After sampling the fourth leaf, the protein was extracted by the same procedure as described above, and the chitinase activity was measured.
  • the gramineous plant disease resistance enhancer of the present invention comprises an amino acid as an active ingredient, is highly safe and can be produced at low cost. Moreover, the disease resistance of gramineous plants can be effectively enhanced by the method of the present invention.

Abstract

The present invention prevents gramineous plant blight disease, gramineous plant blast disease, and other diseases by the application, in gramineous plants, of a gramineous plant disease resistance enhancer containing an amino acid selected from the group consisting of the L or D isomer of glutamic acid, asparagine, aspartic acid, threonine, alanine, valine, arginine, and serine.

Description

イネ科植物病害抵抗性増強剤およびそれを用いたイネ科植物の病害防除法Gramineous plant disease resistance enhancer and disease control method of gramineous plant using the same
　本発明は、アミノ酸を有効成分として、環境負荷が少なくかつ使用者および消費者にとって安全なイネ科植物の病害抵抗性増強剤、及び病害防除方法に関する。 [Technical Field] The present invention relates to a disease resistance enhancer and a disease control method for gramineous plants that contain an amino acid as an active ingredient and have a low environmental load and are safe for users and consumers.
　農作物の病害を防除する方法においては、植物病原菌に対する殺菌作用を有する化学物質や病原菌の感染機能を阻害する薬剤などを有効成分とする化学農薬が主として用いられている。しかしながら、このようなタイプの農薬は標的生物以外にも少なからず影響を及ぼすと考えられている。近年の食料品に対する消費者の安全意識の高まりとともに、化学合成農薬の不使用、使用量の低減、天然系農薬による病害防除技術が強く求められるようになってきている。 In the method for controlling diseases of agricultural crops, chemical pesticides mainly containing chemical substances having a bactericidal action against plant pathogens or drugs that inhibit the infection function of pathogens are used. However, these types of pesticides are thought to have a significant impact other than the target organism. With the recent increase in consumer safety awareness of food products, there has been a strong demand for non-use of chemically synthesized pesticides, reduction of usage, and disease control technology using natural pesticides.
　そのような時流の中で注目されているのが、植物本来の病害抵抗性を増強させる技術である。 What is attracting attention in such a trend is a technology for enhancing the disease resistance inherent in plants.
　植物が有する病原菌に対する抵抗性は、静的（受動的）抵抗性と動的（能動的）抵抗性に大別される。静的抵抗性とは、植物細胞壁の硬さや厚さ、ポリフェノール類の蓄積など、病原菌の感染を受ける前から植物が具備している物理的・化学的な性質による抵抗性を意味する。これに対し、動的抵抗性は、植物細胞または組織が外敵の攻撃やその予兆を察知して積極的に発動する一連の防御応答を意味する。高等植物の動的抵抗性反応としては、活性酸素の生成とそれに伴う過敏感細胞死、パピラの形成、病原菌に対して抗菌活性をもつファイトアレキシン（phytoalexins）合成系酵素をコードする遺伝子群の発現上昇、侵入菌の細胞壁を分解するキチナーゼやグルカナーゼ、あるいはその他のＰＲ（Pathogenesis-related）タンパク質の合成などがある（例えば非特許文献１）。 Resistance to pathogenic bacteria possessed by plants is roughly divided into static (passive) resistance and dynamic (active) resistance. Static resistance means resistance due to the physical and chemical properties of a plant before being infected with pathogenic bacteria, such as plant cell wall hardness and thickness, and accumulation of polyphenols. In contrast, dynamic resistance means a series of defense responses in which plant cells or tissues are activated in response to an enemy attack and signs. The dynamic resistance reaction of higher plants includes the generation of active oxygen and associated hypersensitive cell death, the formation of papillas, and the genes that encode phytoalexins synthase enzymes that have antibacterial activity against pathogens. There are increases in expression, synthesis of chitinases and glucanases that degrade the cell walls of invading bacteria, and other PR (Pathogenesis-related) proteins (for example, Non-Patent Document 1).
　植物と非親和性関係の病原体、またはエリシターなどで植物を人為的に処理することによって植物組織に構築される、物理的・化学的な障壁に基づいた抵抗性を、誘導抵抗性と呼ぶ。この現象は、抵抗性誘導物質と呼ばれる特定の物質で植物を前処理することによって、植物が本来有する動的抵抗性が引き出された結果であるとみなすことができる。 Resistance based on physical and chemical barriers, which is constructed in plant tissues by artificially treating plants with pathogens that have no affinity with plants or elicitors, is called induction resistance. This phenomenon can be regarded as a result of the dynamic resistance inherent in the plant being derived by pretreating the plant with a specific substance called a resistance inducer.
　他方、誘導抵抗性と比べると応答は微弱であるが、病原菌の侵入を受けると、動的抵抗性を迅速かつ強力に誘導できる状態が植物細胞内に作り出される現象をプライミングという。 On the other hand, the response is weak compared to the induction resistance, but when a pathogen is invaded, the phenomenon that a dynamic resistance can be induced quickly and strongly is created in the plant cell is called priming.
　動的抵抗性は、空間的な差異から局部獲得（誘導）抵抗性（Localized Acquired Resistance：ＬＡＲ）と全身獲得（誘導）抵抗性（Systemic Acquired Resistance：ＳＡＲ）に分類される。ＳＡＲとは、基本的には植物体の一部に化学的、物理的または生物的ストレスを与えたとき、その情報が全身に伝わるとともにそのストレスに対する新たな抵抗性が全身に誘導される現象をいう。ＬＡＲはＳＡＲとは区別されている。ＬＡＲが全身的に誘導されたものがＳＡＲとの考えもあるが、ＳＡＲは複数のシグナルを介して伝達されていると考えられ、病害応答遺伝子の発現パターンからも、質的に両者はまったく同じではないと考えられている（例えば非特許文献１）。 Dynamic resistance is classified into Local Acquired Resistance (LAR) and Systemic Acquired Resistance (SAR) based on spatial differences. SAR is basically a phenomenon in which when chemical, physical or biological stress is applied to a part of a plant, the information is transmitted to the whole body and a new resistance to the stress is induced in the whole body. Say. LAR is distinguished from SAR. Although SAR is induced systemically, SAR is thought to be SAR, but SAR is thought to be transmitted through multiple signals, and both are qualitatively identical from the expression pattern of disease-responsive genes. It is thought that it is not (for example, nonpatent literature 1).
　ＳＡＲにおいて中心的な働きをする物質として、サリチル酸（ＳＡ）が古くから注目されており、多くの研究がなされている。このＳＡシグナル経路で最も重要な因子の一つがＮＰＲ１であり、ＮＰＲ１遺伝子が破壊されているシロイヌナズナではＳＡより下流でＳＡＲのシグナル伝達が遮断される上、過剰発現させることによって耐病性が誘導される（非特許文献１）。 Salicylic acid (SA) has been attracting attention for a long time as a substance that plays a central role in SAR, and many studies have been made. One of the most important factors in the SA signal pathway is NPR1, and in Arabidopsis thaliana in which the NPR1 gene is disrupted, SAR signaling is blocked downstream from SA, and disease resistance is induced by overexpression. (Non-Patent Document 1).
　イネでは、ＳＡの下流はＮＰＲ１経路とＷＲＫＹ４５経路に分岐していることが示されている（非特許文献２）。ＷＲＫＹ４５は転写因子をコードし、ＰＲタンパク質遺伝子など病害抵抗性に関わる多くの遺伝子の発現を制御している。ＷＲＫＹ４５遺伝子の強発現株ではイネいもち病菌に対し強い抵抗性を示す（非特許文献２）。 In rice, it is shown that the downstream of SA branches into an NPR1 route and a WRKY45 route (Non-Patent Document 2). WRKY45 encodes a transcription factor and controls the expression of many genes involved in disease resistance such as PR protein gene. A strong expression strain of the WRKY45 gene exhibits strong resistance to rice blast fungus (Non-patent Document 2).
　植物病害を防除する手段として、殺菌剤を散布することによって直接病原体を攻撃する方法の他に、静的抵抗性および動的抵抗性を利用した病害防除方法も複数提案されており、その一部は農業で実用化されている。 In addition to the method of directly attacking pathogens by spraying bactericides as a means of controlling plant diseases, several methods for controlling diseases using static resistance and dynamic resistance have been proposed. Has been put to practical use in agriculture.
　静的抵抗性を高める方法としてはケイ素を施肥する方法が広く知られている。特にイネではケイ素で処理することで細胞壁が物理的に強固になり、イネいもち病菌をはじめとする病原菌の感染を抑制すると考えられている。 The method of fertilizing silicon is widely known as a method for increasing static resistance. Particularly in rice, it is considered that treatment with silicon physically strengthens the cell wall and suppresses infection of pathogenic bacteria such as rice blast fungus.
　イネをケイ素で処理することで病害を抑制する手法としては、シリカゲルを施肥する方法が知られている（非特許文献３）。また、ヒスチジン、リジン、グルタミン、グリシン、イミダゾール、ケイ酸ナトリウムをイネに施用して、ケイ酸の取り込みを促進させる方法が報告されている（非特許文献４）。 As a method of suppressing disease by treating rice with silicon, a method of fertilizing silica gel is known (Non-Patent Document 3). In addition, a method has been reported in which histidine, lysine, glutamine, glycine, imidazole, and sodium silicate are applied to rice to promote uptake of silicic acid (Non-patent Document 4).
　他方、動的抵抗性を利用した薬剤は植物抵抗性誘導剤と呼ばれる。殺菌剤など植物病原体に対して直接作用するタイプの農薬は、継続的に使用すると薬剤に対して抵抗性変異株が出現する場合が多い。これに対し植物抵抗性誘導剤は、直接病原菌に作用するのではなく、植物の抵抗性を誘導することで病害感染を防除することから、これまでにこのタイプの農薬の抵抗性変異株が出現した事例は認められていない。さらに、抵抗性誘導型農薬は生物に対する殺菌作用が少ないために、植物以外の生物を含めた環境への負荷は比較的少ないと考えられている。 On the other hand, a drug using dynamic resistance is called a plant resistance inducer. When a pesticide that directly acts on a plant pathogen such as a bactericide is used continuously, a resistance mutant to the drug often appears. Plant resistance inducers, on the other hand, do not act directly on pathogenic bacteria, but control disease infection by inducing plant resistance. No cases have been accepted. Furthermore, resistance-inducing pesticides are considered to have a relatively low environmental impact, including organisms other than plants, because they have little bactericidal action on organisms.
　これまで植物の病害抵抗性誘導を目的とした農薬として、プロベナゾール(商品名：オリゼメート。明治製薬（株）)、ベンゾチアゾール系(BTH)のアシベンゾラルＳメチル（ASM、商品名：バイオン。シンジェンタ ジャパン（株））、チアジアゾールカルボキサミド系のチアニジル（商品名：ブイゲット。日本農薬（株））が販売されている。 Previously, as pesticides for the purpose of inducing disease resistance in plants, probenazole (trade name: Orizemate, Meiji Pharmaceutical Co., Ltd.), benzothiazole (BTH) acibenzoral S-methyl (ASM, trade name: Vion. Syngenta Sakai Japan ( Co., Ltd.), thiadiazole carboxamide-based thianidyl (trade name: Vuget. Nippon Agricultural Chemicals Co., Ltd.) is sold.
　また、病害抵抗性を誘導する天然物由来の物質としては多糖体分解物（例えば、特許文献１）、セレブロシド類（例えば、特許文献２）、ジャスモン酸（例えば、特許文献３）、キチンオリゴ糖（例えば、非特許文献５）、β-1,3-およびβ-1,6-グルカンオリゴ糖（例えば、非特許文献６）、胆汁酸（特許文献４）、ペプチドグリカン（非特許文献７）、リポポリサッカライド(非特許文献８)などが報告されている。これらの物質は特にエリシターと呼ばれており、これらを処理することによって動的抵抗性が誘導され、ファイトアレキシンの蓄積、キチナーゼやβ－１，３－グルカナーゼなどのＰＲタンパク質の蓄積、過敏感細胞死が誘起されることが知られている（例えば、非特許文献１）。また、イネではケイ素で処理することによってファイトアレキシンの蓄積が増強されるとの報告もある（非特許文献１０）。 In addition, as a substance derived from a natural product that induces disease resistance, a polysaccharide degradation product (for example, Patent Document 1), cerebrosides (for example, Patent Document 2), jasmonic acid (for example, Patent Document 3), chitin oligosaccharide (For example, Non-Patent Document 5), β-1,3- and β-1,6-glucan oligosaccharide (for example, Non-Patent Document 6), bile acid (Patent Document 4), peptidoglycan (Non-Patent Document 7), Lipopolysaccharide (Non-patent Document 8) has been reported. These substances are called elicitors, and dynamic resistance is induced by treatment of these substances, accumulation of phytoalexins, accumulation of PR proteins such as chitinase and β-1,3-glucanase, and hypersensitivity. It is known that cell death is induced (for example, Non-Patent Document 1). In addition, there is a report that the accumulation of phytoalexin is enhanced by treating with silicon in rice (Non-patent Document 10).
　一部の植物抵抗性誘導剤は、プライミング効果物質として働いていると考えられている（例えば非特許文献１）。プライミング効果物質とは、これで植物を事前に処理することによって、エリシターや病原菌に対する細胞応答を著しく高進させ、動的抵抗性を迅速かつ強力に誘導できる状況を植物細胞内に作り出す物質を指す。プライミング効果物質としては、植物抵抗性誘導剤の他にβ－アミノ酪酸（BABA）が報告されている。 Some plant resistance inducers are considered to work as priming effect substances (for example, Non-Patent Document 1). A priming effect substance refers to a substance that, by pre-treating a plant with this, significantly enhances the cellular response to elicitors and pathogens and creates a situation in plant cells that can induce dynamic resistance quickly and powerfully. . In addition to plant resistance inducers, β-aminobutyric acid (BABA) has been reported as a priming effect substance.
　アミノ酸によって病害が抑制されるとの報告はいくつか存在する。プロリン（非特許文献９）がトウジンビエの病害を防除又は抑制することが報告されている。また、含硫アミノ酸（特許文献７、８）、アミノ酪酸（特許文献７）、又はグリシン（特許文献９）は、微生物やグルコース等と組み合わせることによって、植物の病害を防除、又は病害抵抗性を高めることが報告されているが、アミノ酸単独の効果ではない。さらに、アミノ酸を含む混合肥料（特許文献１０、１１、１２）や、アミノ酸発酵液（特許文献５）又は微生物菌体を酸性溶液中で加熱処理することにより得られる抽出液（特許文献６）の施用により植物病害を防除し得ることが報告されているが、これらもアミノ酸単独の効果ではない。また、アミノ酸混合物（プロリン、メチオニン）（特許文献１３）による植物病害防除も報告されているが、単独のアミノ酸による効果ではなく、病害抵抗性誘導によるものでもない。さらに、リジン又はグルタミン酸の発酵液による病害防除について報告されているが（特許文献１４）、データは記載されておらず、有効成分が不明であり、病害抵抗性誘導によるものであるかも明らかではない。また、ＤＬ－フェニルアラニンでシコクビエの葉を処理するとフェノール物質の合成が促進されること、それをイネごま葉枯病菌の胞子発芽と発芽管伸長を阻害することが報告されているが（非特許文献１１）、実際にフェニルアラニン処理が病原菌の侵入や感染を抑制するのかどうかは記載されていない。 There are some reports that diseases are suppressed by amino acids. It has been reported that proline (Non-patent Document 9) controls or suppresses the disease of pearl millet. In addition, sulfur-containing amino acids (Patent Documents 7 and 8), aminobutyric acid (Patent Document 7), or glycine (Patent Document 9) are combined with microorganisms, glucose, and the like to control plant diseases or have disease resistance. It has been reported to increase, but it is not the effect of amino acids alone. Further, mixed fertilizers containing amino acids (Patent Documents 10, 11, and 12), amino acid fermentation broth (Patent Document 5), or an extract obtained by heat-treating microbial cells in an acidic solution (Patent Document 6) Although it has been reported that plant diseases can be controlled by application, these are also not the effects of amino acids alone. Moreover, although plant disease control by an amino acid mixture (proline, methionine) (Patent Document 13) has been reported, it is not an effect by a single amino acid, nor is it due to disease resistance induction. Furthermore, although there has been a report on disease control by fermentation liquid of lysine or glutamic acid (Patent Document 14), no data is described, the active ingredient is unknown, and it is not clear whether it is due to disease resistance induction. . Furthermore, it has been reported that the treatment of sugarcane leaves with DL-phenylalanine promotes the synthesis of phenolic substances and inhibits the spore germination and germ tube elongation of rice sesame leaf blight fungi (Non-patent Documents) 11) It is not described whether phenylalanine treatment actually suppresses invasion or infection of pathogenic bacteria.
　イネ科植物であるイネ（Oryza sativa）に対するアミノ酸単独での病害抵抗性誘導に関しては、Ｌ－ヒスチジン、Ｌ－リジン、Ｌ－グルタミン、又はＬ－グリシンで水田を処理することによってイネの病害を抑制するとの報告あるが、これはケイ酸の取り込みを促進し植物細胞壁、特に葉面の物理的強度を高めるというものである（非特許文献４）。尚、同文献では、グルタミン酸による虫害抑制について記載されているが、病害については記載されていない。 With regard to the induction of disease resistance of rice (Oryza sativa) with an amino acid alone, the disease of rice is suppressed by treating paddy fields with L-histidine, L-lysine, L-glutamine, or L-glycine. Although it is reported, this promotes the uptake of silicic acid and increases the physical strength of the plant cell wall, particularly the leaf surface (Non-patent Document 4). In addition, although the literature describes the insect damage suppression by glutamic acid, it does not describe the disease.
　アミノ酸と病害抵抗性との関連が示されている先行知見では、Ｌ－体が使用されており、他方のエナンチオマー（鏡像異性体）であるＤ－体に関する知見は無い。Ｄ－アミノ酸は真核生物の生体内には極わずかしか存在しておらず、その生物学的意義は不明な部分が多い。 In the prior knowledge showing the relationship between amino acids and disease resistance, L-form is used, and there is no knowledge about D-form which is the other enantiomer (enantiomer). There are very few D-amino acids in the living body of eukaryotes, and there are many parts whose biological significance is unknown.
特開平5-331016号公報JP-A-5-331016 特許第2846610号公報Japanese Patent No. 2846610 特開平11-29412号公報Japanese Patent Laid-Open No. 11-29412 特開2006-219372公報JP 2006-219372 JP 特開平6-80530号公報Japanese Patent Laid-Open No. 6-80530 国際公開第WO/2009/088074号International Publication No. WO / 2009/088074 特開2003-34607公報JP2003-34607 特許第4287515号Patent No. 4287515 中国特許公開第1640233AChinese Patent Publication No. 1640233A 中国特許公報第1316893CChinese Patent Publication No. 1316893C 中国特許公開第1328769AChinese Patent Publication No. 1328769A 中国特許公開第101182270AChina Patent Publication No. 101182270A 中国特許公開第101142925AChina Patent Publication No. 101142925A 中国特許公報第1155543CChinese Patent Publication No. 1155543C
　本発明は、消費者および使用者に対して安全性が高く、安価なイネ科植物の病害抵抗性増強剤とそれを用いたイネ科植物の病害防除法を提供することを課題とする。 The object of the present invention is to provide a disease resistance enhancer for gramineous plants that is highly safe and inexpensive for consumers and users, and a disease control method for gramineous plants using the same.
　本発明者らは、イネの病害抵抗性を誘導する物質を探索したところ、Ｌ－またはＤ－アミノ酸に高い病害抵抗性誘導活性があることを発見した。特に、グルタミン酸、アスパラギン、アスパラギン酸、スレオニン、バリン、アルギニン、アラニンをイネに施用することで、病害に対する動的抵抗性が全身的に誘導されることを見出した。また、これらのアミノ酸処理がイネいもち病菌のような病害に対して高い防除効果を示すことを確認した。さらに、イネ以外のイネ科植物にも同様の現象を引き起こすことが明らかとなった。 The present inventors searched for a substance that induces disease resistance of rice and found that L- or D-amino acids have high disease resistance-inducing activity. In particular, it has been found that by applying glutamic acid, asparagine, aspartic acid, threonine, valine, arginine, and alanine to rice, dynamic resistance to disease is systemically induced. Moreover, it confirmed that these amino acid processing showed the high control effect with respect to diseases, such as a rice blast fungus. Furthermore, it has been clarified that the same phenomenon is caused in other gramineous plants.
　さらに、アミノ酸以外の病害抵抗性誘導剤と前記アミノ酸を併用することにより、病害抵抗性誘導効果を顕著に高めることを見出した。本発明はこれらの知見に基づいて完成されたものである。 Furthermore, it has been found that the disease resistance-inducing effect is remarkably enhanced by using the amino acid in combination with a disease resistance-inducing agent other than amino acids. The present invention has been completed based on these findings.
　すなわち本発明は、以下のとおりである。
（１）グルタミン酸、アスパラギン、アスパラギン酸、スレオニン、アラニン、バリン、アルギニン、及びセリンからなる群から選ばれるアミノ酸を含むイネ科植物の病害抵抗性増強剤。
（２）根圏施用又は葉面散布によりイネ科植物に施用される、前記病害抵抗性増強剤。
（３）イネ科植物がイネ、シバ、又はトウモロコシである、前記病害抵抗性増強剤。
（４）病害が、イネ科植物いもち病、又はイネ白葉枯病である、前記病害抵抗性増強剤。
（５）前記アミノ酸を、合計量で０．２～２００ｍＭ含む、前記病害抵抗性増強剤。
（６）前記アミノ酸がＬ－体である、前記病害抵抗性増強剤。
（７）前記アミノ酸がＤ－体である、前記病害抵抗性増強剤。
（８）さらに、アミノ酸以外の病害抵抗性誘導剤を含む、前記病害抵抗性増強剤。
（９）グルタミン酸を含む、前記病害抵抗性増強剤。
（１０）前記病害抵抗性増強剤をイネ科植物に施用することを特徴とする、イネ科植物の病害を防除する方法。
（１１）前記病害抵抗性増強剤を、前記アミノ酸の量として、０．２～２００ｍＭ／１００～５０００Ｌ／ヘクタールの施用量で施用する、前記方法。 That is, the present invention is as follows.
(1) A disease resistance enhancer for gramineous plants comprising an amino acid selected from the group consisting of glutamic acid, asparagine, aspartic acid, threonine, alanine, valine, arginine, and serine.
(2) The disease resistance enhancer, which is applied to gramineous plants by rhizosphere application or foliar application.
(3) The disease resistance enhancer, wherein the gramineous plant is rice, shiba or corn.
(4) The disease resistance enhancer, wherein the disease is Gramineae blast or rice leaf blight.
(5) The disease resistance enhancer comprising the amino acid in a total amount of 0.2 to 200 mM.
(6) The disease resistance enhancer, wherein the amino acid is L-form.
(7) The disease resistance enhancer, wherein the amino acid is D-form.
(8) The disease resistance enhancer further comprising a disease resistance inducer other than amino acids.
(9) The disease resistance enhancer comprising glutamic acid.
(10) A method for controlling diseases of gramineous plants, wherein the disease resistance enhancer is applied to gramineous plants.
(11) The method as described above, wherein the disease resistance enhancer is applied at an application rate of 0.2 to 200 mM / 100 to 5000 L / ha as the amount of the amino acid.
　殺菌剤のような植物病原菌に対して直接作用するタイプの農薬は、継続的な使用により薬剤に対する抵抗性変異株が出現することが多いが、植物抵抗性誘導剤は、薬剤の抵抗性変異株が出現しにくく、長期間にわたる使用が可能であることが知られている。本発明の病害抵抗性増強剤の有効成分であるＬ－またはＤ－アミノ酸は、病原菌に対する直接的な殺菌作用ではなく、病害抵抗性を誘導することによって病原菌の感染を防ぐものであり、抵抗性変異株が出現しにくく、長期間にわたる使用が可能であることが期待でき、産業上極めて有用である。 Agricultural chemicals that act directly against phytopathogenic fungi such as fungicides often show resistance mutants to drugs with continued use, but plant resistance inducers are resistant mutants of drugs. It is known that is difficult to appear and can be used for a long time. The L- or D-amino acid that is an active ingredient of the disease resistance enhancer of the present invention is not a direct bactericidal action against pathogenic bacteria, but prevents disease infection by inducing disease resistance. Mutant strains are unlikely to appear, and it can be expected that they can be used over a long period of time, which is extremely useful industrially.
　生物はエナンチオマーを区別して認識する。Ｄ－アミノ酸は、Ｌ－アミノ酸と比較すると環境中での微生物による分解は受けにくいと考えられている。本発明の一態様では、Ｄ－アミノ酸を利用することで安定的な病害防除効果の持続が実現できると期待される。 Living organisms recognize and recognize enantiomers. D-amino acids are considered less susceptible to degradation by microorganisms in the environment than L-amino acids. In one embodiment of the present invention, it is expected that a stable disease control effect can be realized by using a D-amino acid.
　既存の植物病害抵抗性誘導剤、例えば農薬登録がなされている植物病害抵抗性誘導剤とアミノ酸を混合使用することによって、防除効果を高めるか、又は植物病害抵抗性誘導剤の使用量を低減させることができる。 By using a mixture of an existing plant disease resistance inducer, for example, a plant disease resistance inducer registered with an agrochemical and an amino acid, the control effect is increased or the amount of the plant disease resistance inducer used is reduced. be able to.
アミノ酸の根圏施用によるイネいもち病防除効果を示す図。The figure which shows the rice blast control effect by the rhizosphere application of an amino acid. アミノ酸の葉面散布によるイネいもち病防除効果を示す図。PBZはプロベナゾールを表す（以下、同様）。The figure which shows the rice blast control effect by the foliar application of an amino acid. PBZ represents probenazole (hereinafter the same). アミノ酸の根圏施用によるイネ根組織における病害抵抗性誘導効果を示す図。cht3はキチナーゼ３遺伝子を、OsNPR1はイネゲノムにおけるNPR1遺伝子を表す。ORZはオリゼメート（プロベナゾールを有効成分とする農薬）を表す。The figure which shows the disease resistance induction effect in the rice root tissue by the rhizosphere application of an amino acid. cht3 represents the chitinase 3 gene, and OsNPR1 represents the NPR1 gene in the rice genome. ORZ represents oryzate (agrochemicals containing probenazole as an active ingredient). 人工培地上で無菌的に栽培したイネのアミノ酸に対する応答を示す図。conはネガティブコントロールを表す（以下、同様）。根組織における病害応答遺伝子の発現量を示す。The figure which shows the response with respect to the amino acid of the rice cultivated aseptically on the artificial culture medium. con represents a negative control (hereinafter the same). The expression level of the disease response gene in the root tissue is shown. プロベナゾールとアミノ酸の併用効果を示す図。根組織における病害応答遺伝子の発現量を示す。The figure which shows the combined use effect of a probenazole and an amino acid. The expression level of the disease response gene in the root tissue is shown. オリゼメートとアミノ酸の併用効果を示す図。controlはコントロールを表す。第４葉における病害応答遺伝子の発現量を示す。The figure which shows the combined use effect of an oryzate and an amino acid. control represents a control. The expression level of the disease response gene in the 4th leaf is shown. アミノ酸のエナンチオマー間での病害抵抗性誘導効果の違いを示す図。The figure which shows the difference in the disease-resistance induction effect between the enantiomers of an amino acid. アミノ酸処理によるシバのキチナーゼ活性上昇を示す図。L-GluはＬ－グルタミン酸を、conはコントロールを各々示す。The figure which shows the chitinase activity rise of a buckwheat by an amino acid process. L-Glu represents L-glutamic acid, and con represents a control. アミノ酸処理によるトウモロコシのキチナーゼ活性上昇を示す図。L-GluはＬ－グルタミン酸を、conはコントロールを各々示す。The figure which shows the chitinase activity rise of the corn by an amino acid process. L-Glu represents L-glutamic acid, and con represents a control.
　以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
　本発明のイネ科植物の病害抵抗性増強剤は、グルタミン酸、アスパラギン、アスパラギン酸、スレオニン、アラニン、バリン、アルギニン、及びセリンからなる群から選ばれるアミノ酸を有効成分として含む。これらのアミノ酸のうち、グルタミン酸、アスパラギン、アスパラギン酸、スレオニン、及びアラニンが好ましい。病害抵抗性増強剤は、各アミノ酸を単独で含んでいてもよく、任意の２種又はそれ以上のアミノ酸を含んでいてもよい。複数種のアミノ酸を含む場合、１種はグルタミン酸であることが好ましい。また、病害抵抗性増強剤は、これらのアミノ酸以外のアミノ酸を含んでいてもよい。 The disease resistance enhancer for gramineous plants of the present invention contains an amino acid selected from the group consisting of glutamic acid, asparagine, aspartic acid, threonine, alanine, valine, arginine, and serine as an active ingredient. Of these amino acids, glutamic acid, asparagine, aspartic acid, threonine, and alanine are preferred. The disease resistance enhancer may contain each amino acid alone, or may contain any two or more amino acids. When a plurality of amino acids are included, one is preferably glutamic acid. Moreover, the disease resistance enhancer may contain amino acids other than these amino acids.
　前記各アミノ酸は、Ｌ－体、Ｄ－体のいずれであってもよく、Ｌ－体及びＤ－体を任意の割合で含む混合物であってもよい。特にセリンは、Ｄ－体が好ましい。 The amino acids may be either L-form or D-form, and may be a mixture containing L-form and D-form at an arbitrary ratio. In particular, serine is preferably D-form.
　アミノ酸は、フリー体でもよく、アンモニウム塩、ナトリウム塩、カリウム塩等の塩であってもよい。 The amino acid may be a free form or a salt such as ammonium salt, sodium salt or potassium salt.
　前記アミノ酸は、そのアミノ酸を含む限り形態は特に制限されず、一般に販売されている試薬、発酵法で製造した精製品又は粗精製品、精製過程で生じる副生物であってもよく、また、海産物からの抽出物、又はタンパク質の加水分解物など、前記アミノ酸を含む組成物であってもよい。 The form of the amino acid is not particularly limited as long as it contains the amino acid, and may be a generally sold reagent, a refined or crude product produced by a fermentation method, a by-product generated in a purification process, and a marine product. It may be a composition containing the amino acid, such as an extract from or a protein hydrolyzate.
　本発明の他の形態は、前記アミノ酸に加えて、アミノ酸以外の病害抵抗性誘導剤を含む。アミノ酸以外の病害抵抗性誘導剤としては、アミノ酸以外の化合物であって、イネ科植物に動的抵抗性又はプライミングを誘導することができる物質であれば特に制限されず、プロベナゾール(商品名：オリゼメート。明治製薬（株）)、ベンゾチアゾール系(BTH)のアシベンゾラルＳメチル（ASM、商品名：バイオン。シンジェンタ ジャパン（株））、チアジアゾールカルボキサミド系のチアニジル（商品名：ブイゲット。日本農薬（株））等の市販されている薬剤が挙げられる。また、多糖体分解物（特開平5-331016号）、セレブロシド類（特許第2846610号）、ジャスモン酸（特開平11-29412号）、キチンオリゴ糖（Yamada A. et. al., Biosci. Biotech. Biochem., 1993, 57(3):405-409）、β-1,3-およびβ-1,6-グルカンオリゴ糖（Yamaguchi, T. et. al., Plant Cell, 2000, 12:817-826）、胆汁酸（特開2006-219372公報）、ペプチドグリカン（Gust, A.A. et. al., J. Biol. Chem., 2007, 282:32338-32348）、リポポリサッカライド(V Raj, S.N. et al., Phytoparasitica, 2004, 32:523-527)などの天然物由来の病害抵抗性誘導物質も使用することができる。 Other forms of the present invention include disease resistance inducers other than amino acids in addition to the amino acids. The disease resistance inducer other than amino acids is not particularly limited as long as it is a compound other than amino acids and can induce dynamic resistance or priming to gramineous plants. Probenazole (trade name: oryzate) Meiji Seiyaku Co., Ltd.), benzothiazole (BTH) acibenzoral S-methyl (ASM, trade name: Vion. Syngenta Sakai Japan Co., Ltd.), thiadiazole carboxamide-based thianidyl (trade name: Vuget. Nippon Agricultural Chemicals Co., Ltd.) And other commercially available drugs. In addition, degradation products of polysaccharides (Japanese Patent Laid-Open No. 5-331016), cerebrosides (Japanese Patent No. 2846610), jasmonic acid (Japanese Patent Laid-Open No. 11-29412), chitin oligosaccharides (Yamada A. et. Al., Biosci. Biotech) Biochem., 1993, 57 (3): 405-409), β-1,3- and β-1,6-glucan oligosaccharides (Yamaguchi, T. et. Al., Plant Cell, 2000, 12: 817 -826), bile acids (JP 2006-219372), peptidoglycan (Gust, AA et. Al., J. Biol. Chem., 2007, 282: 32338-32348), lipopolysaccharide (V Raj, SN et) al., Phytoparasitica, 2004, 32: 523-527).
　尚、本発明において、「病害抵抗性増強剤」と「病害抵抗性誘導剤」の用語は、本発明の病害抵抗性増強剤と、アミノ酸以外の病害抵抗性誘導作用を有する物質を便宜的に区別するものであり、それらの作用を区別するためのものではない。本発明の病害抵抗性増強剤は、病害抵抗性誘導作用を有するものである。したがって、本発明の病害抵抗性増強剤は、病害抵抗性誘導剤と呼ぶこともできる。 In the present invention, the terms “disease resistance enhancer” and “disease resistance inducer” refer to the disease resistance enhancer of the present invention and substances having a disease resistance inducing action other than amino acids for convenience. It is to distinguish, not to distinguish their actions. The disease resistance enhancer of the present invention has a disease resistance inducing action. Therefore, the disease resistance enhancer of the present invention can also be called a disease resistance inducer.
　アミノ酸と、アミノ酸以外の病害抵抗性誘導剤を併用する場合、好ましいアミノ酸はグルタミン酸である。 When amino acid and a disease resistance inducer other than amino acid are used in combination, a preferred amino acid is glutamic acid.
　イネ科植物病害抵抗性増強剤は、前記アミノ酸、及びアミノ酸以外の病害抵抗性誘導剤以外に、任意の成分を含んでいてもよい。このような成分としては、溶媒、担体、pH調整剤、植物体への展着力を高めるための展着剤、植物への浸透性を高めるための界面活性剤等の成分、肥効を高めるためのミネラル等の肥料成分、農薬成分、バインダー、増量剤等が挙げられる。これらの成分としては、本発明の効果を損なわない限り、通常農薬、肥料等に用いられている成分を用いることができる。 The gramineous plant disease resistance enhancer may contain any component in addition to the amino acid and the disease resistance inducer other than amino acids. Such components include solvents, carriers, pH adjusters, spreading agents for increasing the spreading power to plants, surfactants for increasing the permeability to plants, etc., for enhancing the fertilization effect. Fertilizer components such as minerals, agricultural chemical components, binders, extenders and the like. As these components, components usually used for agricultural chemicals, fertilizers and the like can be used as long as the effects of the present invention are not impaired.
　本発明によるイネ科植物病害抵抗性増強剤の剤型は液剤、粉剤、粒剤、乳剤、水和剤、油剤、エアゾール、フロアブル剤等の何れの使用形態でも良く、施用の為の薬剤の形態、その使用形態、施用方法は特に限定されるものではない。 The dosage form of the gramineous plant disease resistance enhancer according to the present invention may be any use form such as solution, powder, granule, emulsion, wettable powder, oil, aerosol, flowable, etc., and the form of the drug for application The use form and application method are not particularly limited.
　担体成分としては、例えば、本発明のイネ科植物病害抵抗性増強剤が底床添加剤又は固形剤である場合には、バーミキュライト、タルク、珪藻土、カオリン、炭酸カルシウム、クレー、水酸化カルシウム、白土、シリカゲル等の無機質や小麦粉、澱粉等の固体担体を用いることができる。また、イネ科植物病害抵抗性増強剤が液剤である場合には、水、キシレン等の芳香族炭化水素類、エタノール、エチレングリコール等のアルコール類、アセトン等のケトン類、ジオキサン、テトラヒドロフラン等のエーテル類、ジメチルホルムアミド、ジメチルスルホキシド、アセトニトリル等の液体担体を用いることができる。 Examples of the carrier component include vermiculite, talc, diatomaceous earth, kaolin, calcium carbonate, clay, calcium hydroxide, clay when the gramineous plant disease resistance enhancer of the present invention is a bottom floor additive or a solid agent. Inorganic substances such as silica gel and solid carriers such as wheat flour and starch can be used. When the gramineous plant disease resistance enhancer is a liquid, water, aromatic hydrocarbons such as xylene, alcohols such as ethanol and ethylene glycol, ketones such as acetone, ethers such as dioxane and tetrahydrofuran , Liquid carriers such as dimethylformamide, dimethyl sulfoxide, acetonitrile and the like can be used.
　また、使用に際して、固体状又は粉体状のイネ科植物病害抵抗性増強剤を、水、アルコール等の溶媒に溶解又は分散させてもよい。また、液状のイネ科植物病害抵抗性増強剤を、水、アルコール等の溶媒で希釈してもよい。 In use, a solid or powdery gramineous plant disease resistance enhancer may be dissolved or dispersed in a solvent such as water or alcohol. Further, the liquid gramineous plant disease resistance enhancer may be diluted with a solvent such as water or alcohol.
　また、本発明の好ましい態様によれば、イネ科植物病害抵抗性増強剤は病害防除剤として用いられるものである。そして、本発明の別の態様によれば、本発明によるイネ科植物病害抵抗性増強剤をイネ科植物に施用する、イネ科植物病害防除方法が提供される。 Moreover, according to a preferred embodiment of the present invention, the gramineous plant disease resistance enhancer is used as a disease control agent. And according to another aspect of the present invention, there is provided a method for controlling a gramineous plant disease, which comprises applying the gramineous plant disease resistance enhancer according to the present invention to a gramineous plant.
　イネ科植物病害抵抗性増強剤におけるアミノ酸の含量は特に制限されず、後述の施用量に応じて適宜設定することができる。例えば、イネ科植物病害抵抗性増強剤におけるアミノ酸の含量は、病害抵抗性増強に有効な量が施用できる限り特に制限されないが、アミノ酸を単独で含む場合は、例えば、通常０．２～２００ｍＭ、好ましくは１～１００ｍＭである。複数のアミノ酸を含む場合の含量は、合計で、０．２～２００ｍＭ、好ましくは１～１００ｍＭである。尚、前記濃度は、植物病害抵抗性増強剤が固形又は粉体状の場合は、使用時に溶液にしたときの濃度である。また、液状のイネ科植物病害抵抗性増強剤を使用時に希釈して用いる場合は、前記濃度は希釈後の濃度である。 The content of amino acids in the gramineous plant disease resistance enhancer is not particularly limited, and can be appropriately set according to the application rate described later. For example, the content of amino acids in the gramineous plant disease resistance enhancer is not particularly limited as long as an effective amount for enhancing disease resistance can be applied, but when it contains an amino acid alone, for example, usually 0.2 to 200 mM, Preferably it is 1 to 100 mM. In the case of containing a plurality of amino acids, the total content is 0.2 to 200 mM, preferably 1 to 100 mM. In addition, the said density | concentration is a density | concentration when it is made into the solution at the time of use, when a plant disease resistance enhancer is solid or a powder form. Moreover, when diluting and using a liquid gramineous plant disease resistance enhancer at the time of use, the said density | concentration is a density | concentration after dilution.
　イネ科植物病害抵抗性増強剤が、アミノ酸以外の病害抵抗性誘導剤を含む場合、その含有量は特に制限されないが、例えば、通常、０．１ｍM～２００ｍM、好ましくは１ｍM～５０ｍMである。農薬登録を受けている病害抵抗性誘導剤については、それぞれの推奨量に依存する。
　また、アミノ酸と、アミノ酸以外の病害抵抗性誘導剤の量比は、例えば、通常、1:1/10000～1：1000、好ましくは1：1/200～1：20である。 When the gramineous plant disease resistance enhancer contains a disease resistance inducer other than amino acids, the content is not particularly limited, but is usually 0.1 mM to 200 mM, preferably 1 mM to 50 mM, for example. For disease resistance inducers that are registered as agricultural chemicals, it depends on the recommended amount.
The amount ratio of the amino acid and the disease resistance inducer other than the amino acid is, for example, usually 1: 1/10000 to 1: 1000, preferably 1: 1/200 to 1:20.
　本発明の病害抵抗性増強剤の対象となる植物はイネ科に属する植物であり、イネ（Oryza sativa）、シバ（Zoysia属植物）、コムギ（Triticum属植物）、トウモロコシ（Zea mays）、オオムギ（Hordeum vulgare）、ライムギ（Secale cereale）、サトウキビ（Saccharum officinarum）などが挙げられる。品種には下記のものが挙げられるが、これらに制限されない。 Plants targeted by the disease resistance enhancer of the present invention are plants belonging to the family Gramineae, and include rice (Oryza sativa), shiba (Zoysia genus plant), wheat (Triticum genus plant), maize (Zea mays), barley ( Hordeum vulgare), rye (Secale cereale), sugarcane (Saccharum officinarum) and the like. Examples of varieties include, but are not limited to:
　イネの品種としては、日本晴、コシヒカリ、ひとめぼれ、ヒノヒカリ、あきたこまち、はえぬき、きらら３９７、キヌヒカリ、ほしのゆめ、つがるロマン、ななつぼし、ゆめあかり、あさひの夢、あいちのかおり、夢つくし、ササニシキ、コー・コー　７号、コー・コー　２３号、コー・コー　１０号、プレー　１、サンパートーン　１、カオドークマリ　１０５、ピサヌローク　１、ピサヌローク　３、ピサヌローク　６０－１、コー・コー　６、ナム・ルー、チャオ・ホー、シウ・メーチャン、Ｒ２５８、ラチサイル、ＩＲ５、ペリタ１－１、シサダネ、シサンガルング、ＩＲ６４、シボゴ、コンデ、シヘラング、ドドカン、ダナウテムペ、シラタ、ＩＲ８、ＴＮ１、ウイダス、ＩＲ２０、ダナウガウング、ペタ、シアプス、メムベラモ等がある。 As rice varieties, Nihonbare, Koshihikari, Hitomebore, Hinohikari, Akitakomachi, Haenuki, Kirara 397, Kinuhikari, Hoshino Yume, Tsugaru Romance, Natsutoshi, Yume Akari, Asahi Dream, Aichi no Kaori, Yume Tsukushi, Sasanishiki, Koh Ko No. 7, Koh Ko No. 23, Koh Ko No. 10, Play 1, Thumper Tone 1, Khaodok Mari 105, Phitsanulok 1, Phitsanulok 3, Phitsanulok 60-1, Koh Ko 6, Nam Lu, Chao Ho, Siu Mae Chan, R258, Latissile, IR5, Perita 1-1, Shisadane, Shisangarung, IR64, Shibogo, Conde, Siherang, Dodokan, Danautempe, Sirata, IR8, TN1, Wiidas, IR20, Danaugaung, Peta, Siaps, Membella And the like.
　シバ品種としては、ヒメコウライシバ、ノシバ、センチピードグラス、バミューダグラス、ティフトンシバ、リビエラ、サンデビルII、ティフウェイ、バヒアグラス、セントオーガンスチングラスなどが挙げられる。 Shiba varieties include Himeko-ryashiba, Noshiba, Centipedegrass, Bermudagrass, Tifton Shiva, Riviera, Sun Devil II, Tiffway, Bahiagrass, St. Augustine Singrass and the like.
　コムギ品種としては、小麦農林10号、ホクシン、農林61号、チクゴイズミ、春よ恋、さぬきの夢2000、シラネコムギ、きたもえ、もち姫、タクネコムギ、オーストラリア産スタンダードホワイト（ASW）、デュラム、プライムハード (PH)、ウエスタンホワイト (WW)、ダークノーザンスプリング (DNS)、カナダウエスタンレッドスプリング (CWRS)などが挙げられる。 As wheat varieties, wheat farm forest No. 10, Hokushin, Norin 61, Chikugoizumi, Spring Yo Koi, Sanuki no Yume 2000, Shiranekomugi, Kitamoe, Mochihime, Takunekomugi, Australian Standard White (ASW), Durum, Prime Hard Rice ( PH), Western White (WW), Dark Northern Spring (DNS), Canada Western Red Spring (CWRS).
　トウモロコシ品種としては、ハニーバンタム、味来390、味来早生130、ピクニックコーン、ゴールドラッシュ、ピュアホワイト、あまいんです、ゆめのコーン、ゆめのコーン85、サンライズコーン、ウッディーコーン、シルバーハニーバンタム、スーパーハニーバンタム、ピーターコーン、ピーター610、ピーター早生1号、カキクケコーン、おひさまコーン、歩味ドリーム、どでかコーン優作、サラダコーン、スイートコーン味来390、キャンベラ86、キャンベラ90、スイート恵味(めぐみ)86、ルーシー90、Sweet歩味86、ゴールデンクロスバンタム、ポップコーン、黄八行うるきび(ロングフェロー)、黒もちきび、白もちきび、黄もちきび、などが挙げられる。 As corn varieties, Honey Bantam, Ajiko 390, Ajiko Hayami 130, Picnic Corn, Gold Rush, Pure White, Sweet, Yume no Corn, Yume no Corn 85, Sunrise Corn, Woody Corn, Silver Honey Bantam, Super Honey bantam, Peter corn, Peter 610, Peter early birth No. 1, oyster corn, sunflower corn, hobby dream, dodge corn masterpiece, salad corn, sweet corn miracle 390, Canberra 86, Canberra 90, sweet taste (Megumi) 86, Lucy 90, Sweet Hobby 86, Golden Cross Bantam, Popcorn, Hakuhachi Rukibi (Long Fellow), Black Rice Cracker, White Rice Cake, Yellow Rice Cake, etc.
　オオムギ品種としては、二条大麦、四条大麦、六条大麦、裸大麦、サチホゴールデンなどが挙げられる。 ¡Barley varieties include Nijo barley, Shijo barley, Rojo barley, bare barley, and Sachiho golden.
　ライムギ品種としては、春香、春一番、キングライ麦、ライ太郎、Petkusなどが挙げられる。 Rye varieties include Haruka, Haruichi, King Rye, Ryotaro, Petkus, etc.
　サトウキビ品種としては、農林８号、農林９号、農林10号、農林11号、農林13号、農林15号、農林19号、農林20号、F161、KY96-189、KY96T-537などが挙げられる。 Examples of sugarcane varieties include agricultural forest 8, agricultural forest 9, agricultural forest 10, agricultural forest 11, agricultural forest 13, agricultural forest 15, agricultural forest 19, agricultural forest 20, F161, KY96-189, KY96T-537, etc. .
　実施例に示すように、アミノ酸施用によってイネの病害応答遺伝子の発現が顕著に上昇することが示され、アミノ酸施用による病害抵抗性は、ケイ酸など他の物質を介さない、直接的作用による動的抵抗性、特に全身獲得抵抗性の誘導によるものであることが示された。植物の動的抵抗性は病原菌に対して非特異的であることから、対象病害としては特に制限されない。例えば糸状菌、細菌、ウイルスを原因とする植物病害が含まれる。 As shown in the Examples, it was shown that the expression of disease-responsive genes in rice was significantly increased by amino acid application, and the disease resistance by amino acid application is not caused by other substances such as silicic acid. It was shown to be due to the induction of mechanical resistance, particularly systemic acquired resistance. Since the dynamic resistance of plants is non-specific to pathogenic bacteria, the target disease is not particularly limited. For example, plant diseases caused by filamentous fungi, bacteria and viruses are included.
　イネの代表的な病害の例としてイネ白葉枯病、イネ炭疽病、イネ紋枯病、イネ苗立枯病、イネ葉鞘褐変病、イネばか苗病、イネ縞葉枯病、イネ黄化萎縮病などが挙げられる。 Examples of typical rice diseases are rice leaf blight, rice anthracnose, rice coat blight, rice seedling blight, rice leaf sheath browning, rice scab, rice stripe disease, and rice yellowing atrophy Etc.
　シバの病害の例としてはシバ萎縮病、シバモザイク病、シバ萎黄病、シバ麦角病、シバ白葉病、シバさび病、シバいもち病、シバ立枯病などが挙げられる。 Examples of diseases of wrinkles include wrinkle dwarf disease, wrinkle mosaic disease, wrinkle dwarf disease, buckwheat horn disease, wrinkle white leaf disease, wrinkle rust disease, wrinkle blast disease, and wrinkle blight.
　コムギの病害としてはコムギ萎縮病、コムギ赤かび病、コムギ赤さび病、コムギ麦角病、コムギ葉枯病、コムギ斑点病、コムギいもち病、コムギ角斑病、コムギ黒点病、コムギ黒さび病、コムギ黄斑病、コムギ炭疽病、コムギ立枯病、コムギうどんこ病などが挙げられる。 The diseases of wheat include wheat dwarf disease, wheat red mold disease, wheat red rust disease, wheat horn horn disease, wheat leaf blight, wheat spot disease, wheat blast disease, wheat horn spot disease, wheat black spot disease, wheat black rust disease, wheat Examples include macular disease, wheat anthrax, wheat blight, and wheat powdery mildew.
　トウモロコシの病害としては、トウモロコシすじ萎縮病、トウモロコシ黄化病、トウモロコシモザイク病、トウモロコシ赤かび病、トウモロコシごま葉枯病、トウモロコシ斑点病、トウモロコシ葉枯病、トウモロコシいもち病、トウモロコシ褐斑病、トウモロコシ紋枯病、トウモロコシ苗立枯病、トウモロコシ南方さび病、トウモロコシさび病などが挙げられる。 Maize diseases include corn streak dwarf disease, corn yellowing disease, corn mosaic disease, corn leaf blight, corn sesame leaf blight, corn leaf blight, corn leaf blight, corn blast, corn brown blight, corn Examples include blight, maize seedling blight, corn southern rust, and corn rust.
　オオムギの病害としては、オオムギうどんこ病、オオムギ輪紋病、オオムギ条斑病、オオムギ立枯病、オオムギすす紋病、オオムギ雲形病、オオムギ小さび病、オオムギ黒変病、オオムギ黒点病などが挙げられる。 Barley diseases include barley powdery mildew, barley ring rot, barley streak, barley blight, barley soot, barley cloud, barley dwarf, barley black spot, barley black spot, etc. Can be mentioned.
　ライムギの病害としては、ライムギ黄葉病、ライムギ麦角病、ライムギ赤さび病、ライムギ斑点病、ライムギうどんこ病、ライムギ黒変病、ライムギ赤かび病、ライムギ条斑細菌病などが挙げられる。 Examples of diseases of rye include rye yellow leaf disease, rye ergot, rye red rust, rye spot disease, rye powdery mildew, rye black spot disease, rye red mold disease, and rye streak bacterial disease.
　サトウキビの病害としては、サトウキビモザイク病、サトウキビ赤すじ病、サトウキビ赤腐病、サトウキビ外皮病、サトウキビ白すじ病、サトウキビべと病、サトウキビわい化病、サトウキビ眼点病、サトウキビ擬似赤すじ病、サトウキビ褐条病、サトウキビ葉枯病、サトウキビ黒穂病、サトウキビ輪斑病、サトウキビさび病などが挙げられる。 Sugarcane diseases include sugarcane mosaic disease, sugarcane red stripe disease, sugarcane red rot, sugarcane peel disease, sugarcane white stripe disease, sugarcane downy mildew, sugarcane dwarf disease, sugarcane eye spot disease, sugarcane pseudo red stripe disease, Examples include sugarcane brown stripe disease, sugarcane leaf blight, sugarcane smut, sugarcane ring spot disease, and sugarcane rust disease.
　これらの中では特に、イネ科植物に広く感染するいもち病菌（Magnaporthe oryzae）によって引き起こされるいもち病に有効である。 Among these, it is particularly effective for blast caused by blast fungus (Magnaporthe oryzae) that is widely infecting gramineous plants.
　また、本発明の植物病害防除法は、病害の予防を主な目的としており、病害が発生する時期に先駆けて使用することが好ましい。ただし、病害の発生後であってもその拡大を抑制したり、病害を減弱する効果は期待できる。 Further, the plant disease control method of the present invention has a main purpose of preventing disease, and is preferably used prior to the time when the disease occurs. However, even after the occurrence of a disease, an effect of suppressing the expansion or attenuating the disease can be expected.
　病害抵抗性増強剤を、イネ科植物に施用することにより、上記のような病害に対する抵抗性を増強することができる。施用の方法は特に制限されず、その剤形および水田または畑の状態、農業従事者の事情に応じた方法で用いられ得る。例えば、植物の生長点のみならず、茎や葉をはじめとする植物体の一部又は全体に液剤や乳剤として散布、滴下あるいは塗布等することができる。またその他にも根圏への施用、例えば土壌への表面散布、潅注、土壌への混合、又は根への浸漬処理が挙げられる。これらの中では、根圏施用が好ましい。
　尚、実施例に示されるように、本発明のイネ科植物病害抵抗性増強剤を根圏に施用した場合、地上部では、全身獲得抵抗性またはプライミングが誘導されることが示唆される。 By applying a disease resistance enhancer to a gramineous plant, resistance to the above diseases can be enhanced. The method of application is not particularly limited, and can be used by a method according to the dosage form, the state of paddy field or field, and the situation of the farmer. For example, it can be sprayed, dripped or applied as a liquid or an emulsion to not only the plant growth point but also a part or the whole of the plant body including stems and leaves. In addition, application to the rhizosphere, for example, surface application to the soil, irrigation, mixing into the soil, or immersion treatment in the root can be mentioned. Of these, rhizosphere application is preferred.
In addition, as shown in the Examples, it is suggested that when the gramineous plant disease resistance enhancer of the present invention is applied to the rhizosphere, systemic acquired resistance or priming is induced in the above-ground part.
　本イネ科植物病害抵抗性増強剤のイネ科植物への投与については、その頻度は投与目的やイネ科植物個体の生育ステージ等により異なる。例えば、田植え前であれば苗床に施用することで幼病の病害を抑制できる。 Regarding the administration of this Gramineae plant disease resistance enhancer to gramineous plants, the frequency varies depending on the purpose of administration, the growth stage of gramineous plants, and the like. For example, if it is before rice planting, the disease of a young disease can be suppressed by applying to a nursery.
　イネ科植物病害抵抗性増強剤の施用量は、有効成分の濃度、施用時期、施用回数、植物の種類、栽培密度、生育段階、施用方法等によっても異なり得る。葉面散布では、施用量は、アミノ酸の量として、例えば、通常０．２～２００ｍＭを１００～５０００Ｌ／ヘクタール、好ましくは２～１００ｍＭを５００～１０００Ｌ／ヘクタールである。複数のアミノ酸を併用する場合は、合計量で通常０．２～２００ｍＭを１００～５０００Ｌ／ヘクタール、好ましくは２～１００ｍＭを５００～１０００Ｌ／ヘクタールが好ましい。また、イネ科植物病害抵抗性増強剤を根圏に施用する場合も、施用量は上記の葉面散布と同様である。
　アミノ酸を含む病害抵抗性増強剤と、アミノ酸以外の病害抵抗性誘導剤は、別々にイネ科植物に施用してもよい。 The application amount of the gramineous plant disease resistance enhancer may vary depending on the concentration of the active ingredient, the application time, the frequency of application, the type of plant, the cultivation density, the growth stage, the application method, and the like. In foliar spraying, the application rate is usually, for example, 0.2 to 200 mM at 100 to 5000 L / ha, preferably 2 to 100 mM at 500 to 1000 L / ha, as the amount of amino acid. When a plurality of amino acids are used in combination, the total amount is usually 0.2 to 200 mM at 100 to 5000 L / ha, preferably 2 to 100 mM at 500 to 1000 L / ha. In addition, when a gramineous plant disease resistance enhancer is applied to the rhizosphere, the application amount is the same as that in the foliar application.
The disease resistance enhancer containing an amino acid and the disease resistance inducer other than the amino acid may be separately applied to the grass family plant.
　以下、実施例をもって本発明をさらに具体的に説明するが、本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
〔実施例１〕アミノ酸によるイネいもち病の防除効果
（１）植物体の栽培と散布方法
　イネ（品種「日本晴」）の栽培は以下のように行った。園芸用培養土（パワーソイル; 呉羽化学工業株式会社）とバーミキュライト（株式会社エス・ケー・アグリ）を４：１の割合で混合した土をポット（ＢＥＥ　ＰＯＴ；キャネロン化工株式会社）に入れ、催芽させたイネを播種し、１４日間栽培した。培養庫内の温度は23～25℃、日周は14時間明期、光強度はおよそ100μmol m-2 s-1で栽培した。
　アミノ酸のイネの根圏への施用は、フードパック（中央化学株式会社、Ｃ－ＡＰフルーツ２００）に３００ｍｌの溶液を入れ、そこにイネをポットごと浸漬することで行った。またアミノ酸の葉面への直接散布は、同様の手順で生育させたイネの第４葉に、ミックスパワー（シンジェンタジャパン）を０．１％（ｖ／ｖ）加えた各溶液を噴霧することにより行った。 [Example 1] Effect of amino acid on control of rice blast (1) Cultivation and application method of plants Rice (cultivar "Nihonbare") was cultivated as follows. Putting soil mixed with cultivated soil for horticulture (Powersoil; Kureha Chemical Co., Ltd.) and vermiculite (S.K. The sowed rice was sown and cultivated for 14 days. The incubator was cultivated at a temperature of 23 to 25 ° C., a daily period of 14 hours light, and a light intensity of about 100 μmol m −2 s −1 .
Application of amino acids to the rhizosphere of rice was performed by putting 300 ml of the solution in a food pack (Chuo Chemical Co., Ltd., C-AP fruit 200) and immersing the rice in the pot there. In addition, the direct application of amino acids onto the leaf surface is performed by spraying each solution with 0.1% (v / v) mix power (Syngenta Japan) onto the fourth leaf of rice grown in the same procedure. went.
（２）イネいもち病防除評価
　１０ｍＭ Ｌ－グルタミン酸ナトリウム（Ｇｌｕ）、１０ｍＭ Ｌ－アラニン（Ａｌａ）、１０ｍＭ Ｌ－アスパラギン（Ａｓｎ）、１０ｍＭ Ｌ－アスパラギン酸ナトリウム（Ａｓｐ）、１０ｍＭ Ｌ－フェニルアラニン（Ｐｈｅ）、１０ｍＭ Ｌ－スレオニン（Ｔｈｒ）、１０ｍＭ Ｌ－バリン（Ｖａｌ）、又は１０ｍＭ Ｌ－アルギニン（Ａｒｇ）を、それぞれ播種して２週間後のイネの根圏、又は葉面に施用した。根圏施用の場合はその48時間後に、葉面施用の場合は24時間後に、イネいもち病菌（Magnaporthe oryzae）をイネに接種した。根圏施用は、前記のとおりポットをアミノ酸溶液に浸漬することにより、葉面施用は直接散布により、各々行った。イネいもち病菌の接種は、分生胞子懸濁液（1×105 分生胞子/mL）を葉面に噴霧することにより行った。噴霧接種後、暗所、湿室下に24時間静置することにより、イネいもち病菌を植物体に感染させた。接種４日後に各処理葉に発生した罹病性病斑数を測定することで、評価を行った。アミノ酸溶液の代りに、ネガティブコントロール区には水を、葉面散布試験ではポジティブコントロールとして0.5mMプロベナゾールを用いた。防除価は以下の式に従って算出した。３～４回実験を繰り返し、それぞれの実験における防除価の平均を図１および図２に示す。 (2) Rice Blast Control Evaluation 10 mM L-glutamate sodium (Glu), 10 mM L-alanine (Ala), 10 mM L-asparagine (Asn), 10 mM L-sodium aspartate (Asp), 10 mM L-phenylalanine (Phe) 10 mM L-threonine (Thr), 10 mM L-valine (Val), or 10 mM L-arginine (Arg) was applied to the rhizosphere or foliage of rice two weeks after sowing. Rice was inoculated with rice blast fungus (Magnaporthe oryzae) 48 hours after rhizosphere application and 24 hours after foliar application. The rhizosphere application was performed by immersing the pot in an amino acid solution as described above, and the foliar application was performed by direct spraying. Inoculation of rice blast fungus was performed by spraying the conidia suspension (1 × 10 5 conidia / mL) on the leaf surface. After spray inoculation, the plant was infected with rice blast fungus by leaving it in a dark place for 24 hours. Evaluation was carried out by measuring the number of diseased lesions occurring on each treated leaf 4 days after the inoculation. Instead of the amino acid solution, water was used in the negative control group, and 0.5 mM probenazole was used as a positive control in the foliar spray test. The control value was calculated according to the following formula. The experiment was repeated 3 to 4 times, and the average control value in each experiment is shown in FIG. 1 and FIG.
　防除価＝（１－各区一葉当たりの平均病斑数／ネガティブコントロール区一葉当たりの平均病斑数）×１００ Control value = (1-average number of lesions per leaf in each ward / average number of lesions per leaf in the negative control ward) x 100
　アミノ酸根圏施用による防除価を図１に示す。ネガティブコントロール区と比較し、いずれのアミノ酸も根圏施用により有意に病原菌の感染を防除できることが示された。また、Ｌ－フェニルアラニン以外の各アミノ酸は、Ｌ－フェニルアラニンよりも高い感性防御効果を示した。 Fig. 1 shows the control value by application of amino acid rhizosphere. Compared with the negative control group, it was shown that any amino acid can significantly control the infection of pathogenic bacteria by applying the rhizosphere. In addition, each amino acid other than L-phenylalanine showed a higher sensitivity defense effect than L-phenylalanine.
　アミノ酸葉面散布による防除価を図２に示す。ネガティブコントロール区と比較し、アミノ酸の葉面散布により有意に病原菌の感染を防除できることが示された。 Fig. 2 shows the control value by spraying amino acid leaves. Compared to the negative control group, it was shown that infection with pathogenic bacteria can be significantly controlled by foliar application of amino acids.
　本実験からアミノ酸による防除効果は葉面散布よりも根圏施用の方がより高いことが示された。 This experiment showed that the control effect by amino acids was higher in the rhizosphere application than in foliar application.
　また、根圏施用により直接アミノ酸が接触していない葉部でも抵抗性が誘導されていることから、全身的に病害抵抗性が誘導されていることが示された。 Moreover, since the resistance was induced even in the leaf portion where the amino acid was not in direct contact with the rhizosphere application, it was shown that disease resistance was induced systemically.
〔実施例２〕病害応答遺伝子の発現上昇を指標とした植物病害抵抗性誘導の評価
（１）植物体の栽培と施用方法
　イネは、培養庫内の温度は２８～２５℃、日周は１４時間明期、光強度はおよそ３００００Ｌｘで栽培した。イネ品種は日本晴を用いた。籾殻を取り除いたイネ種子を２日間、暗下、３０℃で催芽させた。園芸用培養土（パワーソイル; 呉羽化学工業（株））とバーミキュライト（（株）エス・ケー・アグリ）を４：１の割合で混合した土をポット（ＢＥＥ　ＰＯＴ；キャネロン化工（株））に入れ、催芽させたイネを播種し、１４日間栽培した。被検試料のイネの根圏施用は、フードパック（中央化学（株）、Ｃ－ＡＰフルーツ２００）に３００ｍｌの溶液を入れ、そこにイネをポットごと浸漬することで行った。 [Example 2] Evaluation of plant disease resistance induction using the increase in the expression of disease response gene as an index (1) Cultivation and application method of plant body The temperature in the incubator is 28-25 ° C and the daily rate is 14 During the light season, the light intensity was cultivated at about 30000 Lx. Nihonbare was used as the rice variety. Rice seeds from which rice husks were removed were germinated at 30 ° C. in the dark for 2 days. Garden soil (power soil; Kureha Chemical Co., Ltd.) and vermiculite (SK Aguri Co., Ltd.) mixed in a ratio of 4: 1 to pot (BEE POT; Caneron Chemical Co., Ltd.) The sown rice was sown and cultivated for 14 days. The rhizosphere application of rice as a test sample was performed by putting 300 ml of the solution in a food pack (Chuo Chemical Co., Ltd., C-AP fruit 200) and immersing the rice in the pot there.
（２）ＲＮＡ抽出と定量ＲＴ－ＰＣＲ
　イネの病害抵抗性誘導評価は、根および第４葉を用いて行なった。
　病害応答遺伝子の転写量を定量するため、上記のようにして栽培したイネから根又は第４葉をサンプリング後、直ちに液体窒素で凍結し、-80℃で保存した。凍結状態のまま植物破砕機（MM300 MIXER MILL GRINDER；Retsch)により破砕した。EZ1 RNA Tissue Mini Kit（キアゲン社）およびMagtration System 12Gc（キアゲン社）を用いてＲＮＡ抽出を行った後、ＰＣＲ装置（7500Real Time PCR System；Applied Biosystems社）を用い、定量的ＰＣＲによりキチナーゼ３遺伝子、OsＮＰＲ１遺伝子、ＷＲＫＹ４５遺伝子、及びＰＲ１０（ＰＢＺ１）遺伝子の発現量の比較を行った。内部標準には真核生物転写伸張因子ｅＥＦ－１ａ遺伝子を用いた。
　キチナーゼ遺伝子、ＮＰＲ１遺伝子、ＰＢＺ１（ＰＲ１０）遺伝子（分子レベルから見た植物の耐病性－ポストゲノム時代の植物免疫研究－　島本功ら　秀潤社　２００４）、及びＷＲＫＹ４５（Shimono, M. et. al., Plant Cell,2007,19:2064-2076）は、病害抵抗性との関連が知られている。 (2) RNA extraction and quantitative RT-PCR
Evaluation of disease resistance induction of rice was performed using the root and the fourth leaf.
In order to quantify the transcription amount of the disease response gene, the root or the fourth leaf was sampled from the rice cultivated as described above, immediately frozen in liquid nitrogen, and stored at -80 ° C. It was crushed with a plant crusher (MM300 MIXER MILL GRINDER; Retsch) in a frozen state. After RNA extraction using EZ1 RNA Tissue Mini Kit (Qiagen) and Magtration System 12Gc (Qiagen), the chitinase 3 gene, by quantitative PCR using a PCR device (7500 Real Time PCR System; Applied Biosystems) The expression levels of the OsNPR1 gene, the WRKY45 gene, and the PR10 (PBZ1) gene were compared. The eukaryotic transcription elongation factor eEF-1a gene was used as an internal standard.
Chitinase gene, NPR1 gene, PBZ1 (PR10) gene (plant disease resistance from the molecular level-plant immunity research in the post-genomic era-Isao Shimamoto et al., Shujunsha 2004), and WRKY45 (Shimono, M. et. Al. Plant Cell, 2007, 19: 2064-2076) is known to be associated with disease resistance.
　評価試験の際における病害応答遺伝子発現の発現を誘導する物質としては、Ｌ－グルタミン酸ナトリウム、Ｌ－アスパラギン、Ｌ－アスパラギン酸ナトリウム、Ｌ－スレオニン、Ｌ－バリン、Ｌ－アルギニン、Ｌ－アラニン、Ｌ－フェニルアラニン、又は比較のために0.6％オリゼメート（明治製菓（株）の登録商標）を用いた。オリゼメートはSARを誘導する薬剤であるプロベナゾールを有効成分とする農薬（粒剤）である。 Substances that induce the expression of disease response gene expression in the evaluation test include sodium L-glutamate, L-asparagine, sodium L-aspartate, L-threonine, L-valine, L-arginine, L-alanine, L -Phenylalanine or 0.6% oryzate (registered trademark of Meiji Seika Co., Ltd.) was used for comparison. Orizemate is an agrochemical (granule) containing probenazole, a drug that induces SAR, as an active ingredient.
　各種アミノ酸溶液をそれぞれ単独の１０ｍＭ濃度でイネ根圏に施用し、根組織における６時間後の遺伝子の転写量を定量した結果を図３に示す。図３には、それぞれ３反復の実験の平均値と標準誤差（ＳＥ）を示し、グルタミン酸、アスパラギン、アスパラギン酸、スレオニン、バリン、アルギニン、アラニン、又はフェニルアラニンを施用することで根組織におけるキチナーゼ３、OsＮＰＲ１、ＷＲＫＹ４５、ＰＲ１、ＰＲ１０（ＰＢＺ１）の各遺伝子の転写活性が上昇することが明らかとなった。このことは、根組織において病害応答が起こっていることを示している。尚、各アミノ酸による各遺伝子の転写活性の上昇は、Ｌ－フェニルアラニンと同程度がそれ以上であった。 FIG. 3 shows the results of applying various amino acid solutions to the rice rhizosphere at a single concentration of 10 mM, respectively, and quantifying the amount of gene transcription in root tissues after 6 hours. FIG. 3 shows the mean and standard error (SE) of three replicate experiments, respectively, and chitinase 3 in root tissue by applying glutamic acid, asparagine, aspartic acid, threonine, valine, arginine, alanine, or phenylalanine, It was revealed that the transcriptional activity of each gene of OsNPR1, WRKY45, PR1, and PR10 (PBZ1) is increased. This indicates that a disease response is occurring in the root tissue. The increase in the transcriptional activity of each gene by each amino acid was more than that of L-phenylalanine.
　根組織における応答は非常に安定しており、各遺伝子の転写レベルと、いもち病防除効果とが相関していた。このことから、アミノ酸を根圏施用した際に根で起こる応答がいもち病抵抗性を付与する最初の重要なステップであると考えた。
　根圏施用した48時間後の地上部（第４葉）における病害応答遺伝子の転写量を測定したところ、病害応答遺伝子の明確な発現上昇が観察された。WRKY45の高発現株はイネいもち病に対し強い抵抗性を示すことが報告されている（Shimono, M. et. al., Plant Cell,2007,19:2064-2076）。実際にアミノ酸の根圏施用によって、いもち病菌の感染は顕著に抑制されることから、SARが誘導されていると考えられた。 The response in the root tissue was very stable, and the transcription level of each gene was correlated with the blast control effect. From this, it was considered that the response that occurs in the roots when amino acids were applied in the rhizosphere was the first important step in conferring blast resistance.
When the transcription amount of the disease response gene in the above-ground part (4th leaf) 48 hours after application of the rhizosphere was measured, a clear increase in the expression of the disease response gene was observed. It has been reported that a high expression strain of WRKY45 shows strong resistance to rice blast (Shimono, M. et. Al., Plant Cell, 2007, 19: 2064-2076). In fact, the application of amino acids in the rhizosphere significantly suppressed the infection of blast fungus, suggesting that SAR was induced.
〔実施例３〕アミノ酸施用による病害抵抗性誘導の機序
　アミノ酸がケイ酸の吸収を促し、その結果植物の表層の物理的強化により病害抵抗性が付与されるとの報告がある（Voleti, S.R. et al., Crop Protection, 2008, 27:1398-1402）。またケイ酸によってイネ抗菌性物質、ファイトアレキシンの蓄積が誘導されるとの結果が報告されている（Rodorigues, F. A., et al., Phytopathology, 2004, 94:177-183）。 [Example 3] Mechanism of disease resistance induction by amino acid application It has been reported that amino acid promotes absorption of silicic acid and, as a result, disease resistance is imparted by physical strengthening of the surface layer of plants (Voleti, SR et al., Crop Protection, 2008, 27: 1398-1402). It has also been reported that silicic acid induces the accumulation of rice antibacterial substance, phytoalexin (Rodorigues, FA, et al., Phytopathology, 2004, 94: 177-183).
　アミノ酸施用によるSARが、ケイ酸など他の物質を介さない、直接的作用によるものなのかを明らかにするために、下記の実験を行った。 In order to clarify whether the SAR caused by amino acid application is due to direct action not involving other substances such as silicic acid, the following experiment was conducted.
　ムラシゲ・スクーグ培地用混合塩類（日本製薬（株）)４．６ｇ、チアミン塩酸塩０．３ｍｇ、ニコチン酸０．５ｍｇ、ピリドキシン塩酸塩０．０５ｍｇ、ミオイノシトール１００ｍｇ、スクロース３０ｇを１リットルの純水（ミリポア社純水製造装置を使用）に溶解させ、pH5.8に調整後、ゲランガムを３ｇ加えオートクレーブ（１２０℃、２０分）した。滅菌した培養器にオートクレーブ後の培地を所定の量流し込み、冷却させ寒天培地を作製した。イネ（日本晴）の種子を２０％次亜塩素酸ナトリウムで表面殺菌後、寒天培地上に播種し１週間生育させた。クリーンベンチ内でイネを慎重に引き抜き、可能な限り根に付着した培地片を取り除いた後、殺菌済みのプラスチック容器に分注した１０ｍＭ Ｌ－グルタミン酸ナトリウム（Ｇｌｕ）、または１０ｍＭ Ｌ－アスパラギン（Ａｓｎ）に根部を浸した。ネガティブコントロールには水を、ポジティブコントロールには0.5mM プロベナゾールを用いた。４８時間、２８℃、３００００Ｌｘで培養後、根をサンプリングし、上述の方法でRNAを抽出した。ｃＤＮＡを作製後、定量的ＰＣＲによりキチナーゼ３遺伝子、OsＮＰＲ１遺伝子、ＷＲＫＹ４５遺伝子、ＰＲ１０（ＰＢＺ１）遺伝子の発現量の比較を行った。本実験では内部標準にポリユビキチン遺伝子ＰＵＢＱを用いた。 Mixed water for Murashige-Skoog medium (Nippon Pharmaceutical Co., Ltd.) 4.6g, thiamine hydrochloride 0.3mg, nicotinic acid 0.5mg, pyridoxine hydrochloride 0.05mg, myo-inositol 100mg, sucrose 30g in 1 liter of pure water It was dissolved in (using Millipore pure water production apparatus), adjusted to pH 5.8, 3 g of gellan gum was added, and autoclaved (120 ° C., 20 minutes). A predetermined amount of the autoclaved medium was poured into a sterilized incubator and cooled to prepare an agar medium. Rice (Nipponbare) seeds were sterilized with 20% sodium hypochlorite and then sown on an agar medium and grown for 1 week. Carefully pull out the rice in a clean bench, remove as much of the medium as possible from the roots, and then dispense 10 mM L-sodium glutamate (Glu) or 10 mM L-asparagine (Asn) into a sterilized plastic container. Soaked the roots. Water was used for the negative control and 0.5 mM provenazole was used for the positive control. After culturing at 30000 Lx at 28 ° C. for 48 hours, the roots were sampled and RNA was extracted by the method described above. After preparing cDNA, the expression levels of chitinase 3 gene, OsNPR1 gene, WRKY45 gene, and PR10 (PBZ1) gene were compared by quantitative PCR. In this experiment, polyubiquitin gene PUBQ was used as an internal standard.
　その結果を図４に示す。
　人工培地上で無菌的に生育させたイネの根圏にアミノ酸を施用することでも、ＷＲＫＹ４５、OsNPR1、ＰＲ１ｂ、ＰＢＺ１（ＰＲ１０）、キチナーゼ３の各遺伝子の転写活性が上昇した。このことから、アミノ酸が直接イネの組織に作用することによって病害抵抗性を誘導すること、さらにその経路はケイ酸等土壌に含まれる他の物質を介したものではないことが明らかとなった。 The result is shown in FIG.
The transcription activity of each gene of WRKY45, OsNPR1, PR1b, PBZ1 (PR10) and chitinase 3 was also increased by applying amino acids to the rhizosphere of rice grown aseptically on an artificial medium. From this, it was clarified that amino acid induces disease resistance by directly acting on rice tissues, and that the pathway is not mediated by other substances contained in soil such as silicic acid.
　また本実験により、病害抵抗性は微生物の混入や汚染によって誘起されたものではないことが示された。 Also, this experiment showed that disease resistance was not induced by microbial contamination or contamination.
〔実施例４〕既知の植物病害抵抗性誘導剤とアミノ酸の混合施用による病害抵抗性誘導効果の上昇
　アミノ酸と既知の植物病害抵抗性誘導剤を併用することによって、植物病害抵抗性誘導効果が高まるのかを明らかにするため、以下の実験を行った。 [Example 4] Increase in disease resistance inducing effect by application of a mixture of a known plant disease resistance inducer and an amino acid By using together an amino acid and a known plant disease resistance inducer, the plant disease resistance inducing effect is enhanced. In order to clarify whether this is the case, the following experiment was conducted.
　根組織における病害抵抗性誘導を観察するために、上述のとおり、ムラシゲ・スクーグ培地上で無菌的に栽培したイネの根を１ｍＭプロベナゾール、１ｍＭ Ｌ－グルタミン酸ナトリウム（Ｇｌｕ）、および両者を１ｍＭずつ含む溶液に６時間浸漬させた。
　また、第４葉における病害抵抗性誘導を観察するために、実施例２で記載した手順に従ってイネを生育させ、イネの根を０．３％ オリゼメート、５ｍＭ Ｌ－グルタミン酸ナトリウム（Ｇｌｕ）、および両者を前記濃度で含む溶液に４８時間浸漬した。コントロールとして水を用いた。それぞれの組織をサンプリング後、全ＲＮＡを抽出しｃＤＮＡを合成後、上記の手段でキチナーゼ３遺伝子およびＰＲ１０（ＰＢＺ１）、ＷＲＫＹ45、OsＮＰＲ１の各遺伝子の転写量を定量した。 In order to observe the induction of disease resistance in root tissues, as described above, rice roots aseptically grown on Murashige-Skoog medium contain 1 mM probenazole, 1 mM sodium L-glutamate (Glu), and both contain 1 mM each. It was immersed in the solution for 6 hours.
Further, in order to observe the disease resistance induction in the fourth leaf, rice was grown according to the procedure described in Example 2, the rice root was changed to 0.3% oryzate, 5 mM sodium L-glutamate (Glu), and both Was immersed in the solution containing the above-mentioned concentration for 48 hours. Water was used as a control. After sampling each tissue, total RNA was extracted and cDNA was synthesized, and then the transcription amounts of chitinase 3 gene and PR10 (PBZ1), WRKY45, and OsNPR1 genes were quantified by the above-described means.
　根組織における各遺伝子の発現量の変化を図５に示す。
　プロベナゾール、Ｌ－グルタミン酸それぞれ単独の場合に比べ、両者を併用することによって、根組織での各遺伝子発現量が顕著に上昇することが明らかとなった。 FIG. 5 shows changes in the expression level of each gene in the root tissue.
It has been clarified that the expression level of each gene in root tissue is significantly increased by using both in combination with probenazole and L-glutamic acid alone.
　第４葉における各遺伝子の発現量の変化を図６に示す。
　オリゼメートとＬ－グルタミン酸を併用することで、４葉部での各遺伝子発現量が上昇することが明らかとなった。 The change in the expression level of each gene in the fourth leaf is shown in FIG.
It was clarified that the expression level of each gene in the four leaves was increased by using oryzate and L-glutamic acid in combination.
　本実験より、アミノ酸以外の植物病害抵抗性誘導剤とアミノ酸を併用することで、全身的に病害応答遺伝子の発現が上昇することが示された。したがって、アミノ酸以外の植物病害抵抗性誘導剤とアミノ酸の併用によって、植物病害抵抗性誘導効果が高まることが示された。本発見により、農薬である植物病害抵抗性誘導剤に非農薬のアミノ酸を添加することで、農薬の使用量を削減できると考えられる。 This experiment shows that the expression of disease-responsive genes increases systemically by using a plant disease resistance inducer other than amino acids in combination with amino acids. Therefore, it was shown that the plant disease resistance inducing effect is enhanced by the combined use of an amino acid and a plant disease resistance inducer other than amino acids. With this discovery, it is considered that the use of pesticides can be reduced by adding non-pesticide amino acids to the plant disease resistance inducers that are pesticides.
〔実施例６〕アミノ酸のエナンチオマー間での病害抵抗性誘導効果の違い
　タンパク質を構成するＬ－アミノ酸だけでなく、生体内にわずかしか存在しないＤ－体にも病害抵抗性を誘導する効果があるのか評価を行った。 [Example 6] Difference in disease resistance-inducing effect between amino acid enantiomers In addition to L-amino acids constituting proteins, there is an effect of inducing disease resistance not only in D-forms that are present in the living body in a small amount. It was evaluated.
　上述のとおり、ムラシゲ・スクーグ培地上で無菌的に栽培したイネの根を、１ｍＭのＤ－またはＬ－体のグルタミン酸（Ｇｌｕ）、セリン（Ｓｅｒ）またはバリン（Ｖａｌ）溶液にそれぞれ６時間浸漬した。ポジティブコントロールとして１ｍＭプロベナゾールを施用した。サンプリング後全ＲＮＡを抽出しｃＤＮＡを合成後、上記の方法でキチナーゼ３およびＰＲ１０（ＰＢＺ１）、ＷＲＫＹ４５の各遺伝子の転写量を定量した。 As described above, rice roots grown aseptically on Murashige-Skoog medium were soaked in 1 mM D- or L-form glutamic acid (Glu), serine (Ser) or valine (Val) solution for 6 hours, respectively. . As a positive control, 1 mM probenazole was applied. After sampling, total RNA was extracted to synthesize cDNA, and the transcription amounts of chitinase 3, PR10 (PBZ1), and WRKY45 genes were quantified by the above method.
　その結果を図７に示す。
　バリンはＬ－体のほうがＤ－体よりも強く病害抵抗性を誘導したが、グルタミン酸の場合、両者はほぼ同レベルであった。これに対しセリンはＤ－体がより強く病害抵抗性を誘導した。このことは、効果の差はあることしても、Ｌ－体だけでなくＤ－体でも病害抵抗性誘導効果が期待されることを示している。 The result is shown in FIG.
Valine induced disease resistance in the L-form more strongly than the D-form, but in the case of glutamic acid, both levels were almost the same. In contrast, serine induced disease resistance more strongly in the D-form. This shows that disease resistance-inducing effects are expected not only in the L-form but also in the D-form, even though there are differences in the effects.
〔実施例７〕アミノ酸によるシバの病害抵抗性誘導
　イネ以外のイネ科植物においてもアミノ酸によって病害抵抗性が誘導されるのか、西洋シバ（Poa pratensis）を用いて以下の実験を行った。病害抵抗性誘導の指標としてキチナーゼ活性を用いた。 [Example 7] Induction of disease resistance of buckwheat by amino acids Whether disease resistance is induced by amino acids in rice plants other than rice, the following experiment was carried out using Poa pratensis. Chitinase activity was used as an index of disease resistance induction.
　キチナーゼは糸状菌の細胞壁成分であるキチンを分解する酵素であり、植物の防御機構の一端を担っていると考えられている。そのため、その活性上昇は病害応答の指標の一つとして広く認識されている。 Chitinase is an enzyme that degrades chitin, which is a cell wall component of filamentous fungi, and is thought to play a part in the defense mechanism of plants. Therefore, the increase in activity is widely recognized as one of the indicators of disease response.
　ケンタッキーブルーグラス（タキイ種苗株式会社）をイネと同様の条件で3週間栽培し、５ｍＭのＬ－グルタミン酸ナトリウム溶液に根圏を24時間浸漬させた。 Kentucky bluegrass (Takii Tanae Co., Ltd.) was cultivated for 3 weeks under the same conditions as rice, and the rhizosphere was immersed in a 5 mM sodium L-glutamate solution for 24 hours.
　地上部組織をサンプリング後、直ちに液体窒素で凍結し、-80℃で保存した。凍結状態のまま植物破砕機MM300 MIXER MILL GRINDER (Retsch)により破砕し、300μLの抽出バッファー［100 mM NaH2PO4 / Na2HPO4(pH6.0), 1 mM DTT, protease inhibitor/complete mini EDTA free (Roche社)］に懸濁した。10,000 rpm 5分間の遠心分離後の上清を、0.22μmフィルターに通し不溶物を除去した。得られた画分を粗抽出画分とし、Bradford法によるタンパク質濃度測定後、酵素活性測定に用いた。 After sampling the above-ground tissue, it was immediately frozen in liquid nitrogen and stored at -80 ° C. Crush it with a plant crusher MM300 MIXER MILL GRINDER (Retsch) in a frozen state and extract 300 μL of extraction buffer [100 mM NaH 2 PO 4 / Na 2 HPO 4 (pH 6.0), 1 mM DTT, protease inhibitor / complete mini EDTA free (Roche)]. The supernatant after centrifugation at 10,000 rpm for 5 minutes was passed through a 0.22 μm filter to remove insoluble matters. The obtained fraction was used as a crudely extracted fraction, which was used for enzyme activity measurement after protein concentration measurement by Bradford method.
　キチナーゼ活性は、McCreathらによる方法（McCreath, K. et al., J. Microbiol. Methods 14:229-237, 1992）により測定した。基質である4MU-(GlcNAc)3 (4-methylumbelliferyl-β-d-N,N',N''-triacetylchitobiose; SIGMA M5639)は、最終濃度0.4mMになるように50% エタノール中に溶解し、-20℃で保存した。使用時に10倍に希釈し、基質溶液とした。上記粗抽出画分を1μg/μLに調整した。各試料50μLずつを96穴プレート上で37℃10分間プレインキュベーションした後に、基質溶液50μLを添加し37℃で反応を開始した。 Chitinase activity was measured by the method by McCreath et al. (McCreath, K. et al., J. Microbiol. Methods 14: 229-237, 1992). The substrate 4MU- (GlcNAc) 3 (4-methylumbelliferyl-β-dN, N ', N''-triacetylchitobiose; SIGMA M5639) was dissolved in 50% ethanol to a final concentration of 0.4 mM, and -20 Stored at ° C. At the time of use, it was diluted 10 times to obtain a substrate solution. The crude extract fraction was adjusted to 1 μg / μL. After 50 μL of each sample was preincubated on a 96-well plate at 37 ° C. for 10 minutes, 50 μL of the substrate solution was added and the reaction was started at 37 ° C.
　反応開始後60分後、及び120分後に、反応液に100μLの1M Gly/NaOH buffer (pH 10.2)を添加し、反応を停止した。反応、及び反応停止は96ウェルプレート上で行い、最終量200μLとした。液面の泡を完全に除去した後に、蛍光検出用プレートリーダーSpectraMax M2 (Molecular Devices）を用いて蛍光強度を測定した。蛍光測定では、360 nmエキサイテイーション、450 nmエミッションにより測定した。4MU(4-methylumbelliferone)を標準物質として求めた標準値にもとづき、1分間に1μmol反応する酵素量を1ユニットと定義した。 After 60 minutes and 120 minutes after the start of the reaction, 100 μL of 1M Gly / NaOH buffer (pH 10.2) was added to the reaction solution to stop the reaction. Reaction and reaction termination were performed on a 96-well plate to a final volume of 200 μL. After the bubbles on the liquid surface were completely removed, the fluorescence intensity was measured using a fluorescence detection plate reader SpectraMax M2 (Molecular Devices). In the fluorescence measurement, the measurement was performed by 360 nm excitation and 450 nm emission. Based on the standard value obtained using 4MU (4-methylumbelliferone) as a standard substance, the amount of enzyme that reacts at 1 μmol per minute was defined as 1 unit.
　その結果を図８に示す。
　Ｌ－グルタミン酸を根圏処理することによって、地上部組織中のキチナーゼ活性が上昇した。 The result is shown in FIG.
The rhizosphere treatment with L-glutamic acid increased chitinase activity in the above-ground tissue.
　本実験より、シバにおいてもアミノ酸によって病害抵抗性が誘導されることが明らかとなったと同時に、イネ以外の他のイネ科植物においても、アミノ酸によって病害抵抗性が増強される可能性が高いことが示された。 From this experiment, it was clarified that disease resistance was induced by amino acids in shiba, and at the same time, in other grasses other than rice, there is a high possibility that disease resistance would be enhanced by amino acids. Indicated.
〔実施例８〕アミノ酸によるトウモロコシ病害抵抗性誘導
　アミノ酸によって病害抵抗性誘導がイネ科植物に広く観察されるされる現象であるのかさらに検証するため、トウモロコシを用いた実験を行った。 [Example 8] Induction of maize disease resistance by amino acids In order to further verify whether induction of disease resistance by amino acids is a phenomenon widely observed in gramineous plants, experiments using maize were conducted.
　実施例６と同様、キチナーゼ活性の上昇を抵抗性誘導の指標とした。トウモロコシ品種、ハニーバンタム（株式会社サカタのタネ）を播種後２週間栽培し、５ｍＭのＬ－グルタミン酸ナトリウム溶液に根圏を24時間浸漬させた。第４葉をサンプリング後、上述と同様の手順でタンパク質を抽出しキチナーゼ活性を測定した。 As in Example 6, an increase in chitinase activity was used as an index for resistance induction. A corn variety, Honey Bantam (Sakata Seed Co., Ltd.) was cultivated for 2 weeks after sowing, and the rhizosphere was immersed in a 5 mM sodium L-glutamate solution for 24 hours. After sampling the fourth leaf, the protein was extracted by the same procedure as described above, and the chitinase activity was measured.
　その結果を図９に示す。
　Ｌ－グルタミン酸を根圏処理することによって、トウモロコシの葉組織中のキチナーゼ活性が上昇することが明らかとなった。 The result is shown in FIG.
It has been clarified that the chitinase activity in the corn leaf tissue is increased by the rhizosphere treatment of L-glutamic acid.
　本実験より、トウモロコシにおいてもアミノ酸によって病害抵抗性が誘導されること明らかとなり、アミノ酸による抵抗性誘導はイネ科植物に広く起こる現象であることが示唆された。 From this experiment, it became clear that disease resistance was induced by amino acids in corn, and it was suggested that resistance induction by amino acids is a phenomenon that occurs widely in gramineous plants.
　本発明のイネ科植物病害抵抗性増強剤は、アミノ酸を有効成分とするものであり、安全性が高く、安価に製造することができる。また、本発明の方法により、イネ科植物の病害抵抗性を効果的に増強することができる。 The gramineous plant disease resistance enhancer of the present invention comprises an amino acid as an active ingredient, is highly safe and can be produced at low cost. Moreover, the disease resistance of gramineous plants can be effectively enhanced by the method of the present invention.

Claims (11)

  1. 　グルタミン酸、アスパラギン、アスパラギン酸、スレオニン、アラニン、バリン、アルギニン、及びセリンからなる群から選ばれるアミノ酸を含むイネ科植物の病害抵抗性増強剤。 A disease resistance enhancer for gramineous plants comprising an amino acid selected from the group consisting of glutamic acid, asparagine, aspartic acid, threonine, alanine, valine, arginine, and serine.
  2. 　根圏施用又は葉面散布によりイネ科植物に施用される、請求項１に記載の病害抵抗性増強剤。 The disease resistance enhancer according to claim 1, which is applied to gramineous plants by rhizosphere application or foliar application.
  3. 　イネ科植物がイネ、シバ、又はトウモロコシである、請求項１又は２に記載の病害抵抗性増強剤。 The disease resistance enhancer according to claim 1 or 2, wherein the gramineous plant is rice, shiba or corn.
  4. 　病害が、イネ科植物いもち病、又はイネ白葉枯病である、請求項１～３のいずれか一項に記載の病害抵抗性増強剤。 The disease resistance enhancer according to any one of claims 1 to 3, wherein the disease is Gramineae blast or rice leaf blight.
  5. 　前記アミノ酸を、合計量で０．２～２００ｍＭ含む、請求項１～４のいずれか一項に記載の病害抵抗性増強剤。 The disease resistance enhancer according to any one of Claims 1 to 4, wherein the amino acid is contained in a total amount of 0.2 to 200 mM.
  6. 　前記アミノ酸がＬ－体である、請求項１～５のいずれか一項に記載の病害抵抗性増強剤。 The disease resistance enhancer according to any one of claims 1 to 5, wherein the amino acid is L-form.
  7. 　前記アミノ酸がＤ－体である、請求項１～５のいずれか一項に記載の病害抵抗性増強剤。 The disease resistance enhancer according to any one of claims 1 to 5, wherein the amino acid is D-form.
  8. 　さらに、アミノ酸以外の病害抵抗性誘導剤を含む、請求項１～７のいずれか一項に記載の病害抵抗性増強剤。 The disease resistance enhancer according to any one of claims 1 to 7, further comprising a disease resistance inducer other than amino acids.
  9. 　グルタミン酸を含む、請求項８に記載の病害抵抗性増強剤。 The disease resistance enhancer according to claim 8, comprising glutamic acid.
  10. 　請求項１～９のいずれか一項に記載の病害抵抗性増強剤をイネ科植物に施用することを特徴とする、イネ科植物の病害を防除する方法。 A method for controlling a disease of a gramineous plant, comprising applying the disease resistance enhancer according to any one of claims 1 to 9 to the gramineous plant.
  11. 　前記病害抵抗性増強剤を、前記アミノ酸の量として、０．２～２００ｍＭ／１００～５０００Ｌ／ヘクタールの施用量で施用する、請求項１０に記載の方法。 The method according to claim 10, wherein the disease resistance enhancer is applied at an application rate of 0.2 to 200 mM / 100 to 5000 L / ha as the amount of the amino acid.
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